SYSTEMS BIOLOGY OF AMINO ACID USE BY MAMMARY GLAND: MILK PROTEIN SYNTHESIS AND BEYOND

Juan Loor1 and Zheng Zhou2

1Professor, Department of Animal Sciences, Division of Nutritional

Sciences

University of Illinois, Urbana-Champaign2Assistant Professor, Department of Animal and Veterinary Science,

Clemson University, Clemson

EAAP 69th Annual Meeting

Dubrovnik, Croatia

August 27 - 31, 2018

Nutritionally and Energetically costly process

Amino acids

MetLys

Energy (glucose)

Tissue

proteins

ca. 80% ca. 20%

Milk

proteins

➢ 5-fold in energy and protein requirements during lactation in dairy cows

➢ 4-7-fold in translation activity of lactating mammary gland

➢ “Low” efficiency of dietary N milk protein (25-30%)

↑ Metabolizable E intake↑ Insulin↑ Glucose and AA supply↑ Milk protein

Emery R. S., J Dairy Sci, 1978

Nutritionally and Energetically costly process

Amino acids

MetLysIle

Leu

Thr

His

Arg

etc.

Energy (glucose)

Tissue

proteins

ca. 80% ca. 20%

Milk

proteins

Single-limiting AA theory may not always apply

Saturable response in casein synthesis to EAA – efficiency of AA use not fixed

(Arriola-Apelo et al., 2014)

➢ 5-fold in energy and protein requirements during lactation in dairy cows

➢ 4-7-fold in translation activity of lactating mammary gland

➢ “Low” efficiency of dietary N milk protein (25-30%)

~2 week prior to calving

~2 week after calving

✓ Prolactin ➔ JAK/STAT5✓ IGF-1, Insulin ➔ IRS1, AKT1,

mTOR✓ Transcription binding sites for

caseins: >100✓ FEW transcription factors

experimentally verified

“Systems biology” approach: an integrative and iterativeprocess ➔ reconstructing underlying biology

(Papin et al., 2005)

• RNAseq

• “Omics”

2009

6,382 differentially expressed genes (FDR P ≤ 0.001)

Parturition

Other similar research:

❖ Cow milk (Wickramasinghe et al., 2012)

❖ Goat mammary gland (Shi et al., 2015)

Epigenetic “stabilizer” (Bionaz et al., 2012ab)

Most relevant impacted functions during lactation in cow

mammary gland

Central in milk protein synth. regulation

Endocrine signalsNutrient signals

• Well-known that some components of nutrient sensing are conserved across evolution

• Allows for control of cellular growth and metabolism

• These processes can be fine-tuned:

• AA profile?• “Optimal”

concentrations?• How to deliver to

mammary?

PR

OTE

INS

IN P

ATH

WA

Y

↑ 230 g/d

CaseinWater

❖ Role of insulin first proposed in 1966 (Schmidt, JDS 49:381-385)

❖ One mechanism likely involves greater AA uptake by mammary tissue (Park et al., 1979)

❖ Feed restriction plus infusions of starch, casein, or both for 36 hours

❖ Mixed responses to starch with or without casein❖ ↑ protein yield with starch + casein

↑ Starch + casein

60S rLSUeIF2GDP

60S rLSU

eIF4E

mTOR

S2448 T2446

AMPKαP172

4E-BP1PP

Leucine

Raptor GβL

Rheb

14-3-3

IP3KPDK

PKB

AMPK β

TSC1 TSC2

p70S6K

mTOR

S2448 T2446

RaptoreIF3

eIF4B

P

P PP

4E-BP1PP

P

FKBP12

PI3K

IRS1

mTOR

S2448 T2446

Raptor GβL

FKBP12

S235S236

S240 S244S247

p53S15

Insulin

eiF4G

eiF4A

40S rSU

60S rLSU

tRN

A

40S rSU

tRN

Am

et

eIF4E

Translation initiation Translation elongation

eIF2GDP

*

*

*

***

* * *

*

INSR Insulin

*

EEF2K

eEF2

40S rSU eEF1 *

*

*

tRNAsynthetase

*

LARS2

LARS

*

*

Other

rpS6*P

P

*

*

GSK3

AA

*

CAT1

EAAT3

4F2hc

ASCT2

LAT1

Leu

Ile

Ala

Gly

Met

Gln

His

Val

Ser

Cys

Thr

Arg

Asn

Phe

Trp

Lys

Glu

*

*

*

*

*

Tyr

Asp

Pro

*

*

*

Prolactin

*

*

STAT5B

κ-casein

JAK2

P

CSN3ELF5 *

P

STAT5B

STAT5BPP

STAT5B

STAT5B

*

*

*

Milk proteins

STAT5B

LALBA

Milk lactose

*

ATP

PRLR

*

*

*

LKB1

PAT1

Bionaz and Loor, Bioinformatics and Biology Insights, 2011

AA transporters

0

1

2

3

4

5

SLC1A5

SLC3A2

SLC7A5

SLC36A1

mTOR related

Glucose transporters

log

2 m

RN

A f

old

exp

ressio

n r

ela

tive

to

-1

5d

0.0

0.5

1.0

1.5

2.0

2.5SLC2A1

SLC2A3

SLC2A8

Day relative to parturition

-15 11530 60 12

024

0-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

STAT5A

ELF5

Jak2-Stat5 signaling

Insulin signaling

0.0

0.5

1.0

1.5

2.0

2.5 INSR

IRS1

AKT3

PDPK1

0.0

0.2

0.4

0.6

0.8

1.0

Inhibition of mTOR pathway

-15 11530 60 12

024

00.0

0.5

1.0

1.5

2.0

EIF4EBP2

TSC1

GSK3A

mTOR (FRAP1)

Milk protein

Day Relative to Parturition

15 30 60 120 240

Yie

ld (

Kg/d

ay)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Perc

enta

ge

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0Protein yield

Protein %

a a

ab

b

ab

Insulin-mTOR pathways appear to be inhibited

overall, BUT

Inhibition overcome by insulin, AA, and

glucose

Insulin, IGF-1 and regulation

of milk protein synthesis

networks:

“Missing link with nutrition”

➢ Clear role in transcription

and translation

➢ Menzies et al. (2009):

↑ transcription, translation

role for ELF5 (transc. factor)

➢ Translation regulation via

mTOR

➢ Various research groups

over last 10 years

Take home

messages

Glucose and AA are

important

Essential AA are

key

Leucine and

Isoleucine are

required

Translation

machinery turned-

on postpartum

mTOR pathway is

sensitive to

nutrients/hormones

IGF-1 plays role in

mTOR signalling

Ideal ratio Lys:Met 2.5 Lys:Met 2.0

Rela

tive p

rote

in a

bun

dance

0.0

0.5

1.0

1.5

2.0

mTOR p-mTOR p-S6K1

❖ Some tissue “preference” for EAA seems to exist

❖ Mammary utilization of EAA changes with arterial concentration, physiological state, and hormonal status

❖ Transporter affinity differs, blood flow could be a factor, epithelial cell number, etc, etc

a

b

a

b

b

a

ab

c

❖ Some evidence for a response to greater supply of Met when Lys and other EAA are “optimal”

❖ BUT, at mRNA level various AA transporters and casein downregulated

❖ Various AA and other metabolites (e.g. Putrescine) altered

❖ Increased supply of Threonine, Isoleucine, and Valine particularly effective in ↑↑ p-mTOR, p-RPS6, and casein mRNA

❖ Positive effects in spite of ↓ mRNA for various AA transporters

❖ Post-transcriptional regulation appears very important…

Diet

❖ Polyamine synthesis may be responsive to supply of Arginine and Methionine

❖ Importance??TranscriptionCell proliferationmTOR signalingOxidative stress

➢ Metabolizable Methionine supply clearly associated with milk protein % and yield

➢ Is there an additive effect of Methionine and Arginine??

➢ His, Trp, and Thr also may be limiting

Amino acid sensors, energy metabolism, CLOCK, and mTOR – novel mechanisms of protein synthesis regulation

4F2hc

RORE

BMAL1CLOCK

PER1/PER2/PER3

CRY1/CRY2 REV-ERBs

RORs

AMPKα/β

GSK-3β

BMAL1

PPARs

PPRE

MetArg

CRYs

PERsCK1ε/δ

CRYs

PERs

REV-ERBα

RORα

PPARδ/β

SNAT9

GATOR1

mTORC1Rag A/BRegulator

TSC2

SNAT2

NCoR1-HDAC3

FLCN/FNIP2

LKB1

E-box

LAT1

[Ca2+]i/CaM & PI3K

CAT1

EAAT3

ASCT2

GLUT1 4F2hc

AA transporters:LAT1, 4F2hc, ASCT2, etc.

DYRK1A

Fatty acids

GATOR2

CASTOR1/2

NEGATIVE NUTRIENT BALANCE

(Hu and Loor - unpublished)

4F2hc

RORE

BMAL1CLOCK

PER1/PER2/PER3

CRY1/CRY2 REV-ERBs

RORs

βTrCP

AMPKα/β

GSK-3β

FBXL326S proteasome

BMAL1

26S proteasome

SIRT1

NAMPT

NAD+

NMNADP-r

PPARs

PPRE

MetArg

CRYs

PERsCK1ε/δ

CRYs

PERs

REV-ERBα

RORα

PPARδ/β

SNAT9

GATOR1

mTORC1Rag A/BRegulator

TSC2

SNAT2

NCoR1-HDAC3

FLCN/FNIP2

LKB1

Nampt

E-box

LAT1

[Ca2+]i/CaM & PI3K

CAT1

EAAT3

ASCT2

GLUT1 4F2hc

AA transporters:LAT1, 4F2hc, ASCT2, etc.

NAM

AMPKα/β

CK1ε/δ

DYRK1A

GATOR2

CASTOR1/2

Amino acid sensors, energy metabolism, CLOCK, and mTOR – novel mechanisms of protein synthesis regulation

(Hu and Loor - unpublished)

(Hu and Loor - unpublished)

4F2hc

RORE

BMAL1CLOCK

PER1/PER2/PER3

CRY1/CRY2 REV-ERBs

RORs

AMPKα/β

GSK-3β

BMAL1

SIRT1

NAMPT

NAD+

NMNADP-r

PPARs

PPRE

MetArg

CRYs

PERsCK1ε/δ

CRYs

PERs

REV-ERBα

RORα

PPARδ/β

SNAT9

GATOR1

mTORC1Rag A/BRegulator

TSC2

SNAT2

NCoR1-HDAC3

FLCN/FNIP2

LKB1

Nampt

E-box

LAT1

[Ca2+]i/CaM & PI3K

CAT1

EAAT3

ASCT2

GLUT1 4F2hc

AA transporters:LAT1, 4F2hc, ASCT2, etc.

NAM

Gluc uptake &ATP

AMPKα/β

CK1ε/δ

DYRK1A

Glucose➢ Lactation (prolactin?) in mice enhances

stability of the CLOCK-BMAL1 heterodimer ➔↑ metabolism??

TAS1R1/TAS1R3

[Ca2+]i & ERK1/2

Val

GATOR2

CASTOR1/2

POSITIVE NUTRIENT BALANCE

Amino acid sensors, energy metabolism, CLOCK, and mTOR – novel mechanisms of protein synthesis regulation

4F2hc

RORE

BMAL1CLOCK

PER1/PER2/PER3

CRY1/CRY2 REV-ERBs

RORs

AMPKα/β

GSK-3β

BMAL1

SIRT1

NAMPT

NAD+

NMNADP-r

PPARs

PPRE

MetArg

CRYs

PERsCK1ε/δ

CRYs

PERs

REV-ERBα

RORα

PPARδ/β

SNAT9

GATOR1

mTORC1Rag A/BRegulator

TSC2

SNAT2

NCoR1-HDAC3

FLCN/FNIP2

LKB1

Nampt

E-box

LAT1

[Ca2+]i/CaM & PI3K

EAAT3

ASCT2

GLUT1 4F2hc

AA transporters:LAT1, 4F2hc, ASCT2, etc.

NAM

Gluc uptake &ATP

AMPKα/β

CK1ε/δ

DYRK1A

Glucose

GATOR2

CASTOR1/2

CAT1

Amino acid sensors, energy metabolism, CLOCK, and mTOR – novel mechanisms of protein synthesis regulation

(Hu and Loor - unpublished)

➢ Lactation (prolactin?) in mice enhances stability of the CLOCK-BMAL1 heterodimer ➔↑ metabolism??

POSITIVE NUTRIENT BALANCE

mTOR central hub also for lipid synthesis??

Arg

Milk protein genes

STAT5

Insulin

AA transporters

LALBA

TF? Gln

MetLeu

GAAC/ATF4

PPARγ

LCFA

LXRSREBP1

Milk fat genes

CLA

Other TF?

AP

A

A

E

mTOR

E

A

A

EE?

AP

Glucose

GLUT

E?

EA?

Sp1

ChREBP

Spot14

E

E?

Milk lactoseMilk Protein Milk Fat

E?E

E?

E?A

A

E

E

E

E

E?

A?

A?

EA

Lys

AE A?

E

A?

AKT1

AP

E?A?

A

E

E?

Met

Lys

Arg

E

E

E

E

A?

Saline Acetate Casein Glucose Glu + Cas Ace + Cas SEM P-value

Milk fat 1.41 1.58 1.42 1.55 1.51 1.65 - -

ACSS2 0.48 c 0.80 b 0.90 b 1.17 ab 0.95 b 1.51 a 0.17 0.02

ACACA 0.51 c 0.75 b 0.85 b 0.79 b 0.87 b 1.34 a 0.16 0.05

FASN 0.45 c 0.79 b 0.91 b 0.95 b 0.72 bc 1.68 a 0.19 0.02

FABP3 0.01 c 0.47 b 0.01 c 0.06 c 0.01 c 1.80 a 0.62 0.01

Acetate

Acetyl CoA

MalonylCoAAcetate

Long-chain fatty acid

ACSS2ACACA

FASN

Suggestive of an effect of EAA in regulating FA

synthesis and esterification

Acetate response not greater than casein and glucose

(Danes, Batistel, Wattiaux, Broderick, and Loor - unpublished)

• Feed restriction to 85% of ad libitumintake (last 6 d of 14 d periods)

• Feeding 6 times/d• Abomasal infusion of treatments• Mammary biopsy on d 14

FABP3 TAG

Summary and Perspectives

• Single-limiting amino acid theory may not always apply

– Optimal”/”Ideal” intracellular concentrations? How to deliver to

mammary cells?

– Link with lipid synthesis?

– Integrate transcription, translation, post-translational regulation:

Systems approach

• Better description of transcription factor function

– In silico analysis can provide viable candidates

– In vitro culture (primary or immortalized cells) to determine function/s

• Epigenetic mechanisms

– Methylation status of TF binding sites ➔ link with AA nutrition?

– Programming effects of mammary gland ➔ in utero and/or prior to

weaning?