Page 1 of 62 Diabetes JNK activation of BIM promotes hepatic oxidative stress, steatosis and insulin resistance in obesity. Sara A. Litwak1, Lokman Pang1,2, Sandra Galic1, Mariana Igoillo-Esteve3, William J. Stanley1,2, Jean-Valery Turatsinze3, Kim Loh1, Helen E. Thomas1,2, Arpeeta Sharma4, Eric Trepo5,6, Christophe Moreno5,6, Daniel J. Gough7,8, Decio L. Eizirik3, Judy B. de Haan4, Esteban N. Gurzov1,2,a 1St Vincent’s Institute of Medical Research, Melbourne, Australia. 2Department of Medicine, St. Vincent’s Hospital, The University of Melbourne, Melbourne, Australia. 3ULB Center for Diabetes Research, Université Libre de Bruxelles (ULB), Brussels, Belgium. 4Oxidative Stress Laboratory, Basic Science Division, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia. 5CUB Hôpital Erasme, Université Libre de Bruxelles (ULB), Belgium. 6Laboratory of experimental Gastroenterology, Université Libre de Bruxelles (ULB), Belgium. 7Hudson Institute of Medical Research, Clayton, VIC, Australia. 8Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia. aPresent address, to where correspondence and reprint requests should be addressed: Dr Esteban N. Gurzov ULB Center for Diabetes Research Université Libre de Bruxelles Campus Erasme, Route de Lennik 808, B-1070-Brussels-Belgium Phone: +32 2 5556242 Fax: +32 2 5556239 Email: [email protected] Disclosure statement: The authors declare no conflict of interest Running Title: BIM regulates lipid metabolism in hepatocytes. 1 Diabetes Publish Ahead of Print, published online September 19, 2017 Diabetes Page 2 of 62 ABSTRACT The BCL-2 family are crucial regulators of the mitochondrial pathway of apoptosis in normal physiology and disease. Besides their role in cell death, BCL-2 proteins have been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism. It remains unclear, however, whether these proteins have a physiological role in glucose homeostasis and metabolism in vivo. Here we report that fat accumulation in the liver increases JNK-dependent BIM expression in hepatocytes. To determine the consequences of hepatic BIM deficiency in diet- induced obesity, we generated liver-specific BIM knockout (BLKO) mice. BLKO mice had lower hepatic lipid content, increased insulin signalling and improved global glucose metabolism. Consistent with these findings, lipogenic and lipid uptake genes were downregulated and lipid oxidation enhanced in obese BLKO mice. Mechanistically, BIM deficiency improved mitochondrial function and decreased oxidative stress, oxidation of protein tyrosine phosphatases and ameliorated activation of PPAR-γ/SREBP1/CD36 in hepatocytes from high fat fed mice. Importantly, short- term knockdown of BIM rescued obese mice from insulin resistance, evidenced by reduced fat accumulation and improved insulin sensitivity. Our data indicate that BIM is an important regulator of liver dysfunction in obesity, and a novel therapeutic target for restoring hepatocyte function. 2 Page 3 of 62 Diabetes INTRODUCTION Obesity is a major risk factor for the development of severe complications such as non- alcoholic fatty liver disease (NAFLD), cardiovascular disease and diabetes (1). The liver has an essential role in the regulation of glucose homeostasis, producing glucose during fasting periods to prevent hypoglycaemia, maintaining brain function and survival. In obese individuals, excessive nutrients are stored in the liver as fat droplets (hepatosteatosis), which can eventually result in inflammation, insulin resistance and diabetes (2; 3). Obesity triggers activation of the c-Jun N-terminal kinase (JNK) pathway in hepatocytes with subsequent insulin resistance as well as steatosis (4; 5). The mechanisms of obesity-induced JNK activation include endoplasmic reticulum (ER) and oxidative stress, Toll-like receptors (TLRs) and inflammatory cytokines, such as tumor necrosis factor (TNF)-α (6). Hepatocyte‐specific deletion of both JNK1 and JNK2 improved glucose and insulin tolerance, increased hepatic insulin action and lowered fasting blood glucose levels in obese mice (7). However, the molecular pathways and proteins mediating JNK-induced hepatocyte dysfunction remain to be fully elucidated. The BCL-2 proteins are key regulators of apoptotic pathways (8), but growing evidence indicates that these proteins can also play an important role in glucose homeostasis and metabolism (9; 10). We have recently reported that loss of the BCL- 2 protein PUMA influences circulating leptin levels and food intake in mice (11). Moreover, BCL-2-associated death promoter (BAD) phosphorylation activates glucokinase and thus controls hepatic gluconeogenesis and insulin secretion in pancreatic β-cells (12; 13). In addition, BCL-2 proteins have been reported to stimulate glucose consumption (14) and regulate glucose metabolism through 3 Diabetes Page 4 of 62 mitochondrial activity (15) or Ca2+ trafficking (16; 17). This dual role in metabolism/apoptosis of BCL-2 proteins is reminiscent of the action of cytochrome c, a protein that functions as a crucial component for oxidative phosphorylation in the mitochondria, but when displaced to the cytosol induces cleavage of caspases and cell death (9). BCL-2 interacting mediator of cell death (BIM) is a BCL-2 homology 3 (BH3)-only protein widely expressed in tissues (18). Saturated free fatty acids trigger cell death in hepatocytes in culture through BIM up-regulation (19-21). Fat accumulation and hepatocellular carcinoma in liver-specific STAT5 knockout mice is associated with down-regulation of BIM and PUMA (22). In line with these findings, liver apoptosis in BCL-XL- or MCL-1-knockout mice is ameliorated by simultaneous BIM deletion (23). While it is well accepted that BIM has a role in apoptosis induction in hepatocytes, its role in the regulation of hepatic lipid and glucose metabolism in vivo is unclear. Moreover, it remains unknown whether dysregulation of BIM may contribute to the development of metabolic diseases such as liver steatosis and insulin resistance. Here, we show that BIM is induced in hepatocytes during obesity. To clarify the role of BIM, we generated a novel liver-specific BIM-deficient mouse model and observed that BIM deletion decreases fat accumulation, and improves insulin signalling in vivo. These novel observations highlight BIM as a key mediator of liver dysfunction in obesity. RESEARCH DESIGN AND METHODS Human samples. We studied 19 biopsy specimens of patients undergoing a liver biopsy for medical 4 Page 5 of 62 Diabetes reasons in our institution. The clinical characteristics of these patients are shown in Supplementary Figure 1E. The biopsies were collected after approval of the Erasme Hospital Ethics Committee. Written informed consent was obtained from each participant. Mice. Mice were maintained at St. Vincent’s Institute animal care facility on a 12h-light- dark cycle in a temperature-controlled room and obtained food and water ad libitum. Bimlox/lox mice were generated on a C57BL/6 background as previously described (24). Tissue-specific deletion of BIM was generated by crossing Bimlox/lox mice with Alb-Cre (C57BL/6) mice (JAX). Male mice were kept on regular chow (20% protein, 6% fat and 3.2% crude fibre) or placed at 8-10 weeks of age on a high fat diet (SF04- 027 Speciality Feeds, Perth, Western Australia) for 14-20 weeks. The nutritional composition of the high fat diet was 18.4% protein, 23.5% fat and 4.7% crude fibre. The calculated composition of fatty acids in the high fat diet is: 14.31% total saturated fats, 7.54% total mono-unsaturated fats, and 2.07% total polyunsaturated fats. In this diet, 46% of total energy is from lipids, 20% of total calculated energy from protein and the remainder from carbohydrates. All animal studies were conducted at St Vincent’s Institute (Melbourne, Australia) following the guidelines of the Institutional Animal Ethics Committee. Metabolic analysis. After 14-15 weeks on the diet and 24h of acclimatisation, oxygen consumption, energy expenditure, respiratory exchange ratio, activity and food intake were assessed using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus- 5 Diabetes Page 6 of 62 Instruments, OH). Data was averaged for 2 dark and light cycles. Body and tissue composition were determined by MRI scanning (EchoMRI, Houston, TX). After 18- 20 weeks of chow/high fat feeding, mice were sacrificed and tissues were weighed, and collected for further analysis. Serum triacylglycerides (Wako Diagnostic, Richmond, VA), insulin (Merck Millipore, Billerica, MA), and β-hydroxybutyrate (Sigma-Aldrich, Darmstadt, Germany) concentrations were determined using commercial kits and following manufacturer’s instructions. Mice were fasted for 4h before performing intra-peritoneal insulin tolerance test. Insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark), at a dose of 0.65mU/g, was injected intraperitoneally and blood glucose was measured after tail bleeding. Mice were fasted for 6h before an intra-peritoneal glucose (2mg/g, Baxter, Deerfield, IL) or pyruvate (2mg/g, Sigma-Aldrich) tolerance test. Cell culture and treatments. Mouse hepatocytes were isolated by a two-step collagenase A (0.05% w/v; Roche Diagnostics) perfusion method as described previously (25). Where indicated cells were treated with 0.5mM sodium palmitate in the presence of 1% w/v fatty acid free BSA (26). The JNK inhibitor SP600125 (Santa Cruz Biotechnology, CA) was used at 10µM (27). JNKs (JNK1 and JNK2) were knocked down transiently