health promoting effects of bioactive compounds in plants: targeting...

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Health promoting effects of bioactive compounds in plants:

Targeting Type 2 diabetes

PhD Thesis

Sumangala Bhattacharya

Department of food Science

Faculty of Science and Technology

Aarhus University

July, 2013

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Main Supervisor:

Niels Oksbjerg

Senior scientist, Department of Food Science, Aarhus University, Denmark

Co-supervisor:

Jette Feveile Young

Associate Professor, Department of Food Science, Aarhus University, Denmark

Assessment committee:

Marianne Hammershøj (chairperson),

Associate Professor,

Department of Food Science,

Aarhus University,

Denmark.

Lars Bohlin,

Professor,

Department of Pharmacy,

University of Uppsala,

Sweden.

Charles S. Bestwick,

Rowett Institute of Nutrition and Health,

University of Aberdeen,

UK

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Preface

The present PhD thesis is the outcome of research conducted to identify plants with anti-diabetic

properties, and their intrinsic bioactive compound(s) responsible for this activity. An effort to elucidate

the signaling pathways mediated by these compounds has also been made.

This project is part of the main project ‘Health promoting effects of bioactive compounds in

plants’, which was conceived to identify and isolate bioactive compounds from selected plants and

vegetables to address their possible role in lifestyle diseases like T2D and obesity, by exposing them to

a bioassay based screening platform and bioactivity guided fractionation and chromatographic

separation. The project consisted of 6 work packages, of which, this work belongs to the 5th

.

The work performed in this thesis on primary porcine myotube cultures was performed at the

Department of Food Science, Aarhus University (Denmark). As part of the PhD education, a 4-month

stay at The Department of Endocrinology, Aarhus University Hospital, resulted in the work on

pancreatic beta cells.

The project was financed by The Danish Council for Strategic Research (Grant no.: 09-063086)

and Graduate School of Agriculture, Food and Environment (SAFE).


July 2013

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Table of contents Abstract ..................................................................................................................................................... 5

Dansk resumé ............................................................................................................................................ 7

List of attached manuscripts ..................................................................................................................... 9

List of supporting manuscripts ................................................................................................................ 10

Abbreviations .......................................................................................................................................... 11

1. Introduction ..................................................................................................................................... 14

1.1. Muscle loss and T2D ................................................................................................................................ 14

1.2. Need for T2D drug discovery ............................................................................................................... 16

1.3. Tissues and organs involved in T2D ................................................................................................. 16

1.4. Pancreatic beta cells in T2D ................................................................................................................. 18

1.5. Skeletal muscles and insulin resistance .......................................................................................... 18

1.6. Oxidative stress and insulin resistance ........................................................................................... 19

1.7. The insulin signaling pathway ............................................................................................................ 20

1.8. The AMPK signaling pathway .............................................................................................................. 21

1.9. The Glucosamine pathway .................................................................................................................... 23

1.10. Plant kingdom as a drug depot ........................................................................................................... 23

1.11. Plants and experimental models used ............................................................................................. 23

1.11.1. Selected medicinal plants studied.................................................................................. 23

1.11.2. Primary porcine myotube cultures as a model for skeletal muscles ............................. 26

1.11.3. INS 1E cells as a model for pancreatic beta cells ......................................................... 27

1.12. Overview of the main project: Test for bioactivities in different work packages ........... 28

1.13. Hypothesis for WP5 ................................................................................................................................. 29

1.14. Study structure .......................................................................................................................................... 29

2. Study I: Study of plant extracts in satellite cell derived primary porcine myotube cultures .......... 31

2.1. Objective: ..................................................................................................................................................... 31

2.2. Methods: ...................................................................................................................................................... 31

2.2.1. Cell proliferation and myotube viability ........................................................................... 31

2.2.2. Statistics ............................................................................................................................ 32

2.3. Results: ......................................................................................................................................................... 33

2.4. Discussion ................................................................................................................................................... 51

2.5. Short summary:......................................................................................................................................... 55

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3. Study II: Studying the influence of selected elderflower compounds and related polyphenols in

clonal beta cells ....................................................................................................................................... 56

3.1. Objective: ..................................................................................................................................................... 56

3.2. Methods: ...................................................................................................................................................... 56

3.3. Results: ......................................................................................................................................................... 56

3.4. Discussion: .................................................................................................................................................. 57

3.5. Short summary:......................................................................................................................................... 58

4. Study III: Study of the possible signaling pathways behind Naringenin and Falcarinol induced

glucose uptake ......................................................................................................................................... 59

4.1. Objective: ..................................................................................................................................................... 59

4.2. Methods: ...................................................................................................................................................... 59

4.3. Results: ......................................................................................................................................................... 59

4.4. Discussion: .................................................................................................................................................. 59

4.5. Short summary:......................................................................................................................................... 60

5. General Discussion ......................................................................................................................... 61

5.1. Screening of plant extracts, fractions and their secondary metabolites for bioactivity 61

5.1.1. Investigation of oxidative stress by intracellular ROS generation ................................... 61

5.1.2. Study of satellite cell proliferation inducing potential...................................................... 62

5.1.3. Fractionation and further screening of fractions and secondary metabolites .................. 63

5.2. Study of selected phenolic compounds on insulin secretion and gene expression in INS

1E cells 65

5.2.1. Insulin secretion under chronic exposure and glucotoxic conditions ............................... 65

5.2.2. Impact of selected phenolic compounds on beta cell gene expression under glucotoxic

conditions ........................................................................................................................................ 66

5.3. Studying the mechanism behind naringenin and falcarinol induced glucose uptake ... 68

5.3.1. Investigating the dependence on Glut4 and insulin/AMPK signalling ............................. 69

5.3.2. Impact on TBC1D4 and TBC1D1 phosphorylation .......................................................... 71

6. Conclusions and Future perspectives .............................................................................................. 73

7. Reference List ................................................................................................................................. 77

8. Acknowledgements ......................................................................................................................... 89

9. Manuscripts ..................................................................................................................................... 91

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Abstract

Type 2 diabetes (T2D) is a metabolic disorder that has engulfed modern societies in both developed

and developing countries. Insulin resistance in skeletal muscles, resulting in diminished glucose is a

major feature of T2D. Often life-style interventions and exercise regimes are not sufficient to curb such

metabolic disorders, making medication an indispensable part of the treatment. Plant kingdom has

contributed immensely to the modern drug library, and several plants have been used as natural

remedies for diabetes since ancient times.

The primary objective of this study was to screen selected plants with medicinal and/or food

backgrounds for their potential for stimulating glucose uptake in primary porcine myotubes, identify

the inherent compounds responsible, and simultaneously provide a molecular basis for this bioactivity.

The overall study has been divided into three studies. Study I describes the screening of 22

extracts from 8 plants, of which 5 plants, namely, Thymus vulgaris, Daucus carota (bolero, carrots),

Echinacea purpurea, Rhodiola rosea, and Sambucus nigra (elderflowers) were found to stimulate

glucose uptake most prominently. Elderflowers and carrots were chosen for further fractionation, and

the fractions were tested for bioactivity. The bioactive fractions of elderflowers were then separated by

High performance liquid chromatography (by a collaborating group of scientists) and the constituent

compounds together with other related polyphenols were examined for their potential to enhance

glucose uptake. Among these, phenolic acids like caffeic acid, ferulic acid. p-coumaric acid and 5-O-

caffeoylquinic acid; and flavonoids like naringenin and kaempferol showed promising glucose uptake

stimulating potential. Two polyacetylenes know to be present in carrots were separately tested, and

found to be capable of stimulating glucose uptake in myotubes.

In Study II, the phenolic compounds, naringenin, kaempferol, caffeic acid, ferulic acid, p-

coumaric acid, quercetin, and quercetin-3-β-D-glucoside were examined for their insulin secreting

potential in clonal pancreatic beta cells (INS-1E) under acute exposure. Among them, naringenin,

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caffeic acid and quercetin were selected for further investigation under hypoglycemic, hyperglycemic

and glucotoxic conditions. Gene expression studies under glucotoxic conditions were also performed to

analyze the regulation of genes involved in beta-cell functions, stress, survival/apoptosis, and glucose

sensitivity. The genes whose expression was studied were Glut2, Gck, Ins1, Ins2, Beta2, Pdx1, Akt1,

Akt2, Irs1, Acc1, Bcl2, Bax, Casp3, Hsp70, and Hsp90. All three phenolic compounds were found to

increase insulin secretion in INS-1E cells both under hyperglycemic and glucotoxic conditions; and

upregulated insulin, glucokinase, and Hsp 70 and down regulated Acc1 gene expression. The pro-

survival gene Bcl2 was upregulated under normoglycemic conditions but remained unaffected under

glucotoxic conditions by all the phenolic compounds. The phenolic compounds differentially regulated

the gene expression of the other genes studied.

Study III was conducted to provide clues, for elucidation of the mechanism behind the glucose

uptake stimulating potential of naringenin and falcarinol. Glut4 inhibitor indinavir, PI3K inhibitor

wortmannin, and AMPK inhibitor dorsomorphin used in the study indicated the dependence of

naringenin and falcarinol on PI3K and/or its downstream target p-38 MAPK. Their direct dependence

on Glut4 for glucose transport was also demonstrated in this study. The study also confirmed that

active AMPK was required for naringenin to induce glucose uptake in myotubes and revealed that the

same was not true for falcarinol. The phosphorylation/activation of key signaling proteins TBC1D1 and

TBC1D4 (directly involved in Glut4 translocation) by naringenin and falcarinol were also studied.

Naringenin and falcarinol were found to preferentially increase TBC1D1 phosphorylation, as compared

to that of TBC1D4. Wortmannin suppressed naringenin and falcarinol induced phosphorylation,

whereas dorsomorphin suppressed phosphorylation induced by naringenin only; which also confirms

the observation about AMPK-independent glucose uptake by falcarinol.

In conclusion, the results obtained during this PhD study, reveals the multifaceted potential of

certain bioactive plants and their constituent compounds in amelioration of T2D, and provides a

mechanistic clue to elucidate the basis of the observed bioactivity.

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Dansk resumé

Type 2 diabetes (T2D) er en metabolisk sygdom, som omklamrer moderne samfund både i udviklede-

og i udviklende lande. Insulin resistens i skeletmuskulaturen, der resulterer i mindsket

glukoseoptagelse, spiller en stor rolle i T2D. Livsstilsændringer og fysik træning er ikke altid

tilstrækkelige interventioner til at forhindrer sygdommen hvilket gør medicinering nødvendig i

behandlingen af T2D. Planteriget har bidraget markant til en liste af plantemedicin, og i mange år har

adskillige planter været brugt som naturlige værktøjer til behandling af diabetes. Det primære formål

med dette arbejde var at screene udvalgte planter, med medicinsk og/eller fødevarebaggrund, for deres

potentiale m.h.t. at stimulere glukoseoptagelsen i primære porcine myorør i kultur. Videre var formålet,

at identificere plantestoffer som er ansvarlige og samtidig studere det molekylære grundlag for

bioaktivitet. Overordnet inddeles arbejdet i 3 studier.

Studie 1 beskriver screening af 22 ekstrakter fra 8 planter, af hvilke 5 planter, Thymus vulgaris, Daucus

carota (bolero carrots) Echinecia pupurcea, Rhadiola rosea, og Sambucus nigra (hyldeblomst) markant

stimulerede glukoseoptagelsen i myorør. Hyldeblomst og gulerødder blev valgt for yderligere

fraktionering og disse fraktioner blev testet for bioaktivitet. De bioaktive fraktioner af hyldeblomst blev

derefter separeret med højtryksvæske kromatografi (udført af samarbejdspartnere i projektet) og viste at

disse fraktioner sammen med andre relaterede polyfenoler, som caffeic acid, ferulic acid, p-coumaric

acid og 5-O-caffeolytic acid, og flavonoider, som naringinin og kaempferol stimulerede

glukoseoptagelse i myorør. To polyacetylener, som vides at være i gulerødder, stimulerede ligeledes

glukose optagelsen i myorør.

I studie II blev den akutte påvirkning af fenoler som naringinin, kaemferol, caffeic acid, ferulic acid, p-

coumaric acid, quercitin, og quercitin-3-β-D glucoside undersøgt for deres evne til at stimulere insulin

sekretion i pankreas celler (INS-1 celler). Blandt disse blev naringinin, caffeic acid, og quercetin

udvalgt til yderligere undersøgelser under hypo-glukæmiske og glukotoksiske betingelser.

Genekspression under glucotosisk betingelser blev også udført for at undersøge reguleringen af gener,

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der er involveret i beta-celle funktion, stress, overlevelse/celledød og følsomhed over overfor glukose.

De undersøgte gener var: Glut2, Gck, Ins1, Ins2, Beta2, Pdx1, Akt1, Akt2, Irs1, Acc1, Bcl2, Bax,

Casp3, Hsp70 og Hsp90. Alle tre fenoler øgede insulin sekretionen i INS-1 celler under både

hyperglukæmiske og glukotoksiske tilstande, og opregulerede genudtrykkene af insulin, glukokinase og

Hsp70 og nedregulerede Acc1 genudtrykket. Udtrykket af Bcl2 blev opreguleret under

normoglykæmiske betingelser, men forblev uforandret under glukotoksiske betingelser. Fenolerne

regulerede udtrykkene af de øvrige gener i varierende grad.

Studie III blev udført med det formål at undersøge de bagved liggende mekanismer, der er årsag til at

naringinin and falcarinol stimulerer glukoseoptagelsen i myorør. Tilsætning af hæmmeren af Glut4,

indinavir, af PI3K, wortmanin og af AMPK, dorsomorphin antydede, at effekten af naringinin og

falcarinol på glukoseoptagelsen er afhængig af PI3K og/eller dets ”downstream target”, p-38 MAPK.

Betydning af Glut4 transport af glukose blev ligeledes vist efter behandling med naringini og

falcarinol. Studiet viste ligeledes, at aktiveret AMPK er en forudsætning for at stimulere

glukoseoptagelse i myorør efter tilsætning af naringinin og falcarinol. Fosforylering/aktivering af nøgle

signalerende proteiner TBC1D1 og TBC1D4 (påvirker direkte Glut4 translocation) blev også undersøgt

og naringinin og facarinol øgede hovedsageligt TBC1D1. Wortmanin reducerede naringinin og

falcarinol induceret fosforylering, hvorimod dorsomorphin kun reducerede fosphoryleringen efter

tilsætning af naringinin. Dette underbygger observationerne af, at AMPK ikke er nødvendig for

falcarinol induceret glukoseoptagelse.

Overordnet afslører resultaterne fra nærværende afhandling flersidige potentialer af visse planter og

deres indholdsstoffer til fremtidige behandling af T2D og giver desuden indsigt i

reguleringsmekanismer.

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List of attached manuscripts

I. Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine

myotube cultures and reduce fat accumulation in Caenorhabditis elegans

Sumangala Bhattacharya1, Kathrine B. Christensen

2, Louise C. B. Olsen

3, Lars P. Christensen

2, Kai Grevsen

4,

Nils J. Færgeman3, Karsten Kristiansen

5, Jette F. Young

1, and Niels Oksbjerg

1*

(Submitted to ‘The Journal of Agricultural and Food Chemistry’)

II. Caffeic acid, Naringenin and Quercetin enhance glucose stimulated insulin secretion and glucose

sensitivity in INS-1E cells

Sumangala Bhattacharya1, Niels Oksbjerg

1, Jette F. Young

1, and Per Bendix Jeppesen

2

(Submitted to ‘Diabetes, Obesity and Metabolism’)

III. Naringenin and falcarinol stimulate glucose uptake and TBC1D1 phosphorylation in primary porcine

myotube cultures

Sumangala Bhattacharya1, Martin Krøyer Rasmussen

1, Jette F. Young


2, Karsten

Kristiansen2 and Niels Oksbjerg

1*

(To be submitted to ‘Biochemical and Biophysical Communications’)

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List of supporting manuscripts

I. Screening for Bioactive Metabolites in Plant Extracts Modulating Glucose Uptake and Fat

Accumulation

Rime B. El-Houri,1*

Dorota Kotowska,2 Louise C. B. Olsen,

3 Sumangala Bhattacharya,

5 Kathrine B.

Christensen,1 Kai Grevsen,

4 Nils Oksbjerg,

5 Nils Færgeman,

3 Karsten Kristiansen

2 and Lars P. Christensen

1

(Submitted to ‘Plant foods for human nutrition’)

II. Effects of falcarinol and falcarindiol on glucose uptake in adipocytes and muscle cells, PPARγ

transactivation and adipocyte differentiation

Rime B. El-Houri,1*

Dorota Kotowska,2 Sumangala Bhattacharya,

3 Kathrine B. Christensen,

1 Nils Oksbjerg,

3

Karsten Kristiansen2 and Lars P. Christensen

1

(Submitted to ‘Food Chemistry’)

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Abbreviations

5-O-C = 5-O-caffeoylquinic acid

α-LA = alpha-linolenic acid

Acc1 = acetyl CoA carboxylase 1

ADP = Adenosine diphosphate

AICAR = 5-Aminoimidazole-4-carboxamide ribonucleotide

Akt1 = RAC-alpha serine/threonine-protein kinase encoding gene

Akt2 = RAC-beta serine/threonine-protein kinase encoding gene

AMP = Adenosine monophosphate

AMPK = 5' adenosine monophosphate-activated protein kinase

ATP = Adenosine triphosphate

Bax = Bcl-2 associated X protein

Bcl2 = beta-cell lymphoma 2 protein

Beta2 = neurogenic differentiation protein 1

c-AMP = Cyclic adenosine monophosphate

CaA = caffeic acid

CaMKK = Calcium/calmodulin-dependent protein kinase kinase

Casp3 = caspase 3

DM = dorsomorphin

FeA = ferulic acid

FGF = fibroblast growth factor

Gck = glucokinase

GFAT = glutamine: fructose-6-phosphate aminotransferase

Glut1 = Glucose transporter type 1

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GTP = Guanosine triphosphate

HDL = High-density lipoprotein

HGF = Hepatocyte growth factor

Hsp 70 = heat shock protein 70

Hsp90 = heat shock protein 90

I-3-O-G = isorhamnetin-3-O-glucoside

I-3-O-R = isorhamnetin-3-O-rutinoside

Ins1 = insulin 1

Ins2 = insulin 2

IRM = insulin resistant myotubes

IRS = Insulin receptor substrate

IRS1 = insulin receptor substrate 1

K-3-O-R = kaempferol-3-O-rutinoside

LA = linoleic acid

LDL = Low-density lipoprotein

LKB1 = Liver Kinase B1

MAPK = Mitogen-activated protein kinase

p-CA = p-coumaric acid

Pdx1 = pancreatic and duodenal homeobox protein 1

PI3K = Phosphatidylinositide 3-kinase

PKB = Protein kinase B PPAR = Peroxisome proliferator-activated receptor

Q-3-O-6’’-A = quercetin-3-O-6’’-acetylglucoside

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Q-3-O-G = quercetin-3-O-glucoside

Q-3-O-R = quercetin-3-O-rutinoside

ROS = reactive oxygen species

Sirt1 = NAD-dependent deacetylase sirtuin-1

T2D = Type 2 diabetes

TBC1D1 = TBC1 domain family member 1

TBC1D4 = TBC1 domain family member 4

TNF-α = tumor necrosis factor alpha

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1. Introduction

Human beings have treaded the long path of evolution to create a mechanized world for a comfortable

life. However, it is unfortunately associated with its own challenges. Metabolic disorders have been a

part and parcel of developed societies for decades; but as very correctly called ‘an epidemic’ it is

spreading ever so rapidly to the developing countries as well. Type 2 diabetes (T2D) seems to be the

converging point of all the diseases born out of dysfunctional metabolism characterised by insulin

resistance, resulting in glucose intolerance, hyperinsulinemia, hypertension, dyslipidemia and

abdominal obesity (Zinn et al., 2008). T2D predisposes the patient to related ailments like myocardial

ischemia, renal failure, diabetic neuropathy and stroke (Cao and Cooper, 2011; Cheng et al., 2011).

Exercise and dietary restriction has been proved to reduce the detrimental effect of this disease to some

extent, depending on the stage of progression of the disease (Golubovic et al., 2013; Kelley and

Goodpaster, 2001; Potteiger et al., 2012). Nevertheless, anti-diabetic drugs are presently indispensable

for a comprehensive treatment of T2D, especially for physically weak and/or elderly patients and those

who have been detected at an advanced stage of the disease.

1.1. Muscle loss and T2D

Loss of muscle mass and strength is a serious problem, especially observed in the elderly population.

Muscle loss, occurring as a subsequent outcome of chronic diseases such as heart failure, chronic

obstructive pulmonary disorder, cancer, and terminal renal disorder is termed Cachexia (Sakuma and

Yamaguchi, 2012; Thomas, 2007). Loss of muscle mass resulting as a natural consequence of aging, is

known as Sarcopenia (Sakuma and Yamaguchi, 2012). Skeletal muscles, being a major site for glucose

disposal, play a key role in glucose homeostasis. Loss of muscle mass therefore has direct

consequences on glucose utilisation and blood glucose levels. Sarcopenia, together with its common

off-shoot, sarcopenic obesity (where a loss of muscle mass occurs with a simultaneous gain on fat

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mass) has been found to be strongly associated with insulin resistance in adults, irrespective of their

age, implicating a reduction of muscle mass as an autonomous risk factor for T2D and other metabolic

diseases (Srikanthan et al., 2010).

In normal individuals, quiescent satellite cells localized between the basal lamina and

sarcolemma get activated by HGF and FGF during growth or as a response to muscle damage or

physical exercise. Upon activation, they up-regulate the myogenic master transcription factor MyoD

(Berkes and Tapscott, 2005), which marks the beginning of proliferation and finally, either fuse with

each other to form new muscle fibers replacing the damaged fibers, or merger with the existing fibers

to improve muscle protein turnover (Hawke and Garry, 2001). Lack of muscle regeneration and

turnover associated with a sedentary life style (even more so in the elderly), is caused by a decrease in

satellite cell proliferation and differentiation, and has been suggested as a major reason for muscle loss

(Sakuma and Yamaguchi, 2012). Insulin has been found to promote proliferation of skeletal muscle

satellite cell cultures (Dodson et al., 1985). Resistance or strength training, which causes muscle injury,

and thereby induces activation of satellite cells and muscle protein turnover, has also been found to be

beneficial in combating the loss of muscle mass (Evans, 2004). However, amino acid supplementation,

together with resistance training has been found to be more effective in promoting protein anabolism,

resulting in an increase in muscle mass, both in young adults and the elderly (Paddon-Jones et al.,

2004; Yarasheski et al., 1993).

Skeletal muscle consists of different muscle fiber types which differ in their constituent myosin

heavy chain isoforms, and metabolic characteristics. They have been classified into slow-twitch (type

1) and fast-twitch (type 2a, 2x, and 2b) fibers (Schiaffino and Reggiani, 2011). Type 1 and 2a fibers are

oxidative, whereas type 2x and 2b are mainly glycolytic. However the fiber-type characteristics vary

between species (Lefaucheur, 2010; Schiaffino and Reggiani, 2011). Major protein members of the

insulin signaling pathway have been found to be differentially expressed in muscles with different fiber

type combinations, leading to differences in insulin responsiveness (Song et al., 1999). The difference

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in physiology and metabolic characteristics of the fiber types make them respond differentially to

pathophysiological conditions like diabetes, sarcopenia, ageing, denervation sepsis, kidney and heart

failure. The type 1 fibers are more affected by lack of physical activity and denervation-related atrophy.

On the other hand, type 2 fibers are more susceptible to diabetes, heart failure and aging (Macpherson

et al., 2011; Picard et al., 2011; Schiaffino and Reggiani, 2011; von Walden et al., 2012). However,

proteins like PGC1-α, have been found to play a protective role in preventing atrophy in slow-twitch

oxidative fibers (Takikita et al., 2010). Members of the FoxO family, and NF-κB signaling pathway

have also been found to play a key role in fiber-type specific skeletal muscle atrophy (Hunter and

Kandarian, 2004; Sandri et al., 2006).

1.2. Need for T2D drug discovery

Several insulin sensitising drugs, for example, a class of PPAR (peroxisome proliferator-activated

receptors) agonists, known as the thiazolidinediones have been found to be associated with major

adverse side-effects like weight gain, fluid retention in the body, and increased frequency of heart

failure (Shearer and Billin, 2007). Again, anti-diabetic therapies (using for example insulin,

sulfonylureas, or metformin) targeting T2D, by increasing insulin secretion in the secretory pancreatic

beta cells or by inducing glucose uptake in glucose utilizing tissues, have been associated with varying

degrees of contraindications like repeated events of hypoglycemia (Noh et al., 2011). Therefore, the

need for discovery of new compounds that have the potential to be developed into efficient and reliable

drugs is undeniable.

1.3. Tissues and organs involved in T2D

The study of T2D reveals a complex interplay of signals between organs and tissues that takes place in

a metabolically healthy individual, and how the disruption of these signals in T2D patients causes

precipitation of this disease. The skeletal muscles, the adipose tissue, the liver, and the pancreas (figure

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1) are the most important tissues/organs involved in metabolic homeostasis and hence play a major role

in this disease (Corkey and Shirihai, 2012). In the diseased state, insulin resistance in the skeletal

muscles, together with an increase in gluconeogenesis in the liver results in elevated blood glucose

levels compelling the pancreatic beta cells to produce more insulin. This leads to beta cell exhaustion,

and upon prolonged hyperglycemia, beta cell apoptosis (Butler et al., 2003), causing the progression of

the disease. Adipose tissue in obese individuals is infiltrated with macrophages, which secrete certain

cytokines (e.g. TNF-α), that can directly cause insulin resistance in insulin responsive tissues

(Hotamisligil et al., 1993). Literature holds a huge amount of knowledge on this subject. To stay within

the scope of this thesis, only the cells and tissues relevant for the work done in this particular project

will be discussed.

Fig. 1 Major tissues and organs involved in glucose metabolism and T2D [Adopted and modified

from (Dove, 2002)]. Different organs are involved and affected by diabetes, and have been major drug

targets. This figure shows the production and uptake of glucose by different organs and tissues in the

body of healthy subjects. Glucose is released in blood after food intake. This causes insulin secretion

from the pancreas, leading to glucose uptake in skeletal muscles, liver, and adipocytes. Again, during

fasting conditions, glucose levels are maintained in the blood by hepatic gluconeogenesis and

glycogenolysis.

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1.4. Pancreatic beta cells in T2D

Pancreatic islets (Islets of Langerhans) consist of five different kinds of endocrine cells: the glucagon

producing α-cells, the ghrelin producing ξ-cells, the pancreatic polypeptide producing pp- or γ-cells,

the somatostatin producing δ-cells and the insulin producing β-cells (Elayat et al., 1995).

One of the major players involved in the strict regulation of blood glucose levels is the

pancreatic β-cell and is therefore, of critical importance in T2D. In initial stages, the progression of the

disease is asymptomatic. Here, the development and progression of insulin resistance in skeletal muscle

is not manifested by an increase in blood glucose. During this phase of the disease progression, the

pancreatic β-cells compensate the growing insulin resistance by producing more insulin, keeping blood

glucose levels within normal limits (Polonsky, 2000). However, beyond this point, the growing insulin

insensitivity in the target tissues causes β-cell exhaustion, and under extreme insulin resistance, leads

to β-cell apoptosis (Butler et al., 2003), which further deteriorates the glycemic control.

1.5. Skeletal muscles and insulin resistance

Skeletal muscle is a major glucose utilizing tissue, responsible for 75 – 80 % of insulin stimulated

glucose uptake (Saltiel and Kahn, 2001; Thiebaud et al., 1982). Under normal conditions, a

postprandial increase in blood glucose level (usually during food intake), induce insulin secretion from

the pancreatic β-cells. Insulin binds to its receptors on target tissues (skeletal muscles, adipose tissue

and liver), triggering the insulin signalling cascade. This results in the translocation of the glucose

transporter 4 (Glut4; highly expressed in skeletal muscles) into the plasma membrane, where it

participates in the transport of glucose across the membrane into the cells (Huang and Czech, 2007).

The glucose in the cell is then used for energy production (glycolysis) or stored in the form of

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glycogen. In T2D, the insulin responsive tissues (principally the skeletal muscles) become insulin

resistant, leading to an increase in blood glucose levels. Two other glucose transporters, namely Glut1

and Glut12 has been found to cause glucose uptake in skeletal muscles, where the former is responsible

for insulin independent basal transport of glucose and the latter for insulin stimulated glucose transport

(Klip et al., 1996; Stuart et al., 2009). It is thought that Glut12 is an ancestral predecessor of Glut4 and

now functions additionally to the principal Glut4-driven system of glucose transport.

Insulin resistance is a multi-factorial disease, affecting different tissues and is normally

triggered by chronic nutritional surplus. This state of over-nutrition leads to inflammation,

dyslipidemia, and/or gut microbial dysbiosis induced obesity, which feeds into a vicious cycle

contributing to the progression of the insulin resistant state (Johnson and Olefsky, 2013). In

understanding insulin resistance, of key importance are the three major signaling cascades regulating

cellular and whole-body glucose and lipid homeostasis; namely the Insulin, AMPK and Glucosamine

signaling pathway. These signaling pathways mediate a multitude of cellular responses, ranging from

cell growth, differentiation, protein synthesis, to glucose uptake, glycogen synthesis, fatty acid

oxidation, and more (Obici et al., 2002; Taniguchi et al., 2006; Viollet et al., 2009).

1.6. Oxidative stress and insulin resistance

Oxidative stress has been found to be correlated with insulin resistance (Meigs et al., 2007). Increasing

concentrations of glucose has been found to increase oxidative stress in human smooth muscle and

endothelial cells (Ceriello et al., 1996; Du et al., 2000). An increase in oxidative stress, was indicated

by elevated levels of 8-hydroxy-2´ deoxyguanosine (a major dna oxidation product), in mononuclear

cells isolated from whole blood (Dandona et al., 1996), as well as in the urine of diabetic patients, and

could be correlated with the levels of glycosylated hemoglobin (Leinonen et al., 1997). Again the

skeletal muscle of obese insulin resistant subjects showed increased lipid peroxidation (Russell et al.,

2003). Mitochondria, also called ‘the power house of the cell’, are a major source of reactive oxygen

20

species (ROS). ROS are an indispensable part of important cellular processes, normally required to

carry out routine cellular functions. ROS has also been found to function as signaling molecules (Rhee

et al., 2000). Again, anti-oxidant mediated quenching of ROS has been found to inhibit cellular

adaptation to hormetic stressors like physical exercise (Gomez-Cabrera et al., 2008). However

imbalance in the redox equilibrium, leading to elevated levels of ROS, can promote pro-inflammatory

pathways, contributing to abnormal insulin signaling (Wei et al., 2008).

1.7. The insulin signaling pathway

The insulin receptor, when bound to insulin, undergoes auto-phosphorylation, activating its kinase

domain; which leads to the phosphorylation and activation of the insulin receptor substrate (IRS)

proteins (figure 2). The activated IRS proteins are responsible for the initiation of two major signaling

cascades, namely the phosphatidylinositiol-3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway and

the Ras-mitogen-activated protein kinase (MAPK) pathway. The PI3K/Akt pathway mediates most of

the metabolic responses of insulin, where PI3K activates Akt, which causes Glut4 translocation to the

plasma membrane via phosphorylation of Akt substrates of 160 kDa: AS160/TBC1D4 (and TBC1D1,

an isoform of As160) (Sakamoto and Holman, 2008), which is proposed to lead to the activation of a

Rab-GTPase (Sano et al., 2003). Rab proteins in their GTP bound state regulates fundamental

mechanisms in vesicle trafficking. On the other hand the MAPK pathway causes changes in gene

expression and protein activity leading to complex changes in cell behaviour (Avruch, 1998; Taniguchi

et al., 2006).

21

Fig. 2 The insulin signaling pathway [Adopted and modified from (Taniguchi et al., 2006)]. The key

regulatory proteins mediating insulin actions of glucose uptake, glycogen synthesis, inhibition of

gluconeogenesis, protein synthesis, cell growth and differentiation has been highlighted in this figure.

1.8. The AMPK signaling pathway

AMP–activated protein kinase (AMPK), also known as the ‘metabolic master switch’ mediates cellular

responses to the depletion of intracellular ATP levels, caused by hypoxia, heat shock, starvation or

exercise. AMPK when activated (figure 3) down regulates the anabolic pathways driving fatty acid,

cholesterol and protein synthesis; and simultaneously up-regulates the catabolic pathways like

glycolysis, fatty acid oxidation, and biogenesis of new mitochondria in muscles and several other

tissues, resulting in ATP production (Viollet et al., 2009). Two protein kinases, namely, LKB1 and the

β-isoform of Ca2+

/calmodulin-dependent kinase kinase (CaMkkβ) has been shown to be capable of

activating AMPK via phosphorylation of its Thr172 residue (Shaw et al., 2004; Woods et al., 2005).

Research has shown that activation of AMPK in muscles caused both by exercise and AICAR (AMPK

agonist) stimulation leads to an increase in glucose uptake in both diabetic and non-diabetic subjects,

22

through a signaling pathway, distinctly different from the insulin signaling pathway (Koistinen et al.,

2003). The fact that glucose uptake can be enhanced independent of insulin, has made AMPK an

attractive therapeutic target.

It is noteworthy, that in pancreatic β-cells (responsible for insulin secretion) activation of

AMPK causes reduction of glucose stimulated insulin secretion, causing an improvement in glucose

tolerance and β-cell function (Carr et al., 2003). Upon activation, AMPK has been found to

phosphorylate As160 /TBC1D4 and/or TBC1D1, based on the tissue-specific abundance of these

proteins (Cartee and Wojtaszewski, 2007; Taylor et al., 2008), causing an increase in GLUT4

translocation and thereby enhancing glucose uptake. Phosphorylation of the Akt-substrates TBC1D4

and TBC1D1 is therefore the converging point of insulin and AMPK signaling pathways.

Fig. 3 AMPK activation and signaling [Adopted from (Srivastava et al., 2012)]. The figure

demonstrates the activation of AMPK upon ADP or AMP binding by LKB1 and CAMKK2 kinases.

However, till date Thr-172 phosphorylation has only been documented to occur upon AMP binding.

The various cellular actions mediated by activation of AMPK have been depicted here.

23

1.9. The Glucosamine pathway

In insulin responsive tissues, like the skeletal muscles, hyperglycemia leads to the activation of the

Glucosamine pathway, where the enzyme GFAT (glutamine: fructose-6-phosphate amidotransferase)

catalyzes the rate limiting step of converting fructose -6-phosphate to glucosamine-6-phosphate.

Activation of GFAT or the glucosamine pathway acts as an intracellular energy sensor, signaling the

cell to reduce glucose uptake, even in presence of insulin (Marshall et al., 1991; Obici et al., 2002).

1.10. Plant kingdom as a drug depot

Since primitive to modern times, botanicals have been used as a rich source for traditional medicines,

and have served as the foundation of innumerable drug discoveries. Being a rich source of secondary

metabolites, plants have a high potential to influence different cellular mechanisms, including key

signalling pathways balancing energy utilization and storage. Till date, more than 1200 plant species

have been tested for their efficacy against diabetes (Marles and Farnsworth, 1995).

1.11. Plants and experimental models used

1.11.1. Selected medicinal plants studied

Several reviews have collectively described the anti-diabetic and hypo-lipidemic properties of many

natural products (Jung et al., 2006; Marles and Farnsworth, 1995; Yeh et al., 2003); although in many

cases the components and cellular mechanisms responsible for the bioactivity have not been elucidated.

In this study, eight plants were selected, based on either their medicinal background as

traditional anti-diabetics or their significance in the field of food and nutrition. The plants selected were

Echinacea purpurea (purple coneflower), Thymus vulgaris (thyme), Daucus carota (carrots), Brassica

oleracea (broccoli and cabbage), Sambucus nigra (elderflowers), Rhodiola rosea (roseroot), and

Satureja hortensis (summer savory).

24

Different plants of the genus ‘Echinacea’ (belonging to the family Asteraceae) have been

widely studied. Among them E. purpurea, E. pallida, and E. angustifolia are used for medicinal

purposes. These plants are indigenous to different parts of North America, where they are traditionally

used against infections, mild septicemia and snake bites. It has been shown to possess

immunomodulatory, anti-inflammatory, anti-viral, anti-fungal and anti-bacterial properties (Barnes et

al., 2005). In recent studies, extracts and metabolites of E. purpurea, has been found to activate the

peroxisome proliferator- activated receptor gamma (PPARγ, a type II nuclear receptor, regulating fat

storage and glucose metabolism, primarily in adipose tissue) and enhance insulin-stimulated glucose

uptake in adipocytes (Christensen et al., 2009a; Christensen et al., 2009b).

Thymus vulgaris (thyme) is one of the most commonly used aromatic herbs used to enhance the

flavor of food. Other than its use as a condiment, Thymus vulgaris has been used as an alternative

medicine for its anti-oxidant, anti-microbial, anti-spasmodic, and detoxifying properties

(Baranauskiene et al., 2003). Extracts of thyme has been found to ameliorate the lipid profile of

streptozotocin-induced type 1 diabetic rats (Ozkol et al., 2013). However, the ability of thyme extracts

to activate PPARγ and improve insulin-stimulated glucose uptake has been published recently

(Christensen et al., 2009a).

Sambucus nigra (elderflowers), belonging to the family Caprofoliaceae, is indigenous to

Northern Africa, Europe, as well as Western and Central Asia. It is generally used to make wine, juice

and preserves, and is thought to have several beneficial effects on health. Sambucus nigra concoctions

have been used as a traditional medicine as a diuretic, and as a treatment against common cold,

influenza, inflammation and diabetes (Kultur, 2007; Swanstonflatt et al., 1991). Elderberries have been

extensively studied in the past for their anti-viral and immunity boosting effects (Roxas and Jurenka,

2007). But elderflowers have also been found to contain several bioactive metabolites like flavonoids,

phenolic acids and triterpenoids (Christensen et al., 2008; Gray et al., 2000). Moreover, aqueous

25

extracts of elderflowers exhibited insulin-like and insulin–releasing effects in in vitro studies performed

with mouse abdominal muscle cells and clonal pancreatic beta (BRIN-BD11) cells (Gray et al., 2000).

Satureja hortensis (summer savory), belonging to the family Lamiaceae, is native to Atlantic

Canada. The essential oil of S. hortensis has been found to exhibit anti-oxidant, anti-microbial, anti-

nociceptive and anti-inflammatory properties (Dikbas et al., 2012; Hajhashemi et al., 2012; Kotan et

al., 2012). Very recently, supplements of savory plants native to Southern Iran (Satureja khuzestanica),

was used to assess changes in metabolic parameters of hyperlipidemic subjects with T2D. A significant

reduction in total and LDL cholesterol and an improvement in HDL cholesterol levels in blood were

observed (Vosough-Ghanbari et al., 2010).

Rhodiola rosea (rose root), belonging to the family Crassulaceae, is normally found in colder

parts of the world, like Northern and North-Eastern America, Central Asian mountains, and

mountainous regions of Europe. It has been used as a folk medicine in different parts of Asia and

Eastern Europe, and has been found to improve work performance, stimulate the nervous system, and

reduce depression and fatigue (El-Alfy et al., 2012; Ishaque et al., 2012; Mannucci et al., 2012; Noreen

et al., 2013). Moreover it has shown pronounced cardio-protective effects against arrhythmia,

hypertension, and exhibited marked improvement in coronary flow and contractility during post-

ischemic period, and prevention of stress induced cardiac damage (Lee et al., 2012; Li et al., 2006;

Maslov and Lishmanov, 2007). Roseroot extracts has also shown anti-oxidative and as an anti-diabetic

properties in diabetic mice (Kim et al., 2006).

Daucus carota (carrots), belonging to the family Apiaceae, is a commonly used vegetable in

both oriental and occidental cuisines. It contains a large variety of phytochemicals, like the phenolic

compounds, carotenoids, α-tocopherols and polyacetylenes. The relative concentrations of the

phytochemicals vary (among other parameters) within different varieties. For example the purple

carrots (purple haze) generally have relatively higher amounts of anthocyanins, which also contributes

to their higher antioxidant capacity (Metzger and Barnes, 2009). Bioactive secondary metabolites like

26

polyacetylenes have found to have potent antifungal activity, with neurotoxic effects at high

concentrations and cytotoxic effects on cancer cells (Greenwald et al., 2001). The polyacetylenes

falcarinol and falcarindiol have been found to exhibit anti-inflammatory effects and reduce platelet

aggregation (Teng et al., 1989). Moreover falcarinol has shown lipoxygenase inhibitory effects in

mammalian cells (Alanko et al., 1994).

Brassica oleracea (green cabbage and broccoli), belonging to the family Brassicaceae, is also a

commonly consumed vegetable. Broccoli sprouts have been found to exhibit a reduction in hepatic and

plasma cholesterol in hamsters, although a gender dependence was observed, where the reduction in

hepatic cholesterol was higher in females (Rodriguez-Cantu et al., 2011), and triglyceride levels in T2D

patients (Bahadoran et al., 2012) together with a cyto-protective effect against chemical and UV-light

induced carcinogenesis (Dinkova-Kostova et al., 2006; Fahey et al., 1997) in SKH-1 high risk mice.

Green cabbage, too has been shown to have anti-oxidative and anti-proliferative effects on HepG2

(human liver carcinoma) cells (Chu et al., 2002).

1.11.2. Primary porcine myotube cultures as a model for skeletal muscles

Primary cell cultures are believed to serve as better experimental models than cell lines. But obtaining

satellite cells from humans to set up a primary culture is problematic. Due the fact that it is difficult to

obtain a continuous source for human primary myotube cultures, we used porcine primary myotube

cultures (figure 6A) as our experimental model.

Porcine anatomical and physiological characteristics as well as the development of

pathophysiology are very similar to humans (Swindle and Smith, 1998). Moreover, comparable

nutrient requirements, together with metabolic and glycemic control (Larsen et al., 2007) makes it a

viable model for biomedical research.

27

1.11.3. INS 1E cells as a model for pancreatic beta cells

In biochemical research, human pancreatic tissue is scarce and is obtainable only during autopsy.

Moreover, the pancreatic tissue obtained in such conditions is substantially degraded (Butler et al.,

2003). Again the clinical history of the subject going through autopsy is often unavailable. The scarcity

of human pancreatic tissue in health research as well as the complexity involved in handling the fragile

pancreatic endocrine cells individually has led to the development of several beta and alpha cell lines

from rodents. The most commonly used insulin secreting cell lines in beta cell research are INS-1, RIN,

HIT, MIN, and βTC (Skelin et al., 2010). The cell lines vary among themselves in several aspects,

starting from glucose sensitivity, insulin secretion capacity, proliferation rate to sensitivity towards

secretagogues compared to the native pancreatic beta cells (Hohmeier et al., 2000).

The INS-1 cell line (figure 6B) has been used in this project as an experimental model for

pancreatic beta cells. A couple of decades ago, the cell-line was isolated from radiation-induced rat

insulinoma (Asfari et al., 1992).

Fig. 6 Differentiated porcine myotubes and INS-1E cells. Primary porcine myotubes (A) 8 days after

seeding, and INS-1E cells (B) 4 days after seeding.

28

1.12. Overview of the main project: Test for bioactivities in different work packages

The work presented in this thesis is a part of a more elaborate ‘main’ research project, which is

comprised of six work packages (WP). Most of the plants used in the main project was cultivated by

WP1; extraction, bio-assay guided fractionation, chromatographic separation, and characterization was

done by WP2; screening of the extracts were done by a platform of different bio-assays where they

were evaluated for their potential to induce: glucose uptake and PPARγ activation in mammalian

adipocytes (WP3), fat accumulation in C. elegans (WP4), glucose uptake in primary porcine myotube

cultures (WP5), and neuro-protection in organotypic brain slice cultures (WP6). Extracts and fractions

used in this work were made by WP2; otherwise, this thesis is based on independent research done in

WP5. The overview of the main project is given in figure 4. And work flow in WP5 and interlink with

WP2 has been shown in figure 5.

Fig. 4 Structure of the main project. The main project was comprised of six work packages. The

work presented in this thesis is based on the independent work done by WP5.

29

1.13. Hypothesis for WP5

It was hypothesized that the extracts of the plants Thymus vulgaris, Echinacea purpurea, Daucus

carota, Brassica oleracea, Sambucus nigra, Satureja hortensis, and Rhodiola rosea contain bioactive

compounds with anti-diabetic properties, capable of enhancing glucose uptake in skeletal muscles

(tested in myotube cultures) and stimulating insulin secretion in pancreatic beta cells (tested in INS 1E

cell line). The mechanism of action of the bioactive compounds could be related to insulin, AMPK, or

ROS induced glucose uptake, observed in muscles.

Fig. 5 Work flow in WP5. The figure describes the basic workflow and the aim of this project and the

steps where collaboration with WP2 was necessary.

1.14. Study structure

The work presented in the thesis has been divided into three studies. In Study I, majority of the work

involved screening of the plant extracts, and fractions of the selected extracts for glucose uptake in

primary porcine myotubes. The pure compounds present in the bioactive fractions, together with those

known to be inherently present in these plants, as well as some related compounds were screened for

30

their ability to enhance glucose uptake. Part of the work done in this study has resulted in Manuscript I

(attached). The remaining part of the work has been described here in ‘Study I’.

Various bioactive compounds were examined for their ability to increase insulin secretion in

INS 1E beta cell line in Study II. Based on initial screening, some of the compounds were selected for

studying insulin secretion and gene expression under glucotoxic conditions. Most of the work has been

explained in Manuscript II (attached), and the part not included in the manuscript, has been described

under ‘Study II’.

The mechanism of action behind the observed increase in glucose uptake caused by naringenin

and falcarinol was investigated in Study III. In this study, naringenin and falcarinol induced glucose

uptake was tested in presence of different inhibitors, and their impact on phosphorylation of proteins

responsible for Glut4 translocation was examined. This study has been incorporated in Manuscript III.

Additional information has been included, under ‘Study III’.

Studies I, II, and III, each contains a short summary highlighting their respective findings.

31

2. Study I: Study of plant extracts in satellite cell derived primary porcine myotube cultures

2.1. Objective:

To assess the potential of the crude extracts to enhance glucose uptake in primary porcine myotube

cultures, and examine their pro- / anti- oxidative and proliferative potential.

2.2. Methods:

Most of the methods used in Study I have been described in the Manuscript I. Those that have not been

described in Manuscript I have been included here.

2.2.1. Cell proliferation and myotube viability

The isolated satellite cells were seeded on Matrigel (1:50) in 96-well plates. After incubation with

Porcine Growth Medium (PGM; 10% foetal calf serum (FCS), 10% horse serum, 80% DMEM

(Dulbecco’s modified Eagles medium, Life Technologies, Naperville, IL) containing 25 mM Glucose,

and antibiotics (100 IU/mL penicillin, 100 IU/mL streptomycin sulphate, 3 µg/mL amphotericin B, 20

µg/mL gentamycin)), for 72 h, PGM (12.5 mM glucose) containing different treatments were added to

the cells. Fresh media (containing the treatments) was added to the cells during a proliferation period of

4 days. To measure proliferation, cell viability was measured by adding 10 µl of WST-1 reagent

(Roche) per well and incubated for 4hrs. The absorbance was then measured using a microplate reader

(EnVision 2103 multilabel reader, PerkinElmer) at 450 nm, with the reference wavelength being 650

nm. WST-1 is a formazan salt which is cleaved to formazan (dark red) by mitochondrial dehydrogenase

of viable cells. The data was corrected with background measurements of media and WST-1 alone.

In order to estimate cell viability alone, differentiated myotubes were incubated with different

treatments only for 24 h, before WST-1 reagent is added. The rest is performed as stated above.

32

2.2.2. Statistics

The glucose uptake data resulting from the screening of plant extracts were subjected to statistical

analyses, using the ‘Mixed procedure’ of SAS statistical programming software (Ver. 9.2; SAS

Institute Inc., Cary, NC, USA). The model included main effects of factors (plants, plant

extracts/fractions, concentration of plant extracts/fractions, and their interactions. As random effects,

satellite cell cultures from pigs and their replicates were nested within treatments. When overall effects

were significant, LSmeans were separated by pairwise comparison

33

2.3. Results:

Table 1. Plant extracts used in this study, together with the parts of the plants and the solvents used for

extraction*.

Extract

no.

Plant species (Latin) Plant Plant part Solvent Type of

extract

1 Thymus vulgaris Thyme Aerial parts DCM1

Crude

2 Thymus vulgaris Thyme Aerial parts MeOH2

Crude

3 Echinacea purpurea Purple coneflower Roots MeOH1

Crude

4 Echinacea purpurea Purple coneflower Roots DCM2

Crude

5 Echinacea purpurea Purple coneflower Roots DCM1

Crude

6 Echinacea purpurea Purple coneflower Roots MeOH2

Crude

7 Daucus carota (bolero) Carrot Roots DCM1

Crude

8 Daucus carota (bolero) Carrot Roots MeOH2

Crude

9 Daucus carota (purple haze) Carrot Roots DCM1

Crude

10 Daucus carota (purple Haze) Carrot Roots MeOH2

Crude

11 Brassica oleracea Broccoli Aerial parts DCM1

Crude

12 Brassica oleracea Broccoli Aerial parts MeOH2

Crude

13 Brassica oleracea Cabbage Aerial parts DCM1

Crude

14 Brassica oleracea Cabbage Aerial parts MeOH2

Crude

15 Sambucus nigra Elderflower Flowers DCM1

Crude

16 Sambucus nigra Elderflower Flowers MeOH2

Crude

17 Satureja hortensis Summer savory Aerial parts DCM1

Crude

18 Satureja hortensis Summer savory Aerial parts MeOH2

Crude

19 Rhodiola rosea Roseroot Flowers DCM1

Crude

20 Rhodiola rosea Roseroot Flowers MeOH2

Crude

21 Rhodiola rosea Roseroot Roots DCM1

Crude

22 Rhodiola rosea Roseroot Roots MeOH2

Crude

DCM = Dichloromethane, MeOH = Methanol, *Extraction was performed by WP2. 1 and 2 indicates the sequence in which the solvents

were used for extraction. For all plants, other than E. purpurea (where extraction was also made with MeOH as the first and DCM as the

second solvent), DCM was the first and MeOH the second solvent for extraction.

34

To examine the potential of the extracts to enhance glucose uptake, the differentiated myotubes were

either treated with 0.5, 0.7, and 1 mg/mL of the extracts separately, in presence of 750 pM insulin

(figure 6 – 9); or 1 mg/mL extract in absence of insulin (figure 10). A significant increase in glucose

uptake (p = 0.02) was observed when myotubes were treated with 750 pM insulin. All the extracts were

primarily screened on the satellite cells isolated from a single pig, with 6 replicates. The DCM extract

of thyme (T. vulgaris), carrots (D. carota, bolero and purple haze), elderflower (S. nigra) and Roseroot

(R. rosea, flowers) exhibited prominent effects (≥ 50 % at any concentration) on glucose uptake in the

presence of insulin compared to control (750 pM insulin only). An increase of 149.7, 186.6, and 173.5

% (p < 0.001) for thyme (DCM); 222.8, 142.0, and – 20.4 % (p < 0.001) for carrots (bolero, DCM);

67.8, -16.3, and – 68.0 % (p < 0.001) for carrots (purple haze, DCM); 25.6, 65.7, and 65.8 % (p <

0.001) for elderflowers (DCM); and 133.7, 148.0, and 110.5 % (p < 0.001) for roseroot (flowers,

DCM) extracts was observed at 0.5, 0.7, and 1 mg/mL concentrations. A more moderate increase (20 -

50 % at any concentration) of 36.1, 41.2, and 36.2 % (p < 0.001) for DCM2; – 4.1, 119.1, and 28.6 % (p

= 0.6, 0.01, and < 0.001) for MeOH1; and 10.5, 18.0 and 29.9 % (p = 0.1, 0.015, and < 0.001) for

MeOH2 extracts of purple coneflower, compared to control was observed at 0.5, 0.7, and 1 mg/mL

concentrations. Other plant extracts, also showing a moderate increase were elderflower MeOH, and

roseroot DCM extracts, showing an increase of -25.1, 9.4 and 20.4 % (p < 0.001, = 0.18, and 0.009);

and 22.1, 23.9, and 21.8 % (p < 0.002) for 0.5, 0.7, and 1 mg/mL concentrations, respectively.

Glucose uptake experiments when carried out in absence of insulin, the most prominent

increase (> 50 %) was observed for thyme DCM extract (149.4 %, p < 0.001); elderflower, DCM and

MeOH extracts (82.4 and 61.7 %, respectively; p < 0.001); and roseroot DCM extract (104.7 %, p <

0.001). However, a relatively moderate increase (20 - 50 %) was observed for purple coneflower

MeOH1 (21.9 %, p < 0.001) and MeOH

2 (24.3 %, p < 0.001) extracts; roseroot (root) DCM extracts

20.1 %, (p < 0.001). It is important to note here, that E. purpurea DCM2 extract increased glucose

uptake significantly in presence of insulin, but not in its absence.

35

Extracts (mg/mL)

0 0.5 0.7 1

2 -

DO

G u

pta

ke (

% o

f con

trol)

0

50

100

150

200

250

300

3501 T. vulgaris (DCM)

2 T. vulgaris (MeOH)

3 E. pupurea (MeOH1)

4 E. purpurea (DCM2)

5 E. purpurea (DCM1)

6 E. purpurea (MeOH2)

a

b

cd

b b b

a abb

a

bb

b b b

aa

b

Fig. 6 Effect of T. vulgaris and E. purpurea on glucose uptake in presence of 750 pM insulin.

The differentiated myotubes were incubated with 0.0, 0.5, 0.7, and 1 mg/mL of T. vulgaris DCM and

MeOH extract; and E. purpurea MeOH1, DCM

2, DCM

1 and MeOH

2 respectively, for 1 h with 750 pM

insulin, following which, 2-DOG uptake was measured. Values are given as LS means ± SEM of

experiments conducted with satellite cells from 1 pig, expressed as percent of control, where 6

replicates were used per treatment. 1

and 2 indicate the sequence in which the solvents were used for

extraction. For all plants, other than E. purpurea (where extraction was also made with MeOH as the

first and DCM as the second solvent), DCM was the first and MeOH the second solvent for extraction.

The letters a, b, c, and d shows the different significance levels within each extract.

36

Extracts (mg/mL)

0 0.5 0.7 1

2-D

OG

up

take

(%

of

co

ntr

ol)

0

50

100

150

200

250

300

3507 D. carota (bolero, DCM)

8 D. carota (bolero, MeOH)

9 D. carota (purple haze, DCM)

10 D. carota (purple haze, MeOH)

11 B. oleracea (broccoli, DCM)

12 B. oleracea (broccoli, MeOH)

b

c

d

aa a

a

b

c

d

a a

bbbc c

aa

a

Fig. 7 Effect of D. carota and B. oleracea on glucose uptake in presence of 750 pM insulin.

The differentiated myotubes were incubated with 0.0, 0.5, 0.7, and 1 mg/mL of D.carota (varieties

bolero and purple haze) DCM and MeOH extract; and B. oleracea DCM and MeOH extract,

respectively for 1 h with 750 pM insulin, following which, 2-DOG uptake was measured. Values are

given as LS means ± SEM of experiments conducted with satellite cells from 1 pig, expressed as

percent of control, where 6 replicates were used per treatment. The letters a, b, c, and d shows the

different significance levels within each extract.

37

Extract (mg/mL)

0 0.5 0.7 1

2-D

OG

up

take

(%

of

co

ntr

ol)

0

20

40

60

80

100

120

140

160

180 13 B. oleracea (cabbage, DCM)

14 B. oleracea (cabbage, MeOH)

15 S. nigra (DCM)

16 S. nigra (MeOH)

17 S. hortensis (DCM)

18 S. hortensis (MeOH)

a

b bc

c

b

bc

c

b

c c

ac

b

c

b b b

a a

a

Fig. 8 Effect of B. oleracea, S. nigra and S. hortensis on glucose uptake in presence of 750 pM insulin.

The differentiated myotubes were incubated with 0.0, 0.5, 0.7, and 1 mg/mL of DCM and MeOH

extracts of B. oleracea, S. nigra and S. hortensis, respectively, for 1 h with 750 pM insulin, following

which, 2-DOG uptake was measured. Values are given as LS means ± SEM of experiments conducted

with satellite cells from 1 pig, expressed as percent of control, where 6 replicates were used per

treatment. The letters a, b, c, and d shows the different significance levels within each extract.

38

Extracts (mg/mL)

0 0.5 0.7 1

2-D

OG

up

take (

% o

f con

trol)

0

50

100

150

200

250

30019 R. rosea (flowers, DCM)

20 R.rosea (flowers, MeOH)

21 R. rosea (roots, DCM)

22 R. rosea (roots, MeOH)

a

bc

d

b b b

b b b

b b b

Fig. 9 Effect of R. rosea on glucose uptake in presence of 750 pM insulin.

The differentiated myotubes were incubated with 0.0, 0.5, 0.7, and 1 mg/mL of DCM and MeOH

extracts of R. rosea flowers and roots, respectively, for 1 h with 750 pM insulin, following which, 2-

DOG uptake was measured. For R. rosea, two plant parts were used for extraction, as indicated in the

plot. Values are given as LS means ± SEM of experiments conducted with satellite cells from 1 pig,

expressed as percent of control, where 6 replicates were used per treatment. The letters a, b, c, and d

shows the different significance levels within each extract.

39

Extracts

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

2-D

OG

up

take

(%

of

co

ntr

ol)

0

50

100

150

200

250

300GU < 20 %

GU > 50 %

GU 20 - 50 %

***

***

***

***

***

***

***

***

***

***

***

***

**

***

*

***

***

***

**

Fig. 10 Effect of all the extracts on insulin independent glucose uptake

The differentiated myotubes were incubated with 1 mg/mL of all the plant extracts for 1 h, following

which 2-DOG uptake was measured. Values are given as LS means ± SEM of experiments conducted

with satellite cells from 1 pig, expressed as percent of control, where 6 replicates were used per

treatment. C = control (DMSO in media), GU = glucose uptake. Table 1 lists all the names of the

extracts used; *p < 0.05, **p <0.01, ***p < 0.001 vs. control.

The extracts showing > 50 % increase in the preliminary screening experiments were selected for the

verification of the observed bioactivity. Therefore, Thyme DCM extract, roseroot (flower) DCM

extract, carrot (bolero) DCM extract, and elderflower DCM extract were selected for further

experiments conducted on satellite cells of three pigs, using the same concentration range in presence

and absence of insulin (figure 10, 11). At 0.5, 0.7, and 1 mg/mL concentrations, the thyme DCM

extract enhanced glucose uptake by 59.1, 70.5, and 63.4 % (p < 0.001); the roseroot (flower) DCM

40

extract by 60.7, 64.4, and 33.5 % (p < 0.001); carrot (bolero) DCM extract by 79.8, 19.2, and - 62 % (p

< 0.001); and elderflower DCM extract by 48.2, 56.3, and 56.3 % (p < 0.001) respectively (figure 11)

in presence of 750 pM insulin. Here thyme and elderflower DCM extracts showed a stable increase at

all concentrations tested; whereas carrots (bolero) DCM extracts exhibited a sharp fall in glucose

uptake with increasing concentrations. For roseroot (flowers) DCM extract, the increase was stable for

0.5 and 0.7 mg/mL; after which there was a comparative decrease in glucose uptake, although it was

still significantly high compared to control. It can be expected, that a linear increase in glucose uptake

with increasing concentration of extracts could have been observed at a lower concentration range.

When examined independent of insulin at a concentration of 1 mg/mL (figure 12), the DCM extracts of

thyme, roseroot (flowers), carrots (bolero), and elderflowers increased glucose uptake by 65.2 % (p <

0.001), 38.2 % (p < 0.001), - 44.5 % (p < 0.001), and 72.8 % (p < 0.001). The glucose uptake at1

mg/mL concentration was found to be comparable in presence or absence of insulin for the DCM

extracts of thyme, roseroot (flowers), and carrots (bolero); with the exception of elderflowers, where

the insulin- independent glucose uptake was found to be considerably higher.

In order to verify, whether the DCM extracts of thyme, roseroot (flowers), carrots (bolero), and

elderflowers caused oxidative stress or were in any way detrimental to the myotubes, ROS generation

and cell viability in presence of the chosen extracts were determined. Generation of intracellular ROS

in presence of the DCM extracts of thyme, roseroot (flowers), and carrots (bolero) have been illustrated

in figure 13, and that for the elderflower DCM extract has been mentioned in Manuscript I. The

myotubes were treated with 100, 200 and 500 µg/mL of the extracts for both the assays. The thyme and

roseroot extracts reduced intracellular ROS significantly at all concentrations tested (p < 0.001),

whereas the carrots (bolero) decreased intracellular ROS generation only at the highest concentration

(500 µg/mL) tested (p < 0.001), compared to control. None of the extracts showed any significant

increase or decrease in myotube viability (figure 14) compared to their respective controls; except

carrots (bolero), which showed a significant increase and decrease in mitochondrial activity at the

41

concentrations of 200 µg/mL (p = 0.004) and 500 µg/mL (p < 0.001), respectively, where at the latter

concentration, a sharp reduction in myotube viability was observed. However, a tendency (p = 0.07) to

increase the mitochondrial activity of the myotubes was observed for elderflower DCM extract at the

concentration of 500 µg/mL.

Impact on satellite cell proliferation was studied for D. carota (purple haze) MeOH extract and

S. nigra DCM extract at the concentration of 100, 200 and 500 µg/mL for 4 days (figure 15). There was

a significant increase in myoblast proliferation in the presence of 500 µg/mL carrot (purple haze)

MeOH extract, all three days, i.e. after 24 (p = 0.006), 48 (p = 0.008), and 96 (p < 0.001) h of

incubation, compared to their respective controls. An increase was also observed at a concentration of

200 µg/mL (p = 0.02) but only after 96 h of incubation. The treatment containing 500 µg/mL of

elderflower DCM extract showed significant increase in proliferation compared to its control after 48

(p = 0.01) and 96 (p = 0.002) h of incubation.

Based on the results obtained from glucose uptake studies, D. carota (bolero) DCM extract, and

S. nigra DCM and MeOH extract, were selected for further investigation. To facilitate identification of

the potential bioactive components in these three extracts, they were separated by flash

chromatography (fractionation done by WP2). Fractionation of carrot (bolero) DCM extract resulted in

8 fractions (A to H). The elderflower DCM and MeOH extract resulted in 7 fractions each (A to G). All

the fractions were then screened for their potential to induce GU independently at the concentrations of

50 and 100 µg/mL. Subsequently, the bioactive fractions were separated and constituent compounds,

together with some other related polyphenols were tested for their ability to enhance glucose uptake in

myotubes. The results for elderflower extracts, fractions, its constituent compounds and related

polyphenols have been included and discussed in Manuscript I.

None of the carrot fractions showed any significant increase in glucose uptake (figure 16).

Therefore, it was difficult to identify the compounds behind the observed activity of carrot (bolero)

DCM extract with the help of fractionation based on this particular bioassay. However, in glucose

42

uptake experiments conducted in adipocytes (WP3), fractions C and F showed an increase in insulin

stimulated glucose uptake. Upon chromatographic separation, fraction C and F was found to contain

falcarinol and falcarindiol as major components (chromatographic separation done by WP2) and were

purified using semi-preparative HPLC (WP2). Hence, the two carrot polyacetylenes: falcarinol and

falcarindiol were chosen to be tested for their potential to induce glucose uptake in myotubes (figure

17) and were tested at the concentrations of 0.3, 1, 3, 10, and 30 µM in presence or absence of 10 nM

insulin. Falcarinol exhibited an insulin-independent increase in glucose uptake in a dose-dependent

manner, with a maximum increase of 32. 5 % (p < 0.001) at a concentration of 10 µM, compared to

control; whereas, in presence of insulin the highest increase (16.0 %, p < 0.001) was observed at a

concentration of 3 µM, compared to myotubes treated with 10 nM insulin only. Falcarindiol caused an

insulin-independent increase in glucose uptake at all concentrations tested, compared to control;

although the increase was not significantly different between the tested concentrations. At 0.3 and 30

µM (lowest and highest concentration tested), an increase of 11.5 % (p = 0.01), and at 1, 3, and 10 µM

concentrations, an increase of 17.7, 18.9, and 19.7 % (p < 0.001) was observed compared to control.

Like falcarinol, in presence of insulin, the highest increase was observed at 3 µM (12.8 %, p = 0.006)

compared to myotubes treated with 10 nM insulin only.

Naringenin was identified as one of the compounds present in the S. nigra DCM extract

fractions, which could enhance insulin independent glucose uptake in myotubes. Naringenin, at the

concentrations of 3, 10, 30, and 100 µM was examined for its potential to enhance glucose uptake in

presence and absence of 10 nM insulin (figure 18). A significant increase in glucose uptake was

observed at all concentrations tested, in presence or absence of insulin. The highest increase in absence

of insulin was 19.4 % (p < 0.001) at a concentration of 10 µM, compared to control. At all other

concentrations the increase was slightly less, but statistically equivalent to that observed at 10 µM.

However, in presence of insulin a highest increase of 22.9 % (p < 0.001) was observed at a

concentration of 3 µM compared to myotubes treated with 10 nM insulin only. Here, a significant

43

decrease in glucose uptake was observed with increasing concentrations of naringenin (10 and 30 µM)

compared to that observed at 3 µM; although the values were still significantly higher compared to

myotubes treated with 10 nM insulin only. At a concentration of 100 µM, there was a numerical

increase in glucose uptake, but the value was statistically equivalent to that observed at 3, 10 and 30

µM concentrations.

S. nigra (DCM) extract (mg/mL)

0 0.5 0.7 1

2 -

DO

G u

pta

ke

(%

co

ntr

ol)

0

25

50

75

100

125

150

175

200

T. vulgaris (DCM) extract (mg/mL)

0 0.5 0.7 1

2 -

DO

G u

pta

ke

(%

co

ntr

ol)

0

25

50

75

100

125

150

175

200

******

***

A

R. rosea (flower, DCM) extract (mg/mL)

0 0.5 0.7 1

2 -

DO

G u

pta

ke

(%

co

ntr

ol)

0

25

50

75

100

125

150

175

200

*** ***

**

B

D. carota (bolero, DCM) extract (mg/mL)

0 0.5 0.7 1

2 -

DO

G u

pta

ke

(%

co

ntr

ol)

0

25

50

75

100

125

150

175

200 ***

**

C

****** ***

D

Fig. 11 Effect of selected extracts on glucose uptake in presence of 750 pM insulin

The differentiated myotubes were incubated with 0.5, 0.7, and 1 mg/mL of (A) T. vulgaris DCM, (B)

R. rosea (flowers) DCM, (C) D. carota (bolero) DCM, and (D) S. nigra DCM extracts for 1 h,

following which, 2 - DOG uptake was measured. Values are given as LS means ± SEM of experiments

conducted with satellite cells from 3 pigs, expressed as percent of control, where 6 replicates were used

per pig per treatment, *p < 0.05, **p <0.01, ***p < 0.001 vs. control.

44

DCM extracts

C

T. vulga

ris

R. r

osea

(flower

s)

D. c

arot

a (b

oler

o)

S. n

igra

2 -

DO

G u

pta

ke (

% c

ontr

ol)

0

25

50

75

100

125

150

175

200

***

**

***

***

Fig. 12 Effect of selected extracts on insulin independent glucose uptake

The differentiated myotubes were incubated with 1 mg/mL of T. vulgaris DCM, R. rosea (flowers)

DCM, D. carota (bolero) DCM, and S. nigra DCM extracts for 1 h, following which, 2 - DOG uptake

was measured. Values are given as LS means ± SEM of experiments conducted with satellite cells from

3 pigs, expressed as percent of control, where 6 replicates were used per pig per treatment. C = control

(DMSO in media), *p < 0.05, **p <0.01, ***p < 0.001 vs. control.

45

Time (mins)

0 50 100 150 200 250

Flu

ore

scen

ce

15000

20000

25000

30000

35000

40000Control

H2O2

R. rosea flowers, DCM (100 ug / mL)



B

***

]***

Time (mins)

0 50 100 150 200 250

Flu

ore

scen

ce

15000

20000

25000

30000

35000

40000 Control

H2O2

T. vulgaris DCM (100 ug / ml)



A

***

]***

time (mins)

0 50 100 150 200 250

Flu

ore

scen

ce

15000

20000

25000

30000

35000

40000 Control

H2O2 (100 µM)

D. carota, DCM (100 µg / mL)



***

***

C

Fig. 13 Effect of selected extracts on reactive oxygen species generation

The effect of (A) T. vulgaris, (B) R. rosea (flower) and (C) D. carota DCM extracts on the generation

of reactive oxygen species (ROS) determined at three different concentrations by intracellular 2,7-

dichlorofluorescein oxidation. H2O2 was used as positive control. The x-axis shows every 5th data

point. Values are given as LS means ± SEM of experiments conducted with satellite cells from 3 pigs,

expressed as percent of control, where 6 replicates were used per pig per treatment. Control = DMSO

in media; *p < 0.05, **p <0.01, ***p < 0.001 vs. control.

46

DCM extracts (µg/mL)

0 100 200 500

Absorb

ance

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

T. vulgaris

R. rosea (flower)

D. carota (bolero)

S. nigra

***

**

Fig. 14 Effect of selected extracts on myotube viability

The effect of T. vulgaris, R. rosea (flower) and D. carota (bolero) DCM extracts on myotube viability,

where absorbance values (A450 nm-A650 nm) are proportional to the mitochondrial activity in viable

myotubes after exposure to treatment for 24h. Values are given as LS means ± SEM of experiments

conducted with satellite cells from 3 pigs, where 6 replicates were used per pig per treatment. Control =

DMSO in media; *p < 0.05, **p <0.01, ***p < 0.001 vs. control.

47

Day1 Day 2 Day 4

Absorb

ance

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8 Control

Insulin

100 µg/mL

200 µg/mL

500 µg/mL

***

***

Day1 Day 2 Day 4

Absorb

ance

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8 Control

Insulin

100 µg/mL

200 µg/mL

500 µg/mL

***

**

*

BA

*

Fig. 15 Effect of selected extracts on proliferation of myoblasts

The effect of (A) D. carota (purple haze) MeOH extract and (B) S. nigra DCM extracts on satellite

cells isolated from porcine skeletal muscles. The absorbance values (A450 nm-A650 nm) are proportional to

the mitochondrial activity in viable myoblasts after exposure to 100, 200 and 500 µg/mL of the

extracts. Fresh media containing the treatments were added to the cells every day for 4 days. Insulin

(100 nM) was used as a positive control. Values are given as LS means ± SEM of experiments

conducted with satellite cells from 3 pigs, where 6 replicates were used per pig per treatment. Control =

DMSO in media; *p < 0.05, **p <0.01, ***p < 0.001 vs. control. Here, the significant differences have

been indicated for day 4, compared to its respective control.

48

D. carota (bolero) DCM fractions

Control A B C D E F G H

2 -

DO

G u

pta

ke (

% o

f con

trol)

0

20

40

60

80

100

120 50 ug/mL

100 ug/mL

* *

***

***

***

***

*** ***

*

Fig. 16 Effect of D. carota (bolero) DCM fractions on glucose uptake

The differentiated myotubes were incubated with 50 and 100 µg/mL of D. carota (bolero) DCM

fractions: A to H, for 1 h, following which, 2 – DOG uptake was measured. Values are given as LS

means ± SEM of experiments conducted with satellite cells from 3 pigs, expressed as percent of

control, where 6 replicates were used per pig per treatment, *p < 0.05, **p <0.01, ***p < 0.001 vs.

control.

49

Falcarinol (µM)

0 0.3 1 3 10 30

2 D

OG

- u

pta

ke (

% o

f co

ntr

ol)

80

100

120

140

160

a

ccd

de

e e

e

Falcarindiol (µM)

0 0.3 1 3 10 30

2 -

DO

G u

pta

ke (

% o

f co

ntr

ol)

80

100

120

140

160

a

b b

bcbc bcbc

bc

cdd d

ddecd

b bbc

A B

Fig. 17 Effect of falcarinol and falcarindiol on glucose uptake in presence or absence of 10 nM insulin

The differentiated myotubes were incubated with different concentrations of (A) Falcarinol or (B)

Falcarindiol in presence (grey bars) or absence (black bars) of 10 nM insulin for 1 h, following which,

2 - DOG uptake was measured. Values are given as LS means ± SEM of experiments conducted with

satellite cells from 3 pigs, expressed as percent of control, where 6 replicates were used per pig per

treatment, *p < 0.05, **p <0.01, ***p < 0.001 vs. control. [This work has been contributed to the

supporting Manuscript II (Effects of falcarinol and falcarindiol on glucose uptake in adipocytes and

muscle cells, PPARγ transactivation and adipocyte differentiation)]

50

Naringenin (µM)

0 3 10 30 100

2-D

OG

up

take

(%

of

co

ntr

ol)

80

90

100

110

120

130

140

150

a

b b

bb b

c

d

cd

d

Fig. 18 Effect of naringenin on glucose uptake in presence or absence of 10 nM insulin

The differentiated myotubes were incubated with different concentrations of naringenin in presence

(grey bars) or absence (black bars) of 10 nM insulin for 1 h, following which, glucose uptake was

measured. Values are given as LS means ± SEM of experiments conducted with satellite cells from 3

pigs, expressed as percent of control, where 6 replicates were used per pig per treatment, *p < 0.05, **p

<0.01, ***p < 0.001 vs. control.

51

2.4. Discussion

Inadequate GU in major glucose utilizing tissues is a major aspect of T2D. Plants provide a rich source

of an array of potentially bioactive secondary metabolites and have contributed abundantly to the

pharmaceutical and nutraceutical industry.

To take advantage of the differential solubility of the plant compounds, DCM and MeOH were

chosen as extraction solvents, for their capacity to dissolve non-polar and polar compounds,

respectively. It was interesting to note that the extracts showing pronounced increase in glucose uptake

were primarily DCM extracts. DCM being a solvent of low polarity, this observation might indicate

that the compounds or group/class of compounds responsible for the enhancement of glucose uptake

are relatively non-polar.

In order to investigate any synergistic potential, these extracts were evaluated both in presence

and absence of insulin. In in vitro studies, an insulin concentration of ≥ 10 nM is considered to be

unphysiologically high (Li et al., 2005); therefore a biologically viable concentration of insulin (750

pM) (Iwase et al., 2001) was used in the screening studies to make the results physiologically relevant.

Most of the extracts that showed a prominent increase in glucose uptake in presence of insulin

also did the same when tested independently. Out of the 22 extracts screened in this study for increase

in glucose uptake in primary porcine myotube cultures, an impressive increase in glucose uptake was

observed by T. vulgaris (thyme) DCM extract, D. carota (carrot), bolero DCM extract, R. rosea

(roseroot), flower DCM extract and S. nigra (elderflower) DCM extract both in presence and absence

of insulin, with the exception of D. carota (carrot), bolero DCM extract, which did not show an

increase when tested in absence of insulin. This can be explained by the cytotoxicity of this extract at

the concentrations used in this particular experiment. Other plant extracts, which showed a relatively

moderate increase (> 20 % but < 50 %) in glucose uptake in the initial screening experiments, were: E.

purpurea (purple coneflower) MeOH1, DCM

2, and MeOH

2; and R. rosea (roseroot) root, DCM

extracts. E. purpurea DCM2 and DCM

1 are both DCM extracts of purple cone flower. In the case of

52

DCM2, the plant parts were first extracted with MeOH and later with DCM, and in case of DCM

1,

extraction was done in opposite order. But DCM2 is found to be more bioactive than DCM

1. The reason

behind this could be that, due to dissimilar extraction sequence, the fellow components present in the

extracted pool could be different, which means that the possible inhibitory effect of the fellow

compounds in the extracts are not comparable.

Based on the initial screening results, T. vulgaris (thyme) DCM extract, D. carota (carrot),

bolero DCM extract, R. rosea (roseroot), flower DCM extract and S. nigra (elderflower) DCM extract

were selected for further verification of their bioactivity. When verified using satellite cells from three

pigs, there was a reduction in the glucose uptake values for all the selected extracts (still showing an

increase of approx. 50 % and above) compared to those obtained in the initial screening experiments

carried out with satellite cells from one pig. This could be due to the biological variation introduced by

increasing the number of pigs used in the experiment.

Oxidative stress has been implicated as a contributing factor in insulin resistance. Therefore T.

vulgaris (thyme) DCM extract, D. carota (carrot), bolero DCM extract, R. rosea (roseroot), flower

DCM extract and S. nigra (elderflower) DCM extract were tested for their capacity to induce oxidative

stress based on the formation of ROS. Evaluation of intracellular ROS generation by these extracts

revealed significant anti-oxidative potential. The reduced production of intracellular ROS observed

here, could be attributed to the phenolic acid and flavonoid composition of these plants (Wojdylo et al.,

2007).

A decrease in satellite cell proliferation and differentiation is a major cause of Sarcopenia which

is caused by a reduction in skeletal muscle mass. The skeletal muscle being the primary site for glucose

disposal, muscle loss aggravates hyperglycemia, especially in the elderly. Insulin has been found to

enhance cell proliferation and growth (Malaguarnera et al., 2012). D. carota (purple haze) MeOH

extract and S. nigra DCM extract were examined for their potential to promote satellite cell

53

proliferation. Both the extracts were able to improve cell proliferation, also in comparison to insulin.

This is the first account showing effects of S. nigra extracts on cell proliferation.

Considering the priorities of work packages, and the fact that WP2 was responsible for

fractionation of all the prioritised extracts, we decided to continue with two of the four extracts that

showed pronounced effect on glucose uptake in myotubes. D. carota (bolero) DCM, and S. nigra

DCM and MeOH extracts were chosen for fractionation to aid identification of the constituent bioactive

compounds.

S. nigra DCM and MeOH extracts, their fractions, the constituent bioactive compounds, and related

polyphenols have been discussed in Manuscript I. Since among the carrot varieties screened, only

carrot (bolero) has been chosen for fractionation and further studies, carrot (bolero) will be indicated as

carrots, here onwards.

Generally fractionation is expected to concentrate and relatively separate the bioactive

component (s); but a complete loss of bioactivity in carrot fractions was observed compared to the

carrot DCM extract. The loss of bio-activity when fractionated might indicate that part of the increase

in glucose uptake exhibited by the extracts could be due to synergistic effects. Inhibition by some other

compounds present in relatively larger amounts in specific fractions, compared to the extract could also

be a possibility. Such synergistic interactions have been observed in previous studies (Atangwho et al.,

2012; Christensen et al., 2009a). Again the fact that the carrot fractions C and F induced glucose uptake

in adipocytes (in presence of insulin) but not in myotubes (tested independent of insulin), could

indicate that the mode of action of the compounds present in these fractions were cell-type specific,

and/or dependent on insulin stimulation for their bioactivity, and/or the inhibition/activation of the

constituent components are manifested differently in different cell types.

This is the first account, demonstrating the potential of the polyacetylenes, falcarinol and

falcarindiol (isolated from carrots) to enhance glucose uptake. Other than its prominent anti-microbial

properties (Xu et al., 2009), Falcarinol has been mostly studied in relation to its pro-apoptotic potential,

54

and has been shown to have remarkable anti-cancer properties (Zaini et al., 2012a). It has been found to

exhibit both cyto-protective and apoptotic effects on CaCo-2 cells at low and high concentrations,

respectively (Young et al., 2007). Again, both falcarinol and falcarindiol have been found to show

hormetic effects on myotubes; where at relatively high concentrations it caused oxidative stress, but at

low concentrations it showed a cyto-protective effect (Young et al., 2008). Very interestingly, it has

been reported to induce neurite formation, which is an essential process in neuronal development; by

increasing intracellular c-AMP and causing the activation of c-AMP dependent protein kinase A and

mitogen activated protein kinase (MAPK) in PC12D cells (Wang et al., 2006). It is well known that

increase in intracellular AMP levels during low cellular energy levels, activates AMPK, which induces

glucose uptake, glycolysis, and fat oxidation. The activation of AMPK by c-AMP is relatively unclear.

In a previous study, carried out in adipocytes, c-AMP has been found to activate AMPK, although

several other cellular mediators have been identified to be involved in this process (Omar et al., 2009).

Recently, p38 MAPK has been found to be activated by insulin, contraction and other cellular stimuli

that is thought to regulate the activity and not the translocation of Glut4 (Somwar et al., 2002). Again,

inhibition of p38 MAPK has been found to inhibit glucose uptake caused by the plant alkaloid

Barberine. It has recently been shown that p38 MAPK is a downstream target of AMPK, and is

involved in AMPK stimulated glucose uptake in L6 myotubes (Cheng et al., 2006). Therefore the effect

of falcarinol on activation of MAPK and increasing the c-AMP levels could provide a rationale for the

increased glucose uptake observed in primary myotubes. The similarity in the chemical structure of

falcarinol and falcarindiol, could explain the bioactivity observed for the latter, and at least partially

that observed in carrot DCM extract.

Naringenin, the bioactive component identified from S. nigra extracts has been discussed in

Manuscript I.

55

2.5. Short summary:

Out of the 8 different plants screened for their potential to increase glucose uptake, those showing an

increase of ≥ 20% were

T. vulgaris (thyme)

E. purpurea (purple coneflower)

D. carota (bolero)

R. rosea

S. nigra

Among these five bioactive plants, the two plants chosen for fractionation were

D. carota (bolero)

S. nigra

The S. nigra fractions showed bioactivity, whereas the D. carota (bolero) fractions did not.

Pure compounds known to be present in S. nigra and other related polyphenols, together with those

identified in the S. nigra bioactive fractions were studied for their bioactivity.

Among the tested compounds, naringenin and kaempferol showed maximum increase in glucose

uptake.

The two carrot polyacetylenes, falcarinol and falcarindiol were examined for their bioactivity.

It was shown for the first time that falcarinol and falcarindiol can enhance glucose uptake.

56

3. Study II: Studying the influence of selected elderflower compounds and related polyphenols in clonal beta cells

3.1. Objective:

To investigate any possible effect of the selected pure compounds on insulin secretion, and study their

effect on genes involved in beta cell function, survival, apoptosis and stress.

3.2. Methods:

All methods used have been described in Manuscript II. The RT-PCR analysis was done with the help

of Aros Applied Biotechnology A/S.

3.3. Results:

The pure compounds: FeA, CaA, p-CA, naringenin, kaempferol, quercetin and Q-3-O-G were tested

for their acute effects on insulin secretion in clonal INS-1E cells. The effect of FeA, p-CA, kaempferol,

and Q-3-O-G on insulin secretion has been illustrated in figure 19. All the compounds were tested at

0.01 and 1 µM concentrations in presence of 16.7 mM glucose. A significant increase of 13.4 (p =

0.002) and 22.3 (p < 0.001) % was observed at 0.01 and 1 µM concentrations for FeA; and that of 11.7

(p = 0.003) % was observed at 0.01 µM concentrations for Q-3-O-G, compared to their respective

controls (16.7 mM glucose). Kaempferol and p-CA did not show any significant increase in insulin

secretion. The difference in insulin secretion between cells treated with 3.3 and 16.7 mM glucose in

these experiments varied between 331 to 387 %, indicating the sensitivity of the INS-1E cells to

increase in glucose concentrations. Based on the insulin secretion results obtained from the acute-

exposure experiments, the phenolic compounds: CaA, naringenin and quercetin were chosen for further

studies. These phenolic compounds were investigated further for their effects on insulin secretion

following chronic exposure, in the presence of low (3.3 mM) or high (16.7 mM) glucose, and under

glucotoxic conditions. Moreover, the effect of the phenolic compounds on the expression of genes

57

involved in beta cell function, survival, apoptosis and stress were studied under normoglycemic and

glucotoxic conditions. All the studies investigating the effect of CaA, naringenin and kaempferol on

INS-1E cells have been described in Manuscript II.

3.3

mM

G

16.7

mM

G

16.7

mM

G +

0.0

1 uM

16.7

mM

G +

1 u

M

Insu

lin s

ecre

tion

(ng/m

L)

0

20

40

60

80

100

120

140

160 Ferulic acid

Kaempferol

p-Coumaric acid

Q-3-beta-D- glucoside

**

***

**

Fig. 19 Effect of phenolic compounds on insulin secretion in INS-1E cells after acute exposure.

INS-1E cells were grown in media containing 11 mM glucose for 72 h, thereafter incubated for 1 h in

either 3.3 mM glucose or 16.7 mM glucose in the presence or absence of the compounds at 10-8

and 10-

6 M concentrations, following which the supernatant was harvested and insulin content measured. Data

are shown as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. The stars indicate significant

differences in insulin secretion within each compound, compared to their respective controls (16.7 mM

glucose).

3.4. Discussion:

Studies investigating the effect of CaA, naringenin and quercetin on INS-1E cells have been discussed

in Manuscript II.

58

3.5. Short summary:

Seven phenolic compounds among the S. nigra secondary metabolites and the related polyphenols

tested in Study I were selected for further examination of their insulin secreting capabilities in INS-1E

cells. They are:

CaA

p-CA

FeA

Kaempferol

Naringenin

Quercetin

Q-3-O-G

Based on the insulin secretion experiments conducted by acutely exposing the INS-1E cells to these

compounds, the following three compounds were selected for further studies:

CaA

Naringenin

Quercetin

These compounds increased insulin secretion both upon acute and chronic exposure at hyperglycemic,

as well as glucotoxic conditions.

In gene expression studies, these compounds indicated cyto protective effects on beta cells under

glucotoxic conditions.

59

4. Study III: Study of the possible signaling pathways behind Naringenin and Falcarinol induced glucose uptake

4.1. Objective:

To elucidate the possible signaling pathways involved in the observed increase in glucose uptake by

naringenin and falcarinol.

4.2. Methods:

All the methods used in Study III have been described in the Manuscript III. The information excluded

from Manuscript III has been given below.

Note: The antibodies against phosphorylated Akt and AMPK (pAkt and pAMPK) were bought from

Cell Signalling Technology (Danvers, MA, US).

4.3. Results:

The results obtained in Study III has been illustrated and described in Manuscript III. Additionally,

western blotting trials to detect pAkt and pAMPK were made. However, further optimization trials

would be required to obtain satisfactory results.

4.4. Discussion:

All the results obtained in Study III have been discussed in Manuscript III.

60

4.5. Short summary:

Naringenin and falcarinol stimulate glucose uptake in primary porcine skeletal myotube cultures

Naringenin can cause a greater increase in glucose uptake in insulin resistant myotubes compared to

insulin (10 nM)

Falcarinol stimulated glucose uptake in in insulin resistant myotubes is equivalent to that caused by

insulin (10 nM)

The Glut4 inhibitor indinavir and PI3K inhibitor wortmannin, greatly diminishes naringenin and

falcarinol stimulated glucose uptake

The AMPK inhibitor DM attenuated naringenin stimulated glucose uptake, but increases falcarinol

induced glucose uptake

Naringenin and falcarinol preferentially induced the phosphorylation of TBC1D1, compared to

TBC1D4

Naringenin induced TBC1D1 phosphorylation was suppressed by PI3K and p38MAPK inhibitor,

wortmannin; and the AMPK inhibitor DM. This indicates that an active PI3K and/or p38MAPK, as

well as an active AMPK is required for naringenin to enhance phosphorylation of TBC1D1 and

therefore glucose uptake

Falcarinol stimulated phosphorylation of TBC1D1 was inhibited by wortmannin, but not by DM. This

suggests a PI3K and/orp38 MAPK dependent but AMPK independent regulation of glucose uptake by

falcarinol

61

5. General Discussion

5.1. Screening of plant extracts, fractions and their secondary metabolites for

bioactivity

The primary goal of this PhD study was the identification of plants with a promising potential to

increase glucose uptake in primary porcine myotubes. Screening of 22 DCM and MeOH extracts from

8 medicinal and food related plants [T. vulgaris (thyme), E. purpurea (purple coneflower), D. carota

(carrot, varieties: bolero and purple haze), B. oleracea (broccoli and green cabbage), S. hortensis

(summer savory), S. nigra (elderflowers), and R. rosea (roseroot)] for glucose uptake both in presence

and absence of insulin, revealed 5 plants (thyme, purple coneflower, carrots (bolero), elderflowers and

roseroot) with major potential to increase glucose uptake in primary myotubes. Thyme, carrots

(bolero), elderflowers and roseroot (flowers) DCM extracts showed most pronounced increase in

glucose uptake, and were therefore chosen for further studies.

5.1.1. Investigation of oxidative stress by intracellular ROS generation

Plants possess a huge repertoire of secondary metabolites with varying polarity and chemical

structures, like terpenes, alkaloids, phenolic acids, polyisoprenes, plant amines, glycosides and rare

amino acids. Certain plant polyacetylenes like falcarinol and falcarindiol has shown the potential to

accelerate intracellular ROS generation (Young et al., 2008). Again, it has been previously

demonstrated that increase in ROS mediated oxidative stress can cause an increase in glucose uptake

(Lekas et al., 1999). It has been believed for several decades that elevation in ROS levels is detrimental

for cellular well-being, and influence aging, inflammation, dna-damage, and defects in insulin signaling

both in cell systems and in vivo (Harman, 1956; Vina et al., 2007; Vina et al., 2004; Wei et al., 2008).

But the relatively new research has indicated that ROS also function as signaling molecules, and are

necessary for cellular adaptation after physical activity. Therefore, suppression of these molecules with

62

anti-oxidants can prevent cellular adaptation, suggesting that a balance between ROS formation and

quenching is critical for harvesting the hormetic effects of (oxidative) stress (Gomez-Cabrera et al.,

2008; Ristow and Zarse, 2010; Ristow et al., 2009; Yang and Hekimi, 2010). In order to investigate

whether the reason behind the observed increase in glucose uptake is caused due to oxidative stress,

intracellular ROS generation was measured for the DCM extracts of thyme, carrots (bolero),

elderflowers and roseroot (flowers). None of the extracts were found to increase ROS formation in the

myotubes. On the contrary, the basal level of ROS in the myotubes was slightly but significantly

lowered.

At this stage, due to constraints in fractionation possibilities, elderflower and carrot extracts

were chosen for further studies.

5.1.2. Study of satellite cell proliferation inducing potential

Skeletal muscle atrophy is a persistent problem, especially in the elderly. It can also be an outcome of

chronic disorders like cancer, chronic heart failure, pulmonary obstructive disorders; and the loss of

skeletal muscles increases vulnerability towards diabetes (Sakuma and Yamaguchi, 2012; Srikanthan et

al., 2010; Thomas, 2007). However, activation of satellite cells often initiated by muscle damage (e.g.

that caused by strength training), can reduce severe muscle loss, by satellite cell proliferation and

differentiation (Hawke and Garry, 2001; Sakuma and Yamaguchi, 2012). DCM extract of elderflowers

and MeOH extract of carrots (purple haze) were examined for their impact on satellite cell

proliferation. Proliferation of the satellite cells was improved by both the extracts significantly. These

results open up the possibility of a new area of research, studying the effects of bioactive plant

compounds in alleviation of Sarcopenia.

63

5.1.3. Fractionation and further screening of fractions and secondary metabolites

In order to identify the constituent bioactive components, the extracts of the selected plants

(elderflower DCM and MeOH extract; and carrot (bolero) DCM extract) were fractionated by flash

chromatography (WP2). This resulted in 7 fractions (A-G) from each of the DCM and MeOH

elderflower extracts and 8 fractions (A-H) from carrot (bolero) DCM extract. All the fractions were

screened for their ability to independently enhance glucose uptake in myotubes.

Two elderflower DCM fractions D and E were found to increase glucose uptake significantly.

Neither the fractions obtained from elderflower MeOH extract, nor those obtained from carrot (bolero)

DCM extract showed any increase in glucose uptake. Normally it is expected that fractionation will

concentrate the bioactive component and therefore a further increase in bioactivity will be observed.

On the contrary, a loss of bioactivity was observed in elderflower MeOH and carrot (bolero) DCM

fractions. This could be attributed to the fact that fractionation can also lead to increase concentrations

of the inhibitors, increasing antagonism leading to the suppression or loss of bioactivity.

Chromatographic separation (WP2) of the bioactive fractions D and E of the elderflower DCM

extract mainly contained naringenin, α-LA and LA. These three compounds were selected for further

studies, as well as several other known elderflower metabolites (Christensen et al., 2008), together with

certain related polyphenols and carrot polyacetylenes. Overall, the elderflower metabolites tested were:

naringenin, 5-O-CA, Q-3-O-R, Q-3-O-G, Q-3-O-6’’-A, K-3-O-R, I-3-O-G, I-3-O-R, α-LA, and LA;

the related polyphenols tested were: kaempferol, CaA, FeA, and p-CA and the carrot polyacetylenes

tested were falcarinol and falcarindiol. All were tested for their effect on insulin-independent glucose

uptake. Among them, 5-O-CA, naringenin, kaempferol, CaA, FeA, p-CA, falcarinol and falcarindiol

were found to enhance glucose uptake significantly; whereas LA showed a strong tendency.

Most prominent increase in glucose uptake was observed for naringenin, kaempferol, falcarinol

and falcarindiol. Naringenin has been previously found to enhance glucose uptake via AMPK

activation in L6 myotubes (Zygmunt et al., 2010). Contradictory findings have reported inhibition of

64

glucose uptake in MCF-7 breast cancer and U937 cells (Harmon and Patel, 2004; Park, 1999).

However, this differential effect on glucose uptake could be indicative of a mode of action, that is cell-

type specific.

Amongst the related polyphenols tested, kaempferol showed the highest increase in GU. This

study reported for the first time, the glucose uptake enhancing potential of kaempferol in myotubes.

However, in separate studies, kaempferol has previously been shown to inhibit GU in HeLa cells

(Filomeni et al., 2010) but enhance GU in mature 3T3-L1 adipocytes (Fang et al., 2008). Another

contradictory finding reports that kaempferol-3-O-neohesperidoside (glycosylated natural derivative of

kaempferol) was found to increase glucose uptake in L6 myotubes, where the glycon was reported to

be the structure responsible for the activity (Yamasaki et al., 2011).

CaA and 5-O-CA (also known as chlorogenic acid) were both found to enhance glucose uptake

in this study. In an earlier study conducted in rat skeletal muscle in vitro, CaA but not 5-O-CA was

found to enhance glucose uptake by enhancing AMPK phosphorylation (Tsuda et al., 2012). However,

both CaA and 5-O-CA were found to reduce plasma glucose levels in animals and humans (Bassoli et

al., 2008; Hsu et al., 2000). FeA and p-CA (among the related polyphenols tested) were also found to

enhance glucose uptake. In separate studies p-CA has been shown to increase glucose uptake in L6

myotubes via AMPK-phosphorylation (Yoon et al., 2013); whiles FeA increased glucose uptake via a

PI3K-dependent mechanism (Prabhakar and Doble, 2009).

The carrot polyacetylenes falcarinol and falcarindiol were most commonly known for their

cytotoxic, antibacterial and anti-cancer properties (Xu et al., 2009; Zaini et al., 2012b). However, both

polyacetylenes were found to have concentration dependant biphasic effect on cyto-protection,

oxidative stress, dna-damage, cell proliferation and apoptosis (Young et al., 2008; Young et al., 2007).

This is the first study suggesting both falcarinol and falcarindiol are capable of stimulating glucose

uptake in myotubes. This could, at least partially explain the pronounced increase in glucose uptake,

caused by carrot (bolero) DCM extracts.

65

5.2. Study of selected phenolic compounds on insulin secretion and gene expression in

INS 1E cells

The progression of T2D is asymptomatic in its initial stages. This is due to compensation by increasing

insulin production by the pancreatic beta cells. Increasing insulin resistance beyond this point causes

beta cell exhaustion and if left un-intervened, can lead to beta cell failure and apoptosis. Flavonoids and

phenolic acids are known to possess multiple bioactivities. Therefore, it was interesting to investigate if

these bioactive compounds influence insulin secretion as well as expression of genes involved in

insulin secretion, stress and apoptosis in beta cells.

Seven phenolic compounds: CaA, FeA, naringenin, kaempferol, quercetin, Q-3-O-G, and p-CA

were tested for their ability to stimulate insulin secretion in the clonal pancreatic beta-cell line INS 1E,

after acute exposure in presence of low or high glucose (mimicking hypo- and hyperglycemic

conditions). Results obtained, indicated CaA, naringenin and quercetin to be the most promising

candidates and therefore, these compounds were chosen for further studies.

5.2.1. Insulin secretion under chronic exposure and glucotoxic conditions

Upon chronic exposure to the phenolic compounds, and thereafter to low and high glucose, all three

compounds stimulated insulin secretion significantly under exposure to high glucose; whereas a small

increase in insulin secretion was observed when exposed to low glucose in case of CaA and quercetin

at the highest concentration tested. Naringenin did not cause any increase in insulin secretion at low

glucose. This is an important and desirable property for drugs that stimulates insulin secretion in

pancreatic beta cells, as side-effects of insulin secretion stimulating drugs often involve hypoglycemia,

which can have lethal consequences among weak or elderly diabetic patients (Noh et al., 2011). Some

other insulin secretagogues known to have this differential, glucose dependant effect on insulin

secretion are GLP-1 (glucagon-like peptide 1); and the diterpenes, Stevioside and Steviol (Jeppesen et

al., 2000; Meloni et al., 2013).

66

In cells exposed to glucotoxic conditions, insulin secretion in response to increase in glucose

concentrations was significantly reduced. But those treated with the phenolic compounds showed

improved glucose sensitivity and insulin secretion. Prolonged glucotoxicity can lead to beta cell stress

and apoptosis (Kaiser et al., 2003; Robertson et al., 2003).

5.2.2. Impact of selected phenolic compounds on beta cell gene expression under glucotoxic

conditions

Glucotoxicity has been associated with accelerated apoptosis and faulty gene expression of key beta

cell specific genes (Robertson et al., 2004). Therefore, effect of these phenolic compounds on

expression of genes involved in beta cell function, stress, survival and apoptosis under glucotoxic

conditions were studied. The genes analysed were: Glut2, Gck, Ins1, Ins2, Beta2, Pdx1, Akt1, Akt2,

IRS1, Acc1, Bcl2, Bax, Casp3, Hsp70, and Hsp90.

A significant increase in the expression of the Ins1 gene was observed in the presence of

the phenolic compounds both under normoglycemic and glucotoxic condition. This observation was in

line with the increase in insulin secretion observed under these conditions. A significant increase in

Ins2 gene expression was also observed in cells treated with CaA and naringenin, under

normoglycemic but not under glucotoxic conditions. Glut2, has the highest capacity and the lowest

affinity for glucose, which allows glucose uptake in the beta cells only when glucose level is high and

insulin secretion is necessary (Efrat, 2003). Again, Gck (the enzyme involved in the phosphorylation of

glucose molecules) also acts as a glucose sensor (Doliba et al., 2012; Nakamura et al., 2009). A

significant increase in Glut2 expression (induced by naringenin and quercetin) under glucotoxic

conditions; and Gck expression under both glucotoxic and normoglycemic conditions indicates an

increased sensitivity towards glucose in presence of these phenolic compounds and is in line with the

increase observed in Ins1 gene expression.

The expression of the transcription factors Beta2 and Pdx1 was not changed significantly under

glucotoxic conditions (except by naringenin). However all three phenolic compounds significantly

67

increased their expression under normoglycemic conditions. Studying any increase in the activity of

these transcription factors could provide the fundament for the observed increase in Ins 1 gene

expression. However other beta cell specific transcription factors (e.g. Nkx2.2, Pax6, Foxa2, and

Nkx6.1) could also be responsible for such an effect (Habener et al., 2005).

Irs1, Akt1 and 2 are members of the insulin signalling pathway. Irs1 expression was not

increased by any of the phenolic compounds under glucotoxic conditions. Naringenin and quercetin

induced Akt1 gene expression under glucotoxic conditions; whereas CaA and naringenin under

normoglycemic conditions. All three phenolic compounds increased Akt2 expression under

normoglycemic conditions. However, naringenin enhanced Akt2 expression under glucotoxic

conditions. This suggests the augmentation of an otherwise down regulated insulin signalling pathway

by naringenin.

The INS 1E cells exposed to glucotoxic conditions exhibited an increase in the expression of

apoptotic genes like Casp3 and Bax, and down-regulation of the survival gene Bcl2. This could be

indicative of the apoptotic effects of glucotoxicity. In presence of the phenolic compounds, a reduction

in the pro apoptotic genes Casp3 and Bax (a member of the Bcl2 family) was observed, especially for

CaA and naringenin, suggesting a cyto-protective effect of these phenolic compounds on beta cells.

The Bcl2 family consists of both pro and anti-apoptotic proteins. The pro-survival members of this

family prevent apoptosis by inhibiting the pro-apoptotic proteins like Bax, Bak and BH3-only proteins

(Vogler, 2012). The gene expression of Bcl2 was significantly induced by all three phenolic

compounds under normoglycemic conditions, and naringenin showed a tendency under glucotoxic

conditions, indicating an induction of pro-survival genes by naringenin.

Heat shock proteins are molecular chaperones, induced as a response to cellular injury and

stress, and play an important role in cell survival. Hsp70 has been shown to be involved in combating

neurodegeneration, ischemic heart disease and diabetes, while inhibition of Hsp90 has recently been

recognised as an efficient approach in pacifying various forms of cancer (Soti et al., 2005), which

68

indicates its importance in cell survival. A significant increase in Hsp70 gene expression was observed

in presence of all the phenolic compounds at both normoglycemic and glucotoxic conditions, of which

quercetin caused the maximum (two-fold) increase; whereas the gene expression of Hsp90 was

significantly up-regulated by naringenin at both normoglycemic and glucotoxic conditions, indicating

the potential role of these phenolic compounds in beta cell survival during glucotoxicity.

The first step of lipogenesis is catalysed by the enzyme Acc1. Animals deficient in Acc1 show

loss of body weight and continuous lipid oxidation. Therefore reduction in Acc1 expression and

activity has been a therapeutic goal against obesity and related metabolic disorders like T2D (Tong,

2005). It has been found to play an important role in glucose stimulated insulin secretion in INS 1E

cells (Zhang and Kim, 1998). During hyperglycemia, an increase in Acc1 gene expression has been

observed, and is associated with decrease in fatty acid oxidation (Zhang and Kim, 1998). Our study

also exhibited a steep increase in Acc1 gene expression under glucotoxic compared to normoglycemic

conditions. Moreover, long-term inhibition of fatty acid oxidation, due to overexpression of Acc1 in

beta cells during glucotoxicity can induce beta cell dysfunction and apoptosis due to lipotoxicity

induced endoplasmic reticulum (ER) stress (Cnop et al., 2010). In the present study only naringenin

was found to cause a minute but significant increase in Acc1 expression under normoglycemic

conditions. However, under glucotoxic conditions, Acc1 expression was increased by more than 3 fold

(compared to normoglycemic conditions), but in presence of the phenolic compounds the expression

level was reduced to the same level as observed in cells grown under normoglycemic conditions. This

remarkable restoration of the Acc1 mRNA levels by CaA, naringenin, and quercetin underlines their

importance in the field of lipotoxicity induced beta cell dysfunction in T2D.

5.3. Studying the mechanism behind naringenin and falcarinol induced glucose uptake

As discussed earlier, several phenolic acids, polyacetylenes, and flavonoids were found to increase

glucose uptake in myotubes. Of them, the two secondary metabolites naringenin and falcarinol from

69

elderflowers and carrots respectively were chosen for further investigation. Initially, naringenin and

falcarinol were tested for their ability to enhance glucose uptake in presence or absence of insulin.

Glucose uptake caused by naringenin was significantly higher than control, but it did not vary much

between the concentrations tested. Moreover, in presence of insulin there was an increase in glucose

uptake for all the naringenin concentrations, although maximum increase was observed at the lowest

concentration of naringenin. The additive increase observed could indicate dependence on two different

pathways. However, the fact that the total increase in Glucose uptake was slightly reduced at higher

naringenin concentrations in presence of insulin, might suggest sharing of at least some pathway

proteins by naringenin and insulin.

In order to estimate their potential further, their effect on glucose uptake was tested in IRM, as

compared to normal myotubes. Basal and insulin stimulated glucose uptake was significantly reduced

in IRM. The increase in glucose uptake caused by naringenin was equivalent in both normal and insulin

resistant myotubes, indicating that insulin resistance has not affected its mode of increasing glucose

uptake. However, falcarinol behaved in a way similar to insulin, where there was a reduction in glucose

uptake in IRM, compared to normal myotubes. This might suggest dependence on the same signalling

pathway as insulin, or on one or more of the members of this pathway.

5.3.1. Investigating the dependence on Glut4 and insulin/AMPK signalling

To investigate the underlying mechanism behind the observed bioactivity of naringenin and falcarinol,

myotubes were separately incubated with the Glut4 inhibitor indinavir, PI3K inhibitor wortmannin, and

AMPK inhibitor DM.

Indinavir caused a major decrease in basal, as well as insulin and naringenin/falcarinol

stimulated glucose uptake. The level of glucose uptake in presence of insulin, naringenin and falcarinol

was comparable to the basal uptake in indinavir treated myotubes. This indicates that most of the

glucose uptake caused by naringenin and falcarinol is mediated by Glut4. A minute but significant

increase in naringenin treated myotubes was observed at a concentration of 10 µM in indinavir treated

70

myotubes. A possible explanation for this observation could either be that naringenin can use other

glucose transporters (like Glut1) to some extent or it might be capable of slightly increasing the activity

of the small number of uninhibited Glut4 transporters. A small but significant decrease in glucose

uptake was observed for falcarinol at the concentration of 30 µM in indinavir treated cells, indicating

inhibition of glucose uptake at this concentration.

Upon treatment with the PI3K inhibitor, wortmannin, basal, as well as insulin, naringenin and

falcarinol stimulated glucose uptake was significantly decreased. Glucose uptake caused by naringenin

and falcarinol was comparable to the reduced basal uptake in wortmannin treated myotubes; although

insulin induced glucose uptake was reduced to basal glucose uptake in normal myotubes. It can be

speculated that this difference is due the dependence of naringenin and/or falcarinol on one or more

proteins that is inhibited by wortmannin but is not necessary for insulin stimulated glucose uptake. It is

important to note that in earlier studies, wortmannin has also been found to inhibit MAPK (Ferby et al.,

1996a; Ferby et al., 1996b) with an IC50 of 300 nM. Therefore the protein involved in naringenin and/or

falcarinol induced glucose uptake could be a MAPK. It is to be considered, that p38-MAPK has been

implicated as a downstream target of AMPK (Cheng et al., 2006) and has been found to be involved in

full activation of Glut4 (Konrad et al., 2001).

In myotubes treated with the AMPK inhibitor DM, naringenin and AICAR stimulated glucose

uptake decreased and reached the basal level. DM treatment did not affect the basal glucose uptake. It

has been mentioned earlier, that naringenin can increase glucose uptake via AMPK activation in L6

myotubes (Zygmunt et al., 2010). Therefore an inhibition in glucose uptake caused by naringenin is an

expected outcome of AMPK inhibition. Falcarinol, on the other hand exhibited a significant increase in

presence of DM. The reason behind this is unclear, though cross-talk between signalling pathways, and

inhibition/activation of enzymes/pathways responsible for increasing glucose uptake could be a

possibility. Furthermore, DM has been shown to participate in other signalling cascades, independent

of the AMPK pathway (Jin et al., 2009). Nevertheless, further investigation is necessary to explain this

71

observation. However, this shows that unlike naringenin, falcarinol enhances glucose uptake by an

AMPK-independent mechanism. Previous research has shown that falcarinol at low concentrations can

increase ROS formation (Young et al., 2008). Again, ROS has been found to activate p38-MAPK (Kim

et al., 2013), which in turn plays an important role in glucose uptake (Konrad et al., 2001). This could

be a possible explanation to the AMPK-independent glucose uptake caused by falcarinol.

5.3.2. Impact on TBC1D4 and TBC1D1 phosphorylation

TBC1D4 and TBC1D1 are key Rab-GTPase activating proteins (GAPs), which are involved in the

regulation of Glut4 translocation to the plasma membrane. Upon phosphorylation these proteins bind

14-3-3 proteins, leaving the Rab proteins in a GTP bound active state (Sakamoto and Holman, 2008).

The active GAPs then lead to the translocation of Glut4 containing vesicles to the plasma membrane.

TBC1D4 and TBC1D1 have been found to participate both in insulin and contraction-induced glucose

uptake. They are targets of Akt, AMPK, as well as other kinases (Chen et al., 2008; Geraghty et al.,

2007; Kane et al., 2002; Roach et al., 2007; Taylor et al., 2008; Treebak et al., 2006) .

Phosphorylation of TBC1D4 and TBC1D1 by naringenin and falcarinol was studied to further

investigate their role in skeletal muscle glucose metabolism. The activation of TBC1D1 by naringenin

and falcarinol was reported for the first time in this study. An interesting observation of this study was

that insulin stimulated increase in phosphorylation was much higher for TBC1D4 compared to

TBC1D1, and naringenin and falcarinol solely stimulated TBC1D1 phosphorylation. Both TBC1D4

and TBC1D1 have calmodulin binding domains; mutations in which has been found to diminish

contraction induced but not insulin stimulated glucose uptake in TBC1D4 (Kramer et al., 2007). Such

studies are yet to be conducted on TBC1D1. Moreover, mass spectrometry analysis on TBC1D1 from

mouse skeletal muscle revealed several phosphorylation sites, of which majority were found to be

consensus or near consensus sites for AMPK; and AICAR was found to be a stronger regulator of

TBC1D1, causing more phosphorylation on TBC1D1 than insulin (Taylor et al., 2008). This is very

much in confirmation of our observation that naringenin (known to activate AMPK (Zygmunt et al.,

72

2010)) increases TBC1D1 phosphorylation to a much higher degree compared to insulin. Moreover, it

has been shown earlier, that TBC1D1 is abundantly expressed in fast-twitch muscles, while higher

levels of TBC1D4 is found in muscles with slow-twitch characteristics (Taylor et al., 2008).Overall, the

existence of the homologs, together with their altered expression in different muscle fibre types, as well

as the observed differences in the modulation of glucose uptake in TBC1D4 mutants (Kramer et al.,

2007; Thong et al., 2007) by insulin and contraction, and the many different phosphorylation sites in

TBC1D1 encouraging differential phosphorylation (Taylor et al., 2008), suggests the possibility of

differential regulation of these signalling proteins by various regulators like insulin, contraction

(AMPK), and other small molecule activators.

The observed phosphorylation of TBC1D1 by naringenin was inhibited by wortmannin

and DM; whereas that by falcarinol was inhibited by wortmannin, but increased in presence of DM.

This finding substantiates the diminished glucose uptake observed in presence of wortmannin for both

naringenin and falcarinol, and the increase and decrease in glucose uptake exhibited by falcarinol and

naringenin in presence of DM.

73

6. Conclusions and Future perspectives

Reduction in insulin sensitivity leading to diminished glucose transport into the target tissues is a key

feature of T2D. The screening study performed with eight medicinal and food plants brought into focus

five (Thymus vulgaris, Daucus carota (bolero), Sambucus nigra, Echinacea purpurea and Rhodiola

rosea) that exhibited promising potential to increase glucose uptake in myotubes. Some of these plants

were also found to possess potent anti-oxidant properties.

After completion of the screening studies, due to selection of plants based on joint priorities

between work packages it was not possible to investigate plants like Thymus vulgaris, Echinacea

purpurea and Rhodiola rosea, which also exhibited a huge anti-diabetic potential during screening

studies. Literatures validating the basis of the anti-diabetic properties of these plants are limited,

providing an interesting field for future research.

Daucus carota (bolero) (carrot) and Sambucus nigra (elderflower) were selected for fractionation.

Carrot fractions did not enhance glucose uptake in myotubes; but two elderflower fractions were found

to be potent. Separation of these fractions led to the composition of these fractions. On examination of

these and other known elderflower compounds, together with some related polyphenols, gave an

overview of the individual potential of a wide array of pure compounds to enhance glucose uptake in

myotubes (Manuscript I). Of these, glucose uptake exhibited by naringenin and kaempferol was most

pronounced. The polyacetylenes falcarinol and falcarindiol, found (among some other plants) in carrots

were tested separately. These compounds increased glucose uptake in dose dependent manner.

Separation of the constituent compounds in the bioactive extracts and fractions of elderflowers

revealed the presence of certain unknown flavonol glycosides and naringenin-derivatives. Further

investigation is necessary for identification and structure elucidate of these compounds.

Together with glucose uptake, insulin is also responsible for driving glycogen synthesis in

muscles. It would be interesting to assess the potential of the bioactive compounds (that has been found

74

to cause an increase in glucose uptake), to simultaneously stimulate glycogen formation, in order to

evaluate the extent of their insulin-mimetic properties. At the same time, evaluation of any increase in

glycolysis in the muscles would also provide understanding of the fate of glucose.

The elderflower DCM and carrot (purple haze) MeOH extract showed satellite cell proliferating

potential, which suggests that certain plants might possess bioactive compounds that can enhance

muscle cell proliferation. It would be highly interesting to confirm this finding in vivo. In general,

studying the proliferative potential of bioactive compounds, could generate new knowledge, that can be

useful in the mitigation of skeletal muscle atrophy/sarcopenia.

Phenolic compounds are known to possess several health promoting properties and multiple

bioactivities. Seven phenolic compounds were tested for their capacity to enhance insulin secretion

under acute exposure in INS 1E cells, and three (CaA, naringenin and quercetin) were selected for

further studies under chronic exposure, and glucotoxic conditions. Expression analysis of genes

involved in beta cell function, stress and apoptosis/survival was performed. These compounds had

prominent insulin secreting capacity both under hyperglycemic and glucotoxic conditions (Manuscript

II). They were also found to up-regulate the insulin1 gene, genes involved in glucose sensitivity, and

chaperones, and down-regulate the Acc1 gene, responsible for biosynthesis of fatty acids.

In diabetic patients, dysfunctional exocytosis in beta cells has also been reported to cause

defective insulin secretion. The effect of these phenolic compounds in rehabilitation of the exocytosis

machinery could provide vital information about the extent of their efficacy.

Similar to pancreatic beta cells (and other pancreatic cells e.g. alpha-cells), and myocytes

hepatocytes also play a major role in the progression of T2D. Investigating the effect of these

compounds on gluconeogenesis in hepatocytes could provide an important overview of the efficacy or

possible side-effects of these compounds, as supplementary medicines or nutraceuticals.

75

An in vivo study involving potent plant extracts (e.g. the plant extracts found to have anti-

diabetic properties in this study) and their secondary metabolites could be highly useful both for

providing valuable knowledge on their effect on many different tissues and organs (e.g. muscles, liver,

pancreas and adipose tissue) directly involved in T2D, and also for understanding the interactions or

cross talk between them.

Examination of glucose uptake potential of naringenin and falcarinol in myotubes in presence of

different inhibitors like indinavir, wortmannin, and DM, upheld certain mechanistic clues (Manuscript

III). The study indicated the dependence of naringenin and falcarinol on PI3K and/or p38 MAPK. But

further studies using specific inhibitors or small interfering RNAs (siRNAs) against PI3K or p38

MAPK could further ascertain this finding. Their direct dependence on Glut4 for glucose transport was

also demonstrated in this study. The study also confirmed that active AMPK was required for

naringenin to induce glucose uptake in myotubes and revealed that the same was not true for falcarinol.

Several compounds of plant origin, especially the flavonoids have been found to be AMPK

and/or PPAR agonists. A study screening the potent secondary metabolites (discussed in this study, e.g.

naringenin) for PPAR-δ (most abundant in muscles) or AMPK activation could identify compounds

with single/dual activity; and encourage investigation of their activity in different combinations.

Study on phosphorylation of key Rab-GTPase activating proteins TBC1D4 and TBC1D1 by naringenin

and falcarinol showed that these compounds significantly increase TBC1D1-phosphorylation. This

increase was inhibited by wortmannin and DM (except for falcarinol, where TBC1D1 phosphorylation

increased in presence of DM). On the other hand, TBC1D4 but not TBC1D1 phosphorylation was

significantly enhanced by insulin. It was also observed in glucose uptake experiments that glucose

uptake in presence of falcarinol is increased in DM treated cells. It is known that activation of different

kinases including AMPK, can lead to phosphorylation of TBC1D1. At the same time falcarinol is

76

known to increase intracellular ROS formation. Again p38MAPK, which is thought to play an

important role in glucose uptake, has also been found to be activated by ROS. Based on this

information, one might speculate, that an increase in falcarinol stimulated glucose uptake could occur

via ROS mediated p38MAPK activation. However, in order to substantiate the possibilities further

research is required in this area.

Activation of Sirt1 has been found to reduce insulin resistance. Small molecule activators of

Sirt1 are lucrative therapeutic targets for amelioration of the diabetic state. Earlier research has shown

that Sirt1 is not involved in naringenin stimulated glucose uptake. It would be interesting to find out if

falcarinol is capable of Sirt1 activation.

In entirety, the work presented in this thesis shows that plants contain bioactive compounds capable of

inducing glucose uptake in myotubes and enhancing glucose stimulated insulin secretion in beta cells,

via modulation of signaling pathways involved in the regulation of glucose transport.

Endeavors to study plant secondary metabolites for possible bioactivities and understanding of

the underlying mechanism responsible for such bioactivity could be useful in developing new tools to

build a future strategy for the treatment of metabolic disorders like T2D.

77

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8. Acknowledgements

I sincerely express my gratitude towards my main supervisor Niels Oksbjerg, Department of Food

Science, Aarhus University, for the encouragement, guidance and support he has given me all these

years, and for always being there to discuss any problem I had. I genuinely thank my co-supervisor

Jette F. Young, for her useful advice and able guidance and also for being very supportive during the

hard times. Per Bendix Jeppesen is gratefully thanked for his supervision during the beta-cell studies

at the Department of Endocrinology, Aarhus University Hospital.

Karsten Kristiansen, Department of Biology, University of Copenhagen, is gratefully

acknowledged for conjecturing the project and guiding us through it. I would also like to thank Xavier

Fretté for bringing me samples all the way from Odense on his way home.

I wholeheartedly thank Suresh Rattan and Stig Purup for the scientific as well as friendly

discussions.

Special thanks go to Anne-Grete Dyrvig Petersen, Inge lise Sørensen, Bente Andersen,

Kasper Bøgild Poulsen and Dorthe Rasmussen, for their exceptional technical assistance and

kindness.

My office mates Brita Ngum Che, Bjorn Melin Nielsen, Kasper Høck and Allesandro

Spanó are heartily thanked for all the discussions we had together, and for providing a friendly

atmosphere to work in.

During the course of this PhD, life presented me with several challenges. Among them, the

demise of both of my grandparents, who were very dear to me, and whom I had lived with all my life,

was the hardest to bear. Yet, their love and affection still gives me strength. I express my gratitude to

Helle Vestrup, Aase Karin Sørensen, Anne Hjørth Balling and Birthe Ømark Jensen, for lending a

helpful hand and for sharing their sorrows with me to lessen mine, during this period of grief and

hardship. I will always remember you for your kindness.

90

I offer heartfelt gratitude to my husband Kaustuv for taking the burden of running the house-

hold singlehandedly, so that I could concentrate in my work; and my daughter Surabhi, for

understanding when I did not have time to listen or play, and also for giving me a big hug that

rejuvenated me when I returned home late and exhausted. I cannot thank my parents enough for

encouraging me every step of the way, especially when I felt low; for taking the stake of travelling so

far and so often, just to help me when I needed them; and most importantly for believing in me and for

inculcating in me the tenacity to push against all odds.

I earnestly thank my dearest friends Lakshman, Anuja, Gitta, Per Bo, Ripu, Cecelia, Pratyay

and Madhubanti for all the babysitting they have done for me, for their encouragement, and for being

by my side as an integral part of my life.


July 2013

91

9. Manuscripts

Manuscript I

Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine

myotube cultures and reduce fat accumulation in Caenorhabditis elegans

Sumangala Bhattacharya, Kathrine B. Christensen, Louise C. B. Olsen, Lars P. Christensen, Kai Grevsen, Nils

J. Færgeman, Karsten Kristiansen, Jette F. Young, and Niels Oksbjerg

Submitted to: The Journal of Agricultural and Food Chemistry

1

Bioactive components from flowers of Sambucus nigra L. increase glucose

uptake in primary porcine myotube cultures and reduce fat accumulation in

Caenorhabditis elegans

Sumangala Bhattacharya1, Kathrine B. Christensen

2, Louise C. B. Olsen


2, Kai

Grevsen4, Nils J. Færgeman

3, Karsten Kristiansen

5, Jette F. Young

1, and Niels Oksbjerg

1*

Author affiliations:

1Department of Food Science, Aarhus University, Blicher’s Allé 20, Postbox 50, 8830 Tjele, Denmark

2Department of Chemical Engineering, Biotechnology and Environmental Technology, University of

Southern Denmark, Niels Bohrs Allé 1, 5230 Odense M, Denmark

3Department of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, Denmark

4Department of Food Science, Aarhus University, Kirstinebjergvej 10, 5792 Aarslev, Denmark

5Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark

*Corresponding author:

Niels Oksbjerg

Institute of Food Science, Aarhus University,

Blicher’s Allé 20, Postbox 50, 8830 Tjele, Denmark

E-mail: [email protected], Tel: + 45 8715 7809, Fax: +45 8715 4891

mailto:[email protected]

2

Abstract

Obesity and insulin resistance in skeletal muscles are major features of type 2 diabetes. In the present

study, we examined the potential of Sambucus nigra flower (elderflowers) extracts to stimulate glucose

uptake (GU) in primary porcine myotubes and reduce fat accumulation (FAc) in Caenorhabditis

elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the

identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In

addition, polyphenols related to those found in elderflowers were also tested and among these,

kaempferol, ferulic acid, p-coumaric acid and caffeic acid increased GU significantly. FAc was

significantly reduced in C. elegans, when treated with elderflower extracts, their fractions and the

metabolites naringenin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-5’’-

acetylglycoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-

glucoside and the related polyphenols kaempferol and ferulic acid. The study indicates that elderflower

extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for

nutraceutical and pharmaceutical applications.

Keywords: Elderflowers, Elderflower extracts, Type 2 diabetes, Glucose uptake, Fat accumulation,

Obesity, Naringenin, Kaempferol, Polyphenols

3

Introduction

Type 2 diabetes (T2D) and visceral obesity have been strongly implicated in the occurrence of a

collection of interrelated metabolic abnormalities, commonly called the metabolic syndrome (MS) (1,

2). T2D is commonly characterized by hyperglycaemia and hyperinsulinemia; and are often

accompanied by β-cell failure and enhanced gluconeogenesis in the liver (3, 4). Again, insulin

sensitivity has been found to correlate negatively with fat accumulation (FAc), irrespective of age and

genetic background (5). Unfortunately, some of the available medications for T2D are associated with

several undesirable side effects (6). This has increased the need for the discovery of supplementary

nutraceuticals with anti-diabetic properties, capable of modulating both glucose and lipid metabolism.

Flowers of black elder are used in many European countries for their appealing flavour and

native flower aroma, to make extracts, which are consumed as a beverage (7, 8). Sambucus nigra L.

(black elder) concoctions have also been used as an alternative medicine against common cold and

influenza (9). Most of the studies on black elder have been performed on the fruits of the plant

(elderberries) which is known for its anti-viral and immunity-boosting effects (10, 11); but recent

research has revealed that the flowers of black elder (elderflowers) have potential anti-diabetic

properties (12). Elderflower extracts have been found to activate peroxisome proliferator-activated

receptors and enhance insulin-dependent glucose uptake (GU) in adipocytes (13). Moreover, in an

observational study, elderflowers in combination with Asparagus officinalis has shown significant

weight-reducing potential (14).

Skeletal muscle is the primary site for glucose disposal, where about 75 % of the insulin-driven

glucose disposal takes place (15). Therefore, in this study we have examined the potential of

elderflower extracts, fractions and secondary metabolites to stimulate GU in primary myotube cultures.

4

Our investigation resulted in the identification of potential bioactive flavonoids and phenolic acids

from elderflower extracts and fractions. This motivated us to test additional polyphenols that are

structurally related to the flavonoids and phenolic acids found in elderflowers and gather information

on their potential anti-diabetic effects. These additional polyphenols (kaempferol, ferulic acid, p-

coumaric acid and caffeic acid), has been referred to in the article as ‘related polyphenols’.

Stress can result in increased glucose utilization and uptake (16). Again, oxidative stress has

been implicated as a contributing factor in MS (17). We therefore monitored the effect of elderflower

extracts on (a) the transcription regulation of heme oxygenase 1 (HMOX1), a marker of oxidative stress

response; and heat shock protein 70 (HSP70), whose expression has been found to be induced by

different kinds of cellular stress conditions, including nutritional stress (18) and (b) generation of

reactive oxygen species (ROS).

Together with a reduction in blood glucose levels, reduction of body fat too is an important goal

for combating obesity and MS. In the worm Caenorhabditis elegans (C. elegans), most of the more

than 400 genes involved in fat storage are evolutionarily conserved and act in common cellular

pathways (19). It has been used in several studies to show the effect of nutritional perturbations on

obesity and other metabolic diseases (20). The wild type strain, N2, was used in this study to examine

the accumulation of the lipophilic dye, Nile Red. While feeding on E. coli bacteria, the fluorescent

compounds are primarily deposited in the fat storage compartments in the intestine (21). A measure of

the level of fat storage is obtained by comparing the fluorescence levels in untreated and treated

worms. The present study demonstrates the effect of elderflower extracts and its selected secondary

metabolites as well as some related polyphenols on GU in primary porcine myotubes and on FAc in C.

elegans.

5

Materials and Methods

Pigs used for the isolation of satellite cells were treated according to the Danish Ministry of Justice

Law, no. 382 (June 10, 1987).

Preparation of plant extracts

Elderflowers (Caprifoliaceae, Sambucus nigra L. cv. Haschberg; Holunderhof Helle, Thumby,

Germany) were picked in June 2007 and frozen immediately after harvest at -22 °C. The frozen flowers

(5 kg) were homogenized and extracted using dichloromethane (DCM, 12 L) and subsequently

methanol (MeOH, 10 L), overnight in the dark at 5 °C and filtered afterwards. The extracts were dried

under vacuum, yielding 28.4 g and 151.4 g of dry matter respectively. Liquid chromatography (LC)

with photodiode array detector (PDA) and mass spectrometric detection was performed with LC-PDA-

MS settings as previously described (22).

Fractionation of plant extracts

A part of the DCM extract (11 g) was separated by flash CC (70 mm i.d., 400 g silica gel 63-200 µm

Merck) using the following solvent gradient: 100 % hexane (1 L), 10-100 % ethyl acetate (EtOAc) in

hexane in 10 % steps (1 L each), 50:50 EtOAc-MeOH (1 L), yielding 112 fractions (100 mL each). The

collected fractions were analysed by normal phase thin layer chromatography (TLC) and then

combined into 7 fractions (DCM A-G). A part of the MeOH extract (5 g) was separated by flash CC

(40 mm i.d., 100 g RP-18 silica gel) using the following solvent gradient: 100 % H2O (200 mL), 10 %

acetonitrile in H2O (150 mL), 30-90 % acetonitrile in H2O in 20 % steps (300 mL each), 100 %

acetonitrile (300 mL), yielding 39 fractions (50 mL each). The collected fractions were analysed by

reverse phase TLC and then combined into 7 fractions (MeOH A-G).

6

Preparation of samples for bioassays

All extracts, chromatographic fractions and standards were dissolved in dimethyl sulfoxide (DMSO

99.9 %, Merck, Darmstadt, Germany) before they were tested for biological activity. Standards of

naringenin, quercetin-3-O-rutinoside (Q-3-O-R), quercetin-3-O-glucoside (Q-3-O-G), quercetin-3-O-

6’’-acetylglucoside (Q-3-O-6’’-A), kaempferol, kaempferol-3-O-rutinoside (K-3-O-R), isorhamnetin-3-

O-glucoside (I-3-O-G), isorhamnetin-3-O-rutinoside (I-3-O-R), 5-O-caffeoylquinic acid (5-O-C),

caffeic acid (CaA), p-coumaric acid (p-CA), and ferulic acid (FeA) were purchased from Sigma-

Aldrich Chemie GmbH (Steinheim, Germany) or Extrasynthese (Genay, France).

Preparation of myotube cultures

Satellite cells were isolated from semimembranosus muscles of female pigs weighing approx.12 Kg,

essentially as stated in (23) and stored in liquid nitrogen until used. To prepare myotube cultures, the

cells were thawed and evenly seeded on Matrigel matrix (BD Biosciences, cat no. 354230) coated (1:50

v/v) 24, 48, or 96 well plates for RT-PCR studies, GU assay, and 2’, 7’ dichlorodihydrofluorescein

diacetate (H2DCF-DA) oxidation studies, respectively. Cells were proliferated in Porcine Growth

Medium (PGM) consisting of 10 % foetal calf serum (FCS), 10 % horse serum, 80 % Dulbecco’s

modified Eagles medium (DMEM) with 25 mM glucose (Life Technologies, Naperville, IL) and

antibiotics (100 IU/mL penicillin, 100 IU/mL streptomycin sulphate, 3 µg/mL amphotericin B, 20

µg/mL gentamycin). The cells were grown in PGM until they were approximately 80 % confluent in a

CO2-regulated humidified incubator (95 % air and 5 % CO2 at 37°C). Thereafter, the cells were

proliferated to 100 % confluence in media containing DMEM (7 mM glucose), 10 % FCS, and

antibiotics for 24 h and subsequently differentiated into myotubes by incubating with differentiation

media (DMEM containing 7 mM glucose, 5 % FCS, antibiotics, and 1 µM cytosine arabinoside) for at

least 48 h.

7

Glucose uptake assay

The differentiated myotubes were treated with serum free media (DMEM with 7 mM, glucose,

antibiotics, and 1 µM cytosine arabinoside) overnight, followed by incubation with various treatments

for 1 h. The myotubes were then washed with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HEPES buffered saline (20 mM Hepes, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2,

adjusted to pH 7.4) and incubated with 0.1 mM 2-deoxy-[3H] glucose (2 DOG) (250 µL/well) for 30

min; following which they were quickly washed with ice cold phosphate buffered saline (500 µL/well)

and lysed by adding 0.05 M NaOH (37°C, 250 µL/well) and placed on a shaking board for 30 min. The

cell lysate was transferred to a scintillation tube, mixed with scintillation liquid (Ultima Gold,

PerkinElmer Inc.) in 1:10 ratio and counted in a Win spectral, 1414 liquid scintillation counter

(PerkinElmer, Life Sciences). The data was normalised with protein concentration per well.

mRNA extraction and RT-PCR

Twenty-four well plates containing differentiated primary myotubes were exposed to treatment (200

µg/mL of elderflower DCM extract in differentiation media) for different time points in duplicates.

After respective time intervals (1, 2 and 4 h), the myotubes were washed, harvested and stored for later

RNA extraction with RNeasy mini kit (Qiagen, Albertslund, Denmark). RT-PCR analysis was done

with defined primers and probes described elsewhere (24). The mRNA levels of HMOX1 and HSP70

were normalised against the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A

standard curve was generated by serial dilution. Wells containing either water or genomic DNA were

used as negative controls. All samples were measured in duplicate. The relative expression of HSP70

and HMOX1 were calculated according to the ‘Mathematical model for relative quantification in real-

time PCR’ (25).

8

Intracellular ROS

White-walled 96 well plates, containing differentiated primary myotubes were treated and the

measured data processed, essentially as has been mentioned elsewhere (24), with the following

changes. Background was measured before the addition of H2DCF-DA. Myotubes were incubated with

different concentrations of the elderflower DCM extract (0.1, 0.2, and 0.5 mg/mL) and H2O2 (100 µM

in KCl buffer) separately. The intracellular oxidation of H2DCF-DA in the wells was measured in

quadruplicates at 34°C for 4 h, at intervals of 4 min.

Fat accumulation in C. elegans

The wild-type C. elegans strain, N2 was used in this study. For fat staining experiments, Nile Red

(N3013, Sigma-Aldrich, dissolved in acetone (500 mg/mL)) was added to molten nematode growth

medium (NGM, ~55°C) to a final concentration of 0.05 mg/mL, and aliquoted in 24-well plates (1 mL/

well). The wells were seeded with E. coli bacteria (uracil auxotroph strain OP50) in 2 x Yeast extract

Tryptone medium (40 µL/ well). When dry, 25 µL M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1

mL 1 M MgSO4, H2O to 1 L) and either 10 µL DMSO (control) or 10 µL sample in DMSO were

added. Synchronized L1 larvae were put on the plates and grown for 46 h at 20°C until mid-L4 stage.

Worms were then mounted in a drop of 10 mM tetramizole (T1512, Sigma-Aldrich) atop 2 % agarose

pads laid on a microscopy glass slide and overlaid with a cover slip. Fluorescence microscopy

(rhodamine channel) was done using a Leica DMI6000 B microscope equipped with an Olympus DP71

camera. Images were captured using Visiopharm Integrator System software (Visiopharm, Denmark).

All worms were photographed at 200 × magnification and 30 ms exposure time. Images were

quantified using ImageJ (http://rsbweb.nih.gov/ij/).

https://mail.au.dk/owa/redir.aspx?C=-ymn2W8uDkaibkqd9t_f8SXzq-WgX88IqLoxAQ_QLrE5nyLXqU7HGxeVmVx_7Ddywjq6Nk4A7n8.&URL=http%3a%2f%2frsbweb.nih.gov%2fij%2f

9

Data Analysis

For experiments on porcine myotubes, data was analysed, using the ‘Mixed procedure’ of SAS

statistical programming software (Ver. 9.2; SAS Institute Inc., Cary, NC, USA). The model consisted

of treatments and their interactions as fixed effects, and experiments, replicate determinations, and pigs

within treatments as random effects. The level of HSP70 and HMOX1 mRNA expression were

analysed with a model where time points were considered as fixed effects and replicates as random

effects. The number of pigs from which satellite cells were isolated varied between 3 and 4; the number

of replicates was 6.

For studies on C. elegans, Student’s unpaired t-test or Analysis of variance (ANOVA) were

used. Statistical analysis was done with Graph Pad Prism 5 software package (GraphPad Software,

Inc.) on the raw data before normalization of the data. Number of worms used for each treatment varied

between 13 and 19.

Results

The elderflower DCM and MeOH extracts obtained were characterized by LC-PDA-MS. Major

components in the MeOH extract corresponded to those previously reported (22, 26), and the most

prominent ones were 5-O-C, Q-3-O-R, K-3-O-R, and I-3-O-R. Major components of the DCM extract

were α–linolenic acid (α–LA), linoleic acid (LA), naringenin, and some unknown phenolic acid

derivatives. Separation of each of the two extracts resulted in 7 fractions (A to G). Only the DCM

fractions D and E exhibited both increase in GU and decrease in FAc and were analysed by LC-PDA-

MS. Fraction D contained some unknown phenolic acid derivatives, α-LA, LA and epoxy-linalool;

Fraction E contained naringenin and α-LA as the major components, together with minor amounts of

10

naringenin derivatives. When tested for any detrimental effect on prolonged exposure, neither DCM

nor MeOH extract showed any reduction on myotube viability (data not shown).

The DCM and MeOH extracts of elderflowers were tested for their effect on both insulin-

stimulated and insulin-independent GU in primary porcine myotube cultures (Figure 1). Both extracts

were able to increase GU in muscle cells in the presence and absence of insulin. The DCM extract in

particular, caused a significant increase in GU with respect to the control at all concentrations tested;

the only exception being at 1000 µg/mL where the increase was not statistically significant (Figure 1a).

The MeOH extract showed a small but significant increase in GU (other than at 600 and 1000 µg/mL)

in absence of insulin (Figure 1b). For the DCM extract, the increase in GU without insulin was

statistically consistent within the tested concentration range of 6-1000 µg/mL, with a numerical

maximum increase of 36 % (p < 0.0001) at 60 µg/mL compared to control. In the presence of insulin,

an increase of 21 % (p = 0.0006) at 200 µg/mL was observed for the DCM extract. At the highest

tested concentration (1000 µg/mL) of the DCM extract, the increase without insulin was 23 % (p <

0.0001), and there was no significant increase in the presence of insulin. The effect of the elderflower

MeOH extract was less prominent than that of the DCM extract. It did not show a significant increase

in GU in presence of 750 pM Insulin. However, a significant increase of 7 % (p = 0.04) to 11 % (p =

0.0003) in GU, compared to the control at 6 to 200 µg/mL was observed in the absence of insulin.

MeOH extract showed a significant decrease in GU at the maximum concentration used (1000 µg/mL),

both in the presence and absence of insulin. The DCM extract showed much higher response in GU

compared to MeOH, and was therefore chosen for further studies on stress responses.

To examine whether the observed effect of the extracts on GU was caused through the

induction of stress on the myotubes, mRNA abundance of HSP70 and HMOX1 was studied (Figure

2a). Exposure times of 1, 2 and 4 h and a concentration of 200 µg/mL were chosen in accordance with

11

the exposure time for the GU assay. No significant up or down regulation of either HSP70 or HMOX1

mRNA was observed. Additionally, the influence of the DCM extract on the generation of intracellular

ROS was determined at 100, 200, and 500 µg/mL (Figure 2b). A significant reduction in the formation

of ROS was observed with respect to control at all the concentrations tested.

To aid identification of the bioactive components in the two extracts, they were separated by

flash CC. All the 14 fractions obtained were then screened for their potential to induce GU

independently at concentrations of 50 and 100 µg/mL. Two DCM fractions D and E were able to

increase GU significantly at 100 µg/mL, by 17 and 26 %, respectively (Figure 3a). None of the MeOH

fractions showed any effect on GU. The DCM fractions D and E showed no reduction in myotube

viability (data not shown).

The bioactive fractions D and E of the DCM extract contained mainly naringenin, α-LA and

LA. These three compounds were selected for further studies, as well as several other known

elderflower metabolites (22) and related polyphenols to further explore the observed bioactivities of the

extracts and the fractions. Overall, the elderflower metabolites tested were: naringenin, 5-O-CA, Q-3-

O-R, Q-3-O-G, Q-3-O-6’’-A, K-3-O-R, I-3-O-G, I-3-O-R, α-LA, and LA; and the related polyphenols

tested were: kaempferol, CaA, FeA, and p-CA. All were tested for effects on insulin-independent GU

in primary porcine myotube cultures at the concentrations 0.1, 1.0, and 10 µM; other than α-LA and

LA, which were tested at 10, 30, 70 and 100 µM. The compounds exhibiting a significant increase in

GU are illustrated in Figure 3b. Among them, 5-O-CA was identified in the MeOH fractions;

naringenin in the DCM fractions and kaempferol, FeA, p-CA, and CaA belong to the related

polyphenols tested. None of the other compounds showed any effect on GU, although LA (in DCM

fraction) showed a strong tendency (p =0.056) at the concentration of 10 µM. The highest increase was

observed at the concentration of 10 µM for naringenin and kaempferol (24 % and 21 % respectively).

12

The effect of elderflower extracts, fractions, individual elderflower polyphenols as well as

related polyphenols on C. elegans fat storage is illustrated in Figure 4 & 5. Both MeOH and DCM

extracts (200 µg/mL) decreased the Nile Red Fluorescence (NRF) significantly by 25 % and 50 %

respectively, relative to control (Figure 4a & 5a). The DCM and MeOH fractions (A – G) were tested

further. The MeOH fractions C – G (100 µg/mL) showed significant reduction in NRF (23 to 55 %),

with the maximum reduction by fraction F (55 %, p < 0.0001) (data not shown). Among the DCM

fractions tested at three different concentrations (10, 50, and 100 µg/mL), B, C, F, and G exhibited

significant reduction in NRF (13 to 48 %) (data not shown). But the highest reduction in NRF was

observed for fractions D (78 %) and E (87 %) at a concentration of 100 µg/ml (Figure 4b & 5b). All the

elderflower metabolites (other than α-LA, and LA) and related polyphenols mentioned above were

tested for reduction in FAc at the concentration of 50 µM. The compounds exhibiting significant

decrease in NRF are illustrated in Figure 4c. Among them, Q-3-O-R, Q-3-O-G, Q-3-O-6’’-A, K-3-O-R,

I-3-O-G, and I-3-O-R were identified in the MeOH fractions; naringenin in the DCM fractions; FeA

and kaempferol belongs to the related polyphenols tested. None of the other compounds showed any

effect on FAc. The highest reduction was observed for naringenin and kaempferol (65 and 60 %

respectively) compared to control (Figure 4c & 5c).

Discussion

Insulin resistance, manifested by inadequate GU in major glucose utilizing tissues; and visceral obesity

are two key aspects of T2D and MS. Elderflower extracts have previously been shown to induce weight

loss and exhibit anti-diabetic properties. This study showed for the first time that elderflower extracts

can autonomously stimulate GU in primary myotube cultures and reduce FAc in vivo.

13

The elderflower DCM extract produced a much higher increase in GU in porcine myotubes,

compared to the MeOH extract, and only the chromatographic fractions of this extract showed

bioactivity in both GU and FAc studies in porcine myotube cultures and C. elegans respectively.

Although the MeOH extract showed bioactivity none of its fractions caused any increase in glucose

uptake. Nevertheless, in FAc studies with C. elegans, several of the MeOH fractions were found to be

potent. It is noteworthy that although the fractions are normally expected to contain larger

concentrations of individual secondary metabolites compared to the extracts, the increase in GU,

induced by the DCM extract was higher compared to its bio-active fractions D and E. The reduction (in

case of DCM extract) and loss (in case of MeOH extract) of bio-activity when fractionated indicates

that part of the increase in GU exhibited by the extracts could be due to additive or synergistic effects,

together with possible non-ligand activation by some of the compounds present in the DCM and MeOH

extracts, or antagonism by some other compounds present in relative higher concentrations in specific

fractions, compared to the extract. Such observations are in agreement with synergistic interactions

observed in separate studies carried out previously with both elderflowers and other plant extracts (13,

27). However, such reduction of activity was not observed for FAc studies, indicating a different mode

of action and specificity of the elderflower metabolites.

It has been documented from earlier studies that cellular stress can result in an increase in

glucose uptake (16). However, gene expression analysis of HSP70 and HMOX1 genes and

determination of intracellular ROS showed no indication of cellular stress on the myotubes. On the

contrary, the DCM extract exhibited significant anti-oxidative properties by reducing the amount of

intracellular ROS.

It has been observed throughout the tested concentration range of the DCM extract, that

although it exhibits an increase in GU in the presence of a biologically relevant concentration of insulin

14

(750 pM), it shows a higher increase in GU when administered in the absence of insulin. This indicates

the possibility that the signalling molecules responsible for the observed increase in GU, by the

elderflower metabolites might be shared to some extent with the insulin signalling pathway.

Several fractions of the MeOH and DCM extracts showed a reduction in FAc in C. elegans.

Unlike GU, further decrease in FAc was observed upon fractionation. Although many of the

elderflower metabolites present in the MeOH extract showed significant reduction in FAc, naringenin

(identified in the DCM extract) caused the highest reduction (Figure 4c).

The two fractions, D and E of the DCM extract both exhibited an increase in GU and a decrease

in FAc. Among the compounds present in these fractions, were unknown phenolic acids, and further

research is required to identify and elucidate their structures. However, amongst the compounds that

were identified in these fractions, the most prominent increase in GU and decrease in FAc were

observed for naringenin. Both confirming and contradictory findings exist, where naringenin was found

to enhance GU in L6 myotubes via adenosine monophosphate - activated protein kinase (AMPK)

activation (28) but reduce GU in MCF-7 breast cancer cells (29) and U937 cells (30). The reported

inhibitory effects of naringenin on GU can be explained by the different cell types used in these studies,

implying a cell-type specific effect of this compound.

Amongst the related polyphenols tested, kaempferol showed the highest increase in GU and

reduction in FAc. This is the first account demonstrating the ability of kaempferol to enhance GU in

myotubes and reduce FAc in vivo. However, in separate studies, kaempferol has previously been shown

to inhibit GU in HeLa cells (31), but enhance GU in mature 3T3-L1 adipocytes (32), which is similar to

the cell-type dependant response, observed for naringenin. It is important to note, that although

naringenin is a flavanone and kaempferol a flavonol, they are very similar in their chemical structures,

15

i.e., in size and the substitution pattern of their aromatic rings. This could signify the structural

importance of these molecules in their observed regulation of glucose and lipid homeostasis.

In entirety, it could be concluded that extracts, fractions and several secondary metabolites from

elderflowers possess pronounced bioactivities and can be used to modulate glucose and lipid

metabolism. Studies conducted with the extracts, fractions, and their constituent compounds revealed

that among the elderflower metabolites, naringenin is one of the most potent, with major effect on the

enhancement of GU and reduction in FAc. MS is characterized by elevated blood glucose levels and

increase in visceral obesity; and the fact that elderflower extracts exhibit the potential to amend both of

these metabolic defects, qualifies it for further investigation for its application as a supplementary

nutraceutical.

Abbreviations Used

CaA, caffeic acid; FeA, ferulic acid; I-3-O-G, isorhamnetin-3-O-glucoside; I-3-O-R, isorhamnetin-3-

O-rutinoside; K-3-O-R, kaempferol-3-O-rutinoside; 5-O-C, 5-O-caffeoylquinic acid; p-CA, p-coumaric

acid; Q-3-O-G, quercetin-3-O-glucoside; Q-3-O-6’’-A, quercetin-3-O-6’’-acetylglucoside; Q-3-O-R,

quercetin-3-O-rutinoside; ROS, reactive oxygen species; TLC, thin layer chromatography; NRF, nile

red fluorescence; DCM, dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; H2DCF-

DA, 2’, 7’ dichlorodihydrofluorescein diacetate; MeOH, methanol; MS, metabolic syndrome; FAc, fat

accumulation; GU, glucose uptake;

16

Acknowledgements

The authors gratefully acknowledge Xavier Fretté, Department of Chemical Engineering,

Biotechnology and Environmental technology, University of Southern Denmark,

for his assistance during the LC-MS studies.

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22

Figure 1. Effect of elderflower extracts on insulin-stimulated (750 pM) and insulin-independent

glucose uptake in primary porcine myotube cultures exposed to (a) DCM extract and (b) MeOH

extract. Glucose uptake is indicated as per cent of the control (DMSO) which is set at 100. The plotted

values are LSMeans ± SEM. Ins = Insulin. The letters (a, b, c and d) on top of the bars indicate

significant differences in glucose uptake. Number of pigs used = 3; number of replicates taken per pig

= 6

Figure 2. Elderflower extracts on stress responses in the primary porcine myotubes (a) Effect of

elderflower DCM extract (200 µg/mL) on the expression of heme oxygenase 1 (HMOX1) and Heat

shock protein 70 (HSP70) after 1, 2 and 4 h of exposure. The mRNA levels of HMOX1 and HSP70

have been normalized against the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

and are given in fold change relative to their respective controls at the given time points. (b) Effect of

elderflower DCM extract on generation of reactive oxygen species (ROS) determined by intracellular

2,7-dichlorofluorescein oxidation. The DCM extract were tested at 100, 200 and 500 µg/mL

concentrations. H2O2 (100 µM) is used as a positive control. The x-axis shows every 5th

data point.

Plotted values are LSMeans ± SEM. Control = DMSO in media. Number of pigs used = 3; number of

replicates taken per pig = 6. *p < 0.05, **p <0.01, ***p < 0.001 vs. control

Figure 3. Effect of elderflower DCM fractions D and E, selected elderflower metabolites, and related

polyphenols on glucose uptake in porcine myotubes. (a) Influence of the fractions D and E from

elderflower DCM extract at the concentrations 50 and 100 µg/mL. (b) Effect of selected elderflower

metabolites and related polyphenols tested at the concentrations of 0.1, 1.0 and 10 µM. Glucose uptake

23

is indicated as per cent of control (DMSO) set at 100. The plotted values are LSMeans ± SEM. Number

of pigs used = 3; number of replicates taken per pig = 6. *p < 0.05, **p <0.01, ***p < 0.001 vs. control

Figure 4. Effect of elderflower extracts, fractions, selected elderflower metabolites and related

polyphenols on C. elegans fat accumulation. Wild type worms from larval stage L1 to L4 were treated

with extracts, fractions or pure compounds on standard NGM plates containing the lipophilic dye Nile

Red. Control worms only received DMSO. (a) Effect of DCM and MeOH extracts tested at the

concentrations of 200µg/ml. Number of treated worms = 13-19. (b) Effect of elderflower DCM extract

fractions D and E. Worms were treated with 10, 50, and 100 µg/mL of plant material from D and E

fractions. Number of treated worms = 5-10. (c) Effect of elderflower metabolites and related

polyphenols. Worms were treated with the compounds at the concentration of 50 µM. Number of

treated worms = 10. Fluorescence levels are shown as normalized means +/- normalized SEM. *p <

0.05, **p <0.01, ***p < 0.001 vs. control

Figure 5. Effect of elderflower extracts, fractions, and selected polyphenols on staining of lipid stores

in C. elegans. (a) Control (DMSO) and 20 µg/ml raw extracts of elderflower (b) Control (DMSO) and

100 µg/ml fraction D and E of the DCM extraction of elderflower (c) Control (DMSO) and 50 mM of

pure standards Naringenin and Kaempferol. Upper panel in each row show Differential Interference

Contrast microscopy images; lower panel is the corresponding fluorescence images (rhodamine filter).

Scale bar corresponds to 0.1 mm

24

Figure 1.

DCM extract (µg/ml)

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25

Figure 2.

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26

Figure 3.

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27

Figure 4.

Elderflower DCM fractions

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28

Figure 5.

Manuscript II

Caffeic acid, Naringenin and Quercetin enhance glucose stimulated insulin secretion and glucose

sensitivity in INS-1E cells

Sumangala Bhattacharya, Niels Oksbjerg, Jette F. Young, and Per Bendix Jeppesen

Submitted to: Diabetes, Obesity and Metabolism

1

Caffeic acid, Naringenin and Quercetin enhance glucose stimulated insulin

secretion and glucose sensitivity in INS-1E cells

Sumangala Bhattacharya1, Niels Oksbjerg

1, Jette F. Young

1, and Per Bendix Jeppesen

2

Author names and affiliations:

1Department of Food Science, Aarhus University, Blicher’s Allé 20, Postbox 50, 8830 Tjele, Denmark

2Department of Medicine and Endocrinology, Aarhus University Hospital, Aarhus University, Tage-

Hansens Gade 2, 8000 Aarhus C, Denmark


Niels Oksbjerg

Institute of Food Science, Aarhus University,

Blicher’s Allé 20, Postbox 50, 8830 Tjele, Denmark

E-mail: [email protected]

Tel: + 45 8715 6000

Direct: + 45 8715 7809

Mobile: +45 3011 3204

Sponsor: The Danish Council for Strategic Research (Grant no. 09-063086)

2

Abstract

Aims: Caffeic acid, naringenin and quercetin are naturally occurring phenolic compounds (PCs)

present in many plants as secondary metabolites. The aim of this study was to investigate their effect on

glucose stimulated insulin secretion (GSIS) in INS-1E cells and to explore their effect on expression of

genes involved in beta cell survival and function under normoglycemic and glucotoxic conditions.

Methods: For acute studies, INS-1E cells were grown in 11 mM glucose (72 h) and then incubated

with the PCs (1 h) with 3.3/16.7 mM glucose; whereas, for chronic studies, the cells were grown in 11

mM glucose (72 h) with/without the PCs, and then incubated with 3.3/16.7 mM glucose (1 h);

thereafter, GSIS was measured. For GSIS and gene expression studies (GES) under glucotoxic

conditions, two sets of cells were grown in 11/25 mM glucose with/without the PCs (72 h): one was

used for GES, using real time RT-PCR, and the other was exposed to 3.3/16.7 mM glucose, followed

by measurement of GSIS.

Results: The study demonstrated that the PCs can enhance GSIS under hyperglycemic and glucotoxic

conditions in INS-1E cells. Moreover, these compounds can differentially, yet distinctly change the

expression profile of genes (Glut2, Gck, Ins1, Ins2, Beta2, Pdx1, Akt1, Akt2, IRS1, Acc1, Bcl2, Bax,

Casp3, Hsp70, and Hsp90) involved in beta cell stress, survival and function.

Conclusion: The results indicate that the PCs tested enhance GSIS and glucose sensitivity in INS-1E

cells. They also modulate gene expression profiles to improve beta cell survival and function during

glucotoxicity.

Keywords: Beta cells, gene expression, insulin secretion, type 2 diabetes, INS-1E cells, glucotoxicity,

hyperglycemia, phenolic compounds

3

Abbreviations: Glut2: glucose transporter 2; Ins1: insulin 1; Ins2: insulin 2; Akt1: RAC-alpha

serine/threonine-protein kinase encoding gene; Akt2: RAC-beta serine/threonine-protein kinase

encoding gene; IRS1: insulin receptor substrate 1; Pdx1: pancreatic and duodenal homeobox protein 1;

Beta2: neurogenic differentiation protein 1; Acc1: acetyl CoA carboxylase 1; Gck: glucokinase; Casp3:

caspase 3; Bax: Bcl-2 associated X protein; Bcl2: beta-cell lymphoma 2 protein; Hsp 70: heat shock

protein 70; Hsp90: heat shock protein 90

4

Introduction:

Over the past few decades, type 2 diabetes (T2D) has precipitated into a worldwide epidemic, now

spanning both developed and developing societies [1]. The insulin secreting pancreatic beta-cells have

an unequivocal role in the regulation of glucose homeostasis in mammals and therefore, malfunctions

in the insulin secretion patterns in pancreatic beta-cells is fundamental to the development of T2D.

Failure of the beta-cells to produce adequate amounts of insulin to overcome the growing insulin

resistance in muscles and other effector tissues is an essential pre-requisite in the occurrence of this

multi-faceted ailment [2]. Upon chronic exposure to hyperglycemic conditions, beta-cells experience a

state of impaired sensitivity towards fluctuations in glucose levels, which when left untreated can cause

permanent damage to beta-cell function [3]. During initial stages of hyperglycemic exposure, the beta-

cells are temporarily rendered insensitive to high glucose due to depletion of stored insulin caused by

continuous secretion. At this stage, the beta-cells regain their sensitivity towards glucose when allowed

to rest. Hyperglycemic exposure beyond this stage, initially leads to an adaptable expansion of the beta

cells [4,5]; but continuation of the hyperglycemic state without intervention can cause irreversible

damage, characterised among others by, flawed insulin gene expression and increased rate of apoptosis

[6] resulting in an impairment of beta cell survival and function, a process termed glucotoxicity [7].

Medications available for hyperglycemic therapy are associated with various contraindications [8]

including hypoglycemia, having severe consequences in physically weak or aged diabetic patients [9].

Therefore identification of novel, and more reliable insulin secretagogues remains a subject of major

interest.

The plant kingdom has served as a major source of drugs for innumerable ailments till date. As

a result more than a thousand species of plants have been examined for their efficacy against diabetes

alone [10]. In the current study we have evaluated the effect of three naturally occurring phenolic

compounds (PCs) on glucose stimulated insulin secretion (GSIS). These are: caffeic acid (a phenolic

5

acid; source: all plants e.g. Brussels sprouts (Brassica oleracea) [11], naringenin (a flavanone; source:

mainly citrus fruits e.g. grapefruits [12]) and quercetin (a flavonol; source: many fruits and vegetables

e.g. onions and apples [13]), in INS-1E cells. Naringenin and quercetin has been previously reported to

protect beta cells from cytokine induced cell death [14] and, caffeic acid has been found to have strong

anti-oxidant properties [15] making them interesting candidates for studying insulin secretion and gene

expression under glucotoxic conditions (GC).

The rat insulinoma cell line INS 1E has been used as an experimental model in this study for its

capability to secrete insulin under physiological glucose concentrations, and its susceptibility towards

glucotoxicity, as found in pancreatic beta cells in vivo [16,17]. Moreover, this cell line has been

isolated from the parental INS 1 cells based on their insulin content and secretion capabilities [18].

Among the PCs, caffeic acid has been found to induce glucokinase (Gck) mRNA in rat liver

cells in vitro [19]. Moreover, activation of Gck has been shown to have anti-hyperglycemic effects

[20]. It has also been reported to inhibit acetyl CoA carboxylase 1 (Acc1) in skeletal muscle, which

participates in biosynthesis of fatty acids [21] Again naringenin has shown anti-apoptotic effects by

regulating Bcl2/Bax ratio in HaCaT human keratinocytes[22]. Moreover, a down regulation of the

apoptotic effector protein Caspase 3(Casp 3) was exhibited in naringenin and quercetin treated murine

model of hypobaric hypoxia [23].

The effects of the PCs were studied for their effect on insulin secretion after acute and chronic

exposure to low and high glucose levels and under GC. Moreover, the PCs were tested for their

influence on gene expression in these cells under normoglycemic conditions (NC) and GC. Apoptotic

(Casp 3 and Bax), as well as cyto-protective gene expression (Bcl2, Hsp 70 and Hsp 90), together with

genes responsible for insulin secretion and beta-cell function including the glucose sensor Glut2 and

Gck, where the former is responsible for glucose transport and the latter is involved in the

preservation of beta-cell mass [24,25]. Two isoforms of insulin expressed in rats: Ins1 and Ins2; and

6

members of the insulin signaling pathway: Akt1, Akt2, and IRS1, where Akt1/2 are implicated in the

maintenance of beta-cell mass and function, were also analysed [26,27]. Besides, transcription factors

regulating beta-cell function and insulin gene expression: Pdx1, Beta2 [28]; genes involved in

lipogenesis: Acc1 [29] were studied.

Researchers have used different glucose concentrations to mimic GC in in vitro studies [30-32]

ranging from 16.7 to 30 mM. However, glucose concentrations of 30 mM and higher, have been found

to impair beta-cell functions in rodents [30]. In this study we chose to incubate the INS 1E cells in 25

mM glucose for 72 h to induce glucotoxicity. The glucose concentration characterising NC, was chosen

to be 11 mM, as the insulin secretion ability is best observed at this concentration [31].

Materials and Methods:

Incubation of INS-1E cells

Modified RPMI 1640 culture media (GIBCO), supplemented with antibiotics penicillin and

streptomycin (100 iu/ml and 100 µg/mL respectively, GIBCO), 10 % foetal calf serum (FCS), HEPES

(10 mM, Sigma) and β-mercaptoethanol (5 µM, Sigma)[16] and was used to culture the INS-1E cells.

The cells were incubated at a humified (95 %) atmosphere (5 % CO2) at 37°C. Early culture passages

were washed with PBS, trypsinised and incubated for 5 mins. Trypsinisation was stopped by adding

RPMI 1640 media (containing 11 mM glucose).The cells were then harvested and seeded in 6 (for gene

expression studies) or 24-well plates (for insulin secretion studies) (NUNC Brand Products, Roskilde,

Denmark) at a density of 106 or 3 × 10

5 cells/well, respectively. The cells used in the experiments were

from passage 72 to 78.

7

Secretion studies

The PCs, caffeic acid, naringenin, and quercetin were purchased from Sigma (Steinheim, Germany).

For acute studies, INS-1E cells were grown in modified RPMI 1640 media containing 11 mM glucose

for 72 h in absence of the PCs; whereas for chronic studies, with or without the PCs at the

concentrations of 10-10

, 10-8

, and 10-6

M (dissolved in DMSO). Modified Krebs-Ringer Buffer (KRB),

containing 125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl2, 1.2 mM MgCl2, 25 mM HEPES and 0.1 %

BSA (w/v) (all from Sigma) was used for further incubations. The cells were pre-incubated with 1 mL

of KRB for 15 mins, with subsequent incubation for 1 h in KRB, containing (a) 10-10

, 10-8

, and 10-6

M

concentrations of the PCs in 3.3 and 16.7 mM glucose separately for acute secretion studies and (b) 3.3

or 16.7 mM glucose for chronic secretion studies. Control treatments received DMSO.

For glucotoxicity experiments, the cells were incubated with modified RPMI 1640 containing

11 mM and 25 mM glucose with or without the PCs at the concentration of 10-6

M for 72 h. Control

treatments received DMSO. Thereafter, the cells were pre-incubated with KRB for 15 mins, and then

incubated with 3.3 or 16.7 mM glucose in KRB for 1 h. Incubation media (300 µL) was removed,

centrifuged, and 200 µL of the supernatant was frozen for subsequent analysis of insulin content. The

insulin secretion data was normalised with the number of cells present per well; which was determined

by using the fluorescent nucleic acid stain Syto 24 green.

Insulin content determination

Insulin content was measured by a radioimmunoassay, using a guinea pig anti-porcine insulin anti-body

(Novo Nordisk, Bagsvaerd, Denmark) and mono-125

I-(Tyr A14)-labelled human insulin as tracer and

rat insulin as standard (Novo Nordisk A/S). Ethanol was used to separate bound from free radioactivity

[33]. Naringenin, caffeic acid, and quercetin did not interfere with this assay at tested concentrations.

8

Isolation of total RNA

Cells were washed in phosphate buffered saline and RNA extraction was carried out using the RNeasy®

Plus Mini RNA extraction Kit (Qiagen, Sample & Assay Technologies, Copenhagen, Denmark)

following the manufacturer’s instructions. After extraction, the absorbance at 260 and 280 nm was

measured for quantification of the RNA. The quality of the RNA was ensured by visual examination of

the 18s and 28s ribosomal RNAs on an agarose gel.

Real-time RT PCR

We investigated the expression of Glut2, Ins1, Ins2, Akt1, Akt 2, IRS1, Pdx1, Beta2, Acc1, Gck,

Casp3, Bax, Bcl2, Hsp 70 and Hsp 90 by real time RT-PCR. The cells were incubated with modified

RPMI 1640 containing 11 mM and 25 mM glucose with or without the PCs at the concentration of 10-

6 M for 72 h. Control treatments received DMSO. Complimentary DNA (cDNA) was synthesized

using IScript (BioRad, Hercules, CA, USA), in accordance with the manufacturer’s guidelines. A total

RNA of 50 ng/10 µl reaction mixture was used for determining the abundance of the target mRNA.

The real time polymerase chain reaction (PCR) was performed using the ABI 7500 FAST machine

(ABI, Foster City, CA, USA). Ten microliters of real-time PCR reactions consisted of 5 µl 2 ×

TaqMan® FAST Universal Master Mix (P/N 43660783; ABI) 0.5 µl 20 × TaqMan Assay/probe (ABI)

and reverse transcribed cDNA, equivalent to 50 ng of total RNA in 4.5 µl H2O. The thermal FAST

cycle program was as follows: 20 s at 95°C, and subsequently, 40 cycles composed of 3s at 95°C and

30 s at 60°C. Triplicate reactions were set up for each sample and the mRNA abundance was

normalized to eukaryotic 18s ribosomal RNA (assay Hs99999901_s1) expression. No template controls

(NTC) and No enzyme controls (NEC) were used as negative controls for each gene. Assays were

carried out in 96-well plates and were covered with optical adhesive (P/N 4346906 and P/N4311971;

ABI). The 2-∆∆CT

method was used to calculate the relative gene expression. The TaqMan assays used

for the PCR were Glut2 (assay Rn00563565_m1), Ins1 (assay Rn02121433_g1), Ins2 (assay

9

Rn01774648_g1), Akt1 (assay Rn00583646_m1), Akt2 (assay Rn00690900_m1), IRS1 (assay

Rn02132493_s1), Pdx1 (assay Rn00755591_m1), Beta2 (assay Rn00824571_s1), Acc1 (assay

Rn00573474_m1), Gck (assay Rn00561265_m1), Casp3 (assay Rn00563902_m1), Bax (assay

Rn01480160_g1), Bcl2 (assay Rn99999125_m1), Hsp 70 (assay Rn04224718_u1) and Hsp 90 (assay

Rn00822023_g1).

Statistical Analysis

Data are expressed as means ± standard error of mean (s.e.m). Data analysis was performed and plots

were made using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical

significance between two treatment groups was evaluated by a two-tailed Student’s unpaired T-test.

Treatment conditions were compared with their respective controls. A p value of less than 0.05 was

considered to be statistically significant and that of less than 0.1 was considered a tendency.

Results:

Insulin secretion studies

Caffeic acid, naringenin and quercetin were tested on INS-1E cells for their influence on GSIS. Insulin

secretion was measured after acute or chronic exposure and after induction of glucotoxicity.

Acute exposure

Incubation with the PCs did not show any significant increase in insulin secretion in presence of low

glucose (figure 1A, C, and E). However, in the presence of 16.7 mM glucose (figure 1B, D, and F),

caffeic acid showed a significant rise in GSIS at all concentrations tested (figure 1A); showing an

increase of 14.3, 15.7, and 20 % at 10-10

, 10-8

, and10-6

M concentrations respectively. Naringenin

(figure 1D) induced a significant dose dependant increase of 36.8 and 55.4 % at 10-8

and 10-6

M

10

concentrations respectively; whereas a significant increase of 28.8 % was observed in presence of

quercetin at a concentration of 10-6

M (figure 1F).

Chronic exposure

At low glucose concentrations, caffeic acid and quercetin showed a small but significant increase in

insulin secretion at 10-6

M concentrations (figure 2A and E), but naringenin did not show any

significant increase in insulin secretion in presence of low glucose at any of the concentrations tested

(figure 2C). In the presence of high glucose, all the three PCs significantly increased GSIS at all

concentrations tested (figure 2 B, D, and F). Caffeic acid showed a highly significant increase (p <

0.0001) of 43.6, 53.2, and 54.5 % at 10-10

, 10-8

, and10-6

M concentrations, respectively. For naringenin

too, the increase in GSIS was highly significant for all concentrations tested, showing a 33.5, 30.8 and

32.6 % increase at 10-10

(p < 0.0001), 10-8

(p = 0.005), and 10-6

M (p < 0.0001) concentrations

respectively; whereas quercetin showed a significant increase of 22.8, 16.6, and 26.5 % increase in

GSIS at 10-10

, 10-8

(p < 0.05), and 10-6

M (p < 0.001), respectively.

Insulin secretion during glucotoxicity

The glucose concentration of 11 mM and 25 mM were considered as normoglycemic and glucotoxic,

respectively. The molar concentration of 10-6

was chosen for this study, because all the PCs showed

maximum increase in GSIS at this concentration in the chronic exposure experiments. The results have

been illustrated in figure 3.

As expected, the cells incubated with normoglycemic glucose levels, showed a steep rise in

GSIS in response to 16.7 mM glucose; whereas those incubated with glucotoxic levels of glucose,

showed a highly diminished and response to the same. For the cells incubated under NC, caffeic acid

(figure 3A) showed a small but significant increase (p < 0.01) of 21.1 and 29.7 % in GSIS in presence

11

of 3.3 and 16.7 mM glucose respectively. In case of naringenin (figure 3B), a significant increase (p <

0.05) of 46.2 % in GSIS was observed in presence of 16.7 mM glucose; whereas in cells incubated with

quercetin (figure 3C), a significant increase (p < 0.0001 and p < 0.01) of 33.4 and 47.1 % was

observed in presence of 3.3 and 16.7 mM glucose respectively.

For the cells incubated under GC, caffeic acid showed a significant increase (p < 0.0001) of

41.7 and 50.3 % in GSIS when treated with 3.3 and 16.7 mM glucose respectively. For the cells

incubated with naringenin, there was a significant increase (p < 0.05) of 28.6 and 36.2 % when treated

with 3.3 and 16.7 mM glucose respectively. The response of cells incubated with quercetin was similar

to those that were incubated with caffeic acid, where there was a significant increase (p < 0.0001) of

62.2 and 71.6 % in GSIS, when treated with 3.3 and 16.7 mM glucose.

Gene expression analysis

Gene expression analysis of 15 genes related to regulation of GSIS, beta-cell function, stress response,

and apoptosis inhibition and induction were studied under NC and GC in the presence or absence of the

PCs, as illustrated in figure 4, 5, and 6. The change in mRNA abundance of the genes in the PC treated

cells was compared with untreated control cells. The Glut 2 and Gck mRNA was significantly down

regulated under GC compared to that in NC (p = 0.01 and p = 0.003, respectively) indicating a

reduction in glucose sensitivity of the INS-1E cells during long term exposure to high glucose levels.

Under NC, caffeic acid and quercetin did not significantly affect Glut2 expression (although caffeic

acid showed a tendency, p = 0.08), whereas naringenin showed a highly significant increase (p <

0.0001). However, under GC, naringenin and quercetin showed a significant increase in Glut 2

expression, while caffeic acid did not. Again, under NC, caffeic acid showed a tendency (p = 0.1) but

naringenin and quercetin showed a significant increase in Gck expression; whereas a significant

increase in the presence of caffeic acid and quercetin; and a tendency in presence of naringenin (p =

0.06) was observed under GC. The Ins1 gene was up regulated by all three PCs at both NC and GC.

12

The observed increase in Ins1 gene expression correlates well with the observed up regulation of Glut2

and Gck genes. However, neither of the PCs up regulated Ins2 gene expression under GC, although

naringenin showed a tendency (p = 0.09). A significant up regulation of Gck by caffeic acid and

naringenin was observed under NC.

It is noteworthy that the expression of the transcription factors Beta2 was down regulated under

GC compared to NC. All the three PCs significantly increased the mRNA abundance of Beta2 and

Pdx1 under NC, but none of the PCs showed significant increase under GC, except naringenin. The

expression of Akt1 was found to be up regulated by caffeic acid and naringenin under NC, whereas

naringenin and quercetin significantly up-regulated Akt1 expression under GC. Again, Akt2 expression

was increased significantly in presence of all the three PCs under NC, but under GC, only naringenin

was capable of enhancing its expression significantly. Only caffeic acid caused a significant increase in

IRS1 expression under NC, and but none of the PCs caused any change in its expression under GC.

The expression of Casp3 was noticeably increased (1.5 fold) under GC compared to NC,

indicating a pro-apoptotic state of the beta cells under GC. In presence of caffeic acid, there was a

significant decrease in Casp3 expression under GC. Naringenin too showed a significant decrease

under GC. The expression of the anti-apoptotic gene Bcl2 was down regulated under GC compared to

NC, again suggesting a pro-apoptotic state under GC. All the three PCs were found to increase Bcl2

expression under NC, but showed no significant increase under GC; except naringenin, showing a

tendency (p = 0.05). A minute but significant increase in the pro-apoptotic protein Bax was observed in

the presence of naringenin and quercetin under NC, but a significant decrease was observed in presence

of naringenin and caffeic acid under GC. Gene expression of the chaperones Hsp70 and Hsp90, as

expected, were up regulated under GC compared to NC. All the three PCs increased the expression of

Hsp70 both under NC and GC. Caffeic acid caused a significant increase in Hsp90 expression under

NC and naringenin under both NC and GC, whereas quercetin did not have any effect on Hsp90 gene

13

expression. The expression of Acc1 was considerably increased (3 fold) under GC compared to NC,

indicating increased lipogenesis in the beta cells under GC. Most interestingly, all the three PCs

reduced Acc1 expression to a level close to that observed under NC.

Discussion

Un-intervened chronic hyperglycemia can lead to a state of glucotoxicity, characterised by alterations

in expression patterns of genes involved in beta cell survival and function. Besides life-style

interventions, physical activity and controlled food intake, medicinal support is also crucial for a

comprehensive treatment of T2D. Hence, new therapeutic agents reinforcing the survival and insulin

secreting capacity of beta cells during glucotoxic and hyperglycemic conditions are highly coveted. In

the present study, we examined the three naturally occurring phenolic compounds; caffeic acid,

naringenin, and quercetin, for their ability to enhance insulin secretion, during acute and chronic

exposure to hyperglycemia and GC. Influence of these compounds on the gene expression patterns

under NC and GC was also studied.

All the three PCs were found to increase insulin secretion under hyperglycemic conditions.

Naringenin (10-8

and 10-10

M) and quercetin (10-6

M) showed significant increase at higher

concentrations during acute exposure but caffeic acid increased GSIS significantly at all concentrations

tested (figure 1B, C, and D). Similarly, during chronic exposure, all three PCs significantly increased

insulin secretion under hyperglycemic conditions (figure 2B, C, and D); the highest increase was

shown by caffeic acid, at all concentrations tested. Glucose sensitivity was found to be highly

diminished in cells exposed to GC, compared to NC. The experiments illustrated in figure 3 shows that

all three PCs are capable of increasing insulin secretion under GC. However, caffeic acid and quercetin

seem to be more efficient in this particular study compared to naringenin. This is the first study

reporting the potential of caffeic acid as an enhancer of GSIS in INS-1E cells. The fact that, naringenin

14

did not enhance GSIS in the INS-1E cells in the presence of low glucose after chronic exposure,

suggests its possible role in preventing hypoglycemia, and is reminiscent of GLP-1 (glucagon-like

peptide 1); and the diterpenes, Stevioside and Steviol, that has shown a glucose dependent effect on

insulin secretion [34,35].

In the gene expression analyses, a significant increase in the expression of the Ins1 gene in

presence of the PCs at both NC and GC was corroborated by the increase in GSIS observed under these

conditions. A significant increase in Ins2 gene was also observed under NC in cells treated with caffeic

acid and naringenin, but not under GC. Glut2, the glucose transporter in beta cells, has the highest

capacity and the lowest affinity for glucose, leading to glucose uptake in the beta cells only when

glucose level is high and insulin secretion is necessary [36]. On the other hand, Gck too acts as a

glucose sensor in pancreatic beta cells and its agonists can enhance GSIS and reduce gluconeogenesis

in the liver [24]. The significant increase in Glut2 expression (induced by naringenin and quercetin)

under GC; and Gck expression (naringenin, tendency) under GC and NC (caffeic acid, tendency)

indicates an increased sensitivity towards glucose and correlates well with the increase observed in Ins1

gene expression. These observations also suggest that there is a strong tendency of Gck up-regulation

in the PC treated cells under GC.

The expression of the transcription factors Beta2 and Pdx1 was not changed significantly under

GC (except by naringenin), although all three PCs significantly increased their expression under NC.

The increase in insulin gene expression by naringenin correlates well with the increase in the

expression of the transcription factors responsible for the regulation of insulin gene expression.

Several other transcription factors (e.g. Nkx2.2, Pax6, Foxa2, and Nkx6.1) responsible for insulin gene

expression and beta cell function exist [28]. Examination of the gene expression profiles of these

transcription factors under similar conditions could provide further insight into the increase in gene

15

expression of the beta cell specific genes (including insulin) observed in this study by caffeic acid and

quercetin.

Irs1, Akt1and 2 belong to the insulin signalling pathway. Irs1expression was not enhanced by

any of the PCs under GC. Naringenin and quercetin induced Akt1gene expression under GC and by

caffeic acid and naringenin under NC. Akt2 expression was only enhanced by naringenin under GC,

but by all three PCs under NC, suggesting augmentation of an otherwise down regulated insulin

signalling pathway most efficiently by naringenin.

Glucotoxicity has been associated with faulty gene expression of key beta cell specific genes

and accelerated apoptosis [6]. An increase in the expression of apoptotic genes like Caspase3 and Bax,

and down-regulation of the survival gene Bcl2 was observed under GC, compared to NC. This could be

an indication of the apoptotic effects of glucotoxicity. The reduction in the pro apoptotic gene Casp3

under GC suggests a cyto-protective effect of the PCs; especially caffeic acid and naringenin on beta

cells. The gene expression of another pro-apoptotic protein Bax, belonging to the Bcl2 family was

found to be down regulated by caffeic acid and naringenin under GC, indicating a cyto-protective effect

of these compounds under GC. Quercetin did not affect Bax expression under GC, but a minute, yet

significant increase in Bax expression was observed for naringenin and quercetin under NC. The Bcl2

family consists of both pro and anti-apoptotic proteins. The pro-survival members of this family

prevent apoptosis by inhibiting the pro-apoptotic proteins like Bax, Bak and BH3-only proteins [37].

The gene expression of the anti-apoptotic protein Bcl2 (belonging to the same family) was induced

significantly by all the three PCs under NC, and naringenin showed a tendency under GC indicating an

induction of pro-survival genes by naringenin.

The heat shock proteins are molecular chaperones that are induced as a response to cellular

injury and stress, and play an important role in cell survival. Induction of Hsp70 has been shown to be

helpful in combating neurodegeneration, ischemic heart disease and diabetes, while inhibition of Hsp90

16

has recently been recognised as an efficient approach to pacify various forms of cancer [38], indicating

its importance in cell survival. Hsp70 gene expression was significantly induced by all the three PCs

both at NC and GC, of which quercetin caused the maximum (two-fold) increase; whereas the gene

expression of Hsp90 was significantly up-regulated by naringenin at both NC and GC, indicating the

possible role of these PCs in beta cell survival during glucotoxicity.

Acc1 is an enzyme catalysing the first step of lipogenesis. Animals deficient in Acc1 show loss

of body weight and continuous lipid oxidation. Therefore reduction in Acc1 expression and activity has

been a therapeutic goal against obesity and related metabolic disorders like T2D [29]. On the other

hand hyperglycemic conditions have been found to cause an increase in Acc1 gene expression, as is

also observed in our study, where Acc1 gene is highly up-regulated under GC compared to NC. This

increase is found to suddenly increase malonyl coenzyme A in the cytosol, stopping the long chain

fatty acyl coenzyme As (LC FACoAs) from entering the mitochondria for oxidation. The increase of

these LC FACoAs in the cytosol, enhances insulin secretion [39]. As can be observed in the insulin

secretion studies under NC and GC, GSIS is diminished in cells grown under GC, compared to those

grown under NC, although Acc1 is highly expressed in the former group; indicating that an increase in

Acc1 does not improve glucose sensitivity in the beta cells during glucotoxicity. Again, the effect of a

long-term inhibition of fatty acid oxidation, due to constitutive overexpression of Acc1 in beta cells

during glucotoxicity can induce beta cell dysfunction and apoptosis due to lipotoxicity induced ER

stress [40]. In our study only naringenin was found to cause a minute but significant increase in Acc1

expression under NC. Under GC, Acc1 expression was increased by more than 3 fold, but in presence

of the PCs the expression level was reduced to the same level as found in cells grown under NC. This

remarkable restoration of the Acc1 mRNA levels by caffeic acid, naringenin, and quercetin underlines

their importance in the field of lipotoxicity induced beta cell dysfunction in T2D.

17

Previously, naringenin has been shown to induce glucose uptake via AMPK activation in L6

myotubes [41], whereas caffeic acid exhibited insulin mimetic effects on 3T3-L1 cells and insulin

resistant mouse hepatocytes [42,43]. Again, quercetin has been found to reduce insulin resistance in

primary human adipocytes [44]. It is interesting to observe, that these three PCs exhibit multi-potent

capabilities in the amelioration of T2D, making them promising candidates for drug research.

In conclusion, the three naturally occurring PCs used in this study, namely caffeic acid,

naringenin and quercetin appears to possess beneficial effects on insulin secretion under GC. As

expected, their influence on the gene expression levels of all the genes studied was not uniform; but

they appear to have a positive influence on the expression of key beta cell survival and regulatory

genes; improving glucose sensitivity and survival probabilities of INS-1E cells subjected to

glucotoxicity. Furthermore, the PCs have exhibited a promising potential of improving both insulin

secretion and insulin gene expression under GC, together with restoring the regulation of genes

associated with lipotoxicity in beta cells.

Acknowledgements

This work was supported by The Danish Council for Strategic Research (Grant no. 09-063086) and The

Graduate School of Agriculture, Food and Environment (SAFE), Aarhus University. The Institute of

Clinical Medicine, Aarhus University and The Aarhus University Research Foundation. We would like

to thank Dorthe Rasmussen, Lene Trudsø, Kim Glintborg Poulsen, and Ann Overgaard for their skilled

technical support. The authors declare no conflicts of interest.

18

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Fig. 1 Effect of the phenolic compounds, caffeic acid (A and B), naringenin (C and D), and quercetin

(E and F) on glucose stimulated insulin secretion after acute exposure. INS-1E cells were grown in

media containing 11 mM glucose for 72 h, thereafter incubated for 1 h in either 3.3 (A, C, and E) or

16.7 (B, D, and F) mM glucose in the presence or absence of the compounds at 10-10

, 10-8

and 10-6

M

concentrations, following which the supernatant was harvested and insulin content measured. Number

of replicates /treatment = 12. Control cells were incubated with DMSO (vehicle). Data are shown as

mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2 Effect of the phenolic compounds, caffeic acid (A and B), naringenin (C and D), and quercetin

(E and F) on glucose stimulated insulin secretion after chronic exposure. INS-1E cells were incubated

for 72 h in media containing 11 mM glucose in presence or absence of the compounds at 10-10

, 10-8

and

10-6

M concentrations. Thereafter, the cells were treated with either 3.3 (A, C, and E) or 16.7 (B, D,

and F) mM glucose for 1 h, following which the supernatant was collected and insulin content

measured. Number of replicates /treatment = 18. Control cells were incubated with DMSO (vehicle).

Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3 Effect of the phenolic compounds, caffeic acid (A), naringenin (B), and quercetin (C) on glucose

stimulated insulin secretion after exposure to normoglycemic and glucotoxic conditions. INS-1E cells

were incubated for 72 h in media containing 11 or 25 mM glucose in presence or absence of 10-6

M

concentration of the compounds. Thereafter, the cells were treated with 3.3 and 16.7 mM glucose for 1

h, following which the supernatant was harvested and insulin content measured. 11 and 25 mM

represents normoglycemic and glucotoxic condition. Number of replicates /treatment = 18. Control

cells were incubated with DMSO (vehicle). Data are shown as mean ± s.e.m. In the plot G = glucose;

ns = not significant. *p < 0.05, **p < 0.01, ***p < 0.001

25

Fig. 4 mRNA abundance of Glut2, Gck, Ins1, Ins2, Beta2 and Pdx1 in INS-1E cells treated with caffeic

acid, naringenin or quercetin in presence of 11 or 25 mM glucose for 72 h were studied by real time

RT-PCR using TaqMan®

assays. Duplicate samples were taken for each treatment and the samples

were measured in triplicates. Gene expressions were normalised to 18s ribosomal RNA. Difference in

the mRNA abundance was calculated compared to their respective untreated controls. Open and closed

bars represent the cells grown in 11 and 25 mM glucose. C, N, and Q represent caffeic acid, naringenin

and quercetin respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5 mRNA abundance of Akt1, Akt2, and IRS1 genes in INS-1E cells treated with caffeic acid,

naringenin or quercetin in presence of 11 or 25 mM glucose for 72 h were studied by real time RT-PCR

using TaqMan® assays. Duplicate samples were taken for each treatment and the samples were

measured in triplicates. Gene expressions were normalised to 18s ribosomal RNA. Difference in the

mRNA abundance was calculated compared to their respective untreated controls. Open and closed

bars represent the cells grown in 11 and 25 mM glucose. C, N, and Q represent caffeic acid, naringenin

and quercetin respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6 mRNA abundance of Casp3, Bcl2, Bax, Hsp70, Hsp90 and Acc1 genes in INS-1E cells treated

with caffeic acid, naringenin or quercetin in presence of 11 or 25 mM glucose for 72 h were studied by

real time RT-PCR using TaqMan® assays. Duplicate samples were taken for each treatment and the

samples were measured in triplicates. Gene expressions were normalised to 18s ribosomal RNA.

Difference in the mRNA abundance was calculated compared to their respective untreated controls.

Open and closed bars represent the cells grown in 11 and 25 mM glucose. C, N, and Q represent caffeic

acid, naringenin and quercetin respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

26

Fig. 1

0 10-10

10-8

10-6

15

25

35

45

55

Caffeic acid (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

* ***


Insu

lin

(n

g/m

L)

0 10-10 10-8 10-615

25

35

45

55

Naringenin (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

180

200

*

**

Naringenin (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

15

25

35

45

55

Quercetin (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

180

200

*

Quercetin (mol/L)

Ins

uli

n (

ng

/mL

)

A B

C D

E F

27

Fig. 2

0 10-10 10-8 10-625

35

45

55

65

75

*


Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

180

****** ***


Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

25

35

45

55

65

75

Naringenin (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

180

*** ** ***

Naringenin (mol/L)

Ins

uli

n (

ng

/mL

)

0 10-10

10-8

10-6

25

35

45

55

65

75

*

Quercetin (mol/L)

Insu

lin

(n

g/m

L)

0 10-10

10-8

10-6

80

100

120

140

160

180

*****

Quercetin (mol/L)

Ins

uli

n (

ng

/mL

)

A B

C D

E F

28

Fig. 3

0

20

40

60

80

100

120

140

160

180

Caffeic acid 72 h (10 -6 M) - + - + - + - +

Glucose 1h (mM) 3.3 3.3 16.7 16.7 3.3 3.3 16.7 16.7

**

**

***

***

11 mM G 25 mM G

***

ns

Insu

lin

(n

g/m

L)

0

20

40

60

80

100

120

140

Naringenin 72 h (10 -6 M) - + - + - + - +

Glucose 1h (mM) 3.3 3.3 16.7 16.7 3.3 3.3 16.7 16.7

**

11 mM G 25 mM G

****

ns

Insu

lin

(n

g/m

L)

0

20

40

60

80

100

120

140

160

Quercetin 72 h (10 -6 M) - + - + - + - +

Glucose 1h (mM) 3.3 3.3 16.7 16.7 3.3 3.3 16.7 16.7

***

*

******

25 mM G11 mM G

***

ns

Insu

lin

(n

g/m

L)

A

B

C

29

Fig. 4

Glut2

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

p = 0.08

GL

UT

2 m

RN

A (

rela

tive u

nit

s)

Glut2

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5 ****

GL

UT

2 m

RN

A (

rela

tive u

nit

s)

Glut2

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5*

GL

UT

2 m

RN

A (

rela

tive u

nit

s)

Gck

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5*

p = 0.1

Gck m

RN

A (

rela

tive u

nit

s)

Gck

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5**

p=0.06

Gck m

RN

A (

rela

tive u

nit

s)

Gck

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5

2.0

**

*

Gck m

RN

A (

rela

tive u

nit

s)

Ins1

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5 * **

Ins1 m

RN

A (

rela

tive u

nit

s)

Ins1

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5 ** **

Ins1 m

RN

A (

rela

tive u

nit

s)

Ins1

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5*

*

Ins1 m

RN

A (

rela

tive u

nit

s)

Ins2

11 mM 11 mm+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0

*

Ins2 m

RN

A (

rela

tive u

nit

s)

Ins2

11 mM 11 mm+N 25 mM 25 mM+N0.0

0.5

1.0

1.5 **

p = 0.09

Ins2 m

RN

A (

rela

tive u

nit

s)

Ins2

11 mM 11 mm+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5

Ins2 m

RN

A (

rela

tive u

nit

s)

Beta2

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0 *

Beta

2 m

RN

A (

rela

tive u

nit

s)

Beta2

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5

**

p=0.07

Beta

2 m

RN

A (

rela

tive u

nit

s)

Beta2

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5 **

Beta

2 m

RN

A (

rela

tive u

nit

s)

Pdx1

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0 ***

Pd

x1 m

RN

A (

rela

tive u

nit

s)

Pdx1

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5

2.0 ****

Pd

x1 m

RN

A (

rela

tive u

nit

s)

Caffeic acid Naringenin Quercetin

Pdx1

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5

2.0 ***

Pd

x1 m

RN

A (

rela

tive u

nit

s)

30

Fig. 5

Akt1

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0 *

Akt1

mR

NA

(re

lati

ve u

nit

s)

Akt1

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5**

*

Akt1

mR

NA

(re

lati

ve u

nit

s)

Akt1

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5

2.0 *

Akt1

mR

NA

(re

lati

ve u

nit

s)

Akt2

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0

*

Akt2

mR

NA

(re

lati

ve u

nit

s)

Akt2

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5** **

Akt2

mR

NA

(re

lati

ve u

nit

s)

Akt2

11 mM 11 mM+Q 25 mM 25 mM+Q0.0

0.5

1.0

1.5 **

Akt2

mR

NA

(re

lati

ve u

nit

s)

Irs1

11 mM 11 mM+C 25 mM 25 mM+C0.0

0.5

1.0

1.5

2.0***

Irs1 m

RN

A (

rela

tive u

nit

s)

Irs1

11 mM 11 mM+N 25 mM 25 mM+N0.0

0.5

1.0

1.5

Irs1 m

RN

A (

rela

tive u

nit

s)

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Manuscript III

Naringenin and falcarinol stimulate glucose uptake and TBC1D1 phosphorylation in primary porcine

myotube cultures

Sumangala Bhattacharya, Martin Krøyer Rasmussen, Jette F. Young, Lars P. Christensen, Karsten

Kristiansen, and Niels Oksbjerg

To be submitted to: Biochemical and Biophysical Communications

1

Naringenin and falcarinol stimulate glucose uptake and TBC1D1

phosphorylation in primary porcine myotube cultures

Authors: Sumangala Bhattacharya1, Martin Krøyer Rasmussen


3, Jette F. Young

1,

Karsten Kristiansen2, and Niels Oksbjerg

1*

Institutional affiliations:

1Department of Food Science, Aarhus University, Blichers Allé 20, Postbox 50, 8830 Tjele, Denmark

2Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N,

Denmark

3Department of Chemical Engineering, Biotechnology and Environmental Technology, University of

Southern Denmark, Niels Bohrs Allé 1, 5230 Odense M, Denmark


Niels Oksbjerg

Department of Food Science, Aarhus University,

Blichers Allé 20, Postbox 50, 8830 Tjele, Denmark

E-mail: [email protected], Tel: + 45 8715 7809, Fax: +45 8715 4891

mailto:[email protected]

2

Abstract

Insulin resistance in muscles is a major problem associated with Type 2 diabetes. Bioactive compounds

of plant origin have long been known for possessing anti-diabetic properties. We have studied the

effect of the bioactive compounds naringenin (dihydroflavonol) and falcarinol (polyacetylene) on

glucose uptake (GU) in normal and insulin resistant primary porcine myotubes, in the presence and

absence of insulin to identify signaling pathways mediating their effects on GU. The dependence on

glucose transporter type 4 (Glut4) activity, insulin signaling and AMP- activated protein kinase

(AMPK)-signaling was studied by using the Glut4 inhibitor indinavir, the phosphatidyl inositol-3

kinase (PI3K) and p38 mitogen activated protein kinase (MAPK) inhibitor wortmannin, and the AMPK

inhibitor dorsomorphin (DM), respectively. Naringenin and falcarinol stimulated GU was attenuated in

the presence of indinavir and wortmannin, indicating a dependence on Glut4 activity as well as

PI3Kand/or p38MAPK activity. By contrast, DM diminished GU induced by naringenin only,

indicating that falcarinol-stimulated GU was independent of AMPK activity. Finally, we show that

naringenin and falcarinol enhance phosphorylation of TBC1D1 suggesting that these compounds

enhance translocation of Glut4 containing vesicles and thereby glucose uptake via a TBC1D1-

dependent mechanism.

Keywords: Naringenin, Falcarinol, Glucose uptake, Type 2 diabetes, Insulin, TBC1D1, TBC1D4,

As160

3

1. Introduction

The initiation of insulin resistance in muscles, normally occurs asymptomatically, and is compensated

with increased insulin secretion by the pancreatic β-cells. If left uncontrolled, this condition leads to β-

cell exhaustion and failure, causing an increase in blood glucose level, leading to the manifestation of

T2D [1].

Skeletal muscle is the primary site for glucose uptake (GU) and utilisation. About 75 % of the

insulin stimulated glucose disposal takes place in the skeletal muscles [2], where insulin causes GU via

the translocation of glucose transporter type 4 (Glut4) vesicles to the plasma membrane [3]. Insulin

causes activation of the phosphatidyl inositol-3 kinase (PI3K)-Akt/protein kinase B pathway, which

mediates most of the metabolic actions of insulin [4].

Another major signaling pathway responsible for GU and fatty acid oxidation in muscles is the

AMP- activated protein kinase (AMPK) signaling cascade. In mammals, AMPK acts as a metabolic

energy sensor, maintaining the cellular energy balance [5]. It has been shown that activation of AMPK

by agonists such as AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) causes Glut4

translocation [6;7]. Moreover, thiazolidinediones and metformin, have been found to enhance GU in

skeletal muscle through AMPK phosphorylation [8].

The Rab-GTPase activating protein, TBC1D4 (also known as As160) and its homolog TBC1D1

have been found to play a major role in insulin and AICAR stimulated Glut4 translocation [9-11]. They

are also downstream targets for Akt. It has been suggested that phosphorylation of TBC1D4 and

TBC1D1(TBC1D4/1) lead to the activation of small Rab-GTPases which causes cytoskeletal re-

organization leading to the translocation and docking of the Glut4 vesicles to the plasma membrane

[12].

4

Secondary metabolites from different botanicals have been exploited successfully to provide

relief from several ailments, and are able to influence different cellular mechanisms, including key

signalling pathways balancing energy utilization and storage. Hence, more than a thousand plant

species have been tested for their efficacy against diabetes [13]. Naringenin, a flavonol, found in many

citrus fruits (such as grapefruits and oranges) has been found to enhance insulin sensitivity and reduce

plasma glucose levels in diabetic animal models [14], and cause AMPK activation in L6 myotubes

[15]. However, the different steps involved in its mode of action are yet to be elucidated. Falcarinol, a

polyacetylene present (among other plants) in carrots, is mostly known for its anti-cancer and anti-

inflammatory properties [16;17]. However, biphasic behaviour of falcarinol was reported, where it is

found to exhibit cyto protective [18] and growth-stimulatory effects [19]. Falcarinol has not been

studied yet for its efficacy against diabetes.

In the present study, primary porcine myotube cultures were used as a model for skeletal muscle

to test the GU enhancing potential of naringenin and falcarinol in normal and insulin resistant

myotubes. GU was measured in the presence of indinavir (a Glut4 inhibitor), a fungal metabolite

wortmannin (a PI3K-inhibitor), and dorsomorphin (DM; an AMPK inhibitor) separately.

Simultaneously, the effect of these inhibitors on naringenin and falcarinol induced phosphorylation of

TBC1D4/1 was studied.

2. Materials and methods

2.1. Materials

Falcarinol (> purity 98%) was isolated from carrots according to the procedure described elsewhere

[20] and identified by UV, mass spectrometry (MS) [gas chromatography (GC)-MS (EI, 70 eV)], NMR

(1H and

13C NMR, and

1H-

1H and

1H-

13C correlation spectroscopy recorded in CDCl3 with TMS as

5

internal standard), and optical rotation, and the complete spectral data set corresponded fully with

literature values for falcarinol [21-23]. Chemical structures of falcarinol and naringenin are shown in

Fig. 1. Dulbecco’s modified eagles medium (DMEM), fetal calf serum and horse serum (FCS and HS,

respectively) Trypsin-EDTA were from GIBCO Life technologies. The antibiotics (amphotericin,

penicillin/streptomycin and gentamycin), naringenin, DM and phosphatase inhibitor cocktail (PIC) 2

and 3 were from Sigma-Aldrich. [3H] 2-deoxy-D-glucose (2-DOG) and the scintillation mix (Ultima

Gold) were bought from Perkin Elmer Inc. Indinavir, wortmannin, and AICAR were from Santa Cruz

Biotechnology (Texas, USA). Antibodies against phosphorylated TBC1D4/1 were from Cell Signalling

Technology (Danvers, MA, US) and that against α-Tubulin was from Merck Millipore (Darmstadt,

Germany). Goat anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were from Dako

Denmark A/S (Glostrup, Denmark). Enhanced chemiluminescence reagent (ECL) and High

performance chemiluminescence films were from GE healthcare (Buckinghamshire, UK). The

polyvinylidene difluoride (PVDF) membranes were from BioRad (CA, USA), protein molecular

weight markers from Thermo scientific Inc. (Ma, USA) and 4-12 % Bis-Tris gels from Life

technologies (Paisley, UK).

2.2. Preparation of myotube cultures

Satellite cells were isolated from semimembranosus muscles of female pigs weighing approximately12

kg, as described elsewhere [24] and stored in liquid nitrogen until used. For preparation of myotube

cultures, the cells were thawed and evenly seeded on Matrigel matrix (BD Biosciences, cat no. 354230)

coated (1:50 v/v) 6, 48, or 96 well plates for protein analysis, GU assay, and cell viability studies,

respectively. Cells were seeded and proliferated in Porcine Growth Medium (10 % fetal calf serum, 10

% horse serum (FCS and HS respectively, GIBCO Life technologies, Burlington, ON, Canada), 80 %

6

Dulbecco’s modified eagles medium (DMEM, Life Technologies, Naperville, IL), with 25 mM glucose

and antibiotics (100 IU/mL penicillin, 100 IU/mL streptomycin sulphate, 3 µg/mL amphotericin B, 20

µg/mL gentamycin)) in a CO2-regulated humidified incubator (95 % air and 5 % CO2 at 37°C). After

reaching 80 % confluence, the cells were proliferated in media containing DMEM (7 mM glucose), 10

% FCS, and antibiotics for 24 h to 100 % confluence, and subsequently differentiated into myotubes by

incubating with differentiation media (DMEM containing 7 mM glucose, 5 % FCS, antibiotics, and 1

µM cytosine arabinoside) for 48 h.

2.3. Glucose uptake assay

The differentiated myotubes were treated with serum free media (SFM; DMEM with 7 mM, glucose,

antibiotics, and 1 µM cytosine arabinoside, 1 % FCS) for 2-5 h, followed by incubation with various

treatments for 1 h. The myotubes were then washed with (4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid) HEPES buffered saline (20 mM Hepes, 140 mM NaCl, 5 mM KCl, 2.5

mM MgSO4, 1 mM CaCl2, adjusted to pH 7.4) and incubated with 250 µL/well 0.1 mM [3H] 2-deoxy-

D-glucose (2-DOG), for 30 min; washed thrice with ice cold phosphate buffered saline (PBS, 500

µL/well), lysed by adding 0.05 M NaOH (37°C, 250 µL/well) and placed on a shaking board for 30

min. The cell lysate was mixed with scintillation liquid (1:10) and counted in a Win spectral, 1414

liquid scintillation counter. Following serum deprivation, the differentiated cells were pre-incubated

with 1µM wortmannin for 1 h or 10 µM DM for 5 min, prior to treatment addition. Indinavir (100 µM)

was added 5 min prior to 2DOG addition. AICAR (1 mM) and insulin (10 nM) were used as positive

controls (1 h incubation). To make insulin resistant myotubes (IRM), myotubes were incubated with

differentiation media for 24 h and then with differentiation media containing 12 mM as compared to 7

7

mM glucose for the next 24 h. These myotubes were not treated with SFM, before treatment addition.

Controls received DMSO.

2.5. Western blotting

Differentiated cells were treated with various treatments for 2.5 h, washed with PBS, harvested using

0.25 % Trypsin-EDTA, and frozen at -80°C. Lysis buffer containing 4 % SDS, 10 mM Tris-HCl, and 1

mM EDTA was used to lyse the cells. PIC 2 and 3 were added to the lysis buffer to inhibit

phosphatases. Cell lysates were separated by SDS-PAGE using 4-12 % Bis-Tris gels. Lysates

containing equal amounts of protein were loaded during gel electrophoresis. Proteins were transferred

to a PVDF membrane, stained with Ponceau S and visually inspected for equal loading and blotting

efficiency [25]. The membranes were blocked using 2 % (w/v) BSA in 0.1 % TBS-T buffer (0.05 M

Tris-base, 0.5 M NaCl, 0.1 % (v/v) Tween-20, pH adjusted to 7.4) for 1 h, at room temperature, and

washed in 0.1 % TBS-T. The membrane was then incubated with primary antibody at 4 °C, overnight

or 1 h at room temperature (RT), washed, incubated with HRP-conjugated anti-mouse or anti-rabbit

secondary antibody for 1 h at RT, and washed again. All washing steps after blotting were done 6

times, 10 min each. ECL reagent was used to detect the primary antibody and visualised by exposure to

auto-radiographic films, which were scanned and bands were analysed using the ImageJ software. The

relative protein expression was normalised against the α-Tubulin as a reference protein.

2.6. Statistical analysis

Statistical analysis of data was conducted, using the ‘Mixed’ procedure of SAS statistical programming

software (Ver. 9.2; SAS Institute Inc., Cary, NC, USA). Overall the models used included the fixed

effects of treatments as well as their interactions. As random effects, porcine myotube culture (N=3)

8

and replicates nested within fixed and random effects. Data representing Fig. 2A-J were tested

separately. The model included fixed effects of insulin, glucose concentration, indinavir, wortmannin,

DM, AICAR, naringenin, falcarinol and insulin as well as their interactions. Porcine myotube cultures

(n=3) and replicates (n = 4) nested within treatments were included as random effects. When overall

effects were significant, LSMeans was separated by pairwise comparison (pdiff option in SAS).

For the western blot and cytotoxicity analysis, differences between treatments were determined

by Student’s unpaired t-test. Differences with a p < 0.05 were considered statistically significant.

3. Results

Any possible cytotoxic effects of the treatments on myotube viability were investigated. However, no

significant decrease in myotube viability in presence of 10 or 30 µM naringenin or falcarinol, or in

presence of the inhibitors (at concentrations used in this study) was observed.

GU was determined in differentiated myotubes incubated with 3, 10 or 30 µM of naringenin

and falcarinol separately, in presence or absence of 10 nM insulin (Fig. 2A and 2B) for 1 h.

Naringenin significantly increased GU in the absence of insulin at 3, 10 and 30 µM concentrations by

15.6 (p = 0.001), 19.5 (p < 0.001) and 16.4 % (p < 0.001), respectively compared to control (DMSO).

An increase of 23.0 (p < 0.001), 11.5 (p < 0.01), and 9.2 % (p = 0.02) in GU at 3, 10, and 30 µM

naringenin concentrations was observed compared to 10 nM insulin only. Falcarinol significantly

increased GU at 3, 10 and 30 µM concentrations in the absence of insulin by 26.5 (p = 0.001), 26.0 (p

< 0.001), and 7.6 (p < 0.01) %, respectively, compared to control (DMSO); whereas in presence of 10

nM insulin, an increase of 15.8 (p < 0.01), 4.0 (p = 0.1) and a decrease of 15.1 (p < 0.001) % in GU,

was observed at 3, 10 and 30 µM falcarinol, respectively, compared to 10 nM insulin only. Here, the

9

GU stimulating effect in the presence of insulin was only observed at 3 µM, while GU was inhibited at

concentration of 30 µM falcarinol.

Based on the results on glucose uptake in absence of insulin (Fig. 2A and 2B) the

concentrations 10 and 30 µM for both naringenin and falcarinol were chosen for further experiments.

The effects of naringenin and falcarinol on IRM are illustrated in Fig. 2C and 2D. In order to reduce

insulin sensitivity, myotubes were incubated in high glucose (12 mM) for 24 h before the experiment.

An extracellular glucose concentration of 12 mM was used to reduce the insulin sensitivity of the

myotubes, while keeping the cell viability unaffected, as demonstrated elsewhere [26]. Basal GU was

significantly reduced in IRM (18.1 %, p = 0.02) compared to control. A significant increase in GU was

observed in presence of 10 nM insulin (21.8 %, p =0.005), as well as 10 and 30 µM naringenin (37.7,

and 29.0 %; p < 0.001) in IRM. The increase in GU in presence of 10 but not 30 µM naringenin was

significantly higher (p = 0.03) than that caused by 10 nM Insulin. Falcarinol, at the concentration s of

10 (21.0 %, p = 0.007) and 30 µM (13.0 %, p = 0.09), significantly increased GU in IRM. At 10 µM

concentration, the increase in GU caused by falcarinol was not significantly different from that caused

by 10 nM Insulin.

Indinavir, a specific inhibitor of Glut4 mediated glucose transport, directly binds and blocks the

Glut4 transporter [27]. The IC50 of indinavir with respect to GU, for cells expressing Glut4 is 50 to 100

µM [28]. In order to examine whether the increased GU elicited by naringenin and falcarinol was

dependant on Glut4 transporters, myotubes were incubated in presence or absence of 100 µM indinavir

for 35 min (Fig. 2E and 2F). Incubation with indinavir significantly reduced basal GU by 44.7 % (p <

0.001). There was no significant increase in GU in presence of 10 nM insulin or 30 µM naringenin in

the indinavir treated cells; whereas 10 µM naringenin caused a minute, but significant increase in GU

10

(6 %, p = 0.03). GU was unaffected at 10 µM and further reduced at 30 µM falcarinol concentrations in

indinavir treated cells.

Activation of PI3K has been found to be necessary for both basal and insulin stimulated Glut4

translocation to the plasma membrane, which is inhibited by wortmannin [29]. In order to test whether

the GU induced by naringenin and falcarinol is affected by the inhibition of PI3K, myotubes were

incubated with 1 µM wortmannin for 1 h (Fig. 2G and 2H), which significantly decreased the basal and

insulin stimulated GU by 24.2 and 31.8 % (p < 0.001) respectively. Naringenin induced GU at 10 and

30 µM concentrations was reduced by 39.9 and 33.9 % (p < 0.001) respectively. A similar reduction in

GU for 10 and 30 µM falcarinol in wortmannin treated myotubes (40.0 and 31.2 %, p < 0.001) was

observed.

In order to test the AMPK dependence of naringenin and falcarinol, myotubes were incubated

with DM, at a concentration of 10 µM, for 65 min (Fig. 2I, J). The AMPK agonist AICAR (1 mM) was

used as a positive control. There was no reduction in basal GU, in DM treated myotubes, but AICAR

stimulated GU was significantly reduced (12.1 %, p < 0.001) in presence of DM. A significant

reduction of 23.7 and 13.4 % (p < 0.001) was observed for 10 and 30 µM naringenin compared to

vehicle at the same concentration. Both 10 and 30 µM falcarinol showed an increase of 5.0 and 24.3 (p

< 0.001) % in DM treated myotubes compared to vehicle at the same concentration.

Activation of TBC1D4/1 by naringenin and falcarinol was examined, with insulin (100 nM) as

a positive control. Naringenin and falcarinol was found to solely increase TBC1D1 phosphorylation

(Fig. 3), and a tendency to decrease in presence of wortmannin. Insulin was found to significantly

increase the phosphorylation of TBC1D4/1, where the former was induced to a higher degree. Insulin

stimulated TBC1D4 phosphorylation (but not that of TBC1D1) was significantly reduced by

wortmannin. In DM treated myotubes, naringenin stimulated TBC1D1 phosphorylation was

11

significantly reduced, and a similar tendency was observed for TBC1D4 phosphorylation; whereas

falcarinol showed a significant increase in TBC1D4 phosphorylation in the presence of DM.

TBC1D4/1 were found to have an approximate molecular weight of 70 kDa. TBC1D4/1 have a

molecular weight of 160 kDa in humans and mouse [30;31]. The molecular weights of these proteins in

pigs have not yet been established. However, according to Ensemble and NCBI sources, based on their

mRNA transcripts, porcine TBC1D4 and TBC1D1 is predicted to have an approximate molecular

weight of 65 to 70 kDa

(http://www.ensembl.org/Sus_scrofa/Gene/Summary?g=ENSSSCG00000009464;r=11:52202660-

52264604;t=ENSSSCT00000010375 , http://www.ncbi.nlm.nih.gov/nuccore/350587438,). Two other

studies [32;33] have tried to detect TBC1D4 in porcine muscles, but they used a different approach

where phosphorylated Akt substrate antibody was used to detect the protein.

4. Discussion

In the current study, naringenin and falcarinol were found to enhance GU in primary porcine myotube

cultures autonomously. In presence of insulin, both naringenin and falcarinol showed a higher increase

in GU at the lowest concentration tested (3 µM); which was reduced at 10 and 30 µM concentrations.

A possible explanation could be a shift in the sensitivity range of the compounds in the presence or

absence of insulin, due to competition for common pathway proteins at higher concentrations.

In IRM, basal GU was significantly reduced, while the naringenin induced effect was

maintained in IRM. This indicates an insulin-independent mechanism of GU which is in line with a

study on L6 muscle cells, where naringenin was found to activate AMPK [15], but in contrast, a

reduced GU was observed in naringenin exposed MCF-7 breast cancer cells and myelocytic U937 cells

[34;35], indicating a cell-type specific effect of this flavonol. Falcarinol induced GU on the other hand

http://www.ensembl.org/Sus_scrofa/Gene/Summary?g=ENSSSCG00000009464;r=11:52202660-52264604;t=ENSSSCT00000010375

http://www.ensembl.org/Sus_scrofa/Gene/Summary?g=ENSSSCG00000009464;r=11:52202660-52264604;t=ENSSSCT00000010375

http://www.ncbi.nlm.nih.gov/nuccore/350587438

12

was not maintained, in IRM after 10 µM exposure. This diminished GU induction in the presence of 10

µM falcarinol in IRM may be caused by down-regulation of signalling proteins required for falcarinol

stimulated GU.

A significantly reduced GU was observed in indinavir treated cells. In these cells, naringenin

(10 µM) caused a minute but significant increase in GU, although insulin treatment did not. A similar

observation was obtained for naringenin treated IRM, suggesting that although naringenin mostly

depends on Glut4, for GU, it might be capable of partially inducing other glucose transporters (like

Glut1), or enhancing the activity of the small number of Glut4 still available for transport. However,

falcarinol did not increase GU in the indinavir treated cells; which might be indicative of its complete

dependence on Glut4, as is also the case for insulin.

Neither naringenin nor falcarinol increased GU in wortmannin treated cells, suggesting PI3K

dependence. However, it is important to note that in earlier studies, wortmannin has also been found to

inhibit MAPK [36] with an IC50 of 300 nM. This could link GU by naringenin and falcarinol to

MAPK-inhibition as well; since, other than being a downstream target of AMPK [37], p38-MAPK is

involved in full activation of Glut4 [38].

Treatment of the myotubes with DM did not cause any significant change in basal GU; but

AICAR and naringenin mediated GU was diminished in its presence. This also corroborates well with

naringenin induced AMPK activation [15] and unchanged naringenin induced GU in IRM compared to

normal myotubes. The inability of DM to reduce falcarinol induced GU and TBC1D1 phosphorylation

indicates AMPK independence; while the significant DM induced increase in GU observed at 30 µM

falcarinol is surprising. However a cross talk between different signalling pathways could provide a

rationale. Moreover, DM has been shown to participate in other signalling cascades, independent of the

AMPK pathway [39]. Furthermore, intracellular reactive oxygen species (ROS) has been implicated in

13

GU during exercise/muscle contraction [40] and the activation of p38 MAPK [41], which stimulates

GU [38]. The fact that falcarinol induces ROS formation at low concentrations (1.6 to 25 µM) [18]

could explain the falcarinol stimulated AMPK-independent increase in GU.

The activation of TBC1D1 by naringenin and falcarinol was reported for the first time in this

study. TBC1D1 is more abundant in fast-twitch muscles, while higher levels of TBC1D4 is found in

muscles with slow-twitch characteristics [10]. However, TBC1D4 was more responsive to insulin

induced phosphorylation than TBC1D1. Mass spectrometry analysis on TBC1D1 from mouse skeletal

muscle has revealed several phosphorylation sites, most of which were consensus or near consensus

sites for AMPK; and AICAR was found to be a stronger regulator, causing more phosphorylation on

TBC1D1 than insulin [10]. This also explains the inhibition of naringenin induced

TBC1D1phosphorylation by DM, since naringenin is known to activate AMPK [15].

Overall, it can be concluded that both naringenin and falcarinol depend predominantly on Glut4

and PI3K and/or p38MAPK activity for the induction of GU. Naringenin (not falcarinol) induced GU,

is dependent on AMPK activation. Treatment with wortmannin and DM indicate that naringenin and

falcarinol differ in their mechanism of action, but both increase GU via TBC1D1 phosphorylation.

However, it would be interesting to investigate the complicated distribution and regulation patterns of

TBC1D4 and TBC1D1 in order to successfully understand the proximal steps in the regulation of Glut4

translocation.

ACKNOWLEDGEMENTS

This work has been supported by The Danish Council for Strategic Research (Grant no. 09-063086)

and The Graduate School of Agriculture, Food and Environment (SAFE), Aarhus University. The

authors declare no conflicts of interest.

14

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Fig. 1. Chemical structures of naringenin and falcarinol.

Fig. 2. Effect of naringenin and falcarinol on glucose uptake. The differentiated myotubes were

incubated with 3, 10 and 30 µM (A) naringenin and (B) falcarinol, in presence and absence of 10 nM

insulin (closed and open bars respectively) for 1 h; or incubated for 24 h with either 7 mM or 12 mM

glucose (open and closed bars respectively) and then treated with 10 and 30 µM of (C) naringenin and

(D) falcarinol for 1 h, following which glucose uptake was measured. Myotubes were incubated with

indinavir (100 µM) for 5 minutes prior to 2DOG addition (E & F); 1µM wortmannin for 1 h (G & H)

or 10 µM DM for 5 minutes (I & J), prior to treatment addition. For A – H, insulin (10 nM) and for I &

J, AICAR (1 mM) was used as the positive control. N10, N30 = 10 and 30 µM naringenin and F10, F30

= 10 and 30 µM falcarinol respectively; Vehicle = cells treated with DMSO only. In the plot DM =

dorsomorphin. Values are given as ls means ± sem of experiments conducted with satellite cells from 3

pigs, expressed as percent of control. Number of replicates per pig (n) = 6. Different letters indicate

significant differences.

Fig. 3. Stimulation of TBC1D4 and TBC1D1 phosphorylation by naringenin and falcarinol.

Differentiated myotubes were incubated for 2.5 h with 100 nM insulin (Ins), 10 µM naringenin (N10),

and 10 µM falcarinol (F10), in presence or absence of 1 µM wortmannin (W) and 10 µM dorsomorphin

(DM). Cells were harvested and lysed. Thereafter, equal amounts of protein were used for SDS-PAGE

followed by immunoblotting with specific antibodies recognizing phosphorylated (Thr642) TBC1D4

and (Thr590) TBC1D1 (A and B). α-Tubulin was used as the reference protein. Values are given as

Mean ± SEM of experiments conducted with satellite cells from 3 pigs. Levels of significance, *p <

0.05, **p < 0.01, ***p < 0.001.

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Fig.1.

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Fig.2.

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Fig.3.

health promoting effects of bioactive compounds in plants: targeting...

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