SUMMARY Mitofusin 2 (Mfn2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted in loss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establishes Mfn2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for Mfn2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

Our data show that the pattern of circulating miRNAs is modified by defects in glucose metabolism in a similar manner in mice and humans. This circulating miRNA signature for prediabetes could be used as a new diagnostic tool, as well as to monitor response to intervention.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. However, their potential role in the central regulation of whole-body energy homeostasis is still unknown. In this study we show that the expression of Dicer, an essential endoribonuclease for miRNA maturation, is modulated by nutrient availability and excess in the hypothalamus. Conditional deletion of Dicer in POMC-expressing cells resulted in obesity, characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism and alterations in the pituitary-adrenal axis. The development of the obese phenotype was paralleled by a POMC neuron degenerative process that started around 3 weeks of age. Hypothalamic transcriptomic analysis in presymptomatic POMCDicerKO mice revealed the downregulation of genes implicated in biological pathways associated with classical neurodegenerative disorders, such as MAPK signaling, ubiquitin-proteosome system, autophagy and ribosome biosynthesis. Collectively, our results highlight a key role for miRNAs in POMC neuron survival and the consequent development of neurodegenerative obesity.

Aims/hypothesis During obesity, the increment in beta cell mass in response to the rising demand for insulin is essential to maintain normal glucose homeostasis. However, the precise cellular and molecular mechanisms involved in beta cell mass plasticity remain poorly understood. The Wnt signalling pathway has been suggested as one possible modulator of beta cell proliferation, which represents the principal process involved in beta cell mass expansion. Here, we sought to determine the mechanisms involved in beta cell mass proliferation using diet-induced obese rats. Methods Wistar rats aged 8 weeks old were fed a standard or cafeteria diet. Global transcriptomic analysis of pancreatic rat islets was performed using microarray analysis. Genetic lossof-function approaches were performed in dispersed primary rat islets and the beta cell line INS1E. Gene expression was measured by real-time PCR, protein levels by immunoblot analysis, proliferation rates by ELISA and apoptosis by flow cytometry. Results Sfrp5, coding for secreted frizzled-related protein 5, is downregulated in the pancreatic islets of cafeteria-diet-fed rats as well as in the pancreatic islets of human obese patients. We demonstrate that silencing Sfrp5 increases beta cell proliferation, which correlates with activation of Wnt signalling and enhanced levels of proliferation markers. In addition, we show that expression of Sfrp5 in beta cells is modulated by IGF binding protein 3 (IGFBP3) secreted from visceral adipose tissue. Conclusions/interpretation Together, these findings reveal an important role for SFRP5 and Wnt signalling in the regulation of beta cell proliferation in obesity.

Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its antiobesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate-treated lean, untreated obese, and tungstate-treated obese rats. To distinguish direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of analysis of variance and t test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity direct targets involved in Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport, and redox. Protein oxidation was decreased by tungstate treatment, confirming a role in redox processes; however, palmitate oxidation, as a measure of fatty acid ␤-oxidation, was not altered by tungstate, thus questioning its putative function in fatty acid oxidation. Protein network analyses using Ingenuity Pathways Analysis highlighted peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1␣, and its major target, uncoupling protein 1, was also increased as shown by Western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies and suggest that in brown adipose tissue tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1␣ up-regulation, thus contributing to its overall antiobesity effect.

Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in anti-human immunodeficiency virus (HIV) drugs and alcohol-induced liver disease in a significant number of patients infected with HIV. However, the precise mechanism by which the drugs and alcohol cause ER stress remains elusive. We found that ritonavir-boosted lopi-navir (RL) activated two canonical UPR branches without activation of the third canonical activating transcription factor 6 (ATF6) branch in either HepG2 cells or primary mouse hepatocytes. In the RL-treated cells, ATF6 localization in the Golgi apparatus required for its activation was reduced; this was followed by Golgi fragmentation and dislocation/redistri-bution of Golgi-resident enzymes. Severities of Golgi fragmentation induced by other anti-HIV drugs varied and were correlated with the ER stress response. In the liver of mice fed RL, alcohol feeding deteriorated the Golgi fragmentation, which was correlated with ER stress, elevated alanine aminotransferase, and liver steatosis. The Golgi stress response (GSR) markers GCP60 and HSP47 were increased in RL-treated liver cells, and knockdown of transcription factor for immunoglobulin heavy-chain enhancer 3 of the GSR by small interfering RNA worsened RL-induced cell death. Cotreat-ment of pharmacological agent H89 with RL inhibited the RL-induced Golgi enzyme dislocation and ER stress. Moreover , the coat protein complex II (COPII) complexes that mediate ER-to-Golgi trafficking accumulated in the RL-treated liver cells; this was not due to interference of RL with the initial assembly of the COPII complexes. RL also inhibited Golgi fragmentation and reassembly induced by short treatment and removal of brefeldin A. Conclusion: Our study indicates that ER-to-Golgi trafficking is disrupted by anti-HIV drugs and/or alcohol, and this contributes to subsequent ER stress and hepatic injury.

Expanding the mass of pancreatic insulin-producing beta cells through re-activation of beta cell replication has been proposed as a therapy to prevent or delay the appearance of diabetes. Pancreatic beta cells exhibit an age-dependent decrease in their proliferative activity, partly related to changes in the systemic environment. Here we report the identification of CCN4/Wisp1 as a circulating factor more abundant in pre-weaning than in adult mice. We show that Wisp1 promotes endogenous and transplanted adult beta cell proliferation in vivo. We validate these findings using isolated mouse and human islets and find that the beta cell trophic effect of Wisp1 is dependent on Akt signaling. In summary, our study reveals the role of Wisp1 as an inducer of beta cell replication, supporting the idea that the use of young blood factors may be a useful strategy to expand adult beta cell mass.

Objective: Persistent inflammation and impaired adipogenesis are frequent features of obesity and underlie the development of its complications. However, the factors behind adipose tissue dysfunction are not completely understood. Previously it was shown that histone demethylase KDM1A is required for adipogenesis. Design and Methods: Kdm1a expression was knocked down in 3T3-L1 preadipocytes by siRNA transfection and whole-genome expression profiling was performed by microarray hybridization. The role of NF-jb and C/EBPb was analyzed by incubation with the inhibitor parthenolide and by cebpb knockdown, respectively. Results: Knockdown of kdm1a or rcor2 in 3T3-L1 preadipocytes results in impaired differentiation and induction of inflammatory gene expression. Enhanced expression of il6 in kdm1a knocked down preadipocytes is associated with increased recruitment of C/EBPb and the NF-jb subunit RelA to the il6 promoter. Cebpb knockdown attenuates the induction of il6 expression in kdm1a knocked down cells, whereas simultaneous cebpb knockdown and NF-jb inhibition abrogates it. Dietary-induced and genetic mouse models of obesity display decreased KDM1A in adipose tissue, and this correlates with increased expression of proinflammatory genes and C/EBPb. Conclusion: KDM1A represses the expression of inflammatory genes in preadipocytes. Dysregulated kdm1a expression in preadipocytes may thus participate in the development of obesity-associated inflammation.Obesity (2013) 21, E616-E625.