Publications

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 %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.

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.

BACKGROUND: Insulin 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.

METHODS: Tibialis 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.

RESULTS: In 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.

CONCLUSIONS: Collectively, these findings demonstrate that glucose uptake via GLUT4 regulates skeletal myofiber regeneration following acute injury.

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.

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4-7. 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.

On this 100th anniversary of the discovery of insulin, we recognize the critical role that adipocytes, which are exquisitely responsive to insulin, have played in determining the mechanisms for insulin action at the cellular level. Our understanding of adipose tissue biology has evolved greatly, and it is now clear that adipocytes are far more complicated than simple storage depots for fat. A growing body of evidence documents how adipocytes, in response to insulin, contribute to the control of whole-body nutrient homeostasis. These advances highlight adipocyte plasticity, heterogeneity, and endocrine function, unique features that connect adipocyte metabolism to the regulation of other tissues important for metabolic homeostasis (e.g., liver, muscle, pancreas).

Circulating branched chain amino acid (BCAA) levels are elevated in obese humans and genetically obese rodents. However, the relationship of BCAAs to insulin resistance in diet-induced obese mice, a commonly used model to study glucose homeostasis, is still ill-defined. Here we examined how high-fat high-sucrose (HFHS) or high-fat diet (HFD) feeding, with or without BCAA supplementation in water, alters the metabolome in serum/plasma and tissues in mice and whether raising circulating BCAA levels worsens insulin resistance and glucose intolerance. Neither HFHS nor HFD feeding raised circulating BCAA levels in insulin-resistant diet-induced obese mice. BCAA supplementation raised circulating BCAA and branched-chain α-keto acid levels and C5-OH/C3-DC acylcarnitines (AC) in muscle from mice fed an HFHS diet or HFD, but did not worsen insulin resistance. A set of short- and long-chain acyl CoAs were elevated by diet alone in muscle, liver, and white adipose tissue (WAT), but not increased further by BCAA supplementation. HFD feeding reduced valine and leucine oxidation in WAT but not in muscle. BCAA supplementation markedly increased valine oxidation in muscle from HFD-fed mice, while leucine oxidation was unaffected by diet or BCAA treatment. Here we establish an extensive metabolome database showing tissue-specific changes in mice on 2 different HFDs, with or without BCAA supplementation. We conclude that mildly elevating circulating BCAAs and a subset of ACs by BCAA supplementation does not worsen insulin resistance or glucose tolerance in mice. This work highlights major differences in the effects of BCAAs on glucose homeostasis in diet-induced obese mice versus data reported in obese rats and in humans.

Adipose tissue (AT) inflammation contributes to systemic insulin resistance. In obesity and type 2 diabetes (T2D), retinol binding protein 4 (RBP4), the major retinol carrier in serum, is elevated in AT and has proinflammatory effects which are mediated partially through Toll-like receptor 4 (TLR4). We now show that RBP4 primes the NLRP3 inflammasome for interleukin-1β (IL1β) release, in a glucose-dependent manner, through the TLR4/MD2 receptor complex and TLR2. This impairs insulin signaling in adipocytes. IL1β is elevated in perigonadal white AT (PGWAT) of chow-fed RBP4-overexpressing mice and in serum and PGWAT of high-fat diet-fed RBP4-overexpressing mice vs. wild-type mice. Holo- or apo-RBP4 injection in wild-type mice causes insulin resistance and elevates PGWAT inflammatory markers, including IL1β. TLR4 inhibition in RBP4-overexpressing mice reduces PGWAT inflammation, including IL1β levels and improves insulin sensitivity. Thus, the proinflammatory effects of RBP4 require NLRP3-inflammasome priming. These studies may provide approaches to reduce AT inflammation and insulin resistance in obesity and diabetes.