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6th SRP DIABETES-ENDOMET Joint Retreat

Vår Gård Conference Center May 12 - 13, 2016

Program and Abstracts

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Venue information

Venue: Vår Gård Ringvägen 6 SE-133 80 SALTSJÖBADEN TEL: +46(0)8-748 77 00 www.vargard.se Fitness and recreation For your recreation you can choose between a game of boules or pool or go for a walk/run (5 km or 8 km). There is also a sauna available.

Travel Information

With Saltsjöbanan

From Stockholm central/T-centralen take the metro south to Slussen. Then follow the signs to Saltsjöbanan. The train ride to Saltsjöbaden’s end station takes approx. 30 minutes. From the station you can see a red brick building on the other side of the tracks. The walk to the reception takes just a couple of minutes.You can find the time-table for Saltsjöbanan at: www.sl.se There is a suitable train leaving Slussen at 08:16 and arriving Saltsjöbaden at 08:51. Please make sure to buy your ticket BEFORE you enter the train.

By car Take highway 222 east (Värmdöleden) and make a right on the exit to Saltsjöbaden (highway 228). Once you are in Saltsjöbaden follow the signs to Ångbåtsbryggan and Vår Gård Saltsjöbaden.

GPS coordinates Lat: N 59° 16.825 Long: E 018° 18.653

By Taxi

Vår Gård has a contract with Värmdö Taxi and has fixed prices. Phone: +46 8-570 357 00. Reservation code: "Vår Gård".

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Poster session - Best Poster Awards Posters will be on display just outside the seminar hall 1.8 m high and 0.9 m wide. Materials to attach with will be available. 17:15 – 19:00 Poster presentations and cocktails Posters will be shown in two consecutive sessions; in the first session from 17:15 – 18:05 posters with odd numbers will be presented and in the second session from 18:10 – 19:00 posters with even numbers will be presented. This will allow the presenters to see the other posters. An evaluation panel will decide upon awards for best posters in two different categories: Best poster for completed work Best poster for most promising project Awards will be announced during the dinner May 12.

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Program Thursday May 12 9:00 Arrival on your own at Vår Gård. Coffee + sandwich [Note: time given for all talks below include 5 min for questions.] 9:30-9:40 Welcome: Juleen Zierath and Mats Rudling 9:40-10.50 Session 1: Growth Control and Metabolism Chair: Per-Olof Berggren 09.40-10.10 Emilio Hirsch (Dept. of Molecular Biotechnology and health Sciences, Torino) “Class II PI3Ks in metabolic and growth control” 10.15 - 10.30 Mladen Savikj “Normal myogenic programming of skeletal muscle satellite cells from spinal cord injured subjects“ 10.35 – 10.50 Duarte M.S. Ferreira “LMCD1, a novel road to control skeletal muscle mass and metabolism” 10.50-11.15 Coffee Break 11:15-12.10 Session 2: Vascular Complications Chair: Sergiu Catrina 11.15 - 11.30 Peter Stenvinkel “Coronary artery calcification is an independent predictor of poor outcome in end-stage renal disease – Do statins promote vascular calcification?” 11.35-11.50 Sonia Zambrano Sevilla “Role of podocyte receptor podo-GPCR2 in diabetic nephropathy” 11.55-12.10 Vladimer Darsalia “Gliptins (Dipeptidyl peptidase-4 inhibitor)-mediated neuroprotection against stroke requires chronic pre-treatment and is Glucagon-like peptide-1 receptor-independent” 12.15 – 12.55 Session 3: Polycystic Ovary Syndrome Chair: Ylva Bonde 12:15-12:30 Anna Benrick “Elevated levels of adiponectin have a protective role on metabolic functions in a mouse model of PCOS” 12.35-12.50 Dorina Ujvari “Insulin modulates in vitro decidualization of human endometrial stromal cells via transcriptional inhibition of Forkhead box protein O1” 13.00-14.30 Lunch + Mounting posters + networking

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14.30- 17.00 Session 4: Regulation of Islet Function Chair: Olov Andersson 14:30-15:00 Ulf Ahlgren (Umeå University) "Optical Projection Tomography and Vibrational Microspectroscopy as tools for diabetes research” 15.05-15.20 Pim van Krieken “Functional compensation in pancreatic beta cells under high workload” 15.25-15.40 Katarzyna Malenczyk “Secretagogin, a Ca2+-sensor protein, controls cellular heterogeneity and hormonal responsiveness in pancreatic islets” 15.40-15.55 Short break 15.55-16.25 Tina Vilsbøll (University of Copenhagen) "Incretin (patho)physiology and treatment” 16.30-16.45 Meike Paschen “Selective development of pancreatic β-cell insulin resistance upon different diets” 16.50-17.05 Teresa Daraio “Expression of SNAP-25b in pancreatic islets is essential for fine-tuned insulin secretion and synchronized beta cell network” 17:15-19:00 Poster session Poster area just outside the seminar hall. Posters will be shown in two consecutive sessions; in the first session from 17:15- 18:05 posters with odd numbers will be presented and in the second session from 18:10-19:00 posters with even numbers will be presented. This will allow the presenters to see the other posters. Refreshments (drinks/snacks) will be served. 19:30 Dinner and activities – Best Poster Awards ___________________________________________________________________________ Friday May 13 07:00-09:00 Breakfast + checkout 9:00-10.15 Session 5: Obesity and diabetes Chair: Sara Straniero 09.00-09.30 Matthias H. Tschöp (HelmholtzZentrum, München) "Toward Metabolic Precision Medicines for Obesity and Type 2 Diabetes" 09.35-09.55 Myriam Aouadi “Revealing the role of Kupffer cells in insulin resistance through next generation sequencing” 10.00-10.15 Olof Lagerlöf “The nutrient sensor OGT in PVN neurons regulates feeding”

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10:15 – 10:45 Coffee Break 10.45-12.00 Session 6: Adipose Tissue Chair: Jurga Laurencikiene 10:45-11:15 Matthias Blüher (University of Leipzig) “Molecular Determinants of Adipose Tissue Health” 11.20-11.35 Yasmina Belarbi “Comprehensive functional screening of miRNAs involved in human fat cell insulin sensitivity” 11.40-11.55 Paul Petrus “A candidate based RNAi-screen identifies SLC19A1 as a novel epigenetic regulator of adipose inflammation” 12.00-12.10 Doctoral Education 12.00-12.10 Milena Schönke and Teresa Daraio “Doctoral education programme MetEndo – what, why and for whom?” 12:10-13:30 Lunch 13.30-14.40 Session 7: Lipid Metabolism Chair: Mats Rudling 13:30-14:00 Sander Kersten (Wageningen University, Netherlands). “Regulation of metabolism by PPARs and Angiopoietin like protein” 14.05-14.20 Marcela González-Granillo “Regulation of lipid metabolism by estrogenreceptor beta in obesity” 14.25-14.40 Camilla Pramfalk “Genetic depletion of the Soat2 gene diminishes hepatic steatosis and improves insulin sensitivity via downregulation of FSP27/CIDEC” 14:45-15:00 Voting for best short talk

15.00-15.10 Concluding remarks and best short talk award Per-Olof Berggren, Mats Rudling and Stefan Nobel 15.15-15.45 Coffee and departure

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Oral Abstracts Session 1 Growth Control and Metabolism Class II PI3Ks in metabolic and growth control Emilio Hirsch; Dept. of Molecular Biotechnology and health Sciences, Torino, Italy The spatial restriction of phosphorylated phosphoinositides generated downstream activated membrane receptors is critical for proper cell response to environmental cues. In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. We recently discovered that insulin signaling triggers the association of the liver-specific class II PI3K isoform g (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. Similarly to PI3K-C2γ, PI3K-C2α produces phosphatidylinositol-3,4-bisphosphate as well as phosphatidylinositol-3-phosphate. This unexpectedly versatile enzyme was found to selectively control distinct steps of vesicular traffic such as endocytosis, recycling and primary cilium-bound cargo transport. Recent findings indicate that this protein is involved in metabolic control as well as cell proliferation and cancer through unexpected signaling mechanisms. Normal myogenic programming of skeletal muscle satellite cells from spinal cord injured subjects Savikj M1,2, Kostovski E1,2,3, Ruby M4, Iversen PO3,5, Zierath J4,6, Krook A4,6, Widegren U4 1Section for Spinal Cord Injury, Sunnaas Rehabilitation Hospital, Nesoddtangen, Norway; 2Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 3Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; 4Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 5Department of Hematology, Oslo University Hospital, Oslo, Norway 6Department of Physiology and Pharmacology, Section for Integrative Physiology, Karolinska Institutet, Stockholm, Sweden; Aim: Spinal cord injury leads to changes in skeletal muscle physiology, e.g. severe atrophy, a switch in fibre type composition and metabolic changes of the affected skeletal muscles. Satellite cells grown in vitro have been shown to reflect skeletal muscle changes in response to type 2 diabetes, obesity, exercise and neurological diseases. We hypothesized that spinal cord injury affects the ability of the satellite cells to produce a normal muscle phenotype.

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Methods: Human satellite cells were isolated from muscle biopsies of vastus lateralis from eight longstanding spinal cord injured and six able-bodied controls. Primary muscle cultures were grown, differentiated and harvested. Fatty acid oxidation was measured with a radioactive tracer, and the protein content of differentiation markers and protein metabolism regulators was measured by Western blotting. Results: Protein content of the differentiation markers studied; desmin, myosin heavy chain and myogenin, were expressed to a similar extent in the spinal cord injured as in the able-bodied controls, during differentiation from myoblasts to myotubes. No differences in protein synthesis and degradation pathways were observed between the spinal cord injured and the able-bodied myotubes, analysed by phosphorylation and total protein amount of the following signalling molecules; mTOR, S6, 4EBP1, FoxO3a, MAFbx and LC3. Myotubes from the spinal cord injured were able to oxidize fatty acids at a similar rate as the able-bodied controls. Conclusion: Our results indicate that the functionality of satellite cells from spinal cord injured skeletal muscle is retained, and that these cells are able to produce normal myotubes when extracted and grown in vitro. LMCD1, a novel road to control skeletal muscle mass and metabolism Duarte M.S. Ferreira1, Arthur J. Cheng2, Daniel Edsgärd3, Thomas Chaillou2, Margareta Porsmyr-Palmertz1, Manizheh Izadi1, Leandro Z. Agudelo1, Vicente Martínez-Redondo1, Amanda T. Petersson-Klein1, Igor Cervenka1, Gianluigi Pironti1, Daniel C. Andersson1, Olof Emanuelsson3, Johanna T. Lanner2 and Jorge L. Ruas1 Department of Physiology and Pharmacology. 1Molecular & Cellular Exercise Physiology. 2Calcium Signaling and Molecular Muscle Physiology. Karolinska Institutet. Stockholm. Sweden; 3School of Biotechnology. Royal Institute of Technology (KTH). SciLifeLab. Stockholm. Sweden. Skeletal muscle condition has a remarkable impact on systemic health. Changes in muscle metabolism are associated with debilitating conditions such as obesity and diabetes. We have recently identified several transcription regulators with the potential to control skeletal muscle mass and metabolism. Among those is one of the most prominent candidates: LIM and Cysteine-rich Domains 1 (LMCD1). LMCD1 overexpression in cultured myotubes resulted in an increase in cell size and protein synthesis, indicating it can induce muscle hypertrophy in vitro. Surprisingly, we also observed an increase in the expression levels of mitochondrial respiratory complexes and mitochondrial respiration, which is not always associated with hypertrophy. This was also confirmed in vivo, when we performed intramuscular injections of adenovirus expressing LMCD1 into mouse gastrocnemius muscles. Since LMCD1 has been described to interact with calcineurin, we decided to analyse the effects of LMCD1 expression on calcium handling and fibre contractility. To perform that we injected flexor digitorum brevis muscles with the same adenovirus vectors and dissected individual fibres after 7 days. Indeed, LMCD1 increased fibre size and specific force with a lower requirement for sarcoplasmic reticulum calcium release. We have generated a skeletal muscle-specific LMCD1 transgenic mouse, which is currently under phenotypic analysis. A better understanding of the mechanisms that govern muscle LMCD1 and its impact on systemic energy homeostasis, will set the foundation for the identification of new therapeutic targets to prevent or treat muscle dysfunction associated with diabetes and obesity.

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Session 2 Vascular Complications Coronary artery calcification is an independent predictor of poor outcome in end-stage renal disease – Do statins promote vascular calcification? Zhimin Chen1, Abdul Rashid Qureshi1, Paolo Parini2, Eva Hurt-Camejo3 Bengt Lindholm1, Jonaz Ripsweden4, Torkel B Brismar4, Peter Barany1, Olof Heimburger1, Peter Stenvinkel1

1Division of Renal Medicine and Baxter Novum, Dept of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden; 2Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden 3Translational Science, CVMD iMed, AstraZeneca R&D, Gothenburg, Sweden, 4Division of Medical Imaging and Technology, Dept of Clinical Science, Intervention and Technology, Karolinska Institutet, and Dept of Radiology, Karolinska University Hospital, Huddinge, Stockholm, Sweden Background and Aim: In end-stage renal disease (ESRD), coronary artery calcification (CAC) and inflammation contribute to cardiovascular disease (CVD). Statins - despite effectively lowering LDL-cholesterol levels – do not improve survival in ESRD patients and their effect on vascular calcification is not clear. We explored associations between CAC, inflammatory biomarkers, statin therapy and mortality in ESRD patients. Method: In 268 ESRD patients (64% males; median age 56 years) from three cohorts including 103 recipients of living donor kidney transplant (LD-Rtx), 96 incident dialysis and 69 prevalent peritoneal dialysis patients, associations between CAC score (Agatston Units, AUs) by computed tomography (CT), with interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor (TNF), use of statins and all-cause mortality (by multivariable Cox proportional hazard regression analysis) were analysed. Cardiac CT scan repeated in 48 patients after median 1.5 years of renal replacement therapy. Results: Among 268 patients, 142 (53%) had a CAC score >100 AUs. Multivariate analysis revealed that independent predictors of 1-SD higher CAC score were age, male gender, diabetes and use of statins. In Cox regression analysis, the association between CAC score and mortality remained significant after adjustment for age, gender, diabetes, CVD, use of statins, subjective global assessment (SGA) and inflammation. Repeated CAC imaging in 48 ESRD patients showed that statin therapy was associated with greater progression of CAC. Conclusion: Elevated CAC score is a mortality risk factor in ESRD independent of inflammation. As our data suggest that statins promote vascular calcification, studies are needed to resolve if this could contribute to the documented lack of effect on outcome with statin therapy in ESRD.

Role of podocyte receptor podo-GPCR2 in diabetic nephropathy Sonia Zambrano Sevilla, Patricia Q Rodriguez, Jaakko Patrakka. Integrated Cardio Metabolic Center (ICMC), Department of Laboratory Medicine Huddinge. Karolinska Institutet, Stockholm, Sweden. Background and Objective: Podocyte injury is associated with progressive kidney disease and correlate with diabetic kidney disease (DKD) in both type 1 and type 2 diabetes. However, molecular mechanism leading to podocyte injury in DKD is still poorly understood. By using large-scale expressional profiling we have identified a novel highly podocyte-specific G-protein receptor, podo-GPCR2. Previously, this orphan GPCR has been related with the regulation of Wnt3/ β catenin pathway in the neocortex. This pathway has been linked previously to podocyte damage in

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DKD. The aim of this work is to study the function of podo-GPCR2 in podocytes and its role in DKD. Design: In vitro, we generated a stable cell line of human podocytes over expressing podo-GPCR2. In these cells we carried out studies of gene expression of different component of the Wnt3/β catenin pathway, as well as luciferase experiment using reporters for activated β catenin. We used zebrafish and mouse as in vivo models. In zebrafish, podo-GPCR2 was inactivated using morpholinos. In mouse podo-GPCRC2 was inactivated using gene targeting. 6 KO and 6 control mice were challenged with LPS that is known to cause podocyte injury and proteinuria (features mimicking DKD). Results: In cultured cells, we saw that β catenin was significantly upregulated in human podocytes overexpressing podo-GPCR2. Moreover, the gene expression of Axin3 and DKK2 (two components downstream of wnt3 pathway) was elevated in over expressing podocytes. Using a luciferase reporter we could validate that β catenin was activated in podocytes overexpressing podo-GPCR2. In zebrafish, the inactivation of podo-GPCR2 resulted in podocyte foot process effacement, glomerular basement membrane abnormalities and proteinuria – all features of human DKD. In mouse, the absence of podo-GPCR2 did not affect normal development and function of the glomerulus. However, KO animals were more prone to LPS-induced podocyte damage as they developed higher albuminuria after LPS injection than control animals. Conclusion: Podo-GPCR2 is a novel highly podocyte-specific GPCR that seems to be involved in the pathogenesis of podocyte injury through wnt signaling pathway. As GPCRs are known to be good targets for pharmaceutical invention, podo-GPCR2 may be a new target molecule to treat glomerular diseases. In collaboration with AstraZeneca we are making an effort to deorphanize podo-GPCR2. Gliptins (Dipeptidyl peptidase-4 inhibitor)-mediated neuroprotection against stroke requires chronic pre-treatment and is Glucagon-like peptide-1 receptor-independent Darsalia V1, Larsson M1, Lietzau G1,2, Nathanson D1, Nyström T1, Klein T3, Patrone C1. 1Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden. 2Medical University of Gdansk, Department of Anatomy and Neurobiology, Gdansk, Poland. 3Department of CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany. Background and objectives: Gliptins [(dipeptidyl peptidase-4 inhibitors (DPP-4i)] are anti-T2D drugs regulating glycaemia by preventing endogenous glucagon-like peptide-1 (GLP-1) degradation. Interestingly, a chronic pretreatment with gliptins before experimental stroke reduces brain damage and this effect is believed to be mediated by increased GLP-1 levels. These results suggest the potential of gliptins to be developed into also neuroprotective strategies for high stroke-risk T2D patients. However, whether acute gliptins-treatment after stroke is also neuroprotective is unknown. Additionally, other DPP-4 substrates like Gastric inhibitory polypeptide (GIP) and The stromal cell-derived factor 1 alpha (SDF1a) could be involved; hence the gliptins-mediated neuroprotective mechanisms remain largely unknown. The goal of the study was to determine the neuroprotective efficacy of the gliptin Linagliptin administered acutely at stroke onset and the determine whether Linagliptin-mediated neuroprotection was GLP-1-dependent.

Materials and methods: Adult wt and glp-1r-/- mice were subjected to transient middle cerebral artery occlusion (MCAO). 50 mg/kg/bw Linagliptin or vehicle were administered acutely

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intravenously at stroke onset and mice were sacrificed after 1 week. In the chronic setting, 10 mg/kg/bw Linagliptin or vehicle were given per-orally (p. o.) for 4 weeks before stroke and for 3 weeks thereafter. Additional groups of glp-1r-/- and wt mice were similarly chronically treated with GLP-1R agonist Exendin-4 (Ex-4) 0,1 μg/kg/bw). The neuroprotective efficacy was assessed by stroke volume measurement and stereological counting of surviving neurons. Plasma and brain levels of GLP-1, GIP, SDF1a and DPP-4 activity were analysed by ELISA. Results: Acute administration of Linagliptin at stroke time did not result in neuroprotective efficacy after stroke. In contrast, the chronic pre- and post-treatment resulted in significant reduction of ischemic damage.

Chronic treatment with Linagliptin significantly reduced ischemic damage in glp-1r-/- mice. However, no such effect was recorded in glp-1r-/- mice after Ex-4 treatment.

Chronic treatment with Linagliptin did not increased active GLP-1 or GIP levels in the brain. However, the levels of SDF1a were significantly elevated.

Conclusion: These results indicate that linagliptin-induced neuroprotection requires chronic pre-treatment before experimental stroke and is mediated by a GLP-1R-independent mechanism, likely through SDF1a modulation. However other DPP-4 substrates need to be examined in order to identify the exact mechanism of neuroprotection.

Session 3 Polycystic Ovary Syndrome Elevated levels of adiponectin have a protective role on metabolic functions in a mouse model of PCOS Anna Benrick1, Belén Chanclon1, Laila Hadi1, Kerstin Ebefors1, Stephen Franks2, Jenny Nyström1, Ingrid W Asterholm1, Elisabet Stener-Victorin1,3

1 Dept. of Physiology, Inst. of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden. 2 Inst. of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, London, UK. 3 Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden. Keywords: adiponectin, insulin sensitivity, glucose uptake, androgens, PCOS Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects 5-10% of women in their reproductive age. PCOS is related to metabolic disturbances, such as obesity and insulin resistance. Serum adiponectin together with adipocyte size is the strongest factor explaining the decreased insulin sensitivity in women with PCOS. The aim was to study the metabolic function in adiponectin transgenic (tg) and knockout (ko) mice with or without dihydrotestosterone (DHT)-induced PCOS. We hypothesize that adiponectin has a protective effect on the development of metabolic dysfunction in this PCOS model. DHT-pellets were implanted subcutaneously in pre-pubertal female mice to induce PCOS while controls received placebo-pellets. Adiponectin tg, ko and wt mice were divided into 8 groups (n=7-8). Insulin and glucose tolerance, and body composition measurements were performed between 14-16 weeks of age. Wt-DHT mice were insulin resistant and had a decreased glucose tolerance compared to controls while tg-DHT mice had a normal insulin and glucose tolerance. Ko-DHT mice on the other hand

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were more insulin resistant than wt-DHT animals. Both wt-DHT and ko-DHT groups had an impaired glucose tolerance compared to placebo but there was no difference between the groups. Preliminary data shows that wt-DHT animals have larger adipocytes and that tg-DHT mice are protected against this effect. There were only small differences in body weight and body composition. Studies on reproductive function as well as liver, kidney and pancreas histology and gene expression are ongoing. We conclude that elevated levels of adiponectin have a protective role on metabolic functions in this PCOS mouse model. Insulin modulates in vitro decidualization of human endometrial stromal cells via transcriptional inhibition of Forkhead box protein O1 Dorina Ujvari†*1, Ivika Jakson†1, Shabnam Babayeva2, Daniel Salamon1,3, Bence Rethi4, Sebastian Gidlöf1,5, Angelica Linden Hirschberg1 1Department of Women’s and Children’s Health, Karolinska Institutet and Department of Obstetrics and Gynecology, Karolinska University Hospital, SE-171 76 Stockholm, Sweden 2Department of Obstetrics and Gynecology II, Azerbaijan Medical University, AZ1022 Baku, Azerbaijan

3Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden 4Department of Medicine, Karolinska University Hospital, SE-171 76 Stockholm, Sweden 5Department of Clinical Science, Intervention and Technology, Karolinska Institutet, SE-171 77 Stockholm, Sweden Polycystic ovary syndrome and obesity are associated with insulin resistance, hyperinsulinemia and reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1), one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1) and prolactin) were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1 CTGF, INSR, DCN, LEFTY2), except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin modulated the decidualization process by a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on endometrial function via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons to unexplained infertility.

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Session 4 Regulation of Islet Function Optical Projection Tomography and Vibrational Microspectroscopy as tools for diabetes research Ulf Ahlgren, Umeå University The anatomy of the pancreas makes it exceedingly difficult to perform quantitative or biochemical assessments of the gland if the spatial context is to be preserved. A preserved spatial context may be key to accurately interpret how changes in pancreatic β-cell mass and function is manifested. We have developed a series of protocols that enables Optical Projection Tomography based 3D-spatial and quantitative β-cell mass assessments, throughout the volume of the murine pancreas, with islet level resolution. These tools have contributed new insights into the pancreatic anatomy and may be used to study aspects of β-cell destruction, proliferation and regeneration in large cohorts of experimental animals. The ability to analyze biochemical changes directly in the pancreas may facilitate our mechanistic understanding of diabetes development and could aid in prognostic and/or diagnostic assessments. We have developed a new approach for non-destructive, label-free biochemical profiling of the pancreas, which preserves the spatial context of the tissue. By a technique for multivariate image analysis of vibrational microspectroscopic data of the pancreas (including FT-IR and Raman data), chemical compositional analysis of pancreatic cell types may be performed in situ and in vivo. Hereby, new and previously recorded biochemical alterations of the pancreas could be detected in settings of diabetes. By assessments of islets transplanted to the anterior chamber of the eye, the approach provides a novel means by which biochemical alterations of the islets may be studied in response to external stimuli and over extended periods of time in vivo. Functional compensation in pancreatic beta cells under high workload Pim van Krieken1, Andrea Dicker1, Ulf Ahlgren2, Per-Olof Berggren1, 3, 4, Erwin Ilegems1

1) The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, Stockholm, Sweden. 2) Umeå Centre for Molecular Medicine, Umeå University, Umeå, Sweden. 3) Diabetes Research Institute, University of Miami, Miami, USA. 4) Lee Kong Chian School of Medicine, Nanyang Technological University, Imperial College London, Singapore. It has been shown in mouse models of hyperglycemia and obesity that pancreatic beta cells, generally known for their very low turnover, can exhibit a higher proliferative rate to adapt to increased demands for insulin. In addition to a larger pool of insulin-producing cells, beta cells within individual islets can also adapt by increasing their functional status in terms of glucose-induced insulin release. Failure in the mechanisms regulating beta cell mass and function will lead to the development of diabetes. So far these compensatory mechanisms remain unclear and it is unknown to which extent they could occur under normal blood glucose levels. We therefore sought to investigate functional compensation in beta cells in non-obese mice under normoglycemic conditions. We established a mouse model of high beta cell workload by first removing 50% of pancreatic beta cell mass with streptozotocin, as verified by optical projection tomography, and subsequently transplanting isolated islets into the anterior chamber of the eye to revert hyperglycemia. Characterization after one month demonstrated that mice had no altered glucose tolerance as compared to sham-treated mice. Islet grafts were additionally used to report on

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functional parameters in vivo and ex vivo. We could observe significant changes in beta cell size, intracellular Ca2+ oscillation patterns, and mitochondrial metabolism in islets under high workload. These results indicate that mouse beta cells are capable of displaying functional plasticity under normoglycemic and non-obese conditions. Future experiments will focus on elucidating molecular mechanisms driving functional plasticity of beta cells under high workload. Secretagogin, a Ca2+-sensor protein, controls cellular heterogeneity and hormonal responsiveness in pancreatic islets Katarzyna Malenczyk1,2, Fatima Girach1, Ludwig Wagner3, Robert Schnell4, Tomas Hökfelt5 and Tibor Harkany1,2 1Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Vienna, Austria; 2Division of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm; 3University Clinic for Internal Medicine III General Hospital Vienna, Vienna; 4 Division of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm; 5Department of Neuroscience, Karolinska Institutet, Stockholm The role of a controlled intracellular Ca2+ environment shaped prominently by Ca2+-binding/sensor proteins is critical for regulated pancreatic hormone release. Secretagogin (SCGN) is one of the most abundant EF-hand Ca2+-sensors in pancreatic islets, with functions largely unknown.

Combining functional, genetic and cellular analyses of SCGN null mice and acute SCGN silencing in INS-1E and αTC1-6 cells in vitro, we found this EF-hand Ca2+-sensors being involved in the regulation of islet establishment, and consequently, the ability and capacity of hormone release. SCGN knock-out in mice induced age-dependent modifications to the size, as well as the cellular composition of pancreatic islets. By 6 weeks of age, SCGN null mice exhibited enlarged pancreatic islets resulting from α cell hyperplasia. In contrast, by 6 months of age, SCGN null islets were reduced in size, as compared to those in wild-type mice. This was due to their decreased β cell mass even though α cell hyperplasia continued. Alterations in the composition of SCGN null islets was evoked by the differential control of proliferation and apoptosis in α and β cells, respectively, as substantiated by acute SCGN knock-down in INS-1E (β-like) and αTC1-6 (α-like) cells. The functional correlate of morphological islet reorganization was progressive glucose intolerance, culminating in diabetes. Particularly, the age-dependent loss of β cell mass recapitulated key indices of type 2 diabetes. Reduced secretion of insulin and glucagon in SCGN null mice manifested in response to KCl or glucose irrespective of whether the data were corrected for cell numbers. This was corroborated by the reduced density of secretory granules in α and β cells of SCGN null mice, in particular the number of docked vesicles, resulting from i) decreased mRNA expression and protein content for insulin and glucagon; ii) reduced v-SNARE (VAMP2, synaptophysin) and t-SNARE (SNAP25) protein constituents observed in SCGN null mice. The loss of SNARE integrity was recapitulated by SCGN silencing in both INS-1E and αTC1-6 cells. Cumulatively, these data suggest that SCGN developmentally primes cellular heterogeneity in pancreatic islets, while its postnatal functions center on the control of insulin and glucagon expression and regulated exocytosis.

Cellular composition of pancreatic islets, pancreatic hormone content and readiness of the exocytotic machinery in α and β cells reflects their ability to couple metabolic stimuli to hormone release; and whose disturbance can lead to diabetes. Our genetic and functional analyses establishes SCGN as being essential to the maintenance of the ratio of α and β cells, and insulin and glucagon expression and secretion. Thus, SCGN might become an appealing target for pharmacological intervention.

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Incretin (patho)physiology and treatment Tina Vilsbøll, University of Copenhagen Type 2 diabetes ruins the lives of millions. Treatments addressing the entire range of related pathophysiological traits are crucial. Research has changed the understanding of type 2 diabetes pointing towards the gut as a major harbour for undiscovered disease mechanisms. Our research unit and our diabetes clinic represents a possibility to “bring patients to science and science to patients”. Our research work within a national and international network of clinicians and researchers which allows patient-oriented research - ranging from basic pathophysiology to randomised clinical trials. We aim to clarify core mechanisms in type 2 diabetes pathophysiology by including studying the role of the gastrointestinal tract in human physiology and diabetic pathophysiology. Our overall objective is to identify and develop preventive measures and antidiabetic treatment modalities - and, thus, drive diabetology in a direction that will provide benefit for patients. Selective development of pancreatic β-cell insulin resistance upon different diets Meike Paschen, Tilo Moede, Barbara Leibiger, Ingo B. Leibiger, Per-Olof Berggren The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, SE-171 76 Stockholm, Sweden Background and aims: Obesity and type 2 diabetes mellitus (T2DM) have reached epidemic proportions and a connection between eating behavior and disease development is discussed. Besides insulin resistance pancreatic β-cell dysfunction is central to the development of T2DM. Because the pancreatic β-cell itself is a target for insulin action, β-cell insulin resistance can contribute to β-cell dysfunction. Here we used a recently by us developed technique that reports on insulin resistance in pancreatic β-cells in vivo to investigate the dynamics of pancreatic β-cell insulin sensitivity/resistance in the context of bodyweight gain, whole body insulin resistance and glucose tolerance upon different diet interventions. Methods: C57BL/6J mice were fed a High Sucrose Diet (HSD) (32% sucrose in drinking water), High Fat Diet (HFD) (60% of the kcal from fat), High Fat High Sucrose Diet (HFHSD) (HSD+HFD) or control diet (normal chow diet) for 2 months. At different time points of the diet intervention (1 and 2 weeks and 1 and 2 months after treatment start), β-cell insulin resistance was measured by in vivo imaging of pancreatic islets transduced with a fluorescent insulin resistance biosensor and engrafted in the anterior chamber of the eye of recipient animals. The biosensor is based on the different subcellular localization of FoxO1-GFP, i.e. cytosolic in insulin responsive cells and nuclear at insulin resistance, and used to calculate a β-cell insulin resistance index (βIRI) representing the ratio of nuclear and cytoplasmic FoxO1-GFP localization. Glucose and insulin tolerance were measured by intraperitoneal glucose and insulin tolerance tests, respectively. Results: A diet intervention time of 2 months did not change bodyweight, glucose tolerance, whole body and β-cell insulin sensitivity in mice fed a HSD compared to the control diet. In contrast HFD fed mice developed glucose intolerance during the first week of treatment, showed a significant increase in bodyweight after 2 weeks and became whole body insulin resistant at 1 month after treatment start, but they neither developed β-cell insulin resistance within 2 months of treatment. HFHSD caused glucose intolerance during the first week after treatment start, a significant increase in bodyweight, whole body and β-cell insulin resistance at 1 month of treatment (after 1 month of treatment: Control: βIRI = 0.689 ± 0.02; HFHSD: βIRI = 0.786 ± 0.02; p< 0.01). Conclusion: This study provides in vivo evidence that pancreatic β-cell insulin resistance occurs upon HFHSD, but not HSD and HFD treatment. In general, this for the first time illustrates the development of β-cell insulin resistance in the context of increased bodyweight, glucose intolerance and whole body insulin resistance. These data provide the basis for further investigation of the

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molecular mechanisms underlying the different development patterns of organ specific insulin resistance and may lay the foundation for novel treatment strategies in diabetes. Expression of SNAP-25b in pancreatic islets is essential for fine-tuned insulin secretion and synchronized beta cell network Teresa Daraio1, Lidija Križančić Bombek2, Ismael Valladolid-Acebes1, Marko Gosak2, Essam Refai1, Masa Skelin Klemen2, Tomas Hökfelt3, Kerstin Brismar1, Marjan Slak Rupnik2,4, Christina Bark1. 1 Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden 2 Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia 3 Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden 4 Center for Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria Glucose-stimulated insulin secretion from pancreatic beta cells is a highly dynamic process mediated by the SNARE protein complex consisting of SNAP-25, syntaxin and VAMP/synaptobrevin. SNAP-25 exists as two isoforms, SNAP-25a and SNAP-25b, generated by obligate alternative splicing resulting in two proteins that differ in 9 out of 206 amino acids. Although SNAP-25a is the major splicing variant in primary beta cells, both isoforms can participate in the core SNARE complex and mediate insulin release. The possible importance of SNAP-25b in insulin secretion is not fully understood. To address this question we used, as an experimental model, a unique mutant mouse expressing only SNAP-25a. We ran in vivo and ex vivo experiments including glucose tolerance tests, dynamic insulin release assays, calcium imaging in pancreatic slices and immunohistochemistry. SNAP-25b deficiency induced increased insulin secretion during both the first and second phase. These results were associated with a less synchronized beta cell activity when monitoring spatiotemporal intracellular calcium oscillations upon glucose stimulation, which indicated a loss of network integrity in islets lacking SNAP-25b. Interestingly, sex differences disclosed already in wild-type mice were further increased in SNAP-25b-deficient mutants. Taken together, our findings suggest that expression of both SNAP-25 isoforms in pancreatic islets is necessary for proper control of processes that determine the amount and dynamics of insulin release. Moreover, it highlights the importance of taking sex differences into account. Our results may contribute to a better understanding of factors important for the development of metabolic disease and to a tailored treatment for defined patient populations. Session 5 Obesity and Diabetes Toward Metabolic Precision Medicines for Obesity and Type 2 Diabetes Matthias H. Tschöp, HelmholtzZentrum, München After decades of research unraveling complex metabolic control networks, medicines capable of a safe reversal of morbid human obesity and type 2 diabetes are still not available. Historically, complex diseases have repeatedly proven to be defiant to the best mono-therapeutic approaches. Several examples of combination therapies have largely overcome such challenges, notably for the treatment of severe hypertension and tuberculosis. Obesity and its consequences, such as type 2 diabetes, have proven to be equally resistant to therapeutic approaches based on single medicines. Appropriate management of type 2 diabetes often requires adjunctive medications, and the recent

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registration of a few compound mixtures has set the precedent for combinatorial treatment of obesity. On the other hand, double or triple therapeutic combinations are more difficult to advance to regulatory approval. Following an improved understanding of the molecular basis for metabolic benefits following bariatric surgery interventions, several classes of novel unimolecular or independent combination therapeutics were discovered. These new classes of drug candidates are based on gastrointestinal hormones, offer efficacy superior to currently prescribed options and seem to have potential to fully reverse human obesity and type 2 diabetes. Moreover, gut peptide-based cell-specific targeted delivery of small molecules offer additional potential for novel metabolic precision medicines and reduced systemic side effects. In this presentation the discovery, pre-clinical validation and first clinical test of peptide hormone poly-agonist drug candidates as well as of combinatorial single molecule therapeutic candidates will be summarized, including previously unpublished observations. Revealing the role of Kupffer cells in insulin resistance through next generation sequencing Myriam Aouadi1

1Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Center, Department of Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Sweden A key component in the pathogenesis of type 2 diabetes mellitus (T2D) is obesity-induced chronic inflammation. Immune cells produce factors, such as pro-inflammatory cytokines, which can cause insulin resistance in adipose tissue and liver by inhibiting insulin signal transduction leading to T2D. Consistent with this concept, liver and adipose tissue inflammation correlate with systemic insulin resistance. Liver macrophages, denoted Kupffer cells (KCs), are thought to be the major source of hepatic inflammation. However, their contribution to insulin resistance has not been directly tested. Using a new approach to silence genes specifically in KCs, we found that decreasing the expression of the master regulator of inflammation, nuclear factor kappa B (NFkB), improves insulin sensitivity in obese animals. Although these findings demonstrate that NFkB expressed by KCs do play a role in the regulation of insulin sensitivity, an unbiased approach was required to determine which other pathways and downstream NFkB targets contribute to insulin resistance. Using next generation sequencing methods, we discovered new genes and non-coding RNAs that are regulated with insulin resistance in KCs. The role of these genes is now being investigated using our unique technology, which specifically targets genes expressed by KCs in vivo. The nutrient sensor OGT in PVN neurons regulates feeding Olof Lagerlöf1,2,3, Julia E. Slocomb4, Ingie Hong1, Yeka Aponte5, Seth Blackshaw1, Gerald W. Hart2 and Richard L. Huganir1. 1Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A. 2Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A. 3Present address: Department of Neuroscience, Karolinska Institute, Stockholm 17177, Sweden 4National Institute on Drug Abuse + National Institutes of Health/Johns Hopkins University Graduate Partnership Program, Baltimore, Maryland 21224, USA 5Intramural Research Program, Neuronal Circuits and Behavior Unit, National Institute on Drug Abuse, Baltimore, Maryland 21224, USA Maintaining energy homeostasis is crucial for the survival and health of organisms. The brain regulates feeding by responding to dietary factors and metabolic signals from peripheral organs. It

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is unclear how the brain interprets these signals. O-GlcNAc transferase (OGT) catalyzes the posttranslational modification of proteins by O-GlcNAc and is regulated by nutrient access. The O-GlcNAc pathway has been linked genetically to human overweight. Here we show that acute deletion of OGT from αCaMKII-positive neurons in adult mice caused extreme obesity due to voracious overeating. The hyperphagia correlated with total calorie intake and not with any particular dietary nutrient. Whereas meal frequency was not altered, meal size doubled upon loss of OGT. Local deletion of OGT in αCaMKII-positive neurons in the paraventricular nucleus (PVN) of the hypothalamus using stereotactic virus injection recapitulated the increase in both body weight and food intake. Food intake activated αCaMKII-positive neurons in the PVN and this activation depended on OGT. At least in part, whole-cell patch clamp demonstrated that OGT controls the activity of these cells by maintaining their number of glutamatergic synapses. We confirmed this finding biochemically and morphologically in primary cultured neurons. Conversely, optogenetic stimulation of αCaMKII-positive PVN neurons inhibited food intake by decreasing meal size. Within the PVN, OGT is shown to sense nutrient availability directly by taking advantage of fluorescent reporter mice and hypothalamic explants. These results identify O-GlcNAcylation in αCaMKII-positive neurons of the PVN as a novel molecular mechanism that regulates satiation by coupling calorie intake with caloric need. This manuscript was published in Science March 18 2016. Session 6 Adipose Tissue Molecular Determinants of Adipose Tissue Health Matthias Blüher, University of Leipzig Obesity is frequently associated with chronic inflammation, metabolic and vascular alterations which predispose to a variety of comorbid disorders. However, the individual risk for obesity-related diseases is not determined by increased fat mass alone. In humans, we have studied adipose tissue health in the model of insulin sensitive obesity. Heterogeneity of body composition, fat distribution and adipose tissue function may underly the variable risk to develop metabolic and cardiovascular diseases associated with increased body fat mass. Importantly, an inability to increase adipose tissue mass by adipocyte hyperplasia may lead to adipocyte hypertrophy and could induce dysfunction of adipose tissue characterized by decreased insulin sensitivity, hypoxia, inadequate vascularization, impaired unfolded protein response, increased parameters of intracellular stress, increased autophagy and apoptosis and tissue inflammation. As a result, adipocytes and other adipose tissue cells release signals (e.g. adipokines, cells, metabolites) resulting in a proinflammatory, diabetogenic and atherogenic serum profile. These adverse signals may contribute to further adipose tissue inflammation, attraction of immune cells, altered fat distribution and secondary organ damage in target tissues such as liver, brain, endothelium, vasculature, endocrine organs and skeletal muscle. Recently, a specific adipocyte volume threshold has been shown to predict the risk for obesity-associated type 2 diabetes. Most likely, impaired adipocyte function is caused by genetic, behavioural and environmental factors which are not entirely understood. Here, the molecular signatures contributing to adipose tissue dysfunction including the role of developmental (e.g. HoxA5, HoxC9, HoxC10) and bone morphogenetic genes (e.g. BMP2, BMP7), chemerin, Repin1, vaspin, kallikrein 7 and regulators of autophagy are discussed. Elucidating the mechanisms of adipocyte dysfunction may lead to the identification of novel treatment targets for obesity and metabolic diseases.

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Comprehensive functional screening of miRNAs involved in human fat cell insulin sensitivity Ingrid Dahlman, Yasmina Belarbi, Jurga Laurencikiene, Annie M. Pettersson, Peter Arner, Agné Kulyté* Context: MicroRNAs (miRNA) are dysregulated in obesity, a condition that contributes to type 2 diabetes (T2D). The key pathological link between obesity and T2D is insulin resistance but the molecular mechanisms are not entirely identified. Objective: Our aim was to investigate miRNAs role in regulation of insulin sensitivity in subcutaneous white adipose tissue (scWAT). Design: Global miRNA profiling was explored in abdominal scWAT of obese insulin resistance (OIR), -obese insulin sensitive (OIS) and lean women. Eleven miRNAs were overexpressed in human in vitro-differentiated adipocytes followed by assessment of lipogenesis. Setting: Cohort study (n = 48) in an academic hospital. Patients: ScWAT was obtained from 18 OIR, 21 OIS and 9 lean individuals. Subjects were chosen based on the insulin/basal-stimulated lipogenesis response. Intervention(s): There were no interventions. Main Outcome Measure(s): Expression levels of miRNAs in OIR, OIS with lean individuals were evaluated and effects of miRNA overexpression in human adipocytes on lipogenesis and genes/proteins involved in insulin signaling pathway. Results: Eleven miRNAs displayed differential expression between OIR and OIS states. Overexpression of miR-143-3p and miR-652-3p increased insulin-stimulated lipogenesis in human adipocytes differentiated in vitro and affected genes/proteins involved in insulin signaling pathway at transcriptional and/or post-transcriptional levels. Adipose expression of miR-143-3p and miR-652-3p was positively associated with insulin-stimulated lipogenesis in scWAT independently of BMI. Conclusions: Expression levels of miR-143-3p and miR-652-3p are downregulated in OIR subjects and affects insulin-stimulated lipogenesis in adipocytes. Both miRNA are directly or indirectly involved in regulation of insulin signaling pathway genes. A candidate based RNAi-screen identifies SLC19A1 as a novel epigenetic regulator of adipose inflammation Paul Petrus1, Hui Gao2, Ingrid Dahlman1, Agné Kulyute1, Anna Ehrlund1, Ana Gracia3, Sofia Toft1, Jurga Laurencikiene1, Peter Arner1, Niklas Mejhert1, Mikael Rydén1

1 Dept. of Medicine (Huddinge), Karolinska Institutet, Sweden 2 Dept. of Biosciences and Nutrition, Karolinska Institutet, Sweden 3Dept. Nutrition and Food science, University of Basque country, Vitoria, Spain Obesity and type 2 diabetes mellitus (T2DM) may be linked to white adipose tissue (WAT) dysfunction. In particular, a chronic low grade inflammation in WAT has been shown to result in perturbed lipolysis and insulin resistance. We aimed to identify novel regulators of adipocyte function that may play a causal role in T2DM development. A candidate-based high throughput RNAi screen of 125 genes in human primary adipocytes identified SLC19A1 as a novel regulator of lipolysis. Adipose expression of SLC19A1 was lower in obesity and correlated BMI-independently with several clinical parameters including inflammation and insulin resistance. SLC19A1 encodes an uptake carrier for folate, an essential component in the one-carbon pool. This led us to hypothesize that SLC19A1 downregulation may alter gene transcription via effects on DNA methylation. SLC19A1 knock down in human adipocytes did indeed result in a global DNA hyper-methylation. In order to define SLC19A1-regulated CpG loci, we overlapped results from different gene expression and DNA-methylation micro arrays of human

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WAT/adipocytes. This showed that the expression of CCL2, a gene encoding the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 which is central in initiating and sustaining WAT inflammation, increased upon siSLC19A1 treatment; that there was a negative correlation between SLC19A1 and CCL2 expression and that methylation of a CpG locus (cg12698626) in the promoter region of CCL2 correlated positively with gene expression. These data suggested that CCL2 may be upregulated upon attenuated SLC19A1 levels through increased methylation at cg12698626. This notion was confirmed in vitro by pyro-sequencing of human adipocytes with or without SLC19A1 knockdown. Taken together, SLC19A1 is a novel clinically relevant regulator of WAT inflammation through epigenetic mechanisms. Session 7 Lipid Metabolism Regulation of metabolism by PPARs and Angiopoietin like protein Sander Kersten, Wageningen University, Netherlands In recent years, insights into the mechanisms underlying the biological effects of fatty acids have improved considerably and have provided the foundation for the emerging concept of fatty acid sensing. Fatty acid sensing can be interpreted as the property of fatty acids to influence biological processes by serving as signalling molecules. An important mechanism of fatty acid sensing is via stimulation of DNA transcription via activation of peroxisome proliferators-activated receptors. One of the genes that is consistently and very significantly upregulated by fatty acids via PPARs in numerous organs encodes Angiopoietin like protein 4 (ANGPTL4). ANGPTL4 is a potent inhibitor of lipoprotein lipase, thereby blocking plasma clearance of triglycerides and raising plasma triglycerides. Studies over the past couple of years have shown that ANGPTL4 regulates LPL activity during a variety of physiological conditions, including fasting, cold, and exercise. For example, increased expression of ANGPTL4 (originally called fasting-induced adipose factor) leads to a rapid reduction in adipose tissue LPL activity during fasting. The collective data point to a scenario where ANGPTL4 is the central component of a feedback mechanism that regulates plasma triglyceride hydrolysis and subsequent tissue fatty acid uptake in response to changes in lipid availability and cellular fuel demand. Based on our most recent data, it appears that ANGPTL4 mainly has a local role, at least in adipocytes, by stimulating LPL degradation after LPL processing in the ER. Overall, these data demonstrate the crucial role of ANGPTL4 in the transcriptional and metabolic response to fatty acids and the regulation of fat uptake into cells. The importance of ANGPTL4 in regulation of plasma triglycerides in humans is supported by extensive human genetic studies. Regulation of lipid metabolism by estrogenreceptor beta in obesity Marcela González-Granillo1, Christina Savva1,2, Maria Manti3 , Jan-Åke Gustafsson2, Marion Korach-André1. 1Department of Medicine, MedH, Karolinska Institutet, Huddinge, Sweden. 2Department of Biosciences and Nutrition, Karolinska Insitutet, Huddinge, Sweden. 3Department of Physiology and Pharmacology, Reproductive Endocrinology and Metabolism, Karolinska Institutet, Solna, Sweden. Obesity is an abnormal fat accumulation caused by an energy imbalance between intake and expenditure. Estrogens regulate energy homeostasis through the glucose and fat metabolism.

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Estrogen receptors (ERα and ERβ) belong to the family of nuclear hormone receptors and are expressed mainly in metabolic tissues. A number of studies are still debating about the potential role of ERβ as a primary mediator of anti-obesity effects of circulating estrogens. We thus investigate the physiological and molecular mechanism of ERβ activation by a specific synthetic ligand in mice during obesity. Wild type female mice were fed with chow or high fat diet for 10 weeks before being treated with KB101471 an ERβ specific ligand from KaroBio. Tolerance tests (glucose and insulin) and metabolic cages were used to evaluate glucose and energy metabolism, respectively. Lipid metabolism was investigated in vivo before and after treatment using magnetic resonance spectroscopy and imaging, and its activity ex vivo in visceral and subcutaneous fat, and liver using deuterium water. Real-time PCR, western blot and next generation sequencing were used to found target genes involved in these metabolic changes. HFD-fed mice treated with KB101471 showed significant reduction in body weight as well as in visceral and subcutaneous fat accumulation. Hepatic triglycerides were significantly reduced by the ligand. Additionally, KB101471 treatment reduced fasting glucose level together with enhanced insulin sensitivity. These results suggest that ERβ could be a potential target to treat obesity and associated metabolic disorders avoiding the side effects of ERα activation. Genetic depletion of the Soat2 gene diminishes hepatic steatosis and improves insulin sensitivity via downregulation of FSP27/CIDEC Pramfalk Camilla1,2 , Ahmed Osman1,3, Larsson Lilian1, Härdfeldt Jennifer1, Pedrelli Matteo1,2,4, Vedin Lise-Lotte1, Steffensen Knut R1, Eriksson Mats5, Parini Paolo1,2,5 1 Division of Clinical Chemistry, Department of Laboratory Medicine, and 2 Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Stockholm, Sweden, and 3 Department of Biochemistry, Faculty of Medicine, Khartoum University, Khartoum, Sudan, and 4 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden and 5 Department of Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden Nonalcoholic fatty liver disease (NAFLD) is a growing health problem worldwide. Although the underlying pathological mechanisms are not completely understood, overaccumulation of triglycerides and cholesteryl esters inside the hepatocytes (hepatic steatosis) is a prerequisite for the development of NAFLD. Acyl-Coenzyme A: cholesterol acyltransferase 2 (ACAT2, encoded by Soat2) is exclusively expressed in hepatocytes and enterocytes and biosynthesize cholesteryl esters from cholesterol and long-chain fatty acids. Here we sought: 1) to investigate the role of Soat2 in hepatic steatosis and a potential association between Soat2 and insulin sensitivity; and 2) to assess the effects of different dietary regimens. Soat2-/- mice fed a high-fat (but low-cholesterol) diet had less hepatic steatosis, were more insulin sensitive, had lower hepatic expression of glucose transporter 2 and fat specific protein 27 (FSP27, also named Cidec in humans). Similar findings were present in Soat2-/- mice fed a high-carbohydrate diet. In our studies in mice, genetic depletion of Soat2 significantly reduces hepatic steatosis and improves insulin sensitivity via downregulation of hepatic FSP27/Cidec and glucose transporter 2 expression independently of sex and diet regimes; thus, selective inhibition of this enzyme may be a strategy to treat cardiometabolic diseases.

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Poster Abstracts

P1 The effect of androgen excess on maternal metabolism, placental function and fetal growth in obese dams Romina Fornes1, Manuel Maliqueo1,2, Laila Hadi3, Miguel Jimenez 4, Min Hu2 Anna Benrick2, Jenny Nyström2, Fernand Labrie5, Angelica Lindén Hirschberg6, Thomas Jansson7, Elisabet Stener-Victorin1*

1. Department of Physiology, Department of Physiology and Pharmacolocy, Karolinska Institutet, 171 77 Stockholm, Sweden

2. Endocrinology and Metabolism Laboratory, Department of Medicine, West division, University of Chile, Santiago, Chile

3. Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden

4. Unidad Académica Multidisciplinaria Reynosa Aztlán, Universidad Autónoma de Tamaulipas, Reynosa, Tamaulipas, México.

5. Laval University Research Center in Molecular Endocrinology, Oncology and Human Genomics, CHUL Research Center, Quebec, G1V 4G2, Canada

6. Department of Obstetrics and Gynecology, Karolinska University Hospital, 171 76 Stockholm, Sweden 7. Department of Obstetrics & Gynecology, Division of Reproductive Sciences, University Colorado Denver,

Aurora, Colorado 80045, USA Background and Aim: Women with polycystic ovary syndrome (PCOS) are often hyperandrogenic and obese, which negatively affect fetal development and pregnancy outcomes. Herein we induced maternal (prenatal) androgen excess in a model of maternal obesity to investigate maternal metabolism, placental function and fetal growth. Methods: C57Bl6J female mice were fed with control diet (Ctrl) or high fat/sucrose (HFD) for 4-10 weeks. When HFD animals increased 25% of the initial body weight, they were mated with Ctrl males. From gestational day (GD) 15.5 to 17.5 mice were injected with dihydrotestosterone (DHT) or vehicle: Ctrl, HFD, Ctrl+DHT, HFD+DHT. On day 18.5 were blood, placenta, maternal and fetal livers collected. Body composition was measured by DEXA at GD 0 and GD 18.5 and glucose homeostasis by oral glucose tolerance test (OGTT) in a subset of mice. Fetuses were analyzed by body composition analyzer. Results: Circulating DHT was higher in Ctrl+DHT and HFD+DHT group compared with controls. Dams fed HFD with or without DHT exposure had more fat content before mating, whereas only HFD dams had more fat compared with controls (P < 0.05) at GD 18.5. There were no significant differences in glucose homeostasis. Fetal weight was lower in the HFD+DHT group compared with the HFD, with no difference in placenta weight. Liver weigh more and the liver triglyceride (TG) content was higher in dams fed HFD compared with Ctrl, and was higher in the HFD+DHT mice compared with all three groups. The fetal liver TG content was lower in the HFD group vs Ctrl (P < 0.05). Conclusion. Maternal obesity with androgen excess result in enlarged livers accompanied with pronounced increase in liver TG content together with fetal growth restriction and a decreased fetal liver TG content, without any major effect on maternal glucose metabolism.

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P2 Meal composition affects risk markers for kidney disease differently in type 2 diabetes and healthy subjects C. Olofsson1,3, A. Hilding1, N. Orsini2, I-L. Andersson3, K. Brismar1,3, N Rajamand Ekberg1,3

1Department of molecular medicine and surgery, 2 Department of public health sciences, Karolinska Institutet, 3Department of Endocrinology, Diabetes and Metabolism, Karolinska University Hospital , Stockholm, Sweden Background and aims: Hyperglycemia, oxidative stress and inflammation play a role in development of diabetic kidney disease. Hyperglycemia and hyperlipidemia following a meal are speculated to be associated with increased oxidative stress and inflammation but is insufficiently investigated. The aim of current study was to examine the effect of meal composition on postprandial risk markers for kidney disease among those with type 2 diabetes (T2D) and healthy subjects. Material and methods: On four different occasions 21 patients with T2D and 21 healthy subjects ingested an isocaloric lunch (600 kcal) with different compositions of carbohydrate (CH) (54%), CH & fibers (15 g), fat (50%) and protein (40%). Blood samples were taken and urine samples collected before and up to four hours after the meal. B-glucose, insulin, triglycerides, CRP, ox-LDLchol, IL-6, IL-18, and urine albumin/creatinine ratio were analyzed. Those with T2D had a mean age (±SD) of 63 (4) years while healthy subjects had a mean age of 52 (16), p=0.004. T2D subjects had compared to healthy higher BMI (29 vs 24 kg/m2) and greater waist circumference (for men 108 vs 94 cm, for women 99 vs 77 cm), p<0.001 for all. Mean (min, max) HbA1c was 52 (40, 84) mmol/mol for T2D. Statistical method: repeated measure ANOVA and area under the curve (AUC). Results: The ANOVA showed postprandial differences between the meals for glucose, insulin and triglycerides for both groups, all p<0.05. High CH meals resulted in higher peaks of glucose and insulin, while high fat meal resulted in higher peak of triglycerides. For T2D subjects, glucoseAUC were greater for high CH meal than high protein meal (p=0.01), and high CH with fibers were greater than high protein (p=0.003) and high fat (p=0.03) meal. For healthy subjects, glucoseAUC was greater for high CH meal with (p=0.04) or without (p=0.01) additional fibers in comparison to high protein meal. Triglycerides showed an overall increase independent of meal in both groups, but triglycrideAUC was different in T2D where high-fat meal showed greater increase than high CH with fiber meal, p=0.03. Both those with T2D and healthy subjects had an overall increase in IL-6 over time, p=0.003 and p=0.0002, respectively, with no difference between the meals. CRP did not change after any meal. The urine albumin/creatinine ratio did not show any meal related difference, but there was a tendency to increased ratio after high protein compared to high CH with fiber meal in T2D. IL-18 decreased after a high CH meal only in healthy subjects, p=0.003, while no meal related changes were seen in T2D. Ox-LDLchol was affected by the meal composition in T2D. Conclusion: These preliminary results demonstrated that CH rich meal showed higher glucose and high fat meal higher triglyceride levels suggesting risk for oxidative stress in T2D supported by the changes in ox-LDLchol. However, the inflammatory markers (CRP, IL-6, IL-18) and urine albumin excretion were not significantly modulated by the studied meal composition. P3 Analysis Of Mature Mouse Adipocytes By Flow Cytometry Carolina Hagberg, Debajit Bhowmick, Qian Li, Endre Kiss, Viviana Kozina, Kirsty Spalding Department of Cell and Molecular Biology, KI Solna, and Integrated Cardio Metabolic Centre , Department of Medicine, KI Huddinge, Stockholm, Sweden Isolated primary mature adipocytes are difficult to analyse as they are large (50-200 um in diameter), lipid filled, floating and fragile. This is especially true for analysis on a single cell level.

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Our aim is to develop a reliable method to detect and study large numbers of individual mature adipocytes using flow cytometry. Using flow cytometry we have identified a previously unpublished high Forward Scatter (FSC - size) and low Side Scatter (SSC - granularity) population that is unique for mature adipocytes. The cell identity of this FSC/SSC population was verified to be adipocytes using mouse cells from an adiponectin promoter-driven fluorescent reporter mouse line, which specifically stains all mature adipocytes with Tomato-red. Staining of wildtype mouse adipocytes with both Bodipy (to detect lipid droplets) and DNA dye (to detect nuclei) also revealed the same FFS/SSC adipocyte population. We could show that key to the analysis of adipocytes by FC is an in-tube stirring mechanism, and a possibility to detect large objects by using FSC neutral density filters, both present on the MoFlo XDP sorter (Beckmann Coulter) that was used for this study. Today most analysis of individual primary adipocytes is done using microscopy, which is both laborious and time consuming. By applying FC, a more comprehensive and large-scale profiling of mature adipocytes can be achieved in a much shorter amount of time. This method therefore has broad downstream applications for high throughput interrogation of mature adipocytes, allowing the combination of size and granularity profiles with dye or antibody labelling, and is applicable to both single cell and whole population analyses. P4 Beneficial effects on fatty acid composition and indices of fatty acid desaturase activity with a Paleolithic-type diet during a two-year intervention in obese postmenopausal women 1Caroline Blomquist, 1Elin Chorell, 1Mats Ryberg, 1Caroline Mellberg, 2Christel Larsson, 3Bernt Lindahl, 4Ulf Riserus, 1Tommy Olsson 1Departments of Public Health and Clinical Medicine, Medicine, Umeå University, Umeå, Sweden 2Department of Food and Nutrition and Sport Science, University of Gothenburg, Gothenburg, Sweden.

3Departments of Public Health and Clinical Medicine, Environmental Health, Umeå University, Umeå, Sweden 4Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University, Uppsala, Sweden Objectives: To study diet effects of two defined diets on fatty acid (FA) composition in cholesterol esters (CE) and desaturase activity (DA) in plasma and describe their association to reported intake and changes in insulin resistance (IR) in postmenopausal obese women. Subjects and methods: Seventy obese postmenopausal women (BMI= 32.6 ±3.4) were randomized to either an ad libitum Paleolithic-type diet (PD) aiming at 30 energy percent (E%) protein, 30 E% carbohydrates and 40 E% fats including a high content of unsaturated FAs, or a prudent control diet (CD) aiming at 15 E% protein, 55 E% carbohydrates and 30 E% fat for 24 months. Anthropometry, 4-day food records, IR and relative FA composition of CEs in plasma were measured at baseline and after six and 24 months. Surrogate measures of DAs were expressed as ratios of CEs: Δ-9 DA=16:1/16:0; Δ-6 DA=18:3n-6/18:2n-6 and Δ-5 DA=20:4n-6/20:3n-6. FA E% intake was correlated with FA composition in CE using Spearman rank correlation coefficient. Further sample comparison modeling was performed using orthogonal partial least squares (OPLS) to elucidate correlation patterns between IR and FAs in CE. Results: At 24 months the reported intake in the PD group of saturated fatty acids (SFA) decreased with 19% and monounsaturated fatty acids and polyunsaturated fatty acids (PUFA) increased with 47% respectively 71% (all p<0.001). The correlation between reported change of PUFA E% intake

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and the change of PUFA in CE were significant between 0-6 months (r=0.36, p=0.008) and between 6-24 months (r=0.48, p=0.002). Δ-6 and Δ-9 DA decreased significantly more in the PD group than in the CD group after 24 months (p=0.002 and p=0.013, respectively). Δ-5 DA increased in both groups at 6 months, significantly more in the PD group (p<0.001). FAs associated to IR including 14:0, 16:1, 18:3n-6 and 20:3n-6 decreased all in the PD group after 24 months (p<0.014 for difference between groups). Both diets resulted in a similar reduction in body weight and abdominal height (both p<0.001 within groups) after 24 months. We detected a significant pattern of FAs describing IR, via the OPLS analysis, with higher levels of 14:0, 15:0, 16:1, 18:0, 18:3n-3 and 20:3n-6 and lower levels of 20:4n-6, and Δ-5 DA. Conclusions: The biomarkers for FA intake confirm an increased intake of PUFA and decreased intake of dairy products at 6 months in the PD group. The PD reduced specific FAs (14:0; 16:1; 18:3n-6 and 20:3n-6) and DAs (Δ-9 and Δ-6) associated with IR more distinctly than a CD during a 24-month diet intervention. The changes in FA levels associated to PD may have long-term beneficial effects on obesity-related disorders. P5 Identification of transcriptional networks involved in regulation of human adipogenesis and adipocyte metabolism Christel Björk1, Jianping Liu2, Anders Eriksson2, Ingrid Dahlman1, Peter Arner1 and Jurga Laurencikiene1 1. Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden; 2. Department of Biosciences and Nutrition, Huddinge, Karolinska High Throughput Center, Karolinska Institutet, Stockholm, Sweden Gain of white adipose tissue causes changes in adipose tissue metabolism, which plays an important role in development of obesity with complications such as type 2 diabetes and dyslipidemia. We aim to characterize how transcriptional regulators control fat cell development that in turn will influence the metabolic function of human adipose tissue. Therefore we screened for transcription factors (TFs) that would possibly affect human adipocyte differentiation/metabolism using RNA interference. We have selected 148 TFs, whose expression in adipose tissue is regulated by obesity, adipose tissue morphology, insulin resistance or cancer cachexia based on microarray mRNA profiling data. Primary human adipose tissue mesenchymal stem cells were reversely transfected with siRNAs targeting the selected TFs. Lipid content and cell number (cell nuclei) were quantified with Bodipy and Hoechst by Acumen eX3 HCS microplate cytometer. In addition, glycerol release as a measurement of lipolysis was defined in conditioned media. The screen was performed twice in triplicates. After data normalization, hits were selected using Strictly Standardized Mean Difference (SSMD). Effects of selected hits on human adipocyte differentiation and metabolism are currently analyzed using qRT-PCR, ChIP-seq and metabolic measures of adipocyte phenotype. Novel TFs regulating differentiation of human adipocyte progenitor cells will be further analyzed in additional clinical cohorts. This study is not only important to gain further knowledge of the impact of adipogenesis to the phenotype of mature human adipocytes and morphology of adipose tissue but also for identifying novel signaling proteins or pathways which may be targets for pharmacological interventions. This work is supported by Swedish Science Council, NovoNordisk, Karolinska Institutet and Tore Nilsson foundations.

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P6 ERβ As a Key Player in Lipid Metabolism During Obesity Christina Savva1,2,Marcela González-Granillo1, Maria Manti3 , Jan-Åke Gustafsson2, Marion Korach-André1. 1Department of Medicine, MedH, Karolinska Institutet, Huddinge, Sweden. 2Department of Biosciences and Nutrition, Karolinska Insitutet, Huddinge, Sweden. 3Department of Physiology and Pharmacology, Reproductive Endocrinology and Metabolism, Karolinska Institutet, Solna, Sweden. Background: Obesity is caused by an imbalance between energy intake and energy expenditure. Estrogens regulate energy homeostasis through the alteration of glucose and fat metabolism. Estrogen receptors (ERα and ERβ) belong to the family of nuclear hormone receptors and are expressed mainly in metabolic tissues. Studies debate the potential role of ERβ as a primary mediator of anti-obesity effects of circulating estrogens. Aim: To further investigate the physiological and molecular mechanisms of ERβ activation in mice during obesity using a synthetic ERβ specific ligand. Methods: Wild type female mice were fed with chow or high fat diet for 10 weeks before being treated with ERβ specific ligand. Tolerance tests (glucose and insulin) and metabolic cages were used to evaluate glucose and energy metabolism, respectively. Lipid metabolism was investigated in vivo before and after treatment using magnetic resonance spectroscopy and imaging. Using a stable isotope tracer method (H202), lipid metabolic pathway activities were measured ex vivo in visceral and subcutaneous fat, and liver. Real-time PCR, western blot and next generation sequencing were used to find target genes involved in these metabolic changes. Results: HFD-fed mice treated with ligand showed significant reduction in body weight as well as in visceral and subcutaneous fat accumulation. Triglyceride in liver was severely reduced by the ligand. Additionally, treatment with the ligand showed reduction in blood fasting glucose level together with enhanced insulin sensitivity. Conclusion: ERβ could be a potential target to treat obesity and associated metabolic disorders avoiding the side effects of ERα activation. P7 Estrogenic and anti-estrogenic properties of Mondia whitei extract and fractions using transactivation assays in human U2OS cells Elizabeta Zaplatic1,2, Jannette Wober2, Günter Vollmer2, Inga Marijanovic1

1 Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia 2 Chair for Molecular Cell Physiology and Endocrinology, Institute of Zoology, Department of Biology, Dresden University of Technology, Dresden, Germany Phytoestrogens are a broad group of plant-derived compounds of non-steroidal structure that are structurally and/or functionally similar to mammalian estrogens and their active metabolites. For this reason, phytoestrogens can bind to estrogen receptors (ERs) and act as estrogen agonists or antagonists. Accumulating evidence from molecular and cellular biology experiments, animal studies and human clinical trials suggests that phytoestrogens may confer health benefits related to cardiovascular diseases, hormone-dependent cancers, osteoporosis, menopausal symptoms, obesity and type-2 diabetes. The aim of this work was to evaluate estrogenic and antiestrogenic potential of methanol extract and 12 fractions of Mondia whitei. This tropical African plant is used in traditional medicine to treat various conditions, including urinary tract infections, jaundice, headache, diarrhea, but the most commonly cited use is as an aphrodisiac. Test system used in this study were ER-subtype specific transactivation assays in human bone-derived U2OS-ERα and U2OS-ERβ cells.

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These two cell lines were developed from human osteosarcoma ER-negative U2OS cell line and are characterized by doxycycline-inducible expression of ERα and ERβ, respectively. In the examined methanol extract, neither estrogenic nor anti-estrogenic properties have been observed. One fraction of the extract displayed agonistic activity in both cell lines, with concentration of 10 μg/ml as the lowest that produced agonistic activity. None of the fractions exhibited anti-estrogenic potential. Evidence of an existing estrogenic potential of Mondia whitei forms the basis for further research which should elucidate how the compounds of this plant interact with hormonal feedback loop and determine whether a use of these phytoestrogens is beneficial and safe. P8 Enhancer mediated gene regulation of insulin response in subcutaneous adipose tissue Enrichetta Mileti1, Amitha Raman1, Olga Dethlefsen2, Mikael Rydén3, Peter Arner3, Carsten O. Daub1. 1) Department of Biosciences and Nutrition and Science for Life Laboratory, Karolinska Institutet, SE-141 83 Huddinge, Sweden. 2) Department of Biochemistry and Biophysics and Science for Life Laboratory, Stockholm University, 171 21 Solna, Sweden. 3) Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden. Insulin resistance is associated with obesity, but up to one third of obese subjects display a metabolically healthy phenotype with insulin sensitivities comparable to non-obese individuals [1]. The hormone insulin influences the expression of a large number of genes. Impaired insulin response can be caused dys-regulation of gene expression. Enhancers are non-protein coding elements on the genome controlling the precise spatiotemporal expression of genes. We investigated the role of enhancers as gene regulatory mediators for insulin response. Abdominal subcutaneous white adipose (sWAT) tissue was obtained before and after two-hour hyperinsulinemic euglycemic clamp in a well phenotyped cohort of obese insulin sensitive (ISO, n=21), obese insulin resistant (IRO, n=29) and non-obese (NO, n=15) individuals). The sWAT tissue RNA was analyzed using 5’ Cap Analysis Gene Expression (CAGE) sequencing [2]. CAGE allows genome-wide identification of promoter regions as well as the quantification of gene enhancer activity [3]. In the entire cohort, the clamp induces pronounced alterations in gene expression. Most responding genes were down-regulated and associated with the insulin signaling pathway. Using bimodal CAGE expression enhancer signature patterns, we globally identified more than 40,000 enhancer candidates in sWAT tissue together with more than 100 enhancers possibly regulating insulin response. One example is an enhancer regulating Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1), a transcription factor recently reported to be involved in regulating insulin response [4]. [1] N. Klöting, M. Fasshauer, A. Dietrich, P. Kovacs, M. R. Schön, M. Kern, et al., "Insulin-sensitive obesity," Am

J Physiol Endocrinol Metab, vol. 299, pp. E506-15, Sep 2010. [2] H. Takahashi, T. Lassmann, M. Murata, and P. Carninci, "5' end-centered expression profiling using cap-

analysis gene expression and next-generation sequencing," Nat Protoc, vol. 7, pp. 542-61, Mar 2012. [3] R. Andersson, C. Gebhard, I. Miguel-Escalada, I. Hoof, J. Bornholdt, M. Boyd, et al., "An atlas of active

enhancers across human cell types and tissues," Nature, vol. 507, pp. 455-61, Mar 2014. [4] A. Bouchard-Mercier, I. Rudkowska, S. Lemieux, P. Couture, L. Pérusse, and M. C. Vohl, "SREBF1 gene

variations modulate insulin sensitivity in response to a fish oil supplementation," Lipids Health Dis, vol. 13, p. 152, 2014.

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P9 The dipeptidyl peptidase-4 inhibitor Linagliptin partially reverses striatal neuropathology in type 2 diabetic mice Grazyna Lietzau1,2, Emanuel Candeias1, Thomas Nyström1, Thomas Klein3, Vladimer Darsalia1, Cesare Patrone1 1Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden 2University of Gdańsk, Department of Anatomy and Neurobiology, Gdańsk, Poland 3Boehringer Ingelheim, Biberach, Germany Background and aims: Type 2 diabetes (T2D) is associated with increased risk of Alzheimer’s disease (AD). However, T2D patients present a broad range of cognitive/sensorimotor problems which do not progress towards AD and do not necessarily involve the same brain areas as in AD. The pathogenic mechanisms at the basis of these impairments need to be identified. A growing body of scientific evidence indicates that the impairment of GABAergic interneurons positive for parvalbumin (PV) is involved in the development of brain diseases. However, their potential role in T2D-induced complications in the CNS (central nervous system) is unknown. Dipeptidyl peptidase-4 inhibitors (DPP-4i), which are used for the treatment of T2D, have shown neuroprotective properties in preclinical studies. However, their potential efficacy in preventing T2D-induced complications in the CNS has not been thoroughly studied. In this study, we aimed to determine whether: 1) T2D affects PV-positive interneurons and microglial cells in the striatum (an important brain area involved in sensorimotor functions); 2) Chronic treatment with the DPP-4i linagliptin prevents the development of striatal pathologies. Materials and methods: In Study 1, we analysed 2-months-old C57BL/6 mice (n=10) and 14-months-old C57BL/6 mice receiving standard (n=6) or high-fat diet (HFD; n=7) for 48 weeks. We quantified the number of cells (per 1mm2) and the average cell volume (in µm3) of PV-immunoreactive (-ir) neurons in the striatum. We also assessed potential signs of inflammation by counting number of cells and measuring ionized calcium binding adaptor molecule 1 (Iba-1)-ir microglia cell volume, respectively. In Study 2, 2-months-old C57BL/6 mice received HFD for 36 weeks before being treated with vehicle (n=10) or Linagliptin (5 mg/kg b.w./day; n=8) for 12 weeks. The same assessments as in Study 1 were performed. Data were analysed using one-way ANOVA (Study 1) or unpaired t-test (Study 2), and expressed as mean ± SEM. Results: Aging induced a significant decrease in the number (6.646 ± 0.297 vs. 3.431 ± 0.537; p<0.0001) and volume (µm3, 1182.0 ± 27.0 vs. 733.6 ± 55.7; p<0.0001) of PV-ir interneurons. Interestingly, HFD-treated mice showed a further 51% decrease in PV-ir cells versus their aged-matched controls (3.431 ± 0.537 vs. 1.737 ± 0.156; p<0.01). Aging also increased the volume (174.1 ± 12.7 vs. 274.3 ± 12.8; p<0.0001) of Iba-1-ir cells, with no effect of HFD. Linagliptin-treatment partially reversed the HFD-induced effect on PV-ir interneurons (cell number: 14.51 ± 0.63 vs. 18.63 ± 1.01; p<0.005), but also the inflammatory effect (Iba-1 cell volume: 246.1 ± 7.3 vs. 217.0 ± 8.8; p<0.05) induced by aging. Conclusion: In this study, we show that T2D significantly accelerates the aging-induced decrease of PV-ir interneurons in the striatum. The impairment of these cells in this brain area may be one of the pathogenic mechanisms at the basis of decreased sensorimotor functions observed in T2D. We also show that Linagliptin exerts pleiotropic, normalising effects against the impact of T2D and aging. In summary, these results could have implications for the identification of T2D-induced pathogenic mechanisms in the CNS as well as for the development of suitable treatment strategies.

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P10 Systematic identification of transcription factors regulating lipolysis and lipid droplet morphology in human adipocytes Hui Gao1, Niklas Mejhert2, Paul Petrus2, Johan Henriksson2, Jianping Liu1, Thirupathi Pattipaka1, Karin Dahlman-Wright1, Mikael Rydén2, Peter Arner2 1 Department of Biosciences and Nutrition, karolinska Institutet 2 Department of Medicine, Huddinge, karolinska Institutet Adipocytes store lipids, predominantly triglycerides (TGs), in lipid droplets. Upon energy shortage, TGs are hydrolyzed into non-esterified fatty acids and glycerol in an enzymatic process termed lipolysis. In this project, we aimed to systematically map the expression of transcription factors (TFs) during adipocyte differentiation and to assess which of them that are involved in controlling the lipolytic process and/or lipid droplet biology. Using Cap Analysis of Gene Expression, a single molecule RNA sequencing technique, transcriptional start sites and cognate gene expression levels were measured during human adipogenesis. Altogether, 700 TFs were found to be present in in vitro differentiated adipocytes and in order to assess if these TFs regulated lipid catabolism/lipid droplet morphology, a high throughput RNAi screen was performed where individual genes were knocked down and alterations in glycerol release (a proxy for lipolysis) and lipid droplets were measured. This systematic unbiased approach identified several TFs that affect lipolysis and/or lipid droplet morphology, many of which have not previously been associated with adipocyte biology. P11 Genomics, cistromics, epigenomics and transcriptomics to address molecular details of type 2 diabetes Indranil Sinha, Hui Gao and Karin Dahlman-Wright. Department of Biosciences and Nutrition, Karolinska Institutet. Analysis of the genome, cistrome, epigenome and transcriptome provide opportunities to, in a non-hypothesis driven approach, provide molecular details of the development of type 2 diabetes in different tissues in samples from animal models and humans. Genomic analysis includes the association of genetic variation with disease. The cistrome refers to the global analysis of transcription factor binding to DNA in the context of chromatin and can be explored to assess the binding of transcription factors important for insulin signaling under various conditions. In epigenomics, the correlation and causative role of epigenetic modifications for type 2 diabetes can be investigated. In recent years, significant attention has been given to the correlation and potential causal role of the epigenetic modification DNA-methylation in type 2 diabetes and how the epigenome can be modulated. Furthermore, a correlation between epigenetic modifications and gene expression, the transcriptome, is an area of intense investigation. Here we are presenting some of our recent findings in this field. P12 Free fatty acids modulate IGFBP-1 Markus Näslund, Ishrath Ansurudeen, Kerstin Brismar Dept. of Molecular Medicine and Surgery, Rolf Luft Center for Diabetes Research Karolinska Institutet, Karolinska University Hospital, Solna. Background: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder associated with metabolic disorders like type 2 diabetes mellitus (T2D), metabolic syndrome (MetS) and obesity. It is characterized by intrahepatic fat accumulation which could be reversed by changes in the diet and

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physical exercise. The abnormal accumulation of lipids can be caused by excessive alcohol consumption or intake of western diet. IGFBP-1, the insulin like growth factor binding protein is a marker of hyperinsulinemia and hepatic insulin resistance. It plays an important role in glucose homeostasis by rapidly responding and modulating the bioavailability of IGF-I following a meal or changes in blood glucose levels. Altered serum concentrations of IGFBP-1 have been observed in patients with different liver pathologies and T2D. Studies have shown that both obese and non-obese patients with NAFLD have decreased serum IGFBP-1 levels and IGFBP-1 is inversely associated with the degree of steatosis. However not much is known about role of IGFBP-1 in hepatic steatosis and whether the changes in the IGFBP-1 levels are causative factors or are a result of lipid loading. Aim: The current study was aimed at studying the role of IGFBP-1 in hepatic steatosis and involvement of the IGF system in NAFLD. Methods: HepG2 cells were cultured and treated with combination of 1 mM oleic acid (O) and palmitic acid (P) in varying molar ratios respectively (3:0, 2:1, 1:1, 1:2, 0:3) for 24h and analysed for fatty acid accumulation, programmed cell death and oxidative stress. The changes in gene expression were studied using qPCR. Results: The cells treated with the fatty acids showed significant changes in morphology and showed enhanced accumulation of intracellular lipids with oil Red O staining in all of the treatments. Flow cytometric analysis for ROS (reactive oxygen species) generation showed no significant changes in the treatments with higher ratios of oleic acid O3, O2:P1 and O1:P1. However, there was a significant increase of ROS generation in the P3 (1mM palmitic acid) treatment. Similarly, no visible signs for apoptosis were found in all the treatments except in the P3 samples. IGFBP-1 gene expression was significantly induced by the fatty acids after 24h and caused an increase in the expression of IGFBP-1 mRNA by 2-16 folds. Interestingly, replacing the fatty acids containing medium with fresh medium for the next 24h decreased IGFBP-1 expression and the IGFBP-1 levels fell down significantly by 50% in all treatments except for P3. Conclusions: We observe that IGFBP-1 gene expression increases with fatty acid treatment and decreases drastically after replacing the medium with fatty acid free medium suggesting a direct regulation of IGFBP-1 by fatty acids except for P3. P13 Contribution of non-coding RNAs expressed by Kupffer cells in insulin resistance. Jennifer Jager, Cecilia Morgantini, Xidan Li, Emelie Barreby, Laura Levi, Joar Franzén, Marcela Aparicio-Vergara, Myriam Aouadi. Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Center, Department of Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, 141 57 Stockholm, Sweden. The activation of liver resident macrophages, called Kupffer cells (KCs), could contribute to obesity-induced insulin resistance. Indeed, we have previously shown that silencing the master regulator of inflammation, nuclear factor kappa B (NFkB), specifically in KCs improved insulin sensitivity in obese mice while reducing the expression of its target interleukine-1β (IL-1β). Although IL-1β had been extensively described as a key inflammatory player in the impairment of liver insulin sensitivity in obesity, we found that silencing IL-1β failed to improve insulin sensitivity in obese mice. Here we hypothesize that NFkB target genes other than il1β could be involved in the regulation of insulin sensitivity.

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In order to identify transcripts directly regulated by NFkB in KCs, we applied Global Run-On sequencing (GRO-seq), a genome-wide technique to measure nascent transcripts. Preliminary experiments suggest that coding as well as non-coding RNAs, such as microRNAs, long non-coding RNAs and enhancer-derived RNAs, are differentially expressed in KCs from lean vs. obese animals. We are now testing whether silencing NFkB specifically in KCs of obese mice affects the expression of these transcripts. Finally, using our unique method to deliver small interfering RNA specifically in KCs, we will investigate whether our identified targets could regulate insulin sensitivity in obese mice. P14 Methodologies for human podocyte isolation to examine cell turnover using radiocarbon dating Jones CV1,2, Alkass K3, Bernard S4, Salehpour M5, Druid H2, Spalding KL1,2

1 Department of Cell and Molecular Biology, Karolinska Instititutet, Stockholm, Sweden 2 Integrated CardioMetabolic Centre, Department of Medicine, Karolinska Institutet, Huddinge, Sweden 3 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden 4 Institut Camille Jordan, University of Lyon, Villeurbanne, France 5 Department of Physics and Astronomy, Ion Physics, Uppsala University, Sweden Central to the progression of kidney disease is the damage and loss of podocytes – the specialized epithelial cells of the glomerular filtration barrier. Established dogma states that due to the absence of proliferation or a stem cell population, podocytes are not replaced during our lifetime. However, evidence of clinical and experimental restoration of filtratory function, coupled with recent controversial reports of a putative podocyte progenitor, has sparked interest in the field that there may be a capacity for podocyte replacement. This project will utilize the radiocarbon cell dating method (Spalding et al. 2005) to examine podocyte turnover kinetics in kidney health and disease, and conclusively address whether podocyte regeneration occurs in humans. In brief, by correlating radiocarbon levels in the DNA of cells with atmospheric levels at the time the cell was formed, an accurate timeframe of cell age is revealed. Critical aspects of the project include development of a method to isolate podocytes from postmortem human kidneys; ensuring the high yield and purity required for radiocarbon analysis. Cell and nuclei FACS isolation, based on markers including Podocin and WT1, are being examined. The outcomes of this study have clinical importance for the development of podocyte regeneration as a strategy to halt kidney disease progression. P15 Human adipose tissue macrophages: which types are we talking about? Juan R. Acosta, Iyadh Douagi, Daniel P. Andersson, Jesper Bäckdahl, Mikael Rydén, Peter Arner, and Jurga Laurencikiene

Karolinska Institutet, Stockholm, Sweden We investigated the inflammatory status of adipose tissue in a metabolically well-characterized group of patients: non-obese controls, non-obese type 2 diabetic patients and obese patients with and without insulin resistance. We used 13-colour flow cytometry to identify the constituent cell types in the stroma vascular fraction (SVF) of subcutaneous adipose tissue focusing on macrophage phenotypes. We have defined CD45+/CD14+/CD206+/CD11c+ cells as M1 and CD11c- as M2 macrophages. We found that, in the entire cohort of non-obese patients with and without diabetes

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M1/M2 macrophage ratio correlated positively with fat cell size, HbA1C, TNF-α secretion, and lipolysis. M1 macrophages correlated positively with HbA1c and TNF-α, while M2 macrophages negatively correlated with age. However, earlier studies report that adipose tissue resident macrophages show a mixed phenotype and a functional relevance of different macrophage populations to adipose tissue metabolism is still not fully elucidated. To more deeply characterize macrophages in the SVF of human adipose tissue in vivo we employed additional macrophage markers CD163 (for M2), CD197 (for M1), CD50 (for monocytes), ABCA1 and CD36 (for metabolically activated macrophages) and separated several different macrophage phenotypes using FACS. By combining cell sorting based on these different macrophage markers, mRNA expression and cytokine secretion profiling, single cell RNA sequencing data and metabolically characterized patient cohorts we aim to define a truly pro-inflammatory macrophage phenotype in human adipose tissue that would be a marker of WAT inflammatory environment and insulin resistance. The study is ongoing and preliminary data will be presented. This work is supported by Swedish Science Council, NovoNordisk, Karolinska Institutet and Tore Nilsson foundations. P16 The cell-type specific transcriptome in human adipose tissue and influence of obesity on adipocyte progenitors Anna Ehrlund1, Christel Björk1, Iyadh Douagi2, Juan R Acosta1, Per Hèden3, Peter Arner1, Jurga Laurencikiene1

1 Lipid laboratory, Dept. of Medicine Huddinge, Karolinska Institutet, 2 Center for Hematology and Regenerative Medicine (HERM), Dept. of Medicine Huddinge, 3Akademikliniken, Storängsvägen 10, 115 42 Stockholm, Sweden Obesity affects gene expression and metabolism of white adipose tissue (WAT). However, WAT is a heterogeneous organ containing many different cell types that might respond differently to obesity-induced changes. We performed flow cytometry sorting and RNA expression profiling by microarray of major adipose tissue cell types (adipocytes, progenitors, macrophages, leukocytes) and the total stroma vascular fraction, which enabled us to assign known obesity-regulated genes to specific cell fractions. In addition, we compared transcriptome of adipose progenitor cells obtained from lean and obese individuals. Functional studies identified A2M, CNTN3, ENPEP, HAS2 and C10orf10 as novel obesity-regulated factors that possibly are important for progenitor differentiation/expansion. In addition, we illustrate that relationship between clinical phenotypes and gene expression are different if correlations are performed in intact WAT or a specific WAT-resident cell type. Taken together, we provide a detailed gene expression atlas for clinical/basic research and identify novel regulators of WAT progenitor cell function. P17 Role of podocyte protein Pod-RA1 in the pathogenesis of diabetic nephropathy Katja Möller-Hackbarth and Jaakko Patrakka KI/AZ Integrated Cardio Metabolic Centre (ICMC), Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden. Objective:Podocyte cells are injured and eventually lost in diabetic nephropathy (DN). When lost, they cannot be replaced and podocytopenia develops. This is thought to be a major driver for the progression of glomerular disease processes (including DN). TGF-B plays key role in diabetic

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podocyte damage. This is speculated to result in epithelial-mesenchymal transition (EMT) and apoptosis. Pod-RA1 gene has been detected to be overexpressed in human DN glomeruli by microarray analysis. Previously, Pod-RA1 has been reported to associate with cell proliferation, differentiation, inflammation and fibrosis, processes that are critical for the progression of DN. In this study, we wanted to analyze Pod-RA1 in the glomerulus with the aim to establish its role in the pathogenesis of DN. Methods:We study the expression of Pod-RA1 in human and mouse kidney using RT-PCR, qPCR, immunohistochemistry and Western blotting. In cultured podocytes, we over/underexpress Pod-RA1 and analyze effects on cell morphology, survival and signaling under normal and pathological conditions. We induce podocyte damage in cell culture using TGF-β1, a growth factor known to drive podocyte injury in DN. In vivo, the role of Pod-RA1 will be studied by generating and characterizing a knockout mouse line. Main Outcome Measurements and Results:Pod-RA1 was highly expressed by podocytes (and not by other glomerular cells) and is localized in both, foot and major processes. In cell culture, Pod-RA1 promoted differentiation as overexpression resulted in more arborized morphology and expression of podocyte markers. When exposed to TGF-β1, Pod-RA1 overexpressing cells were more susceptible to EMT and apoptosis, whereas knock-down of Pod-RA1 had opposing effects. Furthermore, Pod-RA1 seemed to induce the nuclear factor (NF)-κB signaling cascade constitutively in podocytes by activating the tyrosine receptor kinase (TRK) family member Axl. Finally, the knockout mouse line for Pod-RA1 was successfully generated and its analysis is in progress. Conclusions:Pod-RA1 is so far the only podocyte gene that seems to be upregulated in DN. Based on our data, we speculate that the induction of Pod-RA1 expression may promote fibrogenic actions. Due to its enrichment to podocytes, Pod-RA1 may be a good target to develop cell-specific therapy for DN. Our further studies will assess the role of Pod-RA1 in vivo and explore whether modulating Pod-RA1 levels can influence DN progression in mice. P18 Transfer of prediabetic islets to a healthy environment improves survival Authors: Karin Åvall, Andrea Dicker, Erwin Ilegems, P-O Berggren and Lisa Juntti-Berggren The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet Aim: To study the effect of the surrounding environment versus the genetic and immunologic programming on the destruction of pancreatic islets in diabetes prone (DP) Bio Breeding (BB) rats, an animal model of type-1 diabetes (T1D). Methods: Islets from diabetes-resistant (DR) BB-rats were transplanted into the anterior chamber of the eye (ACE) of DP rats and islets from DP-rats were transplanted into DR animals. As control, islets from DR-rats were transplanted to DR recipients. The islets were thereafter followed non-invasively over time by confocal microscopy. Additionally, islets from DP rats treated with antisense against apoCIII were transplanted into DP rats, also treated with the antisense, to evaluate if a limited time with decreased apoCIII affects the outcome of the islets. Results: Islets from DR rats transplanted into the prediabetic milieu in the ACE of DP rats showed a similar decrease in size as seen in the endocrine pancreas of DP rats. When moving islets from DP to DR rats the transplanted islets adopted the behavior of the normal islets in the pancreas of the recipient. The temporary decrease of apoCIII in both donor and recipients improved the survival of the islets.

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Discussion: These data suggest that the islet itself does not decide its fate, but instead it is the surrounding prediabetic or healthy environment that has a major influence. Temporarily changing the interior milieu by reducing the levels of the pro-diabetic lipoprotein apoCIII has beneficial effects on the islets. P19 Dynamics of preadipocyte turnover in humans Keng-Yeh Fu1, Christina Jonas1, Peter Arner2, Kirsty Spalding1

1Department of Cell and Molecular Biology, Karolinska Institutet. 2Department of Medicine, Karolinska University Hospital

The expansion of human white adipose tissue (WAT) results from both hypertrophy of preexisting adipocytes and, in severe obesity, hyperplasia due to the formation of new adipocytes. Because adipocytes are considered to be postmitotic and no longer capable of undergoing cell division, the increase in adipocyte number therefore is thought to originate solely from the proliferation/differentiation of adipocyte progenitor cells (preadipocytes). Although such a process is crucial for the maintenance and enlargement of WAT mass, little is known about the in vivo cell turnover dynamics of human preadipocytes because of the difficulties in studying cell turnover in humans. In this study, we will investigate whether the preadipocyte population is largely quiescent, whether it proliferates and differentiates only when needed, or whether the population is constantly turning over. We will study using a carbon 14 dating, a method previously used to determine human adipocyte turnover (Spalding et al., 2008). Preadipocytes, defined as CD31-/CD34+/CD45-/CD235a- stromal vascular cells, will be isolated from human subcutaneous abdominal and omental fat depots and sorted via FACS. Preadipocyte DNA will be extracted and subsequently purified for carbon 14 analysis. Our aim is to investigate preadipocyte population size and turnover rate, across a range of ages and BMIs, in both the subcutaneous and visceral fat depots. Moreover, whether obesity-associated adipocyte hyperplasia results from larger preadipocyte population size in obese individuals, or from an increased preadipocyte proliferation rate in obese individuals will be analyzed. With the understanding of preadipocyte dynamics, we propose that preadipocyte turnover may represent a new avenue of therapeutic intervention against obesity. P20 Fatty acid-induced cross-talk between internal clock genes and insulin sensitivity in skeletal muscle cells. Laura Sardon-Puig, Nicolas J. Pillon, Anna Krook and Juleen Zierath Karolinska Institutet, Department of Physiology and Pharmacology (FYFA), Integrative Physiology Background and aims. Circadian rhythms occur in all species and modulate fundamental physiological processes. Day-night cycles align the phase of circadian rhythms to earth rotation, but most cells of the body follow an endogenous circadian clock independent of light exposure. Disruption of circadian cycles is associated with metabolic imbalance, and leads for instance to increased risk of diabetes in individuals working night shifts. This suggests that alterations in circadian rhythms could contribute to the worldwide epidemic of metabolic syndrome, but the pathophysiological mechanisms are poorly understood. Skeletal muscle is the major determinant of post-prandial glycaemia, and glucose disposal in muscle is a large determinant of whole-body

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insulin sensitivity. Skeletal muscle is also engaged in mechanical activity, and glucose uptake and metabolism in this tissue are exquisitely regulated by insulin and contractile activity. Surprisingly, very little is known about the cross-talk between metabolism and the internal clock in skeletal muscle and subsequent impact on whole body insulin sensitivity. The aim of this project is to study if and how metabolic challenges impair the skeletal muscle internal clock leading to disturbances in glucose and fatty acid metabolism. Materials and methods. Primary human skeletal muscle were isolated from muscle biopsies collected at the Karolinska Hospital. Satellite cells were grown and differentiated into myotubes by serum depletion. Muscle cells were synchronized using dexamethasone shock (200nM, 1h) and exposed to 0.2mM of palmitate (PA), oleate (OL) or a combination of both for up to 64h following synchronization. Samples were collected every 4-8h for RNA or protein extraction followed by qPCR and western blot for the analysis of gene expression and signalling pathways. Results. Primary skeletal muscle cells exhibit a circadian rhythm (period~24h) on the expression of several genes of the clock machinery (REVERB, CLOCK, PER1/2) as well as on some metabolism and inflammation-related genes (PGC1a, IL6). Palmitate or oleate reduced the amplitude of PER2 cycling while increasing the amplitude of CLOCK, suggesting that fatty acids have the ability to modulate the core clock. In parallel, palmitate, but not oleate impaired insulin-induced phosphorylation of Akt and glycogen synthesis in skeletal muscle cells, which occurred only after 24h exposure to fatty acids and concomitant with the induction of clock gene disturbances. Conclusion. Fatty acids have the ability to discretely affect elements of the core clock in skeletal muscle. Future experiments will analyse the interaction between insulin resistance and clock impairments to decipher the cause-consequence relationship between these two events. Identification of pathways linking metabolic and clock disturbances could lead to new pharmacological targets to target fatty acid-induced insulin resistance. P21 Role of maternal androgen excess and obesity in the development of metabolic and anxiety-like behavior in female and male offspring Manti M1*, Folmerz E1*, Qi X1,2*, Fornes R1, Lindén-Hirschberg A3, Jansson T4, Maliqueo M1,5, Benrick A6, Stener-Victorin E1. 1 Department of Physiology and Pharmacology, Karolinska Institutet, 17177 Stockholm, Sweden

2 Department of Physiology, Qiqihar Medical University, Qiqihar, Heilongjiang Province, 161006 P.R. China 3 Division of Obstetrics and Gynecology, Karolinska Institutet, Stockholm, Sweden 4 Department of Obstetrics & Gynecology, Division of Reproductive Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 5 Endocrinology and Metabolism Laboratory, Department of Medicine, West division, University of Chile, Santiago, Chile 6 Institute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden * Contributed equally. Background: Polycystic ovary syndrome (PCOS) have a 3-7 fold increased risk of developing type 2 diabetes and over 60% of women with PCOS are diagnosed with at least one psychiatric disorder such as anxiety or depression. Although a genetic basis for PCOS has been suggested, an emerging piece of evidence propose the intrauterine environment to play a fundamental role in the PCOS pathogenesis. Aim: To determine the effects of maternal androgen excess and high fat diet (HFD) on metabolic, reproductive and behavioural function in adult offspring in a sex-dependent manner. Methods: Female C57BL/6J mice were fed HFD or control diet for 10 weeks and then mated. To mimic maternal androgen excess, pregnant mice were injected with 250 µg of dihydrotestosterone in sesame oil (DHT) or sesame oil alone (Vehicle) on days 16–18 of gestation. Male and female

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offspring were subjected to measurements at 10 weeks of age and these will be repeated at 20 weeks of age, including body composition (DEXA), fasting blood glucose and insulin measurements and anxiety-like behaviour testing. Two-way ANOVA was applied to test interaction between mother’s diet, injection (DHT or Vehicle), offspring diet and sex. Preliminary results: The study is ongoing and part of the 10 weeks measurements are presented. Female offspring exposed for maternal androgen excess displayed disrupted estrus cycles and earlier puberty onset compared with vehicle injected mothers on control diet, which was not further altered by the maternal HFD. At 21 days of age, female and male offspring of mothers fed HFD weigh more than controls. Maternal androgen exposure in mice fed HFD lowered body weight in both female and male offspring compared with HFD + vehicle. At 10 weeks of age, differences in body weight were diminished in females, but not in the males. Maternal obesity increased total fat in male offspring with no effect of androgen exposure. No difference was observed in female offspring. Maternal androgen exposure reduced lean mass in male offspring compared with male offspring from vehicle injected mothers on control diet. Finally, both male and female offspring from mothers fed HFD and injected with DHT spent more time in closed arms and less time in the open arms of the elevated plus maze compared with control diet and DHT injection indicating that the maternal obesity causes an anxiety like behaviour. In the open field, female offspring from mothers on HFD and injected with DHT spent less time in the centre compared with the vehicle group on HFD, demonstrating that maternal androgen excess cause an anxiety like behaviour. Conclusion: This is the first study to combine a maternal androgen excess model with a maternal obesity model to investigate the developmental effects on reproductive, metabolic and behavioural functions. No conclusion is drawn from these preliminary findings. P22 Single acupuncture increases whole body glucose uptake and induces epigenetic and transcriptional changes in adipose tissue in polycystic ovary syndrome Milana Kokosar1, Anna Benrick1, Alexander Perfilyev2, Emma A Nilsson2, Manuel Maliqueo3, 4, Carl Johan Behre5, Antonina Sazonova6, Kurt Højlund7, Charlotte Ling2 and Elisabet Stener-Victorin1*

1Department of Physiology, University of Gothenburg, Sweden, 2Epigenetics and Diabetes, Department of Clinical Sciences, Lund University Diabetes Centre, Sweden, 3Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 4Endocrinology and Metabolism Laboratory, University of Chile, Santiago, Chile, 5The Wallenberg Laboratory, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 6Department of Obstetrics and Gynecology, University of Gothenburg, Sweden, 7Department of Endocrinology, Odense University, Denmark Aim: Impaired glucose regulation, hyperinsulinemia and insulin resistance are common features of polycystic ovary syndrome (PCOS) and we have shown that these metabolic abnormalities might be due to the aberrant DNA methylation and gene expression in subcutaneous adipose tissue (Kokosar et al, Sci Rep, 2016; Mar 15;6:22883. doi: 10.1038/srep22883). A single acupuncture treatment increases whole body glucose uptake in women with PCOS. Whether this effect is mediated via epigenetic and transcriptional changes in adipose tissue is unknown. Objectives: Does single acupuncture treatment remodels genome-wide expression and methylation pattern abnormalities in adipose tissue from 21 women with PCOS? Method: Subcutaneous adipose tissue biopsies were obtained before and after 45 minutes of acupuncture treatment with combined manual and low-frequency electrical stimulation. For analysis of gene expression, Illuminas direct hybridization whole-genome expression assay (HumanHT-12

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Expression Bead Chip) was used. DNA methylation in adipose tissue was analyzed by using Illumina Infinium HumanMethylation450 BeadChip covering 485,577 CpG sites. Differentially expressed genes were integrated with known pathways using Ingenuity Pathway Analysis (IPA, Qiagen). Whole body glucose uptake was measured by an euglycemic-hyperinsulinemic clamp. Results: In total 2471 individual genes changed expression before vs after acupuncture after false discovery rate corrections (FDR) Q <0.05. Out of the 2471 unique genes, 44.7% were up-regulated and 54.1% were down-regulated. Examples of genes with significant changes by a singly bout of acupuncture are ADIPOR2, TWIST2 and PPARG. Ingenuity pathway analysis indicates that acute phase response signaling, coagulation system, TNFR2 Signaling, LXR/RXR activation and Tec Kinase Signaling are the top five canonical pathways activated by a single acupuncture in adipose tissue. In total, methylation of 17 510 CpG sites changed in response to single acupuncture treatment (Q =0.11). Further analysis of the genome-wide gene expression and methylation data is being performed. Conclusion: A single acupuncture treatment increases whole body glucose uptake, an effect that, at least in part, may be mediated via epigenetic and transcriptional changes in adipose tissue in women with PCOS. P23 In vitro modelling of the influence of extracts from Hermetia illucens larvae on wound healing Gutzeit H.O., Adam A., Henker Y., Majnarić M. University of Zagreb, Croatia; Dresden University of Technology, Germany; Karolinska Institutet, Sweden

Larvae of black soldier fly, Hermetia illucens, are scavengers who live in extremely unpleasant and harsh environments, such as manures and compost, populated by bacteria and fungi. Carcasses of dead animals and rotting plants could be degraded by larvae of black soldier fly. These biological characteristics suggest that the larvae of black soldier fly are rich in antimicrobial peptides (AMPs) and other substances which possess the activity to fight against resistant bacterial strains. Also, secretions from Hermetia illucens larvae have proven to be useful in the fight against MRSA (Methicillin-resistant Staphylococcus aureus).

Wound healing is a complex process consisting of four overlapping but strictly defined phases: hemostasis, inflammatory phase, proliferative phase and the remodelling phase (maturation and epithelialization). All phases of wound healing are dependent on the secretion of a variety of cellular compounds such as growth factors, chemokines, cytokines, proteinases, and extracellular matrix proteins. AMPs are aimed to target different wounds but especially the chronic ones such as: foot ulcers which are one of the main diabetes complications. In vitro process of wound healing could be monitored using these different cellular compounds as molecular markers.Focus of this research is enrichment of extracts from Hermetia illucens larvae in order to obtain purified AMPs and other antibiotic compounds and in vitro monitoring of the impact of extracts from Hermetia illucens larvae on the expression of cellular molecules that are characteristic for the process of wound healing. Methods that were used and are still used in this study are: Methanol extraction of AMPs, Cytotoxicity assays, Bacterial Growth Assay, RNA extraction, RT-PCR, Western blot.

This research showed that different kinds of extracts provided such as: H20 Phase extract, Chloroform Phase extract and Ethyl-Acetate Phase extract inhibit growth of 4 different bacterial strains. Moreover, concentrations of extracts that inhibited bacterial growth didn't show toxicity when applied to both HepG2 and NHDF cells.

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Further research includes: Application of extracts to cells and monitoring the changes of expression of molecular markers related to wound healing process.

P24 Diet-induced metabolic disease in SNAP-25b-deficient mutant mice correlates to inflammation and functional changes in brain Muhammad Irfan1, Ismael Valladolid-Acebes1, Teresa Daraio1, Patric K. Stanton 2, Kerstin Brismar1, Tomas Hökfelt3, Christina Bark1. 1 Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden 2 Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, U.S.A. 3 Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden Assembled SNARE core-complex from SNAP-25, syntaxin and VAMP is required for activity-dependent neurotransmitter and neuro-endocrine exocytosis. We have engineered a mouse in which the major splice variant of SNAP-25, SNAP-25b, is replaced by the early expressed variant, SNAP-25a. This substitution predisposes these mutant mice to metabolic disease: obesity and type-2 diabetes, conditions aggravated by western diet (WD). We have now investigated the impact of this substitution, with and without WD, on the expression levels of other members of the SNARE core-complex, on components of down-stream insulin signaling pathway and on the cAMP signal transduction molecule ATF-3 in cerebellum, frontal cortex, hippocampus and hypothalamus. Male mutant mice on control diet exhibited upregulation of total SNAP-25, syntaxin and VAMP, an effect enhanced by WD. This diet also caused brain-region-specific upregulation of a novel SNARE protein, SNAP-47, in both wild-type and mutant-male mice. Female mutants on control diet exhibited upregulation of individual SNAREs but, in contrast to male mice this effect was reversed by the WD in cerebellum and frontal cortex. SNARE levels were unaffected in mutant male mice in hippocampus, but upregulated in female mutants fed either control or WD. We also observed region and sex specific changes in ATF-3 levels, downregulation of BDNF and total plus phosphorylated forms of AMPK, AKT and STAT-3 in response to both mutation and the WD. Changes in functional, metabolic and activating transcriptional factor protein expression levels were accompanied by inflammation, evidenced by astrocyte/microglial activation in brain and impaired performance in a battery of behavioral tests.

P25 Grandpaternal high-fat diet transgenerationally impacts the skeletal muscle unfolded protein response in F2-female offspring Authors: Petter S Alm1, Thais de Castro Barbosa1, Romain Barrès3, Anna Krook1, Juleen Zierath1,2,3. 1Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden 2Department of Molecular Medicine and Surgery, Section of Integrative Physiology, Karolinska Institutet, 171 76 Stockholm, Sweden 3The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark Background: Obesity is increasing worldwide. Parental overweight and obesity can disturb metabolism in the offspring in a transgenerational manner. We have previously shown that grandpaternal high-fat diet (HFD) impaired whole-body glucose metabolism in F2-female offspring. Here we aim to determine how the skeletal muscle is affected.

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Methods: Male Sprague–Dawley rats were fed a HFD or a control diet (CD) for 12 weeks, and bred with CD-fed females, creating the F1 generation. Male F1 rats on CD were bred with a new control female to create the F2 generation. Both generations were further subdivided and given either a HFD or a CD. The EDL muscle was dissected and analyzed by Affymetrix rat gene 1.1 ST microarray. Results: No differences were observed in muscle transcriptomics after correcting for multiple testing when comparing F2 offspring fed a CD. F2 offspring from a HFD-fed grandfather consuming a HFD showed a significant upregulation in the unfolded protein response. Genes selected for RT-qPCR validation confirmed this in F2 EDL, but none in F1. Next we investigated whether DNA methylation in the promoter region of the validated genes were altered with MBD-capturing technique. No significant changes were observed. Conclusion: Our preliminary results demonstrate that grandpaternal HFD consumption transgenerationally impacts F2-female offspring response to HFD in skeletal muscle transcriptomics. This indicates that an additional stressor is needed to induce a response from the grandpaternal legacy and highlights how our own lifestyle choices have a stronger impact than our parental heritage. P26 Genetic impact on obesity and the response to calorie restriction using the 5:2 method for 6 months Rixin Xiong1,2, Harvest F. Gu1, Kerstin Brismar1 1Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden 2 Department of Cardiology, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, P. R. China Background: Obesity is a non-communicable disease, reflecting a complex interplay between genetic variation and environmental factors. Calorie restriction may be an alternative treatment for obese subjects. The present study aimed to investigate the genetic impact on obesity and response to calorie restriction and also to understand the related gender differences. Subjects and methods: A total of 105 Swedish subjects (65 women and 40 men) were recruited via media and from outpatient clinics for diabetes care at Karolinska University Hospital and Sophiahemmet Hospital in Stockholm. Calorie restriction was conducted by using the 5:2 method i.e. 500 calories for women and 600 calories for men 2 days a week and 5 days of normal calorie intake for 6 months. Clinical parameters, including BMI, waist, fat composition etc., were examined. Genotypes of ADCY3, ADRB2, BDNF, FTO, NPY and PPARγ2 genetic polymorphisms were determined with TaqMan allelic discrimination protocol. The allele frequencies and genotype distributions of the studied polymorphisms associated with obesity and reported in Caucasians and other ethnic groups were included into the comparative analyses. Results: Reduction of BMI, waist and fat composition after calorie restriction for 6 months were observed. Compared with men, women had lower waist and higher fat percent in body. According to the genotypes, clinical parameters including BMI, waist and fat composition were found to be decreased in both men and women per see. In the ADCY3 gene, the G allele of SNP rs19684682 confers the risk susceptibility to obesity. The carriers with this allele after calorie restriction for 6 months were observed to be reduced significantly in waist among men (P=0.028) but not women (P=0.979). Summary: Preliminary results from this study suggest that calorie restriction according to the 5:2 method may be an effective treatment for obese and over-weight subjects. Gender difference and genetic influence have to be taken into the evaluation.

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P27 Effects of exercise on metabolic functions in dihydrotestosterone or letrozole induced mice models of polycystic ovary syndrome Rodrigo R. Marcondes1,2, Manuel Maliqueo1,3, Romina Fornes1, Anna Benrick4, Min Hu1, Niklas Ivarsson1, Mattias Carlström1, Samuel W. Cushman5, Karin G. Stenkula6, Gustavo A. R. Maciel2, Elisabet Stener-Victorin1

1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. 2Disciplina de Ginecologia, Laboratório de Ginecologia Estrutural e Molecular (LIM 58), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. 3Endocrinology and Metabolism Laboratory, Department of Medicine, West division, University of Chile, Santiago, Chile. 4Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. 5Experimental Diabetes, Metabolism, and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States of America 6Department of Experimental Medical Sciences, Lund University, Lund, Sweden Aims: To test the hypotheses that gene expression of Notch and browning signaling pathways, adipogenesis, lipid metabolism, and sympathetic activity are dysregulated in the adipose tissue of dihydrotestosterone (DHT)-, and letrozole-induced polycystic ovary syndrome (PCOS) mice models, and whether voluntary exercise restore these dysfunctions. Methods: PCOS phenotypes were induced by implantation of slow-releasing DHT or letrozole (LET) pellets mimicking hyperandrogenemia. Five weeks after pellet implantation, 4 to 5 weeks of voluntary exercise started: Sedentary controls, sedentary DHT, DHT+exercise (DHT+EX), sedentary LET, and LET+EX. Body weight, body composition, adipocyte morphology, insulin and glucose tolerance were assessed. Inguinal (sc) and mesenteric (visceral) adipose tissue mRNA expression were evaluated by RT-PCR. Results: The LET exposed mice weigh more, had an increased fat mass, were insulin intolerant, and displayed an increased inguinal mRNA expression of Prdm16, and increased mesenteric expression of Bmp7, Slc2a4, Lpl and Lipe compared with controls. The DHT exposed mice weigh more, displayed increased number of large inguinal adipocytes and mRNA expression of Bmp2, and lower mesenteric expression of Notch1 compared with controls. In LET exposed mice, exercise restored fat mass and insulin intolerance, and restore altered expressed genes in inguinal and mesenteric fat, except for Slc2a4. In addition, exercise decreased inguinal expression of Notch1 (indicating activation of browning), and mesenteric expression of Rbpj (Notch pathway) and genes related to lipid metabolism. In DHT exposed mice, exercise reduced fat mass and restored the number of large inguinal adipocytes and the expression of Bmp2. In addition, the mesenteric and inguinal expression of Notch1 and the mesenteric expression of Rbpj were decreased. Other genes that was upregulated by exercise in DHT exposed mice were Bmp7 and Cidea, indicating browning in inguinal fat, and Lpl and Lipe in mesenteric fat. Conclusions: Four to five weeks of voluntary exercise restored altered adiposity and insulin intolerance in the LET induced mice and altered adipose tissue morphology in the DHT induced mice. In these PCOS mice models the effect of exercise on metabolic and adipose tissue function may, at least in part, be explained by regulation of pathways related to Notch, browning and lipid metabolism.

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P28 Distinct impact of maternal diet on metabolic response in male and female offspring. Vasiliki Karagianni1, Marcela Gonzalez-Granillo1, Christina Savva1, Marion Korach-André1 1 Department of Medicine, Karolinska Institutet, Karolinska University Hospital, NOVUM, Integrated Cardio Metabolic Center, SE-14157 Huddinge, Sweden

Aims: To investigate the effect of maternal and early neonatal diet on male and female offspring’s physiology and metabolism, using a longitudinal in vivo approach. Methods: C57BI/6 female mice were placed on a chow diet (C) or a high- fat diet (HF) before mating, during pregnancy and weaning. After weaning, female and male offspring from chow diet mothers were fed chow diet (CC) and offspring from high-fat fed mothers were fed either chow (HFC) or high-fat diet (HFHF). Body weight was measured weekly. At 3 and 6 months of age, in vivo magnetic resonance imaging and spectroscopy were conducted for fat distribution and lipid composition assessment in liver, visceral and subcutaneous adipose tissue. Oral glucose tolerance test and insulin tolerance test were performed. Offspring were sacrificed at 24 weeks of age. Plasma and several tissues were isolated for future analysis. Results: Body weight and fat deposition were increased in HFC offspring compared to CC and even more in HFHF offspring. HFC males accumulated more visceral and less subcutaneous fat than CC. HFC females stored proportionally more saturated fat in liver, while HFC and HFHF males stored less than CC. HFC and HFHF males developed hyperglycemia and insulin resistance, whereas females showed the same trend to a lower extent. HFHF offspring showed higher levels of glycemia and insulin secretion compared to HFC and CC in both sexes. Conclusions: Maternal high fat diet can induce metabolic and physiological deregulations in offspring and these effects are aggravated in combination with high fat offspring diet. P29 Mapping Of Brown Adipose Tissue In Different Depots Of Adult Humans Kozina V.1, Slomianka L.2, Ottosson A.3, Spalding, KL.1 1 Department of Cell and Molecular Biology, Karolinska Institute and Integrated Cardio Metabolic Centre, Department of Medicine, Huddinge, Stockholm, Sweden 2 Division of Functional Neuroanatomy, Institute of Anatomy, University of Zürich, Switzerland 3 National Board of Forensic Medicine, Swedish Unit for Forensic Medicine, Department of Clinical Sciences, Lund University, Sweden With the global economic impact of obesity estimated to be around 2 trillion dollars per year, the scientific community is striving to find new ways to address this epidemic. One possible therapeutic target is brown adipose tissue, proven to be present in adults several years ago. To determine whether the ability of brown adipose tissue to burn energy rather than store it is a relevant candidate to fight obesity, we first have to answer some fundamental questions. Does brown adipose tissue exist in all adults? What is the prevalence of brown adipose tissue and how does this relate to various human parameters (such as age, BMI, sex)? In order to answer these questions, we are compiling a comprehensive map of brown adipose tissue prevalence in humans. Adipose tissue samples have been collected from all regions previously reported to contain brown adipocytes: neck, thyroid, supraclavicular, heart, pericardial, adrenal, renal and paravertebral. Our aim is to quantify the number and size of white, putative beige (unilocular UCP1 positive) and brown adipocytes per unit volume in each depot using unbiased stereology. Furthermore, the correlation between white adipocyte cell size and the amount of brown fat in each depot will be assessed. Of interest is also whether the amount of brown adipose tissue correlates with seasonal temperatures at death, as brown fat is known to be stimulated by cold exposure.

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The study has collected postmortem adipose samples from 60 individuals, with varying age, sex and BMI. Tissue was fixed in 4% PFA and embedded in paraffin. Slides with 5µm sections were stained with anti-UCP1 antibody and counterstained with PAS and haematoxylin to visualize adipocyte cell borders. White, brown, vascular and interstitial area are estimated using a combination of area fraction fractionator and nucleator probe stereological methods, using a bright field microscope equipped with Stereoinvestigator® software. Preliminary assessment has revealed brown in 61% of neck samples (31 of 51), thyroid 29% (14/49), supraclavicular 59% (33/56), heart 5/59 (8%), pericardial 45% (24/53), adrenal 81% (44/54), renal 63% (30/48) and paravertebral 100% (6/6). Quantification of the ratio of brown to white within each depot is ongoing. Despite significant therapeutic interest in brown adipose in the fight against obesity, the prevalence of brown adipose amongst people of different age, BMI and gender without cold or adrenergic stimulation is unknown. Results of this ongoing study will assess this missing piece of the puzzle. P30 HIF-1 attenuates oxidative stress and ameliorates renal function in diabetes Xiaowei Zheng, Ileana Ruxandra Botusan, Sampath Narayanan, Cheng Xu, Marianna del Sole, Jacob Grünler, Elisabete Forsberg, Vivekananda Gupta Sunkari, Kerstin Brismar, Sergiu-Bogdan Catrina Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.

Background and aims: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) and is a major cause of mortality in patients with diabetes. Increased reactive oxygen species (ROS) production from mitochondria is proposed as pathogenic mediator for DN. Recently, hypoxia has been shown to play an important pathogenic role for the development of DN. Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the adaptive cellular responses to hypoxia. Under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase domain proteins (PHD). The hydroxylated HIF-1α is then recognized by the von Hippel-Lindeau (VHL) protein, which targets HIF-1α for proteasomal degradation. Under hypoxic conditions, HIF-1α is stabilized and binds to the hypoxia response element (HRE) in the promoter of HIF-1 target genes and activates gene transcription. Despite profound hypoxia in diabetic organs, HIF-1 signalling is inhibited in diabetes. However, the role of HIF-1 in the development of DN is still controversial. In this study, we intend to investigate the role of HIF-1 in regulating mitochondrial ROS production and renal function in diabetes.

Materials and Methods: Renal mIMCD-3 (ATCC) were cultured in normoxia or hypoxia, and exposed to either normal or high glucose levels for 24h. Mitochondrial ROS production was detected by FACS after incubation with MitoSOX, and apoptosis was detected by FACS using Annexin V / 7-AAD kit. Endogenous HIF-1 function was detected as HRE-driven luciferase reporter activity. HIF-1 target gene expression were detected using Real-Time PCR. DMOG (Dimethyloxalyl Glycine) was used to stabilize HIF-1α both in vitro and in vivo. Db/db diabetes mice were obtained from Janvier Labs. Kidney-specific VHL knockout mice were generated by breeding VHL loxP/loxP mice with Cdh16-cre transgenic mice. Diabetes was induced with streptozotocin. Albuminuria and renal ROS production were detected using ELISA methods.

Results: Mitochondrial ROS production and apoptosis increased when renal mIMCD-3 cells were exposed to high glucose levels in hypoxia. High glucose levels suppressed endogenous HIF-1 function in hypoxia as shown by reduced HRE-driven luciferase reporter activity and HIF-1 target gene expression. Recovery of HIF-1 function, through either inhibition of PHD with DMOG or by silencing VHL, attenuated the mitochondrial ROS production and the rate of apoptosis under concomitant exposure to hypoxia and hyperglycemia. These results suggest a potential

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renoprotective role of HIF-1 in diabetes. In order to explore the role of HIF-1 for DN we have induced HIF-1 in diabetic animals using either pharmacological approach with DMOG or genetical approach by knocking down VHL specifically in kidney. DMOG treatment of db/db mice for one month reduced albuminuria and attenuated ROS production in the kidney. Similar beneficial effects of HIF-1 on diabetes-induced albuminuria and renal ROS production was observed in kidney-specific VHL knockout mice where HIF-1 is activated in the kidney, confirming the renoprotective role of HIF-1 in diabetes nephropathy.

Conclusion: HIF-1 attenuates renal oxidative stress and ameliorates renal function in diabetes. Increasing renal HIF-1 function is a promising therapeutic strategy for diabetes nephropathy.

Grants: VR, Family Erling-Persson Foundation, SLL, Tore Nilssons Foundation

P31 Differential expression of genes influencing skeletal muscle metabolism in women with polycystic ovary syndrome Emma Nilsson1, Anna Benrick2, Milana Kokosar2, Carl Johan Behre3, Antonina Sazonova4, Kurt Højlund5, Charlotte Ling1, Elisabet Stener-Victorin6

1Epigenetics and Diabetes, Department of Clinical Sciences, Lund University Diabetes Centre, Lund University, Clinical Research Centre, Malmö, Sweden 2Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 3The Wallenberg Laboratory and Sahlgrenska Center for Cardiovascular and Metabolic Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 4Department of Obstetrics and Gynecology, Reproductive Medicine, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 5Department of Endocrinology, Odense University, Denmark 6Department of Physiology and Pharmacology, Karolinska Institutet, 17177 Stockholm, Sweden Background and aim: Insulin resistance in skeletal muscle is a major risk factor for the development of type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the underlying molecular mechanisms are largely unknown. The aim of this study was to investigate the genome-wide gene expression patterns in skeletal muscle from PCOS women and healthy controls. Material and Methods: Skeletal muscle biopsies were obtained from 17 women with PCOS and 14 controls matched for age, weight and BMI. For analysis of gene expression, Illuminas Human HT-12 Expression BeadChip was used. Results: Women with PCOS had more antral follicles, larger ovaries and higher circulating testosterone, LH and LH/FSH ratio and lower circulating prolactin than controls. Fasting triglycerides, C-peptide, calculated C-peptide index, and HOMA-B were higher in women with PCOS than in controls indicating metabolic aberration and insulin resistance. After FDR-correction, 85 genes were found to be differentially expressed in skeletal muscle from PCOS women versus controls (q<0.05). 66% of the genes were up-regulated and 34% were down-regulated in PCOS women and the absolute expression differences were 21-186%. Several of the identified genes are suggested to play a role in muscle function and metabolism, including DYRK1A, TTN, ZBTB16, FOXO3 and SCP2. According to a gene set enrichment analysis, there was a significant gene expression disruption to pathways involved in the immune system in PCOS women. Also, several GWAS candidate genes for insulin resistance (e.g. APOE and PPARG) were differentially

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expressed in PCOS women versus controls. Analyses of genome-wide DNA methylation are on-going. Conclusion: We here demonstrate that PCOS is associated with gene expression aberrations in skeletal muscle which may explain some of the metabolic abnormalities seen in these women. P32 Charting the gut microbiome in polycystic ovary syndrome (PCOS): A pilot study Lisa Lindheim1, Mina Bashir1, Julia Münzker1, Christian Trummer1, Verena Zachhuber1, Bettina Leber2,3, Thomas R. Pieber1,3, Gregor Gorkiewicz4,5, Vanessa Stadlbauer3,6, Barbara Obermayer-Pietsch1,3

1Department of Internal Medicine, Division of Endocrinology and Diabetology, Medical University Graz, Austria 2Department of Surgery, Division of Transplantation Surgery, Medical University Graz, Austria 3Center for Biomarker Research in Medicine (CBmed), Graz, Austria 4Institute of Pathology, Medical University Graz, Austria 5Biotechmed, Interuniversity Cooperation, Graz, Austria 6Department of Internal Medicine, Division of Gastroenterology and Hepatology, Medical University Graz, Austria Background and Aims: Polycystic ovary syndrome (PCOS) is a common female endocrinopathy of unclear origin exhibiting hyperandrogenism, oligo-/anovulation, and ovarian cysts, frequently accompanied by dysglycemia, dyslipidemia, and inflammation. The gut microbiome is a putative regulator of body weight, insulin sensitivity, and gut barrier function. Endotoxinemia resulting from a leaky gut can cause inflammation, perpetuating insulin resistance, fat accumulation, and hyperandrogenemia. In this pilot study, we aimed to describe the stool microbiome, gut permeability, and systemic inflammation in women with PCOS compared to healthy controls. Methods: 16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analyses were conducted in mothur and QIIME. Gut barrier integrity and inflammatory status were evaluated using surrogate markers in serum and stool. All study subjects were characterized regarding reproductive, metabolic, and anthropometric parameters. Results: The stool microbiome of PCOS patients showed a 15 % diversity reduction and phylogenetic shifts in community composition compared to controls. Three taxa, RF39 and ML615J-28 from the phylum Tenericutes, and S24-7 from the phylum Bacteroidetes, were significantly reduced in PCOS patients. Serum zonulin, blood leukocytes, and blood lymphocytes were significantly higher in PCOS patients. Other markers of gut permeability and inflammation were unchanged between the two groups. Conclusion: PCOS patients exhibit decreased stool bacterial diversity and shifts in microbiome profiles with decreased abundance of taxa associated with leanness. The gut barrier may be impaired in PCOS, but endotoxinemia and inflammation were not driving factors in our pilot cohort.

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P33 A new class of therapeutic agents that targets PGC-11 to treat metabolic disease Pettersson-Klein AT 1, Izadi M 1, Ferreira DMS 1, Martinez-Redondo V 1, Correia JC 1, Agudelo L 1, Cervenka I 1, Porsmyr-Palmertz M 1, Sustarsic EG 2, Cameron M 3, Otrocka M 4, Lundgren B 5,Jenmalm-Jensen A 4, Gerhart-Hines Z 2, Griffin PR 3, Ruas JL 1. 1 Molecular & Cellular Exercise Physiology. Department of Physiology and Pharmacology, Karolinska Institutet, Sweden. 2 Section for Metabolic Receptology, The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark. 3 The Scripps Research Institute, Florida. 4 The Laboratories for Chemical Biology at Karolinska Institutet, Sweden. 5 Science for Life Laboratory, Sweden. The peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) regulates genes involved in energy metabolism and is expressed in energy-demanding tissues like fat, muscle, liver and brain. Activation of PGC-1α1 has been suggested to have beneficial effects on whole-body metabolism by driving browning of adipose tissue, increasing energy expenditure, and improving skeletal muscle fuel handling. PGC-11 has also been shown to have potent anti-inflammatory and anti-atrophic properties when expressed in murine models. PGC-1α1 activation is a highly regulated process and can be achieved by several mechanisms. However, protein stabilization is always a limiting step as the protein has a short half-life at baseline. We designed a cell-based platform to screen for stabilizers of PGC-1α1 protein in a high-throughput format. After screening a subset of a small molecule compound library, positive hits were validated in brown fat cells by western blot and quantitative real-time PCR to assess PGC-1α1 protein stabilization and activation of target gene expression, respectively. Compounds that robustly induced PGC-1α1 protein and activated target genes in the brown fat cell line were further tested in primary white and brown adipocytes from mouse. The effect of compound treatment was evaluated by ability to induce uncoupling of mitochondrial respiration (using a Seahorse Extracellular Flux Analyzer), PGC-1α1 target genes as well as beige and/or brown gene programs (quantitative real-time PCR). Successful compounds will be tested in primary human white adipocytes for their ability to induce beige gene programs. Ultimately, promising compounds will be tested for efficacy in vivo in mice. P34 Transcriptomic profiling of Kupffer cells in obese insulin resistant mice highlights the role of vascular regulation rather than inflammation Cecilia Morgantini1, Jennifer Jager1, Xidan Li1, Laura Levi1, Emelie Barreby1, Marcela Aparicio-Vergara1, Kjell Hultenby2, Myriam Aouadi1 1Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Center, Department of Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Sweden. 2Department of Laboratory Medicine (LABMED), Division of Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.

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Immune cells, such as macrophages, have been shown to be an important component in the pathogenesis of insulin resistance. Liver resident macrophages, namely Kupffer cells (KCs), constitute most of the macrophage population in the body. The direct contribution of KCs to insulin resistance has not yet been tested. The aim of this study is to discover pathways in KCs regulating insulin sensitivity. KCs have been isolated from two models of obese insulin-resistant mice (mice fed high-fat diet (HFD) and Ob/Ob mice), and from aged matched insulin-sensitive lean mice (ND). To isolate KCs we used a protocol developed by our laboratory including a combination of enzymatic digestion, density gradient centrifugation and selective adherence. After RNA isolation, next generation sequencing (RNAseq) was performed. Two hundred and eighty-seven genes were found up-regulated both in HFD and Ob/Ob, when compared to ND mice. Surprisingly, we did not observed any significant change in the expression of inflammatory markers such as interleukine IL-1α, or chemokines CXCL2, CCL7 and CCL4. Unexpectedly, our RNAseq data obtained from a pure population of KCs showed an increase in the expression of genes involved in endothelial regulation and vessels permeability. Interestingly, we observe an increase in the size of endothelial pores, called fenestrae, in obese compared to lean mice. Using a unique technology to silence genes in KCs we will test whether silencing newly identified genes regulate endothelial function and consequently insulin resistance. P35 Coro2b is a novel actin regulating protein of podocyte foot processes that may play a role in the pathogenesis of diabetic nephropathy Angelina Schwarz and Jaakko Patrakka KI/AZ Integrated Cardio Metabolic Centre (ICMC), Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Objective: In diabetic nephropathy (DN) podocyte cells are injured and eventually lost. Morphologically, podocytes exhibit foot process effacement. This change is considered to be a central mechanism in the pathogenesis of proteinuria. The effacement is caused by the reorganization of foot process actin cytoskeleton. Therefore, finding new ways to stabilize foot process cytoskeleton can open up possibilities to treat DN and other proteinuric diseases. In this study, we aim to identify new podocyte-enriched molecules that regulate the actin cytoskeleton in foot processes. We want to characterize their role in the biology and diseases using various in vitro and in vivo models. Design: To identify new podocyte-enriched actin-regulators we perform RNA sequencing of isolated glomeruli as well as analyze publicly available transcriptome databases. We validate the expression of candidate molecules in podocytes using RT-PCR, qPCR, immunohistochemistry and Western blotting. We analyze their function by inactivating them in zebrafish and mouse, as well as analyze them in cultured podocytes through over/underexpression experiments. In addition, we analyze their expression in human DN podocytes using immunohistochemistry. Results: We identified several novel podocyte-enriched molecules that were, based on their amino acid sequence, associating with actin. Of these, we analyzed in detail coronin 2b (Coro2b). By RT-PCR, qPCR and immunohistochemistry Coro2b expression in the kidney was detected only in podocytes. In cultured podocytes the overexpression of Coro2b resulted in the reorganization of actin cytoskeleton. In zebrafish, the inactivation caused podocyte abnormalities and proteinuria. In human patients with DN, we detected decreased expression of Coro2b which did not seem to be related to podocytopenia. Finally, we generated a KO mouse line for Coro2b and the phenotypical analysis is in progress.

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Conclusions: Coro2b is a highly podocyte-specific actin regulating molecule that is differentially expressed in human DN and important for the podocyte function in zebrafish. We speculate that Coro2b modulates the organization of foot process cytoskeleton and play a role in the pathogenesis of proteinuria. P36 Loss of corepressor GPS2 sensitizes macrophage activation upon metabolic stress in obesity and type-2 diabetes Rongrong (Rocky) Fan1, Amine Toubal2, Saioa Goñi1, Zhiqiang Huang1, Anastasios Damdimopoulous1, Ning Liang1, Nicolas Venteclef2 and Eckardt Treuter1 1Karolinska Institutet, Department of Biosciences and Nutrition, S-14183 Huddinge, Sweden. 2Université Pierre et Marie-Curie, INSERM, UMR_S 1138 Cordeliers Research, Paris, France Obese humans differ in their susceptibility to develop insulin resistance and type-2 diabetes (T2D), which may in part relate to the extent of adipose tissue inflammation. Macrophage activation states play a central role in this process and are driven by epigenome alterations linked to gene expression, yet the underlying mechanisms and regulatory components are currently poorly defined. Here we demonstrate that an anti-inflammatory corepressor complex containing the core subunits GPS2 (G-protein pathway suppressor 2), NCOR2 (nuclear receptor corepressor 2, also known as SMRT) and HDAC3 (histone deacetylase 3), critically controls the macrophage epigenome during metabolic activation. The study of adipose tissue from obese versus non-obese humans revealed correlations between reduced GPS2 expression in macrophages, elevated inflammation, and diabetic status. The causality of this relationship was confirmed using macrophage-specific Gps2 knockout mice, where inappropriate corepressor complex function causes increased enhancer activity, pro-inflammatory gene expression (in particular Ccl2) and hypersensitivity towards metabolic signals. In contrast, overexpression of GPS2 by bone marrow transplantation in two obese mouse models (ob/ob, diet-induced obesity) reduced inflammation and improved insulin sensitivity. Thus, our data reveal a potentially reversible disease mechanism that links corepressor-dependent epigenomic alterations in macrophages to adipose tissue inflammation and the development of T2D. Since the underlying mechanisms appear conserved between mice and humans, therapeutic possibilities to target the GPS2-inflammation-axis may emerge, for example via restoring GPS2 expression and function in diabetic subjects.

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List of Participants

First name

Last name Email address

Juan Acosta juan.acosta@ki.se Ulf Adamson Ulfadamson@gmail.com Leandro Agudelo leandro.agudelo@ki.se Ulf Ahlgren ulf.ahlgren@umu.se Ozgur Aldemir aldemir.ozgur@gmail.com Amani Al-Khaifi amani.al-khaifi@ki.se Petter Alm petter.alm@gmail.com Renato Alves renato.alves@ki.se Filipa Alves fcaeiroalves@gmail.com Daniel P Andersson Daniel.P.Andersson@ki.se Olov Andersson olov.andersson@ki.se Björn Anderstam bjorn.anderstam@ki.se Bo Angelin bo.angelin@ki.se Ishrath Ansurudeen ishrath.ansurudeen@ki.se Peter Arner peter.arner@ki.se Myriam Aouadi myriam.aouadi@ki.se John Axelsson john.axelsson@ki.se Christina Bark Christina.Bark@ki.se Christopher Barker chris.barker@ki.se Afroditi Alexandra Barouti afroditi.barouti@stud.ki.se Emelie Barreby emelie.barreby@ki.se Yasmina Belarbi Yasmina.belarbi@ki.se Anna Benrick anna.benrick@gu.se Magnus Bentinger magnus.bentinger@ki.se Per-Olof Berggren per-olof.berggren@ki.se Debajit Bhowmick debajit.bhowmick@ki.se Christel Björk christel.bjork@ki.se Anneli Björklund anneli.bjorklund@ki.se Marie Björnholm marie.bjornholm@ki.se Patrik Blomquist patrik.blomquist@kiinnovations.se Caroline Blomquist caroline.blomquist@umu.se Matthias Blüher matthias.blueher@uniklinik-leipzig.de Ylva Bonde ylva.bonde@ki.se Kerstin Brismar kerstin.brismar@ki.se Christian Broberger Christian.Broberger@ki.se Sergiu Catrina sergiu-bogdan.catrina@ki.se Igor Cervenka igor.cervenka@ki.se Jeremie Charbord jeremie.charbord@ki.se Alexander Chibalin alexander.chibalin@ki.se Lianhe Chu lianhe.chu@ki.se

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Patricia Corrales patricia.corrales@urjc.es Jorge Correia jorge.correia@ki.se Shamim Dadvar shamim.dadvar@stud.ki.se Ingrid Dahlman ingrid.dahlman@ki.se Lu Dai dailuemma@163.com Teresa Daraio teresa.daraio@ki.se Elisabetta Daré Elisabetta.Dare@ki.se Vladimer Darsalia vladimer.darsalia@ki.se Carsten Daub carsten.daub@ki.se Andrea Dicker Andrea.Dicker@ki.se Helena Edlund helena.edlund@umu.se Ewa Ehrenborg ewa.ehrenborg@ki.se Anna Ehrlund anna.ehrlund@ki.se Rongrong Fan rongrong.fan@ki.se Duarte Ferreira duarte.ferreira@ki.se Rachel Fisher rachel.fisher@ki.se John Flanagan john.flanagan@ki.se Elin Folmerz elin.folmerz@gmail.com Romina Fornes romina.fornes@ki.se Keng-Yeh Fu keng-yeh.fu@ki.se Brendan Gabriel brendan.gabriel@ki.se Hui Gao hui.gao@ki.se Alfredo Gimenez-Cassina Alfredo.gimenez-cassina@ki.se Marcela Gonzalez Granillo marcela.gonzalez@ki.se Ana Gracia anajadraque@gmail.com Harvest Gu harvest.gu@ki.se Tibor Harkany Tibor.Harkany@ki.se Emilio Hirsch emilio.hirsch@unito.it Tomas Hökfelt Tomas.Hokfelt@ki.se Erwin Ilegems erwin.ilegems@ki.se Muhammad Irfan muhammad.irfan@ki.se Nasren Jaff nasren.jaff@ki.se Jennifer Jager Jennifer.Jager@ki.se Christina Jones Christina.Jones@ki.se Arja Kants arja.kants@ki.se Vasiliki Karagianni vasso_karagianni@yahoo.gr Mutsumi Katayama mutsumi.katayama@ki.se Yurika Katsu Jiménez yurikakatsu@gmail.com Sander Kersten sander.kersten@wur.nl Lars Ketscher lars.ketscher@ki.se Amanda Klein amanda.klein@ki.se Milana Kokosar milana.kokosar@ki.se Marion Korach-André marion.korach-andre@ki.se Viviana Kozina viviana.kozina@ki.se Anna Krook Anna.Krook@ki.se Agné Kulyté agne.kulyte@ki.se Maria Kutschke maria.kutschke@ki.se Olof Lagerlöf olof.lagerlof@gmail.com

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David Lassiter david.gray.lassiter@ki.se Jurga Laurencikiene jurga.laurencikiene@ki.se Ingo Leibiger ingo.leibiger@ki.se Barbara Leibiger Barbara.Leibiger@ki.se Laura Levi laura.levi@ki.se Grazyna Lietzau grazyna.lietzau@ki.se Eva Lindgren eva.lindgren@ki.se Lisa Lindheim lisa.lindheim@ki.se Ka Cheuk Liu benjamin.liu@ki.se Leonidas Lundell leonidas.lundell@ki.se Zuheng Ma zuheng.ma@ki.se Mislav Majnaric mislav.majnaric@gmail.com Katarzyna Maleńczyk katarzyna.malenczyk@ki.se Maria Manti maria.manti@ki.se Rodrigo Marcondes rodrigo.marcondes@ki.se Vicente Martínez-Redondo vicente.martinez-redondo@ki.se Julie Massart julie.massart@ki.se Enrichetta Mileti enrichetta.mileti@ki.se Tilo Moede tilo.moede@ki.se Cecilia Morgantini cecilia.morgantini@ki.se Jonathan Mudry jonathan.mudry@ki.se Katja Möller-Hackbarth katja.moller-hackbarth@ki.se Emma Nilsson emma_a.nilsson@med.lu.se Stefan Nobel stefan.nobel@ki.se Eduardo Nolasco eduardo.nolasco@ki.se Elisabeth Norén-Krog elisabeth.noren-krog@ki.se Erik Näslund Erik.Naslund@ki.se Markus Näslund markus.naslund@stud.ki.se Camilla Olofsson camilla.olofsson@ki.se Tommy Olsson Tommy.G.Olsson@umu.se Paolo Parini paolo.parini@ki.se Meike Paschen meike.paschen@ki.se Jaakko Patrakka jaakko.patrakka@ki.se Cesare Patrone cesare.patrone@ki.se Teresa Pereira teresa.pereira@ki.se Paul Petrus paul.petrus@ki.se Nicolas Pillon nicolas.pillon@ki.se Margareta Porsmyr Palmertz Margareta.Porsmyr.Palmertz@ki.se Neil Portwood Neil.Portwood@ki.se Camilla Pramfalk camilla.pramfalk@ki.se Xiaojuan Qi qi.xiaojuan@ki.se Subu Surendran Rajasekaran subu.rajasekaran@ki.se Essam Refai essam.refai@ki.se Jorge Ruas jorge.ruas@ki.se Max Ruby maxwellruby@ki.se Mats Rudling mats.rudling@ki.se Mats Ryberg mats.ryberg@umu.se Mikael Ryden mikael.ryden@ki.se

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Laura Sardon laura.sardon.puig@ki.se Mladen Savikj mladen.savikj@ki.se Christina Savva christinasavva7@gmail.com Angelina Schwarz angelina.schwarz@ki.se Milena Schönke milena.schonke@ki.se Indranil Sinha indranil.sinha.2@ki.se Kirsty Spalding Kirsty.spalding@ki.se Pär Steneberg par.steneberg@umu.se Elisabet Stener-Victorin elisabet.stener-victorin@ki.se Peter Stenvinkel peter.stenvinkel@ki.se Sara Straniero sara.straniero@ki.se Rona J Strawbridge rona.strawbridge@ki.se Barbro Svensson barbro.svensson@ki.se Lingjie Tao lingjie.tao@ki.se Michael Tekle michael.tekle@ki.se Eckardt Treuter eckardt.treuter@ki.se Matthias H. Tschöp matthias.tschoep@helmholtz-

muenchen.de Dominika Tworus dominika.tworus@ki.se Dorina Ujvari dorina.ujvari@ki.se Pim van Krieken pim.van.krieken@ki.se Carmela Vazquez Calvo carmela.vazquez.calvo@stud.ki.se Francisco Verdeguer francisco.verdeguer@ki.se Emma Vernersson Lindahl emma.vernersson-lindahl@umu.se Ulrika Widegren ulrika.widegren@ki.se Tina Vilsbøll d299731@dadlnet.dk Montserrat Visa Majoral montse22_visa@hotmail.com Anna Witasp anna.witasp@ki.se Rixin Xiong xiongrixin@163.com Sonia Zambrano Sevilla sonia.zambrano.sevilla@ki.se Elizabeta Zaplatic elizabeta.zaplatic@ki.se Xiaowei Zheng xiaowei.zheng@ki.se Juleen Zierath juleen.zierath@ki.se Karin Åvall karin.avall@ki.se Claes-Göran Östenson claes-goran.ostenson@ki.se

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