FAHFAs are a family of bioactive lipids. A subclass of these, palmitic acid hydroxy stearic acids (PAHSAs) have antiinflammatory and anti-diabetic effects. Adipose tissue PAHSA levels are upregulated with increased de novo lipogenesis and fasting and downregulated with insulin resistance and obesity. Adipose triglyceride lipase (ATGL) regulates FAHFAs through two distinct mechanisms: hydrolysis of triacylglycerol (TG)-containing FAHFAs and catalyzing formation of the ester bond found in all FAHFAs through a transacylase reaction. ATGL mediates the increase of PAHSAs with fasting in white adipose tissue (WAT), but the mechanism for this has not been determined. Here, we show that multiple FAHFAs are dynamically regulated with fasting and short-term refeeding in both perigonadal (PG) and subcutaneous (SQ) WAT due to ATGL transacylase activity. Our in vivo studies with stable isotopes demonstrate that de novo FAHFA synthesis is upregulated with fasting. This observation along with the fact that FAHFA-TGs are unchanged (SQ WAT) or increased (PG WAT) with fasting and FAHFA hydrolysis is unchanged, suggests that the primary mechanism by which FAHFAs increase in WAT with fasting is de novo synthesis. Using adipose tissue-specific ATGL knock out mice, we show that ATGL is required for the fasting-induced upregulation of endogenous levels and de novo synthesis of multiple FAHFAs. Altogether, this study shows that fasting upregulates multiple FAHFAs by increasing ATGL-mediated synthesis of FAHFAs, inferring that fasting, a physiological state that is classically known to activate the lipase activity of ATGL, also stimulates its transacylase activity.

There is a growing need for early biomarkers for Type 2 diabetes (T2D). Fatty-Acid-Hydroxy-Fatty-Acids (FAHFAs) are bioactive lipids with >580 regioisomers in human tissues. FAHFAs such as Palmitic Acid Hydroxy Stearic Acids (PAHSAs) are anti-diabetic and anti-inflammatory. PAHSA concentrations in human serum and adipose tissue strongly correlate with insulin sensitivity. Since PAHSAs and palmitic acid hydroxy oleic acids (PAHOAs) are among the most abundant FAHFAs in human serum, we investigated whether they predict worsening glucose tolerance in first-degree relatives of people with T2D. All participants had normal glucose tolerance (NGT) at baseline; 27 remained NGT (NGT-NGT) and 21 developed impaired glucose tolerance (NGT-IGT). In NGT-NGT, total PAHSA and PAHSA regioisomer concentrations were unchanged from baseline to follow-up, while in NGT-IGT participants, most PAHSA regioisomers decreased. The initial total PAHSAs, 5-PAHSA, and 9-PAHSA, and changes in these correlated inversely with worsening glucose tolerance. Low total PAHSA concentrations at baseline and the decrease in total PAHSAs, 5-PAHSAs, and 9-PAHSAs over time predicted IGT independent of initial BMI or %body fat, change in BMI or in %body fat, initial fasting glucose, fasting insulin, or triglyceride/HDL ratio. In contrast, baseline and follow-up total PAHOA and PAHOA regioisomer levels were higher in NGT-IGT than NGT-NGT, and some PAHOA regioisomers increased during follow-up in NGT-IGT. Higher initial total PAHOAs predicted IGT independent of the same clinical variables. Thus, lower serum PAHSAs and higher PAHOAs predict worsening glucose tolerance/IGT independent of BMI, %body fat, or change in these parameters, even in lean, relatively young people.

Introduction: Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs) are signaling lipids with beneficial metabolic effects. Serum and adipose tissue (AT) PAHSA levels correlate highly with insulin sensitivity in people. PAHSAs enhance insulin sensitivity in diabetic mice. Thus, low PAHSA levels could contribute to the etiology of insulin resistance and T2D. PAHSAs are regulated by synthesis, degradation and incorporation into other lipids. We aimed to identify candidate enzymes that regulate PAHSAs using genetic mouse models in which large changes in PAHSAs are associated with major changes in glucose tolerance and/or insulin sensitivity. Methods: AT PAHSA levels are increased in mice with Adipose-specific Glucose Transporter 4 overexpression (AG4OX) which induces expression of the lipogenic transcription factor, Carbohydrate-response element-binding protein (ChREBP). Thus, we performed lipidomics and bulk RNA sequencing in perigonadal AT from wildtype, AG4OX, ChREBP knockout and AG4OX crossed to ChREBP KO which reverses the massive elevation of PAHSAs in AG4OX. We also measured PAHSAs in 8 genetically inbred mouse strains. We merged these datasets to identify genes strongly associated with PAHSAs in both models. A subsequent GWAS query for lipid-related SNPs delivered a discrete number of genes to validate as important PAHSA regulators. Results: All PAHSA regioisomers increase in AT with upregulation of glucose transport and decrease with ChREBP downregulation. PAHSA levels are higher in insulin-sensitive genetic strains. Many genes that strongly associate with 9-PAHSA belong to lipid metabolism pathways. Our single-gene queries in human GWAS support a role for 27 genes in the regulation of lipid metabolism in humans and, potentially, in PAHSA regulation. Conclusions: These studies provide novel insights into the metabolic pathways and enzymatic machinery responsible for PAHSA metabolism, which may be targeted to prevent or treat T2D in humans. Disclosure A. Santoro: None. M. Keller: None. Z. Chen: None. A.D. Attie: None. D. Siegel: None. G.A. Churchill: None. A. Saghatelian: None. B.B. Kahn: Advisory Panel; Janssen Pharmaceuticals, Inc. Consultant; Vida Ventures Advisors, Arrowhead Pharmaceuticals, Inc. Funding K01 DK128075 (Anna Santoro), NIH P30 DK135043 (Barbara B. Kahn), NIH R01 DK106210 (Barbara B. Kahn and Alan Saghatelian), and JPB foundation (Barbara B. Kahn)

Dietary lipids play an essential role in regulating the function of the gut microbiota and gastrointestinal tract, and these luminal interactions contribute to mediating host metabolism. Palmitic Acid Hydroxy Stearic Acids (PAHSAs) are a family of lipids with antidiabetic and anti-inflammatory properties, but whether the gut microbiota contributes to their beneficial effects on host metabolism is unknown. Here, we report that treating chow-fed female and male germ-free (GF) mice with PAHSAs improves glucose tolerance, but these effects are lost upon high fat diet (HFD) feeding. However, transfer of feces from PAHSA-treated, but not vehicle-treated, chow-fed conventional mice increases insulin sensitivity in HFD-fed GF mice. Thus, the gut microbiota is necessary for, and can transmit, the insulin-sensitizing effects of PAHSAs in HFD-fed GF male mice. Analyses of the cecal metagenome and lipidome of PAHSA-treated mice identified multiple lipid species that associate with the gut commensal Bacteroides thetaiotaomicron ( Bt ) and with insulin sensitivity resulting from PAHSA treatment. Supplementing live, and to some degree, heat-killed Bt to HFD-fed female mice prevented weight gain, reduced adiposity, improved glucose tolerance, fortified the colonic mucus barrier and reduced systemic inflammation compared to HFD-fed controls. These effects were not observed in HFD-fed male mice. Furthermore, ovariectomy partially reversed the beneficial Bt effects on host metabolism, indicating a role for sex hormones in mediating the Bt probiotic effects. Altogether, these studies highlight the fact that PAHSAs can modulate the gut microbiota and that the microbiota is necessary for the beneficial metabolic effects of PAHSAs in HFD-fed mice.

BackgroundInsulin resistance and type 2 diabetes impair cellular regeneration in multiple tissues including skeletal muscle. The molecular basis for this impairment is largely unknown. Glucose uptake via glucose transporter GLUT4 is impaired in insulin resistance. In healthy muscle, acute injury stimulates glucose uptake. Whether decreased glucose uptake via GLUT4 impairs muscle regeneration is presently unknown. The goal of this study was to determine whether GLUT4 regulates muscle glucose uptake and/or regeneration following acute injury.MethodsTibialis anterior and extensor digitorum longus muscles from wild-type, control, or muscle-specific GLUT4 knockout (mG4KO) mice were injected with the myotoxin barium chloride to induce muscle injury. After 3, 5, 7, 10, 14, or 21 days (in wild-type mice), or after 7 or 14 days (in control & mG4KO) mice, muscles were isolated to examine [3H]-2-deoxyglucose uptake, GLUT4 levels, extracellular fluid space, fibrosis, myofiber cross-sectional area, and myofiber centralized nuclei.ResultsIn wild-type mice, muscle glucose uptake was increased 3, 5, 7, and 10 days post-injury. There was a rapid decrease in GLUT4 protein levels that were restored to baseline at 5-7 days post-injury, followed by a super-compensation at 10-21 days. In mG4KO mice, there were no differences in muscle glucose uptake, extracellular fluid space, muscle fibrosis, myofiber cross-sectional areas, or percentage of centrally nucleated myofibers at 7 days post-injury. In contrast, at 14 days injured muscles from mG4KO mice exhibited decreased glucose uptake, muscle weight, myofiber cross sectional areas, and centrally nucleated myofibers, with no change in extracellular fluid space or fibrosis.ConclusionsCollectively, these findings demonstrate that glucose uptake via GLUT4 regulates skeletal myofiber regeneration following acute injury.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with anti-inflammatory and anti-diabetic effects. PAHSAs markedly reduce the incidence and delay the onset of autoimmune T1D in NOD mice. PAHSAs attenuate autoimmune responses by lowering mature T helper cell activation and B cell number and increasing regulatory T cell activity in the islets of NOD mice. Toll-like receptors and NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3) inflammasome pathways play an essential role in T1D pathogenesis. NLRP3 is critical for inducing expression of chemokines and their receptors which stimulate pathogenic T cell migration into islets. Here, we aimed to determine the mechanisms by which PAHSAs attenuate immune responses to prevent T1D. Results: Adoptive transfer of splenocytes from PAHSA-treated NOD mice delays and attenuates T1D incidence in immunodeficient NOD.SCID mice. These effects are associated with decreased expression of NLRP3 inflammasome components by 25-55% and chemokines by 60-70% without altering chemokine receptor expression in immune cells isolated from PAHSA-treated NOD mouse islets. In human islets from normal and type 2 diabetic donors, clonal pancreatic β-cells (MIN6 cells) and bone marrow-derived macrophages, PAHSAs attenuate pro-inflammatory cytokine and chemokine gene expression and secretion in response to LPS or Nigericin (NLRP3 inflammasome activator). In co-culture studies of MIN6 cells and CD4+ T cells, PAHSAs attenuate LPS and Nigericin-induced CD4+ T cell migration by 65%. Conclusions: PAHSAs’ protective effects against T1D can be conferred by transfer of immune cells from PAHSA-treated mice. Inhibition of NLRP3 inflammasome activation and T cell migration into islets are key mechanisms by which PAHSAs protect islets from immune-mediated attack. Thus, PAHSAs may serve as therapeutic agents to prevent and/or treat T1D. Disclosure I. Syed: None. V.G. Magupalli: None. P. Aryal: Employee; Cellarity. A. Santoro: None. D. Siegel: None. B.B. Kahn: Advisory Panel; Janssen Pharmaceuticals, Inc. Consultant; Vida Ventures Advisors, Arrowhead Pharmaceuticals, Inc. Funding NIH K01 DK118041 (IS).Joslin Pilot & Feasibility Grant P30 DK046200 (IS).NIH R01 DK106210 (BBK).JPB Foundation (BBK).

Maturity onset diabetes of the young type 8 (MODY8) is caused by genetic mutations in the CEL gene which is expressed primarily in pancreatic acinar cells. MODY8 patients develop pancreatic exocrine and endocrine dysfunction. How the enzymatic function of mutant (MUT) CEL contributes to the development of diabetes in MODY8 is unknown. Palmitic Acid Hydroxy Stearic Acids (PAHSAs) are signaling lipids that augment glucose-stimulated insulin secretion (GSIS). CEL is the major PAHSA hydrolytic enzyme in the pancreas. Aim: To determine whether CEL regulates insulin secretion and whether the increased PAHSA hydrolytic activity of MUT CEL contributes to MODY8 pathogenesis. Methods: We overexpressed wildtype (WT) or MUT CEL in acinar cells in vitro and in vivo. In vivo, we used 2 approaches both with AAV8 driven by an acinar cell specific promoter: intraductal AAV injections in wildtype mice and intraperitoneal AAV injections in CEL KO mice. CEL overexpression was detected exclusively in acinar cells. Results: 9-PAHSA augments GSIS in human pancreatic beta cells. Recombinant CEL inhibits the PAHSA effect. MUT CEL overexpression in acinar cells increases 9-PAHSA hydrolytic activity compared to WT CEL. In vivo, MUT CEL expression in acinar cells of wildtype mice markedly impairs glucose tolerance. In CEL KO mice, expression of MUT CEL in acinar cells impairs GSIS. Both WT and MUT CEL reduce total pancreatic PAHSA levels in CEL KO mice. However, 12/13-PAHSAs were reduced only with MUT CEL expression. Conclusions: 1) CEL in acinar cells alters PAHSA hydrolysis and modulates insulin secretion. 2) MUT CEL potentially contributes to the development of diabetes by increasing PAHSA hydrolysis and thereby limiting the normal PAHSA-induced augmentation of GSIS. 3) These data highlight the critical role of acinar-beta cell interactions and the physiologic role of PAHSAs in insulin secretion and provide opportunities for developing strategies to treat MODY8 and other forms of diabetes. Disclosure A. Santoro: None. S. Kahraman: Employee; Boehringer-Ingelheim. G. Basile: None. K. El Jellas: None. J. Hu: None. R. Tarpey: None. B.B. Johansson: None. E. Dirice: None. I. Syed: None. D. Siegel: None. A. Molven: None. B.B. Kahn: Advisory Panel; Janssen Pharmaceuticals, Inc. Consultant; Vida Ventures Advisors, Arrowhead Pharmaceuticals, Inc. R. Kulkarni: Advisory Panel; Novo Nordisk, Biomea Fusion, Inc. Consultant; Inversago Pharma. Advisory Panel; REDD Pharmaceutical. Funding K01 DK128075 (Anna Santoro), U01DK135095 (Rohit N. Kulkarni), R01DK067536 (Rohit N. Kulkarni), NIH R01 DK106210 (Barbara B. Kahn), JPB foundation (Barbara B. Kahn), and NIH P30 DK135043 (Barbara B. Kahn). Anna Santoro, Sevim Kahraman, Barbara B. Kahn and Rohit N. Kulkarni contributed equally.

Obesity-induced inflammation causes metabolic dysfunction, but the mechanisms remain elusive. Here we show that the innate immune transcription factor interferon regulatory factor (IRF3) adversely affects glucose homeostasis through induction of the endogenous FAHFA hydrolase androgen induced gene 1 (AIG1) in adipocytes. Adipocyte-specific knockout of IRF3 protects male mice against high-fat diet-induced insulin resistance, whereas overexpression of IRF3 or AIG1 in adipocytes promotes insulin resistance on a high-fat diet. Furthermore, pharmacological inhibition of AIG1 reversed obesity-induced insulin resistance and restored glucose homeostasis in the setting of adipocyte IRF3 overexpression. We, therefore, identify the adipocyte IRF3/AIG1 axis as a crucial link between obesity-induced inflammation and insulin resistance and suggest an approach for limiting the metabolic dysfunction accompanying obesity.

There is a growing need for early biomarkers for Type 2 diabetes (T2D). Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs) are bioactive lipids with >580 regioisomers in human tissues. Some FAHFAs such as Palmitic Acid Hydroxy Stearic Acids (PAHSAs) have anti-diabetic and anti-inflammatory effects. PAHSA levels are lower in serum and adipose tissue of insulin-resistant versus insulin-sensitive humans and strongly correlate with insulin sensitivity. Since PAHSAs and palmitic acid esters of hydroxy oleic acids (PAHOAs) are among the most abundant FAHFAs in human serum, we aimed to determine whether these FAHFAs predict worsening glucose tolerance in people with an increased T2D risk, i.e. first-degree relatives of people with T2D. All participants had normal glucose tolerance (NGT) at baseline. Throughout a rolling enrollment period, 27 participants remained NGT (NGT-NGT) and 21 developed impaired glucose tolerance (NGT-IGT). In the group that remained NGT, followup serum PAHSA regioisomers and total PAHSA levels were similar to baseline. In contrast, total serum PAHSA and most regioisomers concentrations decreased in participants who developed IGT. The change in total PAHSAs (R=-0.476; p=0.0006), 5-PAHSA (R=-0.506; p=0.0002), and 9-PAHSA (R=-0.356; p=0.013) correlated tightly with worsening glucose tolerance. Multivariate analysis showed the decrease in PAHSAs predicted worsening glucose tolerance independent of BMI and age. The levels of total PAHOAs and most PAHOA isomers were similar at enrollment and follow-up in the NGT-NGT group. In NGT-IGT, initial and followup PAHOA levels were higher than in participants who remained NGT. Initial total PAHOA levels were: NGT-NGT (15.63±0.79) vs NGT-IGT (28.19±2.27) p<4.48E-07. Followup total PAHOA levels NGT-NGT (14.70±0.62) vs NGT-IGT (31.07±2.73) p<2.88E-08. Conclusion: A fall in PAHSA levels serves as a biomarker for worsening glucose tolerance while high PAHOA levels predict worsening glucose tolerance and IGT. Disclosure I. Syed: None. K. Sluis: None. P. Aryal: Employee; Cellarity. Z. Solomon: None. R. Patel: Employee; Novo Nordisk. S. Konduri: None. D. Siegel: None. U. Smith: None. B.B. Kahn: Advisory Panel; Janssen Pharmaceuticals, Inc. Consultant; Vida Ventures Advisors, Arrowhead Pharmaceuticals, Inc. Funding NIH K01 DK118041 (IS).Joslin Pilot & Feasibility Grant P30 DK046200 (IS).NIH R01 DK106210 (BBK).JPB Foundation (BBK).

Adipose tissue glucose transport and GLUT4 levels regulate systemic insulin sensitivity and whole-body energy balance, which have implications for the pathogenesis and treatment of obesity and diabetes.

Recent research has shed light on the cellular and molecular functions of bioactive lipids that go far beyond what was known about their role as dietary lipids. Bioactive lipids regulate inflammation and its resolution as signaling molecules. Genetic studies have identified key factors that can increase the risk of cardiovascular diseases and metabolic syndrome through their effects on lipogenesis. Lipid scientists have explored how these signaling pathways affect lipid metabolism in the liver, adipose tissue, and macrophages by utilizing a variety of techniques in both humans and animal models, including novel lipidomics approaches and molecular dynamics models. Dissecting out these lipid pathways can help identify mechanisms that can be targeted to prevent or treat cardiometabolic conditions. Continued investigation of the multitude of functions mediated by bioactive lipids may reveal additional components of these pathways that can provide a greater understanding of metabolic homeostasis.

Disruption of the intestinal mucosa contributes to insulin resistance in obesity and Type 2 diabetes. Probiotic supplementation, which restores gut microbiota composition and fortifies intestinal barrier function, has therapeutic potential to treat obesity and metabolic disease. Treatment of mice on a chow diet or high fat diet (HFD) with Palmitic Acid Hydroxystearic Acids (PAHSAs) improves insulin sensitivity. These effects are mediated, at least in part, by changes in the gut microbiome. We used this paradigm to identify new microbes that may have beneficial metabolic effects. Metagenome sequencing of the cecum from mice treated with PAHSAs identified Bacteroides thetaiotaomicron (Bt) to be most strongly associated with insulin sensitivity. Here, we aimed to determine whether Bt supplementation in mice on a HFD diet has beneficial effects on energy balance and/or glucose homeostasis. Daily oral Bt supplementation in female mice fed a HFD resulted in less weight gain, lower adiposity, and improved glucose tolerance compared to vehicle-treated mice. Additionally, Bt supplementation preserved colonic mucus thickness, an indicator of gut health; increased the number of Goblet cells which are the cells that secrete mucin2, the predominant mucus protein that forms the intestinal mucosal barrier; and increased protein levels of mucin2 in the distal gut compared to vehicle-treated controls. These improvements in distal gut barrier architecture were associated with a reduction in circulating levels of pro-inflammatory lipopolysaccharide (LPS) and interleukin-6 (IL-6) . These beneficial effects of Bt were observed in female and not in male HFD-fed mice. These studies indicate that Bt has sex-specific effects to improve host metabolism in the setting of dietary obesity. Bt represents a next-generation probiotic and novel gut-based therapeutic strategy which could improve metabolic health in women. Disclosure J.Lee: None. K.Wellenstein: None. B.B.Kahn: Advisory Panel; Janssen Pharmaceuticals, Inc., Consultant; Mellicell, Other Relationship; Alnylam Pharmaceuticals, Inc. Funding NIH KDK114162; JPB Foundation

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals 1,2 . A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic 1,3 . Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum 1 . PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases 1,4–7 . The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80–90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

Senescence in pancreatic beta cells plays a major role in beta cell dysfunction, which leads to impaired glucose homeostasis and diabetes. Therefore, prevention of beta cell senescence could reduce the risk of diabetes. Treatment of nonobese diabetic (NOD) mice, a model of type 1 autoimmune diabetes (T1D), with palmitic acid hydroxy stearic acids (PAHSAs), a novel class of endogenous lipids with antidiabetic and antiinflammatory effects, delays the onset and reduces the incidence of T1D from 82% with vehicle treatment to 35% with PAHSAs. Here, we show that a major mechanism by which PAHSAs protect islets of the NOD mice is by directly preventing and reversing the initial steps of metabolic stress-induced senescence. In vitro PAHSAs increased Mdm2 expression, which decreases the stability of p53, a key inducer of senescence-related genes. In addition, PAHSAs enhanced expression of protective genes, such as those regulating DNA repair and glutathione metabolism and promoting autophagy. We demonstrate the translational relevance by showing that PAHSAs prevent and reverse early stages of senescence in metabolically stressed human islets by the same Mdm2 mechanism. Thus, a major mechanism for the dramatic effect of PAHSAs in reducing the incidence of type 1 diabetes in NOD mice is decreasing cellular senescence; PAHSAs may have a similar benefit in humans.

In obesity, anorectic responses to leptin are diminished, giving rise to the concept of "leptin resistance." Increased expression of protein tyrosine phosphatase 1B (PTP1B) has been associated with the attenuation of leptin signaling and development of cellular leptin resistance. Here we report that hypothalamic levels of the tyrosine phosphatase TCPTP are also elevated in obesity to attenuate the leptin response. We show that mice that lack TCPTP in neuronal cells have enhanced leptin sensitivity and are resistant to high-fat-diet-induced weight gain and the development of leptin resistance. Also, intracerebroventricular administration of a TCPTP inhibitor enhances leptin signaling and responses in mice. Moreover, the combined deletion of TCPTP and PTP1B in neuronal cells has additive effects in the prevention of diet-induced obesity. Our results identify TCPTP as a critical negative regulator of hypothalamic leptin signaling and causally link elevated TCPTP to the development of cellular leptin resistance in obesity.