Cynthia T. Luk

Regulation of obesity-related insulin resistance with gut anti-inflammatory agents.

Obesity has reached epidemic proportions, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during high-fat diet (HFD) feeding and is a functional regulator of obesity-related insulin resistance (IR) that can be exploited therapeutically. Obesity induces a chronic phenotypic pro-inflammatory shift in bowel lamina propria immune cell populations. Reduction of the gut immune system, using beta7 integrin-deficient mice (Beta7(null)), decreases HFD-induced IR. Treatment of wild-type HFD C57BL/6 mice with the local gut anti-inflammatory, 5-aminosalicyclic acid (5-ASA), reverses bowel inflammation and improves metabolic parameters. These beneficial effects are dependent on adaptive and gut immunity and are associated with reduced gut permeability and endotoxemia, decreased visceral adipose tissue inflammation, and improved antigen-specific tolerance to luminal antigens. Thus, the mucosal immune system affects multiple pathways associated with systemic IR and represents a novel therapeutic target in this disease.

Hepatocyte-specific Deletion of Janus Kinase 2 (JAK2) Protects against Diet-induced Steatohepatitis and Glucose Intolerance

Background: JAK2 mediates signaling by a number of cytokines in the liver. Results: Hepatic JAK2 KO mice developed spontaneous steatosis but were protected from high fat diet-induced steatohepaitits and insulin resistance. Conclusion: Hepatic JAK2 is required for the development of diet-induced steatohepatitis and glucose intolerance. Significance: Understanding the role of JAK2 in metabolism will provide insights into the pathogenesis of the metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as 2 weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in β-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.

In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic β-Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking

Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic β-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)–driven Cre-loxP recombination system to specifically delete FAK in pancreatic β-cells. These RIPcre+fakfl/fl mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced β-cell viability and proliferation resulting in decreased β-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal–related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient β-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca2+ influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.

DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice

Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinsons disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders.

Perforin Is a Novel Immune Regulator of Obesity-Related Insulin Resistance

Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1null) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1null mice have increased accumulation of proinflammatory IFN-γ–producing CD4+ and CD8+ T cells and M1-polarized macrophages in VAT. CD8+ T cells from the VAT of Prf1null mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8+ T cells from Prf1null mice into CD8-deficient mice (CD8null) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell–deficient mice (NKnull) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.

Pten deletion in RIP-Cre neurons protects against type 2 diabetes by activating the anti-inflammatory reflex

Inflammation has a critical role in the development of insulin resistance. Recent evidence points to a contribution by the central nervous system in the modulation of peripheral inflammation through the anti-inflammatory reflex. However, the importance of this phenomenon remains elusive in type 2 diabetes pathogenesis. Here we show that rat insulin-2 promoter (Rip)-mediated deletion of Pten, a gene encoding a negative regulator of PI3K signaling, led to activation of the cholinergic anti-inflammatory pathway that is mediated by M2 activated macrophages in peripheral tissues. As such, Rip-cre+ Ptenflox/flox mice showed lower systemic inflammation and greater insulin sensitivity under basal conditions compared to littermate controls, which were abolished when the mice were treated with an acetylcholine receptor antagonist or when macrophages were depleted. After feeding with a high-fat diet, the Pten-deleted mice remained markedly insulin sensitive, which correlated with massive subcutaneous fat expansion. They also exhibited more adipogenesis with M2 macrophage infiltration, both of which were abolished after disruption of the anti-inflammatory efferent pathway by left vagotomy. In summary, we show that Pten expression in Rip+ neurons has a critical role in diabetes pathogenesis through mediating the anti-inflammatory reflex.

JAK2 promotes brown adipose tissue function and is required for diet- and cold-induced thermogenesis in mice

Aims/hypothesisNon-shivering thermogenesis in adipose tissue can be activated by excessive energy intake or following cold exposure. The molecular mechanisms regulating this activation have not been fully elucidated. The Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathway mediates the signal transduction of numerous hormones and growth factors that regulate adipose tissue development and function, and may play a role in adaptive thermogenesis.MethodsWe analysed mRNA and protein levels of uncoupling protein 1 (UCP1) and JAK2 in different adipose depots in response to metabolic and thermal stress. The in vivo role of JAK2 in adaptive thermogenesis was examined using mice with adipocyte-specific Jak2 deficiency (A-Jak2 KO).ResultsWe show in murine brown adipose tissue (BAT) that JAK2 is upregulated together with UCP1 in response to high-fat diet (HFD) feeding and cold exposure. In contrast to white adipose tissue, where JAK2 was dispensable for UCP1 induction, we identified an essential role for BAT JAK2 in diet- and cold-induced thermogenesis via mediating the thermogenic response to β-adrenergic stimulation. Accordingly, A-Jak2 KO mice were unable to upregulate BAT UCP1 following a HFD or after cold exposure. Therefore, A-Jak2 KO mice were cold intolerant and susceptible to HFD-induced obesity and diabetes.Conclusions/interpretationTaken together, our results suggest that JAK2 plays a critical role in BAT function and adaptive thermogenesis. Targeting the JAK–STAT pathway may be a novel therapeutic approach for the treatment of obesity and related metabolic disorders.

In Vivo Knockdown of Adipocyte Erythropoietin Receptor Does Not Alter Glucose or Energy Homeostasis

The growing prevalence of obesity and diabetes necessitate a better understanding of the role of adipocyte biology in metabolism. Increasingly, erythropoietin (EPO) has been shown to have extraerythropoietic and cytoprotective roles. Exogenous administration has recently been shown to have beneficial effects on obesity and diabetes in mouse models and EPO can modulate adipogenesis and insulin signaling in 3T3-L1 adipocytes. However, its physiological role in adipocytes has not been identified. Using male and female mice with adipose tissue-specific knockdown of the EPO receptor, we determine that adipocyte EPO signaling is not essential for the maintenance of energy homeostasis or glucose metabolism. Adipose tissue-specific disruption of EPO receptor did not alter adipose tissue expansion, adipocyte morphology, insulin resistance, inflammation, or angiogenesis in vivo. In contrast to the pharmacological effects of EPO, we demonstrate that EPO signaling at physiological levels is not essential for adipose tissue regulation of metabolism.

PTEN deletion in pancreatic α-cells protects against high-fat diet-induced hyperglucagonemia and insulin resistance.

An aberrant increase in circulating catabolic hormone glucagon contributes to type 2 diabetes pathogenesis. However, mechanisms regulating glucagon secretion and α-cell mass are not well understood. In this study, we aimed to demonstrate that phosphatidylinositol 3-kinase (PI3K) signaling is an important regulator of α-cell function. Mice with deletion of PTEN, a negative regulator of this pathway, in α-cells show reduced circulating glucagon levels and attenuated l-arginine–stimulated glucagon secretion both in vivo and in vitro. This hypoglucagonemic state is maintained after high-fat–diet feeding, leading to reduced expression of hepatic glycogenolytic and gluconeogenic genes. These beneficial effects protected high-fat diet–fed mice against hyperglycemia and insulin resistance. The data demonstrate an inhibitory role of PI3K signaling on α-cell function and provide experimental evidence for enhancing α-cell PI3K signaling for diabetes treatment.

FAK signalling controls insulin sensitivity through regulation of adipocyte survival

Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.