Vidisha Raje

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes

Significance Adipose tissue maintains metabolic homeostasis during fasting and fed conditions. When nutrients are plentiful, anabolic signaling is mediated by insulin, stimulating adipocytes to take up glucose for energy storage. In the absence of nutrients, catabolic signaling initiates lipolysis, or the release of lipids for energy use, and is mediated by catecholamines. These opposing pathways are evolutionarily conserved and prevent futile cycling, but can lead to metabolic disorders such as insulin resistance if not properly regulated. Here we define a novel mechanism whereby lipolysis inhibits insulin-stimulated glucose uptake in adipocytes. This signaling mechanism likely contributes to insulin resistance when lipolysis is active, such as during high stress or obesity, and this new understanding may lead to novel treatment approaches for hyperglycemia. Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic–mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase–Akt–mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.

Activation of murine pre-proglucagon–producing neurons reduces food intake and body weight

Peptides derived from pre-proglucagon (GCG peptides) act in both the periphery and the CNS to change food intake, glucose homeostasis, and metabolic rate while playing a role in anxiety behaviors and physiological responses to stress. Although the actions of GCG peptides produced in the gut and pancreas are well described, the role of glutamatergic GGC peptide–secreting hindbrain neurons in regulating metabolic homeostasis has not been investigated. Here, we have shown that chemogenetic stimulation of GCG-producing neurons reduces metabolic rate and food intake in fed and fasted states and suppresses glucose production without an effect on glucose uptake. Stimulation of GCG neurons had no effect on corticosterone secretion, body weight, or conditioned taste aversion. In the diet-induced obese state, the effects of GCG neuronal stimulation on gluconeogenesis were lost, while the food intake–lowering effects remained, resulting in reductions in body weight and adiposity. Our work suggests that GCG peptide–expressing neurons can alter feeding, metabolic rate, and glucose production independent of their effects on hypothalamic pituitary-adrenal (HPA) axis activation, aversive conditioning, or insulin secretion. We conclude that GCG neurons likely stimulate separate populations of downstream cells to produce a change in food intake and glucose homeostasis and that these effects depend on the metabolic state of the animal.

Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduce mitochondrial ROS production

Stress depletes STAT3 in the mitochondria, and restoration of this STAT3 pool suppresses stress-induced ROS production. Dynamic regulation of mitochondrial STAT3 In cytokine-stimulated cells, the transcription factor STAT3 translocates from the cytoplasm to the nucleus to transcriptionally activate genes involved in stress responses. STAT3 also functions in the mitochondria to regulate cellular respiration. Meier et al. found that oxidative stress and cytokines depleted the mitochondrial pool of STAT3, and restoration of this pool required the chaperone protein cyclophilin D. Moreover, the restored STAT3 suppressed the stress-induced generation of reactive oxygen species in the mitochondria. These results suggest that the mitochondrial pool is dynamically regulated similar to the cytoplasmic and nuclear pools of STAT3 and that the mitochondrial pool is responsive to external stimuli. Signal transducer and activator of transcription 3 (STAT3) is associated with various physiological and pathological functions, mainly as a transcription factor that translocates to the nucleus upon tyrosine phosphorylation induced by cytokine stimulation. In addition, a small pool of STAT3 resides in the mitochondria, where it serves as a sensor for various metabolic stressors including reactive oxygen species (ROS). Mitochondrially localized STAT3 largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). It has been assumed that the amounts of STAT3 in the mitochondria are static. We showed that various stimuli, including oxidative stress and cytokines, triggered a signaling cascade that resulted in a rapid loss of mitochondrially localized STAT3. Recovery of the mitochondrial pool of STAT3 over time depended on phosphorylation of Ser727 in STAT3 and new protein synthesis. Under these conditions, mitochondrially localized STAT3 also became competent to bind to cyclophilin D (CypD). Binding of STAT3 to CypD was mediated by the amino terminus of STAT3, which was also important for reducing mitochondrial ROS production after oxidative stress. These results outline a role for mitochondrially localized STAT3 in sensing and responding to external stimuli.

The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1 -/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1 -/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1 -/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1 -/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1 -/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1 -/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1 -/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1 -/- mice.

Dual activities of ritanserin and R59022 as DGKα inhibitors and serotonin receptor antagonists.

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzymes affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro.Graphical abstract Figure. No Caption available. Abstract Diacylglycerol kinase alpha (DGK&agr;) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGK&agr; was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small‐molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGK&agr;. Ritanserin, originally characterized as a serotonin (5‐HT) receptor (5‐HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGK&agr; kinase activity while increasing the enzyme’s affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGK&agr; inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5‐HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGK&agr; and 5‐HTRs in vitro.

Kinase inactive Tyrosine kinase (Tyk2) Supports Differentiation of Brown fat Cells.

It has been known for decades that brown adipose tissue (BAT) plays a central role in maintaining body temperature in hibernating animals and human infants. Recently, it has become evident that there are also depots of brown fat in adult humans, and the mass of brown fat is inversely correlated with body weight. There are a variety of transcription factors implicated in the differentiation of classical Myf5+ brown preadipocytes, one of the most important of which is PRDM16. We have recently identified that in addition to PRDM16, the tyrosine kinase Tyk2 and the STAT3 transcription factor are required for the differentiation of Myf5 positive brown preadipocytes both in cell culture and in mice. Tyk2 is a member of the Jak family of tyrosine kinases, which are activated by exposure of cells to different cytokines and growth factors. In this study we report the surprising observation that a mutated form of Tyk2, which lacks tyrosine kinase activity (Tyk2KD) restores differentiation of brown preadipocytes in vitro as well as in Tyk2-/- mice. Furthermore, expression of the Tyk2KD transgene in brown fat reverses the obese phenotype of Tyk2-/- animals. Treatment of cells with Jak-selective inhibitors suggests that the mechanism by which Tyk2KD functions to restore BAT differentiation is by dimerizing with kinase active Jak1 or Jak2. These results indicate that there are redundant mechanisms by which members of the Jak family can contribute to differentiation of BAT.

STAT3 suppresses Wnt/β-catenin signaling during the induction phase of primary Myf5+ brown adipogenesis

HighlightsSTAT3 is required during the induction phase for differentiation of primary brown pre‐adipocytes.Deletion of STAT3 after the induction period does not affect UCP1 protein levels and differentiation.STAT3 KO cells can be rescued through inhibition of the canonical Wnt/&bgr;‐Catenin pathway or by knock down of &bgr;‐catenin.STAT3 KO cells upregulate Wnt ligands during the induction phase. Abstract Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK‐STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3−/− preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical &bgr;‐catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of &bgr;‐catenin can fully rescue adipogenesis and UCP1 expression in the STAT3−/− adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3−/− adipocytes, potentially explaining the increased levels and activity of &bgr;‐catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/&bgr;‐catenin pathway during the early stages of in‐vitro adipogenesis.

Adipose triglyceride lipolysis - adding fuel to the fire

Recent studies have shown the presence of functional brown adipose tissue (BAT) in adult humans and it role in thermogenesis. This study by Blondin et al published in Cell Metabolism shows that similar to rodents; adult human brown adipose tissue uses lipids as its cellular fuel during thermogenesis. Inhibition of triglyceride lipolysis was associated with a shift in the skeletal muscle mediated shivering instead and a shift in fuel utilization from lipids to glucose. This study provides yet another confirmatory role of a functional brown fat in adult humans that is fueled by fatty acids.

Brown adipose tissue affects lipid metabolism in humans

Recent studies have shown the presence of functional brown adipose tissue (BAT) in adult humans in glucose and energy metabolism....