24th G2L2 Meeting

Lyon, FRANCE

19 May 2017

Amphithéâtre du Pavillon Médical, Bâtiment 1A

Centre Hospitalier Lyon Sud, 165, chemin du grand Revoyet

69930 Pierre-Bénite

Program

The quays of the Rhone, The Museum of Confluences Saint Jean and Fourvière Hill

Welcome… By Etienne Lefai (CarMeN Laboratory, Lyon)

We are very pleased to welcome you in Lyon for the 24th Meeting of the G2L2 that

brings researchers from Lyon, Lausanne, Grenoble and Geneva.

The preliminary program is to be filled with plenary lectures (one for each city) and short

presentations for students and young researchers.

Valerie Large, from CarMeN laboratory (U1060) is deeply involved in the preparation of

the meeting, and she will do her best to offer you the opportunity to share your latest and

nicest results.

A senior researcher from each city is welcome to suggest his name for plenary lectures.

Looking forward to seeing you,

Preliminary program

(The meeting will be conducted in English)

8:30 - 9:00 Welcome – coffee 9:00-9:10 Introduction 9:10-9:40 Plenary session 1: Dr. H. VIDAL (CarMeN director, U1060, Lyon) “Anti-diabetic action

of probiotics: Strain specific regulation of the gut-pancreas incretin axis by Lactobacillus plantarum” 09:4-10:25 Short communications 1: “Effects of exogenous molecules on metabolic diseases” (presentation 10’ + discussion 5’)

B. JULIEN (CarMeN lab, U1060, Lyon) “Metabolic effects of a mixture of low-dose pollutants

in a mouse model of estrogen deficiency”

L. MONTEILLET (U1213, LYON) “Renoprotective effects of fenofibrate in glycogen storage

disease type 1a in mice”

E. BERNARDES MONTEIRO (CarMeN lab, U1060, Lyon) “AÇAÍ polyphenols: a potent

strategy to prevent inflammation and endothelial dysfunction associated with chronic kidney

disease and uremia”

10:25-10:45 Coffee break 20 min 10:45-11:15 Plenary session 2: Pr. N. CHERRADI (Biologie du Cancer et de l'Infection,

Grenoble) "MicroRNAs in adrenocortical cancer: function and clinical potential".

11:15-12.00 Short communications 2: “Cancer, preeclampsia, infertility” (presentation 10’ + discussion 5’)

A. VARGAS (U1209, Grenoble) “New genes involved in multiple morphological abnormalities of

the sperm flagella: study in mouse models”

L. MARTINEZ-CARRIERES (Center for Integrative Genomics, Lausanne) “CDK4 is a

lysosomal regulator via mTORC1”

T. KIEU ( U1036, Grenoble) “Characterization of the role of STOX1 protein in preeclampsia”

12:00-13:30 lunch 13:00-13:30 Bureau Meeting (restricted)

13:30-15h00 Short communications 3: “Diabetes, insulin-resistance and lipid metabolism” (presentation 10’ + discussion 5’)

F. DINGREVILLE (CarMeN lab, U1060, Lyon) “Modulation of endoplasmic reticulum/ mitochondrial interface and mitochondrial dynamic by glucotoxicity in pancreatic beta cells”

F. BERTHOU (Cell Physiology and Metabolism, Geneva) “Circulating factors secreted from PTEN-deficient hepatocytes promote muscle insulin sensitivity and restrain triglycerides anabolism in adipocytes”

J. CASTILLO ARMENGOL (Center for Integrative Genomics, Lausanne) “CDK4,

a new player in brown adipose tissue biology and adipose stem cell fate”

M. SOTY (U1213, LYON) “Intestinal glucose production activates the leptin signal cascade

to promote its metabolic benefits”

A.S. AY (Cell Physiology and Metabolism, Geneva) “miR-22 is a master regulator of hepatic lipid metabolism”

A. GIRALT (Center for Integrative Genomics, Lausanne) “E2F1 promotes hepatic gluconeogenesis during insulin resistance”

15h00-15:30 Plenary session 3: Dr. U. LOIZIDES (Division of Endocrinology, Diabetes and Nutrition,

Geneva) “"Diurnal lipid oscillations in human skeletal muscle persist in cultured myotubes implicating

regulation by the endogenous circadian clock”

15:30-16:20 coffee break and poster session 16:20-16:50 Plenary session 4: Dr. P-D DENECHAUD (Center for Integrative Genomics, Lausanne)

“Cell cycle regulators control metabolism: importance of CDK4-E2F1 pathway in liver physiology and pathology associated”

16h50-17h00 Conclusions and poster award

Plenary session 1

Dr. Hubert VIDAL

U1060 INSERM CarMeN/Univ Lyon 1 /INRA 1397

Faculté de medicine, Centre hospitalier Lyon Sud

165 Chemin du grand Revoyet, 69310 Pierre Bénite

Phone: +33 (0)4 26 23 59 18 hubert.vidal@univ-lyon1.fr

Anti-diabetic action of probiotics: Strain specific regulation of the gut-

pancreas incretin axis by Lactobacillus plantarum. It is now well established that gut

microbiota is a major actor of host metabolism. However, how specific bacterial species contribute to

the regulation of host metabolic homeostasis is still poorly known. The increasing interest in the use of

probiotics for health benefit clearly requires a better understanding of these mechanisms. Based on the

recently discovered influence of certain strains of Lactobacillus on juvenile growth, both in Drosophila

and in mice (Schwarzer et al., Science 2016), we postulated that these bacteria may carry the property

to modulate host metabolism. We demonstrated that daily administration of LpWJL, in a strain-specific

manner, increases insulinemia, improves glucose tolerance, reduces gluconeogenesis, and limits

hepatic triglycerides in mice fed a standard diet. Enhanced insulin production results from a stimulation

of the incretin axis, with increased entero-endocrine cell number and GLP1 secretion, associated with

pancreatic islet enlargement and increased glucose-stimulated insulin secretion. In insulin-resistant

mice fed a high-fat high-sucrose diet, treatment with LpWJL corrects the main metabolic alterations

through the same mechanism. This effect of LpWJL on the gut-pancreas incretin axis reveals a novel

mechanism by which specific gut bacterial strains can modulate host metabolism and be beneficial for

metabolic diseases. These results pave the way for the development of novel probiotic strains of

commensal bacteria based on demonstrated metabolic properties.

Plenary session 2

Pr. Nadia CHERRADI

Laboratoire Biologie du Cancer et de l'Infection

Unité mixte de Recherche INSERM-CEA-UGA UMR-1036

Institut de Biosciences et Biotechnologies de Grenoble

CEA Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 09 France

Tél : 00 33 438 78 35 01 E-mail : nadia.cherradi@cea.fr

MicroRNAs in adrenocortical cancer: function and clinical potential Adrenocortical

carcinoma (ACC) is a rare but highly aggressive malignancy with poor prognosis

and limited therapeutic options. Over the last decade, integration of gene expression profile

data with exome sequencing, SNP array analysis, methylation and microRNA (miRNA) profiling led to

the identification of subgroups of malignant tumors with distinct molecular alterations and clinical

outcomes. However, our understanding of the contribution of deregulated miRNAs to the pathogenesis

of ACC is still in its infancy. We have identified tumor as well as serum microRNA signatures that carry

potential diagnostic and prognostic values for ACC. Using integrated analyses combining transcriptomic

and miRnomic data, we demonstrated that two overexpressed microRNAs, miR-483-5p and miR-139-

5p, targeted two genes belonging to N-Myc Downstream-Regulated Gene family and play a yet

unrecognized role in adrenocortical cancer aggressiveness. Moreover, we showed that circulating miR-

483-5p and miR-139-5p represent promising non-invasive biomarkers of malignancy or recurrence in

ACC patients. In the light of recent insights into the role of extracellular miRNAs in shaping the tumor

microenvironment, we evaluated the impact of extracellular miR-483-5p and miR-139-5p on endothelial

cell (EC) function. Both microRNAs were transferred to EC via exosomes and positively modulated their

angiogenic capacities. Characterization of the molecular mechanisms involved in the angiogenic

response of EC to adrenocortical cancer cell-derived miR-483-5p and miR-139-5p is currently under

investigation. Collectively, our data suggest that miR-483-5p-/miR-139-5p-regulated pathways might be

promising targets for therapeutic intervention in ACC with poor outcome.

Plenary session 3

Dr. Ursula LOIZIDES

Division of Endocrinology, Diabetes, Hypertension and Nutrition

Department of Cell Physiology and Metabolism

University of Geneva, Rue Michel-Servet, 1CH-1211 Geneva 4

Phone. +41 22 379 5339 ursula.loizides-mangold@unige.ch

Diurnal lipid oscillations in human skeletal muscle persist in cultured myotubes

implicating regulation by the endogenous circadian clock Circadian clocks play an

important role in lipid homeostasis with impact on various metabolic diseases. Due to the central role of

skeletal muscle in whole-body metabolism we aimed at studying muscle lipid profiles in a temporal

manner. Whether lipid oscillations in peripheral tissues such as skeletal muscle are driven by diurnal

cycles of rest/activity and food intake or are able to persist in vitro in a cell autonomous manner has not

been shown. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo,

and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids and sphingolipids

exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism.

Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in

vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue

and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated

in their biosynthesis. Notably, lipids enriched in the outer leaflet of the plasma membrane oscillated in a

highly coordinated manner in vivo and in vitro. Taken together, our data suggest a cell-autonomous

control of the endogenous human skeletal muscle clock on lipid metabolism independent of external

synchronizers such as physical activity or food intake.

Plenary session 4

Dr. Pierre-Damien DENECHAUD

Center for integrative Genomics (CIG), FBM

Quartier UNIL- Sorge

Batiment Génopode

1015 Lausanne Suisse

Phone : +41 21 692 41 39 Pierre-Damien.Denechaud@unil.ch

Cell cycle regulators control metabolism: importance of CDK4-E2F1 pathway in

liver physiology and pathology associated. Over the past 30 years, in parallel with the

increasing epidemic of obesity and diabetes, non-alcoholic fatty liver diseases (NAFLD) have

progressively become a serious health concern. NAFLD is a spectrum of diseases that ranges from an

excessive accumulation of lipids in the liver, with benign prognosis, to a more severe form, the non-

alcoholic steatohepatitis (NASH) that may lead to fibrosis and cirrhosis. Identifications of molecular

mechanisms involved in liver physiology and NAFLD will help in the development of new therapeutic

strategies.

In our laboratory we are particularly interested to highlight the role of cell cycle regulators in metabolism.

We found that the transcription factor E2F1 is implicated in liver physiology by participating in liver

glucose production, de novo lipid synthesis and cholesterol metabolism. More importantly, we revealed

that E2F1 is involved in the development of hyperglycemia, liver steatosis and liver fibrosis. As a

consequence E2F1 is an important player in the development of NAFLD, revealing new possible

therapeutics.

Short Communication 1-1

METABOLIC EFFECTS OF A MIXTURE OF LOW-DOSE POLLUTANTS IN A

MOUSE MODEL OF ESTROGEN DEFICIENCY

B. Julien, C. Pinteur, N. Vega, E. Labaronne, H. Vidal, D. Naville, B. Le Magueresse-Battistoni ;

CarMeN laboratory, Inserm U1060, INRA U1397, Université Claude Bernard Lyon 1, Charles Mérieux

Medical School, Oullins, France

Background and aims: Solid evidences have demonstrated that environmental pollutants contribute to the etiology of

obesity and related metabolic disorders. Using a model of lifelong exposure to a mixture of low-dose pollutants, we

recently observed aggravation of glucose intolerance and hepatic insulin resistance of adult females but not of males fed

a high-fat high-sucrose diet (HFSD). Conversely, lifelong exposure to the pollutant mixture induced alleviation of

glucose intolerance and enhanced insulin sensitivity of immature female mice also HFSD-fed (all occurring in the

absence of body weight changes). Thus, we hypothesized that the mixture of pollutants may induce estrogeno-mimetic

activities. Indeed, it is well established that estrogens protect females against metabolic disorders at physiological levels

but that insulin resistance develops following overstimulation of estrogen receptors or in conditions of estrogen

deficiency. To further validate the hypothesis, we evaluated if the mixture of pollutants could alleviate the deleterious

metabolic effects induced by ovariectomy.

Materials and methods: All procedures were performed with the approval of the Regional Committee of Ethics for

Animal Experiments. C57BL/6J female mice were exposed from preconception until adulthood to a HFSD containing

(HFp) or not (HF0) a mixture of persistent organic (2,3,7,8-tetrachlorodibenzo-ρ-dioxin, polychlorinated biphenyl 153)

and short-lived (bisphenol A, diethylhexyl phtalate) pollutants, each used in the range of its tolerable daily intake

reference dose. At 5 weeks of age, mice underwent ovariectomy (OVX) or were sham-operated, generating a total of 4

groups (HF0-sham/HF0-OVX/HFp-sham/HFp-OVX). Weight and food intake were weekly recorded. Several blood

parameters, glucose tolerance, plasma lipids and hepatic expression of estrogen receptor α (Esr1) were measured.

Results: OVX resulted in body weight increase with no modification of food intake. Mice fed HFSD showed glucose

intolerance and high plasma insulin levels which worsened in OVX mice. Pollutant exposure did not impact body

weight, food intake or plasma insulin levels. However, it resulted in a decrease in plasma triglyceride level (0.71±0.019

mM vs 0.83±0.033 mM; p=0.008) and improvement of glucose tolerance in OVX females. In addition, fasting plasma

insulin levels measured at the peak of response after the injection of glucose during the GTT (i.e. 15 minutes) were 1.6-

fold higher in HFp-OVX mice than in HF0-OVX mice. This effect was not observed with sham mice. Thus, and

although the pvalue=0.06, it may explain alleviation of glucose tolerance by pollutants in OVX conditions. Furthermore,

in HFp-OVX mice, the mixture of pollutants led to a 1.4-fold increase in the expression of Esr1 gene (p=0.038) as

compared to non-exposed mice that underwent OVX.

Conclusion: Collectively, these results illustrate the cocktail effect resulting from exposure to a mixture of

environmental pollutants, each used at a dose supposedly without effect. Consistently with our working hypothesis on

the estrogeno-mimetic activity of the mixture of pollutants, we observed beneficial metabolic effects of the pollutant

mixture in conditions of estrogen deficiency. Because of the very low doses of pollutants used in mixture, these findings

may have strong implications in terms of understanding the potential role of environmental contaminants in the

development of metabolic diseases.

Short Communication 1-2

RENOPROTECTIVE EFFECTS OF FENOFIBRATE IN GLYCOGEN STORAGE

DISEASE TYPE 1A IN MICE

Monteillet Laure, Gjorgjieva Monika, Silva Marine, Stefannuti Anne, Mithieux Gilles, Rajas Fabienne

Inserm 1213-Université Lyon 1, 7 rue Guillaume Paradin, Lyon cedex08, France

Introduction: Glycogen Storage Disease type 1a (GSD1a) is a rare metabolic disease due to mutations of the catalytic

subunit of glucose-6 phosphatase (G6PC). GSD1a is characterized by severe hypoglycaemia during short fasting periods

since patients are not able to produce endogenous glucose. Moreover, several long-term complications occur, such as the

development of hepatocellular adenomas and carcinomas as well as chronic kidney disease (CKD), which can progress

to renal failure. The nephropathy is in part due to the renal accumulation of glycogen and lipids that leads to tubular

injuries and renal fibrosis. Here, we tested the effect of fenofibrate, a lipid-lowering drug, on the CKD development in a

mouse model of GSD1a with a specific deletion of G6pc, encoding the catalytic subunit of glucose-6 phosphatase, in the

kidneys (K.G6pc-/-

mice).

Method: After 6 months of G6pc deletion, K.G6pc-/-

mice were fed a standard diet containing 0.2% of fenofibrate for 3

months. C57Bl/6J mice were used as control. CKD was evaluated by renal histological analyses and the determination of

urinary biomarkers. Lipid metabolism and fibrosis development were analyzed by RT-qPCR and western blot. Glycogen

and lipid contents were determined in the renal cortex.

Results: As expected, fenofibrate treatment enhanced lipid oxidation, which was highlighted by activation of PPARα-

target gene expression (Fabp1, Cyp4a10…). Concomitantly, renal glycogen and lipid content was also significantly

decreased by fenofibrate. Interestingly, this resulted in a normalization of CKD urinary markers, such as albumin and

lipocalin2. Accordingly, renal histological observations showed that fenofibrate prevented fibrosis development and

tubular damages in the kidney of K.G6pc-/-

mice. Moreover, molecular pathways involved in renal fibrosis development,

such as the TGFβ1 pathway, were strongly toned down by fenofibrate treatment. Concomitantly, fenobibrate also

prevented the epithelial mesenchymal transition in renal cortex.

Conclusion: This study has proven that fenofibrate can prevent renal injuries, such as glomerulosclerosis and tubular

damages, by decreasing both renal lipid and glycogen contents. Thus fenofibrate treatment appears to be a very

interesting strategy to prevent CKD before the first deleterious signs appear in GSDI patients.

Short Communication 1-3

AÇAÍ POLYPHENOLS: A POTENT STRATEGY TO PREVENT INFLAMMATION

AND ENDOTHELIAL DYSFUNCTION ASSOCIATED WITH CHRONIC KIDNEY

DISEASE AND UREMIA

Elisa BERNARDES MONTEIRO1,2

, Elaine R. SOARES2, Christophe O. SOULAGE

1 and Julio

BELTRAME DALEPRANE2

1CarMeN, UMR INSERM 1060, INSA Lyon, Université de Lyon, 69100, Villeurbanne, France

2 Laboratory for Study of Interactions between Nutrition and Genes, U.E.R.J, Rio de Janeiro, Brazil

The plant Euterpe oleracea Mart., often referred to under the popular name of Açaí, is widely diffused in the Amazon

region of Brazil. Açaí seed is a major contributor of the fruit total weight and is commonly discarded; limited

investigation has been performed on açaí by-products. Recent studies from our group demonstrated that the Açaí seed

extract (ASE) exhibits a potent endothelium-dependent vasodilator effect, as well as antioxidant and antihypertensive

action. ASE also prevented the endothelial dysfunction and vascular structural changes in renovascular hypertensive rats.

In chronic kidney disease (CKD), numerous deleterious compounds accumulate in body fluids, as a result of decreased

renal clearance, and may impair endothelial function. Protein bound uremic toxins, such as p-cresyl sulfate (pCS) or

indoxyl-sulfate (IS), exert major toxic effects because of poor removal by the common dialysis techniques. The main

goal of our study was to evaluate the putative role of uremic toxins in endothelial dysfunction biomarkers using a human

endothelial cell line, featuring the beneficial health effects possibly exerted by ASE. Human umbilical vein endothelial

cells (HUVEC) were incubated with pCS and IS (in concentrations found in uremic patients) alone or in concomitance,

with or without the presence of ASE. The percentage of cell death was evaluated by Trypan blue exclusion assay and

reduction of Alamar blue. The expression of pro-inflammatory cytokines (MCP-1) and adhesion molecules (VCAM-1,

ICAM-1) was evaluated by qT-PCR and endothelial cell migration assay was performed. Preliminary data pointed out

beneficial effects of ASE in ameliorating some of the harmful changes promoted by these toxins. ASE prevented

endothelial cell death induced by indoxyl sulfate and in cells treated with both toxins in concomitance, reduced

expression of pro-inflammatory cytokines (MCP-1) and adhesion molecules (VCAM-1, ICAM-1) and restored

endothelial cell migration capacity for indoxyl sulfate-treated endothelial cells. These findings indicate that ASE could

be beneficial to prevent some of the harmful changes promoted by uremic toxins in endothelial cells.

Short Communication 2-1

NEW GENES INVOLVED IN MULTIPLE MORPHOLOGICAL ABNORMALITIES

OF THE SPERM FLAGELLA: STUDY IN MOUSE MODELS.

Alexandra VARGAS1, Zine-Eddine KERRAF

1, Amir AMIRI-YEKTA, Charles COUTTON

1,2, Béatrice

CONNE4, Aminata TOURE

3, Jessica ESCOFFIER

1,4, Serge NEF

4, Serge P. BOTTARI

1,2, Pierre RAY

1,2

and Christophe ARNOULT1

1: Team Génétique, Epigénétique et Thérapies de l’Infertilité, Institut pour l’avancée des Biosciences,

INSERM U1209, CNRS UMR5309, Grenoble F-38000, France.

2: CHU de Grenoble, Grenoble F-38000, France.

3: INSERM U1016, Institut Cochin, Paris 75014, France

4: University of Geneva, Department of Genetic Medicine and Development, Geneva, Switzerland

Male infertility is a major health issue as it concerns more than 20 million men in the world. Infertility is a multifactorial

disease, but few genetic causes are known despite the fact that there are a large number of genes potentially involved. In

this study, we focused our research on genetic causes of sperm cells presenting with multiple morphological

abnormalities of flagellum (MMAF). In order to find new genes involved in morphological abnormalities of the sperm

flagella, whole exome sequencing was performed on patients presenting this phenotype and coming from

consanguineous families. From this study, two potential genes WDRa and WDRb have been identified. It is known that

WDR proteins are often implicated in protein complexes in sperm cells. In order to validate the involvement of these

genes in MMAF and to investigate the associated molecular defects, corresponding KO mice were generated with

CRISPR/Cas9 technology. Sperm cells from these two mouse strains are totally immobile. WDRa KO sperm cells

display the same defects as in human, i.e. short, thick and/or coiled flagella whereas WDRb KO sperm cells have normal

size and shape, but are completely immotile. The structure of their flagella was assessed by immunofluorescence and

their ultrastructure by electronic microscopy (EM). For WDRa KO sperm, most of the flagella are resumed to

cytoplasmic bags and when a short tail is present, EM indicates a complete disorganization of the axoneme. For WDRb,

mitochondria are strongly disorganized and EM results indicate numerous ultrastructural defects: supernumerary dense

fibers, lack of peripheral doublets of the axoneme, uneven distribution of the fibrous sheath and especially defects in the

longitudinal columns.

Taken together these results emphasize the importance of WDR proteins in the biogenesis and the structure of the sperm

flagella.

Short Communication 2-2

CDK4 IS A LYSOSOMAL REGULATOR VIA mTORC1

Martinez-Carreres L., Orpinell M., Giralt A., López-Mejía IC, Fajas L.

Université de Lausanne, Lausanne, Switzerland.

Cyclin Dependent Kinase 4 (CDK4) is a serine/threonine kinase that belongs to the CDK kinase family, which only

exerts its function when associated to a Cyclin partner. When CDK4 forms a complex with the D-type cyclin in G1

phase of the cell cycle, is able phosphorylate protein targets such as the Retinoblastoma (Rb) protein. Phosphorylated Rb

releases the transcription factor E2F1, which is then able to activate its target genes and hence drive the transition from

the G1 to S phase of the cell cycle.

Given that CDK4 is highly mutated in many forms of human cancer (e.g. malignant melanoma, glioma, sarcoma and

breast, colon, lung, ovary and oral cavity carcinomas) deregulating cell division and promoting cell proliferation,

inhibitors of CDK4-cyclin D complex such as Palbociclib (PD0332991), Ribociclib (LEE011) and Abemaciclib

(LY2835219) have been developed and tested in clinical trials for cancer treatment, also in combination with other

drugs, such as Mammalian Target of Rapamycin (mTOR) inhibitor, Insulin Growth Factor Receptor (IGFR) inhibitor or

Phosphoinositide-3-kinase (PI3K) inhibitor.

Despite the role of CDK4 in the cell cycle progression has been extensively studied, work from our lab has provided

extensive proof that CDK4 plays alternative but crucial roles in Insulin Signaling Pathways as well.

In the present study, using cancer cell lines as a model, we show that CDK4 have a direct impact on lysosomal biology.

CDK4 inhibition or depletion leads not only to an increase in lysosomal size, which correlates with an induction of

autophagy, but also leads to an increase of lysosomal number, suggesting lysosomal biogenesis. Given that autophagy

and lysosomal biogenesis are both regulated by mTORC1, we analyzed its activity. Indeed, when CDK4 is inhibited or

depleted, mTORC1 is not active, as evidenced by a decrease in the phosphorylation of its downstream target S6K. In the

presence of amino acids, mTORC1 translocates to the lysosomal membrane where it is activated by its modulator Rheb.

CDK4 inhibitor or depletion does not allow the binding of mTORC1 in the lysosomal membrane, mimicking starvation

conditions. However, we are still identifying potential CDK4 phosphorylation targets in mTORC1 pathway.

The discovery of this novel regulatory role for CDK4 will bring a better understanding of cancer metabolism, which in

turn will help to open new perspectives for cancer therapies.

Short Communication 2-3

CHARACTERIZATION OF THE ROLE OF STOX1 PROTEIN IN PREECLAMPSIA

Trinh-Le-Vi Kieu1, Déborah Reynaud

1, Roland Abi Nahed

1, Mohamed Benharouga

2, D. Vaiman

3 and

Nadia Alfaidy1.

1- Laboratoire de Biologie du Cancer et de l'Infection, unité mixte INSERM-CEA-UJF U1036, BIG

CEA-Grenoble, Grenoble, France; 2- Laboratoire Chimie et Biologie des Métaux UMR 5249 BIG-

CEA/CNRS/université Grenoble Alpes; 3 INSERM UMR-S1016 CNRS UMR8104 Institut Cochin -

Université Paris Descartes

Preeclampsia (PE) is the most threatening pathology of human pregnancy. PE development is thought to be due to a

failure in the invasion of trophoblast that establish the fetomaternal circulation. We have recently demonstrated that EG-

VEGF (Endocrine gland derived vascular endothelial growth factor), an angiogenic factor secreted from the placenta is

increased in PE and that EG-VEGF overexpression in gravid mice causes PE development. EG-VEGF acts via two

GPCR receptors, PROKR1 and PROKR2. EG-VEGF increased levels in PE suggested that antagonisation of its effects

could be considered as a therapy for PE women.

Recent studies have also demonstrated that PE is associated with an increase in the expression of the transcription factor

STOX1 (Storkhead box 1). A trophoblast cell line and a mouse model overexpressing STOX1 have been developed by

Dr Vaiman. Both the cell line and the mouse model exhibited all hallmarks of PE features. Interestingly, a microarray

analysis of the STOX1 placentas indicated an increase of PROKR1 and PROKR2 expression.

In collaboration with Dr Vaiman we obtained STOX1 cells (AA6 cells) to investigate the levels of expression of EG-

VEGF and its receptors and determine whether the antagonisation of its receptors attenuate hallmarks of PE including

inflammation. Our preliminary results demonstrate that EG-VEGF and its receptors PROKR1 and PROKR2 are

increased both at the mRNA and protein levels. Comparison of cells proliferation using Incucyte® device showed that

STOX1 cells exhibited higher proliferation and lower migration rates compared to control cells and that treatment of the

cells with PROKR2 antagonist reversed these effects in STOX1 cells.

More importantly, our results demonstrate stronger expression of the machinery that activate the NLRP7 (NLR Family

Pyrin Domain Containing 7) inflammasome. These include NLRP7, caspase 1 and ASC (apoptosis-associated speck-like

protein containing CARD). Also STOX1 cells overexpressed interleukin 1Lb protein, a cytokine that reflect the

inflammasomes activity.

Altogether these data demonstrate for the first time that the cell line that represent the best a PE model, exhibited higher

levels of PROKR1 and PROKR2 receptors, confirming the microarray finding in the placentas of the STOX1

overexpressing mice. This suggest that EG-VEGF system might be a target for the treatment of PE.

More importantly, we demonstrate for the first time that the NLRP7inflammasome complex is deregulated in PE. Further

studies are ongoing to confirm and characterize the underlying mechanism using STOX1 animal model of PE.

Short Communication 3-1

MODULATION OF ENDOPLASMIC RETICULUM/MITOCHONDRIAL

INTERFACE AND MITOCHONDRIAL DYNAMIC BY GLUCOTOXICITY IN

PANCREATIC BETA CELLS

F. Dingreville, Y. Gouriou, S. Ducreux, M. Alam, M.-A. Chauvin, F.Van Coppenolle, C. Thivolet, J.

Rieusset, A.-M. Madec. INSERM U1060 CarMen Oullins, France.

Background and aims: Mitochondria network plays a pivotal role in insulin release in pancreatic beta cells by its ability

to product ATP, and is controlled by calcium, a key regulator of fission/fusion event and oxidative energy production.

Alterations of mitochondria and endoplasmic reticulum (ER) have been shown to contribute to metabolic disorders such

as type 2 diabetes (TD2). To determine how glucotoxicity alters insulin secretion we focus on mitochondria fusion and

fission dynamics and on ER/mitochondria interactions in studying MAMs (Mitochondria Associated endoplasmic

reticulum Membrane) in beta cells. MAMs are conserved structures with crucial role for cell survival and death through

the transfer of calcium, lipid and metabolite exchange. Increasing evidence in liver tissue indicates that dysfunctional

MAMs can be an important precursor of disease in TD2.

Materials and methods: Human islets and INS-1 rat beta cells were exposed during 72h to moderate glucotoxicity (16.5

mM and 22.5mM Glucose). ER stress was measured by qPCR, mitochondrial respiration by oxygraphy and insulin

secretion in response to glucose (GSIS) by ELISA. Mitochondrial and reticulum calcium contents were respectively

analyzed using 4mtD3CPV and ERGAP1 probes, and mitochondrial dynamic using mitotracker Green. MAMs were

quantified using the In situ Proximity Ligation Assay (PLA) between IP3R2 and VDAC1, respectively ER and

mitochondria calcium channels implicated in MAM tethering. All statistical tests have been done with unpaired Mann-

Whitney test and paired Student t-test.

Results: Chronic exposure of human islets and INS-1 cells to moderate glucotoxicity induced an increase in ATF4 and

spliced-XBP1 mRNA expression (P<0.01) without modification of CHOP mRNA expression, an ER stress marker of

apoptosis way, reflecting a mild ER stress as in TD2, and a decrease in ER calcium content (P<0.0001). A decrease in

mitochondrial respiration (p<0.0001) and insulin secretion (P<0.001) was parallely observed. Mitochondria

fusion/fission dynamics were disrupted in favor of a fission state (P<0.0001), in synergy with a decrease in

mitochondrial calcium content (P<0.0001) in INS-1 cells. In the same conditions, we showed an up-regulation of the

contacts between ER and mitochondria highlighted by the increase of IP3R2-VDAC1 interactions (P<0.05) in INS-1

cells.

Conclusion: We confirmed that glucotoxicity induces a metabolic stress in both ER and mitochondria in human islets

and rat beta cells. The decrease in mitochondrial calcium content associated with mitochondrial fission were consistent

with an alteration of beta cell function as respiration, calcium homeostasis and insulin

secretion. Interestingly we observed for the first time an increase in ER/mitochondria tethering in beta cells in response

to glucotoxic conditions. The modulation of these interactions could help us to clarify the role of MAMs in pancreatic

beta cells and their dysfunction in TD2 and may open a new potential way of treatments in TD2.

Short Communication 3-2

CIRCULATING FACTORS SECRETED FROM PTEN-DEFICIENT HEPATOCYTES

PROMOTE MUSCLE INSULIN SENSITIVITY AND RESTRAIN TRIGLYCERIDES

ANABOLISM IN ADIPOCYTES

Flavien Berthou, Cyril Sobolewski, Nicolas Calo, Christine Maeder, Margot Fournier, Michelangelo Foti Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva

Background and Aims : Hepatic downregulation of the lipid/protein phosphatase PTEN, as observed in patients with

fatty liver disease, promotes the development of steatosis in mice. However, steatosis induced by PTEN deficiency in

mice is paradoxically associated with improved muscle insulin sensitivity and decreased adiposity. The aim of this study

is to identify the PTEN-dependent molecular mechanisms promoting liver-to-muscle/adipocytes crosstalk and improving

metabolic homeostasis in these organs.

Methods : In vitro murine C2C12 muscle cells and 3T3-L1 preadipocytes were treated with conditioned media (CM) of

primary hepatocytes isolated from wild-type and liver-specific PTEN knockout (LPTENKO) mice. Protein and gene

expressions were analysed by Western-blot and RT-qPCR. Lipid droplets were stained by BodiPy and gene silencing

was achieved by siRNAs.

Results : Insulin-induced Akt activation and glucose uptake in C2C12 myotubes were increased upon exposure of these

cells to CM of LPTENKO primary hepatocytes as compared to controls. Differentiation and fatty acids biosynthesis in

3T3-L1 adipocytes were decreased following treatment with CM from LPTENKO primary hepatocytes. Further analyses

indicated that hepatocytes-derived circulating factors mediating these effects were proteins. Screening of hepatokines

expression in wt and PTEN-deficient hepatocytes, as well as in the liver of obese and diabetic mouse models (e.g. diet-

induced obesity, ob/ob, db/db mice), pointed to several candidate hepatokines potentially involved in this liver-to-

peripheral organs crosstalks, including FGF-21. Deregulation of the expression of these hepatokines was either

dependent of PTEN signaling or associated with metabolic stress of the liver.

Conclusions : These data highlighted hepatokines which potentially improve peripheral metabolic homeostasis although

the presence of hepatic steatosis. Identification of these liver-secreted factors could provide new potential therapeutic

targets for the treatment of obesity and/or type 2 diabetes

Short Communication 3-3

CDK4, A NEW PLAYER IN BROWN ADIPOSE TISSUE BIOLOGY AND ADIPOSE

STEM CELL FATE

Judit Castillo Armengol1, Isabel Lopez-Mejia

1, Honglei Ji

1, Sylviane Lagarrigue

2, Lluis Fajas

1

1. Center for Integrative Genomics, University of Lausanne CH-1015 Lausanne

2. Department of Physiology, University of Lausanne CH-1005 Lausanne

White adipose tissue is known for its role in fat storage and whole body lipid/energy homeostasis. On the other hand,

brown adipose tissue generates heat through the activity of uncoupling protein 1 (UCP1). More recently, attention has

been placed into a third category of specialized heat-producing adipocytes, that can not only store lipids, but also prevent

the onset of the metabolic phenoytpe. These adipocytes have been named brite (brown-in-white) adipocytes. The fact

that the activities of brown and brite fat cells can limit metabolic diseases in mice, and correlate with leanness in humans

underlines the importance of research in this recent field. Numerous genes and pathways that regulate brown and beige

adipocyte biology have now been identified, however the role of cell cycle regulators in the development and the

function of these oxidative adipose depots has not been thoroughly studied yet. We now aim to determine the role of the

CDK4 in the function and differentiation of brown and brite adipocytes. Preliminary data suggests that CDK4 activity is

inversely correlated with oxidative function in brown adipose tissue (BAT) and with browning in subcutaneous adipose

tissue (scWAT). lndeed both morphological and gene expression data show that mice lacking CDK4 exhibit decreased

lipid content and increased brown and oxidative gene expression in BAT. Under chow diet, these animals also display

browning and increased oxidative gene expression in scWAT. Our preliminary data strengthen the need to study the

molecular mechanisms by which CDK4 controls energy metabolism in brown and brite adipose cells. A better

understanding of those processes might open up new therapeutic perspectives in the control of metabolic diseases such as

diabetes or obesity.

Short Communication 3-4

INTESTINAL GLUCOSE PRODUCTION ACTIVATES THE LEPTIN SIGNAL

CASCADE TO PROMOTE ITS METABOLIC BENEFITS

Maud Soty, Margaux Raffin, Fanny Vulin-Bouilloux, Gilles Mithieux

INSERM U1213, Faculté de Médecine Laënnec, Bât B, 1er étage, Rue Guillaume Paradin, 69372 Lyon

Intestinal glucose production (IGP) is a new function, which positively controls glucose and energy homeostasis.

The appearance in the portal vein of glucose produced by the intestine initiates a nervous signal (“portal glucose signal”)

resulting in the activation of important hypothalamic areas. However, the central mechanisms involved in the beneficial

effects of IGP have not yet been deciphered. Leptin exerts its metabolic effects by activating the same central targets

than portal glucose and the absence of IGP induces a central defect in leptin sensitivity.

In view of these data, we propose that central leptin signaling might be a target by which IGN might

influence energy and glucose homeostasis at the hypothalamic level.

In control mice, we studied the effect of portal glucose infusion directly in the portal vein (to mimic IGN) on

food intake and plasma glucose and insulin concentrations. To evaluate the involvement of the leptin signaling pathway

(JAK2/STAT3) in the beneficial effects of IGP: 1/ we studied the effect of portal glucose on the phosphorylation level of

STAT3 in the hypothalamus, and 2/ we studied the effect of a specific inhibitor of JAK2 (AG490). To further question

the potential role of leptin, we studied the effect of portal glucose in leptin deficient (ob/ob) mice.

In control mice, the infusion of glucose in the portal vein induced a marked decrease of about 30% in food

intake. Interestingly, portal glucose also improved glucose homeostasis by decreasing blood glucose and increasing

insulin secretion. Moreover, these effects were associated with an increased the phosphorylation of STAT3 in the

hypothalamus. This was independent of any change in the plasma leptin concentration. Importantly, mice treated with

AG490 did not show a significant decrease in food intake in response to portal glucose infusion compared with mice

treated with vehicle. It is noteworthy that glucose infusion in the portal vein counteracted the hyperphagic behavior of

ob/ob mice, reducing significantly their food intake by about 20%. Moreover, despite the absence of leptin, portal

glucose induced the phosphorylation of STAT3 in the hypothalamus of ob/ob mice. Interestingly, as in WT mice, portal

glucose improved glucose control since ob/ob mice infused with glucose in the portal vein showed decreased blood

glucose.

These results strongly suggest that IGP via the induction of “portal glucose signal” modulates food intake and

glucose metabolism using the leptin signaling pathway, independently of the presence of the hormone. The underlying

mechanism remains to be deciphered and deserves further investigation.

Short Communication 3-5

miR-22 IS A MASTER REGULATOR OF HEPATIC LIPID METABOLISM

Anne-Sophie Ay, Dorothea Portius, Daniel Abegg, Margot Fournier, Pia Rantakari, Fu-Ping Zhang,

Matti Poutanen, Didier Picard, Serge Nef, Alexander Adibekian and Michelangelo Foti

Background: With obesity and diabetes, miR-22 is dysregulated in the liver suggesting an important role for this

specific microRNA in hepatic metabolic disorders. The aim of this study was to investigate in vivo the role of miR-22

deficiency in diet-induced obesity, insulin resistance and non-alcoholic fatty liver diseases.

Methods: miR-22 knockout (miR22KO) mice were generated and fed an obesogen diet for 4 weeks or 3 months. Mice

were metabolically phenotyped and histological and molecular analyses were performed on explanted tissues and

primary hepatocytes. High throughput proteomic analysis was performed with liver tissues of CTL and miR22KO mice.

Results: MiR-22 constitutive deletion in mice breed in normal conditions was completely asymptomatic with no

phenotypical changes in hepatic and white adipose tissues. However, when mice were fed a high fat-containing diet for a

short or long period (4 weeks/3 months), miR-22 deficiency dramatically exacerbated fat mass gain, glucose intolerance,

hepatomegaly and liver steatosis as compared to CTL mice. Proteomic analysis of hepatic tissues from CTL and

miR22KO mice indicated that miR-22 exerts in vivo a major translational repression activity on key regulators of lipid

metabolism thereby fostering hepatic steatosis in miR22KO mice. Further in vitro analyses demonstrated that FASN,

ACC, CD36 and SCD1 are direct targets of miR-22 in hepatic cell line.

Conclusions: miR-22 deficiency fosters the development of systemic and hepatic metabolic disorders associated with

diet-induced obesity.

Short Communication 3-6

E2F1 PROMOTES HEPATIC GLUCONEOGENESIS DURING INSULIN

RESISTANCE

Albert Giralt, Pierre-Damien Denechaud, Isabel C. Lopez-Mejia, Caroline Bonner, Francois Pattou,

Jean-Sébastien Annicotte and Lluis Fajas

Center for Integrative Genomics (CIG), Université de Lausanne, Switzerland

The liver plays a unique role in the maintenance of global lipid and glucose homeostasis. The elucidation of the

molecular mechanisms that regulate liver metabolism in normal and pathological conditions could lead to the

identification of new therapeutic targets to treat metabolic disorders. We have recently demonstrated that the cdk4-Rb-

E2F1 pathway participates in the regulation of glycolysis and lipogenesis in the liver. Here, we report that E2F1 also

participates in the regulation of hepatic gluconeogenesis, a pathway that is abnormally upregulated during insulin

resistance and that critically contributes to hyperglycaemia in this state. E2f1 -/- mice show decreased gluconeogenesis

in vivo and reduced expression of liver gluconeogenic enzymes. Accordingly, E2F1 -/- primary hepatocytes show

reduced glucose production and gluconeogenic gene expression. Cdk4 typically increases E2F1 transcriptional activity

by releasing its repression by pRb. Cdk4 -/- primary hepatocytes show decreased gluconeogenesis, while on the contrary,

a hyperactive form of Cdk4 (R24C) led to an E2F1-dependent increased gluconeogenesis. Adenoviral E2f1

overexpression increased gluconeogenic gene expression and glucose production in primary hepatocytes. ChIP analysis

shows that E2F1 binds to the promoter of Pepck1, while luciferase reporter assays demonstrate that Pepck1 promoter

activity is increased in response to E2F1. Liver biopsies from obese subjects showed a correlation between Pepck1 and

E2f1 mRNA expression, suggesting that E2F1 could contribute to the increased gluconeogenesis observed during insulin

resistance. In agreement with this, decreasing E2F1 activity in two mice models of insulin resistance ameliorates

hyperglycaemia though a decrease in hepatic gluconeogenesis. Taken together, our results show that E2F1 participates in

the regulation of both glucose and lipid metabolism in the liver, suggesting that E2F1 inhibition could be used as a

therapeutic approach for the treatment of type 2 diabetes.

POSTER 1.

ROLE OF CYCLIN DEPENDENT KINASE 7 IN ADIPOSE TISSUE METABOLISM

Honglei Ji, Pierre-Damien Denechaud, Isabel C. Lopez-Mejia, Judit Castillo Armengol, Lluis Fajas Coll

Center for Integrative Genomics, University of Lausanne

Cyclin-dependent kinase 7 (CDK7) is a member of the cyclin-dependent protein kinase (CDK) family, which are

important regulators of cell cycle progression and more recently, metabolism. It forms a trimeric complex with cyclin H

and MAT1 (Ménage-à-trois 1), which functions as a Cdk-activating kinase (CAK). It is also an essential component of

the transcription factor TFIIH, which is involved in transcription initiation and DNA repair. This protein is thought to

serve as a direct link between the regulation of transcription and the cell cycle.

In our project, we generated adipose tissue specific CDK7 knockout (CDK7-atKO) mice by crossing CDK7 floxed mice

with ap2-cre mice. After the validation of CDK7 specific invalidation in adipose tissue, we aimed to characterize the

metabolic phenotype of these mice. We found that CDK7-atKO mice have decreased body weight and fat mass

compared with control mice. In vitro culture of SVF cells from subcutaneous WAT has impaired differentiation capacity,

which is consistent with our in vivo data. These mice did not exhibit insulin or glucose intolerance. We did indirect

calorimetry to measure energy expenditure however without difference between groups. We also found brown adipose

tissue of knockout mice are smaller and more “white”, which indicated an impaired BAT function. Therefore we

challenged these mice with cold exposure, there is no difference in response to cold under normal condition (with food

supply). However, interestingly, if food is deprived during cold exposure, the knockout mice cannot maintain their body

temperature. In parallel, we detected decreased p-HSL in white adipose tissue of knockout mice, which means decreased

lipolysis in WAT. So here we hypothesize that the brown adipose tissue from knockout mice cannot utilize fatty acid or

do not have enough FFA supply from WAT for thermogenesis.

We are still trying to validate our hypothesize and finally we aim to identify new targets of CDK7 and the signaling

pathways that drive its activity in adipose tissue, which will help deepen our understanding in combating obesity and

diabetes.

POSTER 2.

THE ROLE OF NOVEL KINASES IN ADIPOSE TISSUE BIOLOGY

Anita Nasrallah, Group of Prof. Lluis Fajas Coll,

Center for Integrative Genomics, University of Lausanne

Recently, the occurrence rate of obesity, which is the excessive accumulation of body fat, has dramatically increased.

Adipose tissue dysfunction is a primary defect, linking obesity to numerous health problems, including insulin resistance

type 2 diabetes, hypertension, dyslipidemia, atherosclerosis, cancer, etc. However, not all obese individuals develop

cardiovascular or obesity-related metabolic disorders. This might be due to maintained normal adipose tissue function

and

It is well established that pro-inflamatory cytokines, such as IL-6 and TNF-α, affect insulin signalling, which in turn is

essential to maintain glucose homeostasis and to regulate its metabolism in the liver, muscle, and adipose tissues. This

leads to the stimulation of downstream protein kinases, thus activating and crosslinking numerous pathways, potentially

resulting in insulin resistance. Consequently, insulin resistance status is determined by the type of activated

inflammatory pathways, abnormalities of lipid metabolism, as well as in the type of activated kinases and their

downstream targets. The second part of our project revolves around the role of novel kinases in SAT and VAT of

patients that are insulin resistant (IR) or insulin sensitive (IS).

Several of the known protein kinases involved in the onset of insulin resistant are AMP-activated protein kinase

(AMPK), IκB kinase (IKK), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), etc. Identifying new

and specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug

therapies to minimize insulin resistance in patients.

POSTER 3.

NLRP7 EXPRESSION IN NORMAL AND FGR PREGNANCIES: PHYSIOLOGICAL

AND PHYSIO-PATHOLOGICAL IMPLICATIONS.

Roland Abi Nahed1, Déborah Reynaud

1, Sophie Ndagijimana

1, Wael Traboulsi

1, Mohamed Benharouga

2,

and Nadia Alfaidy1

1- Laboratoire de Biologie du Cancer et de l'Infection, unité mixte INSERM-CEA-UJF U1036, BIG

CEA-Grenoble, Grenoble, France

2- Laboratoire Chimie et Biologie des Métaux UMR 5249 BIG-CEA/CNRS/université Grenoble Alpes

Fetal growth restriction (FGR) is a leading cause of neonatal mortality. FGR pregnancies are commonly associated with

placental insufficiency. Recent studies have shown that excessive placental inflammation is associated with several

pathological conditions, including stillbirth and FGR. Inflammation can be triggered by endogenous mediators, such

damage associated molecular patterns or DAMPs, known to activate the inflammasomes pathway.

NLRP7 is a member of a family of proteins involved in inflammatory processes. Nevertheless, neither the role nor the

biological functions of NLRP7 are known in normal and FGR pregnancies. Using both normal trophoblast cells (HTR

cells), placental explant model and a large cohort of sera and placentas collected from FGR patients, we determined

NLRP7 expression and regulation during early pregnancy and compared its mRNA and protein levels in FGR and in age

matched controls (AMC).

We demonstrated that NLRP7 was more expressed during the hypoxic period of placental development and that hypoxia

increased NLRP7 expression in HTR cells. NLRP7 invalidation increased trophoblast invasion both in HTR cells and in

the 3D culture model, i.e. Placental explants. Analyses of the normal and FGR placentas showed differential localization

of NLRP7 within the placentas and increased expression of NLRP7 at the mRNA and protein levels. Comparison of

circulating IL1b, an indicator of NLRP7 activity, in FGR and AMC showed higher levels in FGR patients.

Altogether our results demonstrate that NLRP7 is highly expressed and positively regulated by oxygen tension in the

human placenta. More importantly, NLRP7 controls key parameters of trophoblast invasion, suggesting its potential

involvement in the development of FGR pregnancies.

POSTER 4.

CHARACTERIZATION OF NLRP7 PROTEIN IN NORMAL AND TUMOR

PREGNANCIES: CASE OF CHORIOCARCINOMA

Déborah Reynaud², Roland Abi Nahed², Wael Traboulsi², Mohamed Benlahfid4, Mohamed

Benharouga3and Nadia Alfaidy²

1- Service de Gynécologie-Obstétrique, CHU de Grenoble, France. 2- Laboratoire de Biologie du Cancer

et de l'Infection, unité mixte INSERM-CEA-UJF U1036, BIG CEA-Grenoble, Grenoble, France ; 3-

Laboratoire Chimie et Biologie des Métaux UMR 5249 BIG-cea/CNRS/université Grenoble Alpes ; 4-

CHU Ibn Rochd de casablanca, Faculté de médecine et de pharmacie de Casablanca.

Choriocarcinoma is a veritable placental cancer that develop upon abnormal pregnancies, such Hydatidiform moles

(HMs). Choriocarcinoma can metastasis into multiple maternal organs, such as the lung, the brain and the liver. Recent

studies established an association between recurrent HMs and mutations in a protein called NLRP7. NLRP7 is a member

of a new family of inflammasome proteins. Nevertheless, its role and biological functions remain to be elucidated in

tumor placentation. Using normal trophoblast cells (HTR cells), the choriocarcinoma cell line (JEG3 cells) and a

distinctive cohort of placental tissue and sera collected from HM patients and age matched control pregnant

women(AMC), we investigated the role of NLRP7 in human placental tumorigenesis.

Our results demonstrate that NLRP7 is more expressed in tumor cell lines compared to normal trophoblast cells. This

was observed both at the mRNA and protein levels. We also demonstrated that NLRP7 expression is increased by

hypoxia in and downregulated by b-HCG in JEG3 cell line. Hypoxia and hCG are two parameters that have been

reported to be increased in choriocarcinoma. Using SiRNA strategy, we demonstrated that NLRP7 promotes JEG3 cells

proliferation as well as their invasion.

Comparison of NLRP7 mRNA and protein levels showed a significant increase in this protein, both in placentas

collected from HM and CC compared to normal placentas. Also, analyses of circulating pro- and anti-inflammatory

cytokines showed strong dysregulations in HM patients compared to AMC.

Altogether our results demonstrate that NLRP7 is highly increased in HM and CC patients and that this protein controls

key aspects of placental development. These results suggest that dysregulations in NLRP7 protein might be associated to

choriocarcinoma development. Its direct control of tumor trophoblast cells invasion strongly suggest its potential

involvement in choriocarcinoma progression. Further studies are ongoing to elucidate the role of this new protein in

placental tumorigenesis.

POSTER 5.

SPINK2 DEFICIENCY PREVENTS ACROSOME BIOGENESIS AND CAUSES

AZOOSPERMIA IN MEN AND MICE

Zine-Eddine Kherraf1,2,4

, Marie Christou-Kent1,2

, Amir Amiri-Yekta1,2,3

, Guillaume Martinez1,2

, Emeline

Lambert2, Charles Coutton

,2,4, Pierre F. Ray

1,2,4, Christophe Arnoult

1,2 and Serge P. Bottari

1,2,4

1 Equipe "Génétique, Epigénétique et Thérapies de l'Infertilité", Institut pour l'Avancée des Biosciences

(IAB), INSERM 1209, CNRS UMR 5309, Grenoble, France

2Université Grenoble-Alpes, UFR de Médecine, Grenoble, France

3 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for

Reproductive Biomedicine, ACECR, Tehran, Iran

4 CHU de Grenoble, Grenoble, France

Azoospermia, characterized by the absence of spermatozoa in the ejaculate is a com-mon and usually untreatable cause

of male infertility with a poorly characterized etiology. Genetic analysis of men with azoospermia and oligozoospermia

allowed us to identify mutations in the serine peptidase inhibitor, Kazal type 2 (SPINK2) gene coding for a serine

protease inhibitor, confirming the importance of the family of SPINK proteins in human pathologies. In agreement with

these findings we observed that homozygous Spink2 KO mice replicated the azoospermia phenotype witnessed in

mutated men.

We further demonstrate that in the absence of Spink2, protease-induced stress pre-vents acrosome biogenesis and

initiates Golgi fragmentation associated with a micro-autophagy-like process, contributing to the arrest of spermatid

differentiation and cell detachment from the seminiferous epithelium. These results demonstrate that failure of post

meiotic cellular events is a cause of human azoospermia. We further show that sperm from heterozygous men and

heterozygous Spink2 KO mice exhibit morphologi-cal defects and low mobility, defects which however remained

compatible with sponta-neous fertility. These results show that SPINK2 deficiency induces a pathological continuum

ranging from oligoteratozoospermia with incomplete penetrance in heterozygotes to azoospermia in homozygotes.

POSTER 6.

CHRONIC AICAR TREATMENT IMPROVES STIMULATED GLUCOSE

TRANSPORT IN CARDIOMYOCYTES EXPOSED TO FREE FATTY ACIDS

Christelle Viglino, Carolina Shore, Christophe Montessuit

Department of Pathology and Immunology, University of Geneva Medical School

Background: Myocardial infarction is not only more frequent, but also more severe in patients with type 2 diabetes. The

stimulation of glucose transport by metabolic stress is an important determinant of myocardial susceptibility to ischemia

and reperfusion injury. Stimulation of glucose transport is markedly impaired in cardiomyocytes chronically exposed to

excess free fatty acids (FFA), as occurs in vivo in type 2 diabetes.

Aim: To determine whether chronic activation of AMP-activated kinase (AMPK) with the AMP analog precursor

AICAR improves glucose transport in cardiomyocytes exposed to FFA.

Methods: Isolated cultured cardiomyocytes were chronically exposed to FFA ± AICAR. Glucose transport was

measured during acute metabolic stress provoked by inhibition of the mitochondrial ATP synthase with oligomycin.

Glycolysis was inferred from acidification of the extracellular medium. Cell signaling was assessed by Western blots.

Intracellular neutral lipids were fluorescently labeled with Bodipy 493/503 and examined by confocal microscopy.

Results: Chronic treatment with AICAR improved basal and oligomycin-stimulated glucose transport in FFA-exposed,

but not in control cardiomyocytes. Similarly, basal and oligomycin-stimulated glycolysis was reduced in FFA-exposed

cardiomyocytes, but markedly increased by chronic AICAR treatment. Curiously, whereas acute treatment of

cardiomyocytes with AICAR increased phosphorylation of the AMPKα subunit on residue Thr172, a classical marker of

AMPK activation, chronic AICAR treatment completely obliterated Thr172 phosphorylation. Nevertheless

phosphorylation of the AMPK substrate Acetyl-CoA Carboxylase (ACC) was slightly increased by chronic AICAR

treatment, suggesting dissociation between AMPK phosphorylation and activity. Chronic AICAR treatment also

markedly reduced the global pool of active and recruitable PKC. Finally chronic AICAR treatment induced in FFA-

exposed cardiomyocytes the biogenesis of numerous lipid droplets.

Conclusions: chronic AICAR treatment induces a metabolic shift in FFA-exposed cardiac myocytes, characterized by

improved glucose transport and glycolysis and redirection of fatty acids towards neutral storage. The mechanism may

involve reduction of FFA-stimulated PKC activity. Such metabolic changes in vivo could protect the hearts of patients

with type 2 diabetes against ischemia-reperfusion injury.

POSTER 7.

HEPATIC METABOLISM IN GLYCOGEN STORAGE DISEASE TYPE 1A

M. Gjorgjieva, M. Silva, G. Mithieux, F.Rajas

U1213 « Nutrition, Diabete et Cerveau » Université Lyon 1 Laennec, Lyon

Introduction: Glycogen storage disease type 1a (GSD1a) is a metabolic disease due to mutations in the catalytic subunit

(G6pc) of glucose-6 phosphatase. Consequently, patients are unable to produce glucose and thus suffer from

hypoglycemic episodes. In the liver, excessive accumulation of glycogen and fat leads to chronic NAFLD-like

complications. Thus a large proportion of adult patients develop hepatocellular adenomas (HCA), with a 10% incidence

of transformation to hepatocellular carcinomas (HCC). Since perturbed metabolism in GSD1a leads to tumorigenesis, it

is crucial to decipher the metabolism of GSD1a hepatocytes and to identify what triggers their tumoral transformation. In

this study, we characterized metabolic dysregulations during tumorigenesis in the liver of mice with G6pc deletion

(L.G6pc-/-

mice). These mice develop all hallmarks of GSD1 hepatic pathology, including the development of HCA and

HCC over time.

Methods: Liver samples were obtained at pre-tumoral (4 months) and tumoral (9 months) stages from L.G6pc-/-

mice,

fed a high fat / high sucrose (HF/HS) diet, which accelerates the development of HCA and HCC. Tumors were

characterized by histology. Gene expression was analyzed by RT-qPCR and western-blot.

Results: Several metabolic pathways are responsible for the NAFLD-like hepatic complications in GSD1a, rendering

GSD1a livers probably more favorable to tumorigenesis. The glycolysis pathway is hyperactivated,

leading to

overproduction of lactic acid in L.G6pc-/- mice. Interestingly, the expression of glucokinase was decreased at the

pretumoral and tumoral stage, due to large G6P availability. The expression of pyruvate kinase isoform-M2, which is an

isoform usually expressed in cancer, was increased only in tumors. Since hepatic steatosis is a major problem in GSD1a,

lipid metabolism was investigated. Lipid synthesis was increased (increase of Fas, Acaca, Elovl6 and Scd1) and lipid

oxidation was drastically decreased (inhibition of the expression of Ppara, Cpt1, Cyp4a10 and Cyp4a14) in both non-

tumoral and tumoral L.G6pc-/- tissue. Interestingly, at the pretumoral stage, lipid synthesis was mediated by increased

Chrebp expression, whereas at the tumoral stage, a decreased expression of Chrebp was observed, revealing a potential

metabolic switch during tumorigenesis. Although nucleotide synthesis, facilitating tumor proliferation, was never

observed in the case of GSD1, increased uric acid concentrations in circulation indicates activation of the pentose

phosphate pathway and thus a possible increase in nucleotide anabolism. Finally, the expression of HNF1α, PTEN and

p53, known tumor suppressors, were decreased not only in the tumors, but also in the non-tumoral L.G6pc-/- liver.

In conclusion, the metabolic perturbations linked to the deficiency in glucose-6 phosphatase activity involve a Warburg-

like metabolic switch, liver steatosis and a decrease in tumor suppressor defenses. The consequent hyperactivation of

specific metabolic pathways renders GSD1 hepatocytes susceptible to tumor development, presumably by providing the

building blocks and energy required for cell proliferation.

POSTER 8.

INDOXYL SULFATE, ARYL HYDROCARBON RECEPTOR (AHR) ACTIVATION

AND METABOLIC RISK IN CHRONIC KIDNEY DISEASE (CKD)

Dan YI, Milena BARCZA STOCKLER PINTO, Fitsum GUEBRE-EGZIABHER and Christophe O.

SOULAGE

CarMeN, UMR INSERM 1060, INSA Lyon, Université de Lyon, 69100, Villeurbanne, France

Background:Chronic kidney disease (CKD) is associated with an increased risk of cardio-vascular mortality and

morbidity and is also frequently associated with peripheral insulin resistance which is a also major cardiovascular risk

factor. The uremic syndrome is attributed to the progressive retention of numerous compounds, which in healthy

individuals are normally excreted or metabolized by the kidneys. Indoxyl sulfate (IS) is one of these compounds, poorly

removed by hemodialysis (HD) and that accumulates in CKD patients. IS is derived from tryptophane metabolism by

intestinal microbes, but little is known about its adverse effects on metabolism. Recent studies identified IS as an

endogenous agonist for aryl hydrocarbon receptor (AhR). The aim of the present project is therefore to investigate

whether IS, through activation of AhR could contribute to the development of insulin resistance and other metabolic

disorders associated with CKD.

Methods:

In vitro study: C2C12 myotubes were incubated with 250µM of IS (a concentration chosen to mimic that encountered in

CKD patients) for 30 min and stimulated by 100 nM insulin for 20 min. In order to detect IS intra- or extra- cellular

effects, C2C12 myotubes were preincubated with probenecid (1mM, 1h), an potent inhibitor of organic anion transporer-

3(OAT-3), prior to incubation with IS (250 µM, 30 min) and stimulation with insulin (100 nM, 20 min). Alpha-

naphtoflavone (ANF), a potent inhibitor of AhR was used to prevent AhR activation in muscle cells. C2C12 myotubes

were incubated with alphanaphtoflavone (1 mM, 1 h),prior to incubation with IS (250 µM, 30 min) and stimulation with

insulin (100 nM, 20 min). The insulin signaling pathway was explored by Western Blotting using the phosphorylation of

Protein kinase B (PKB/Akt).

In vivo study: To induce chronic kidney disease, mice were fed for 4 weeks with a diet containing adenine (0.25% w/w).

HPLC-Fluorescence assay was used to determine the plasma concentration of IS. The disposition of IS was explored

after an intraperoitoneal injection of IS (100mg/kg).

Results: Exposition of C2C12 myotubes to IS at CKD-relevant concentration triggered insulin resistance, through also a

direct activation of AhR. C2C12 myotubes pretreated with AhR inhibitor ANF or probenecid prevented the development

of insulin resistance. We developed and validated a non surgical model of CKD in rodents and demonstrated that IS

accumulates in CKD mice as observed in human. We further demonstrated that plasma IS concentration can be

experimentally raised in mice.

Conclusion: IS, through activation of AhR, disrupts insulin signaling pathways and lead to the development of insulin

resistance associated with CKD IS appears to be a very important uremic retention solutes that could contribute to insulin

resistance in patients with CKD.

POSTER 9.

CONTRIBUTION OF OBESE ADIPOSE TISSUE-DERIVED STEM CELLS TO

HEPATO-OR BREAST-CARCINOMA INFLAMMATION, THROUGH PROMOTION

OF TH17 CELLS AND ACTIVATION OF IL-1B BY MONOCYTES

M. Chehimi1, L. Delort

3, M. Robert

2,, H. Vidal, Caldefie-Chezet

3F

1, A. Eljaafari

1,2.

1 CARMEN Laboratory, INSERM U1060 and Claude Bernard University, Pierre Bénite, France

2 Hospices Civils de Lyon, France.

3. Ecrein Unit, UMR 1019 INRA-UdA Clermont-Ferrand, France

Introduction: As opposed with lean adipose tissues (AT), obese AT are heavily infiltrated with variety of inflammatory

cells such as macrophages, or Th17 cells. Obesity-mediated chronic low-grade inflammation is known to contribute to

tumor progression in various cancers, including hepatic and breast cancers. Because we have previously demonstrated,

using co-culture experiments, that obese AT-derived stem cells (obASC) contribute to AT inflammation through

promotion of Th17 cells, and activation of IL-1 secreting monocytes (Diabetes, 2015; Adipocyte, 2016), we postulated

herein that such an inflammatory environment could contribute to tumor progression in cancer-suffering obese patients.

Materials and Methods: Human ASC were isolated from AT of obese donors. Mononuclear cells (MNC) were

collected from healthy blood donors. Co-cultures of ASC and MNC were activated for 48 hours with

phytohemagglutinin A ( PHA), a T cell mitogen, or not. Conditionned media (CM) were collected, and added for 24h to

cultures of HUH7( hepatocarinoma cell line) or of two breast carcinoma cell lines, i.e MCF-7, or MDA-MB-231.

Levels of inflammatory or angiogenic gene expression were evaluated by qRT-PCR. Expression of CXCR4 (a marker of

invasiveness) was measured by flow cytometry in the HuH7 cell line.

Results: CM from PHA-activated-obASC/MNC co-cultures enhanced IL-1b, IL-8 and VEGFa mRNA expression in

HuH7 cells by 1942.2, 45.7 and 6.1 -fold, respectively, as compared with no treatment. A putative effect of CM on

HuH7 invasiveness was supported by a 2- and 3-fold increase in MMP-9, and CXCR4 expression, respectively. In

addition, IL-1b, IL-8 and VEGF-a mRNA expression were increased by 34.3, 33.2, and 2.97 fold respectively in MCF-7

cells, and by 85.8, 52.0 , and 1.34 fold respectively, in MDA-MB-231 cells. These results indicated thus a differential

sensitivity of cancer cell lines to CM from PHA-activated-obASC/MNC co-cultures, with a preponderant increase of IL-

1b mRNA levels in hepatocarcinoma cells versus a similar increase of IL-1b and IL-8 gene expression in breast

carcinoma cells.

Conclusion: Our results suggest that in cancer-suffering obese patients, interaction of obASC with AT infiltrating

immune cells contribute to the establishment of an inflammatory environment, propitious to tumor inflammation and/or

tumor migration. Whether this inflammation could occur through propagation of obese AT inflammatory environment

towards tumors, or through migration of ASC inside tumors and then interaction with tumor infiltrating immune cells,

remain to be explored.

POSTER 10.

GUT, THE FORGOTTEN ORGAN IN CHRONIC KIDNEY DISEASE…

Elsa Hoibian, Christophe Soulage, Hubert Vidal

CarMeN, UMR INSERM 1060, INSA Lyon, Université Lyon 1, 69100 Villeurbanne (Lyon)

Background: Chronic kidney disease (CKD) is associated with the retention of many soluble wastes called uremic

toxins or uremic retention solutes. A large number these uremic toxins is generated in the intestine as by-products of the

metabolism of the gut microbiota. We hypothetized that in CKD, there is an alteration of the intestine structure and

function and an increased intestinal permeability that could promote the diffusion of uremic toxins into the bloodstream.

Material and method: We explored the impact of uremia on the gut structure and function in vivo in a mice model of

CKD (induced non-surgically by a adenine diet rich) and in vitro in monolayers of CACO-2 cells incubated with uremic

sera. In mice, intestinal permeability was measured using FITC-Dextran and through the measurement of expression and

protein content of the main proteins involved in tight junctions. In vitro, CACO-2 cells were incubated for 24 hours with

plasma from healthy volunteers or from hemodialysis patients and permeability was explored by measurement of

transepithelial electric resistance (TEER) and FITC-Dextran. Expression and protein content of the main proteins

involved in tight junctions were explored by RT-qPCR and western blotting, respectively.

Results: CKD mice exhibited a longer transit time (+50%) and a striking increase in intestinal permeability (+500%). In

good agreement, CACO-2 cells incubated with uremic sera also exhibited a strong increase in permeability (+60%)

estimated by TEER measurements as well as with FITC dextran. Ultrafiltration (MWCO 10 kDa) of uremic sera

prevented its effect on intestinal permeability suggesting that the causal agent could belong to the class of the protein

bound uremic toxins. Expression analysis and protein quantification, in vivo and in vitro, demonstrated that the increase

of intestinal permeability in CKD was associated with an increase of Claudin-1 expression (+50%) and Claudin-1 protein

content (+150%).

Conclusion: Although largely underestimated in the literature, an increased intestinal permeability could contribute to

the accumulation of uremic toxins observed in CKD patients.

Organizing Committee

Etienne LEFAI (CarMeN)

Brigitte LE MAGUERESSE (CarMeN)

Jean-Marc VANACKER (IGFL)

Hubert VIDAL (CarMeN)

Camille LEFEVRE (CarMeN)

Amandine GAUTIER-STEIN (U1213)

Valérie LARGE (CarMeN)

Ours Sponsors

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