Beneficial metabolic effects of PAHSAs depend on the gut microbiota in diet-induced obese mice.

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. PAHSAs are a class of lipids with anti-diabetic and anti-inflammatory properties, but whether the gut microbiota contributes to their beneficial effects on host metabolism is unknown. Here, we report that treating high fat diet (HFD)-fed germ-free mice with PAHSAs does not improve insulin sensitivity. However, transfer of feces from PAHSA-treated, but not Vehicle-treated, chow-fed mice increases insulin-sensitivity in HFD-fed germ free mice. Thus, the gut microbiota is necessary for and can transmit the insulin-sensitizing effects of PAHSAs in HFD-fed germ-free mice. Functional 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. Bt supplementation in HFD-fed female mice prevented weight gain, reduced adiposity, improved glucose tolerance, fortified the colonic mucus barrier and reduced systemic inflammation versus chow-fed controls, effects that 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 probiotic effects. Altogether, these studies highlight the fact that lipids can modulate the gut microbiota resulting in improvement in host metabolism and that PAHSA-induced changes in the microbiota result in at least some of their insulin-sensitizing effects in female mice.

PAHSAs reduce cellular senescence and protect pancreatic beta cells from metabolic stress through regulation of Mdm2/p53.

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.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids.

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.

Bioactive lipids and metabolic syndrome-a symposium report.

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.

High-throughput mediation analysis of human proteome and metabolome identifies mediators of post-bariatric surgical diabetes control.

To improve the power of mediation in high-throughput studies, here we introduce High-throughput mediation analysis (Hitman), which accounts for direction of mediation and applies empirical Bayesian linear modeling. We apply Hitman in a retrospective, exploratory analysis of the SLIMM-T2D clinical trial in which participants with type 2 diabetes were randomized to Roux-en-Y gastric bypass (RYGB) or nonsurgical diabetes/weight management, and fasting plasma proteome and metabolome were assayed up to 3 years. RYGB caused greater improvement in HbA1c, which was mediated by growth hormone receptor (GHR). GHR's mediation is more significant than clinical mediators, including BMI. GHR decreases at 3 months postoperatively alongside increased insulin-like growth factor binding proteins IGFBP1/BP2; plasma GH increased at 1 year. Experimental validation indicates (1) hepatic GHR expression decreases in post-bariatric rats; (2) GHR knockdown in primary hepatocytes decreases gluconeogenic gene expression and glucose production. Thus, RYGB may induce resistance to diabetogenic effects of GH signaling.Trial Registration: Clinicaltrials.gov NCT01073020.

Distinct biological activities of isomers from several families of branched fatty acid esters of hydroxy fatty acids (FAHFAs).

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. Each FAHFA family consists of esters with different acyl chains and multiple isomers with branch points at different carbons. Some FAHFAs, including palmitic acid hydroxy stearic acids (PAHSAs), improve insulin sensitivity and glucose tolerance in mice by enhancing glucose-stimulated insulin secretion (GSIS), insulin-stimulated glucose transport, and insulin action to suppress hepatic glucose production and reducing adipose tissue inflammation. However, little is known about the biological effects of other FAHFAs. Here, we investigated whether PAHSAs, oleic acid hydroxy stearic acid, palmitoleic acid hydroxy stearic acid, and stearic acid hydroxy stearic acid potentiate GSIS in ß-cells and human islets, insulin-stimulated glucose uptake in adipocytes, and anti-inflammatory effects in immune cells. We also investigated whether they activate G protein-coupled receptor 40, which mediates the effects of PAHSAs on insulin secretion and sensitivity in vivo. We show that many FAHFAs potentiate GSIS, activate G protein-coupled receptor 40, and attenuate LPS-induced chemokine and cytokine expression and secretion and phagocytosis in immune cells. However, fewer FAHFAs augment insulin-stimulated glucose uptake in adipocytes. S-9-PAHSA, but not R-9-PAHSA, potentiated GSIS and glucose uptake, while both stereoisomers had anti-inflammatory effects. FAHFAs containing unsaturated acyl chains with higher branching from the carboxylate head group are more likely to potentiate GSIS, whereas FAHFAs with lower branching are more likely to be anti-inflammatory. This study provides insight into the specificity of the biological actions of different FAHFAs and could lead to the development of FAHFAs to treat metabolic and immune-mediated diseases.

Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle.

Denervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [3H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt; p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1; or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.

Insulin action in adipocytes, adipose remodeling, and systemic effects.

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).

BCAA Supplementation in Mice with Diet-induced Obesity Alters the Metabolome Without Impairing Glucose Homeostasis.

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.

Retinol binding protein 4 primes the NLRP3 inflammasome by signaling through Toll-like receptors 2 and 4.

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-1ß (IL1ß) release, in a glucose-dependent manner, through the TLR4/MD2 receptor complex and TLR2. This impairs insulin signaling in adipocytes. IL1ß 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 IL1ß. TLR4 inhibition in RBP4-overexpressing mice reduces PGWAT inflammation, including IL1ß 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.

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3.

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.

RBP4 increases lipolysis in human adipocytes and is associated with increased lipolysis and hepatic insulin resistance in obese women.

Retinol-binding protein-4 (RBP4) is elevated in serum and adipose tissue (AT) in obesity-induced insulin resistance and correlates inversely with insulin-stimulated glucose disposal. But its role in insulin-mediated suppression of lipolysis, free fatty acids (FFA), and endogenous glucose production (EGP) in humans is unknown. RBP4 mRNA or protein levels were higher in liver, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) in morbidly obese subjects undergoing Roux-en-Y gastric bypass surgery compared to lean controls undergoing elective laparoscopic cholecystectomy. RBP4 mRNA expression in SAT correlated with the expression of several macrophage and other inflammation markers. Serum RBP4 levels correlated inversely with glucose disposal and insulin-mediated suppression of lipolysis, FFA, and EGP. Mechanistically, RBP4 treatment of human adipocytes in vitro directly stimulated basal lipolysis. Treatment of adipocytes with conditioned media from RBP4-activated macrophages markedly increased basal lipolysis and impaired insulin-mediated lipolysis suppression. RBP4 treatment of macrophages increased TNFα production. These data suggest that elevated serum or adipose tissue RBP4 levels in morbidly obese subjects may cause hepatic and systemic insulin resistance by stimulating basal lipolysis and by activating macrophages in adipose tissue, resulting in release of pro-inflammatory cytokines that impair lipolysis suppression. While we have demonstrated this mechanism in human adipocytes in vitro, and correlations from our flux studies in humans strongly support this, further studies are needed to determine whether this mechanism explains RBP4-induced insulin resistance in humans.

PAHSAs enhance hepatic and systemic insulin sensitivity through direct and indirect mechanisms.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet-fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs' effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein-coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.

PAHSAs attenuate immune responses and promote ß cell survival in autoimmune diabetic mice.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are endogenous antidiabetic and antiinflammatory lipids. Here, we show that PAHSAs protect against type 1 diabetes (T1D) and promote ß cell survival and function. Daily oral PAHSA administration to nonobese diabetic (NOD) mice delayed the onset of T1D and markedly reduced the incidence of T1D, whether PAHSAs were started before or after insulitis was established. PAHSAs reduced T and B cell infiltration and CD4+ and CD8+ T cell activation, while increasing Treg activation in pancreata of NOD mice. PAHSAs promoted ß cell proliferation in both NOD mice and MIN6 cells and increased the number of ß cells in NOD mice. PAHSAs attenuated cytokine-induced apoptotic and necrotic ß cell death and increased ß cell viability. The mechanism appears to involve a reduction of ER stress and MAPK signaling, since PAHSAs lowered ER stress in NOD mice, suppressed thapsigargin-induced PARP cleavage in human islets, and attenuated ERK1/2 and JNK1/2 activation in MIN6 cells. This appeared to be mediated in part by glucagon-like peptide 1 receptor (GLP-1R) and not the G protein-coupled receptor GPR40. PAHSAs also prevented impairment of glucose-stimulated insulin secretion and improved glucose tolerance in NOD mice. Thus, PAHSAs delayed the onset of T1D and reduced its incidence by attenuating immune responses and exerting direct protective effects on ß cell survival and function.

Discovery of FAHFA-Containing Triacylglycerols and Their Metabolic Regulation.

FAHFAs are a class of bioactive lipids, which show great promise for treating diabetes and inflammatory diseases. Deciphering the metabolic pathways that regulate endogenous FAHFA levels is critical for developing diagnostic and therapeutic strategies. However, it remains unclear how FAHFAs are metabolized in cells or tissues. Here, we investigate whether FAHFAs can be incorporated into other lipid classes and identify a novel class of endogenous lipids, FAHFA-containing triacylglycerols (FAHFA-TGs), which contain a FAHFA group esterified to the glycerol backbone. Isotope-labeled FAHFAs are incorporated into FAHFA-TGs when added to differentiated adipocytes, which implies the existence of enzymes and metabolic pathways capable of synthesizing these lipids. Induction of lipolysis (i.e., triacylglycerol hydrolysis) in adipocytes is associated with marked increases in nonesterified FAHFA levels, demonstrating that FAHFA-TGs breakdown is a regulator of cellular FAHFA levels. To quantify FAHFA levels in FAHFA-TGs and determine their regioisomeric distributions, we developed a mild alkaline hydrolysis method that liberates FAHFAs from triacylglycerols for easier detection. FAHFA-TG concentrations are greater than 100-fold than that of nonesterified FAHFAs, indicating that FAHFA-TGs are a major reservoir of FAHFAs in cells and tissues. The discovery of FAHFA-TGs reveals a new branch of TG and FAHFA metabolism with potential roles in metabolic health and regulation of inflammation.

Adipose Tissue, Inter-Organ Communication, and the Path to Type 2 Diabetes: The 2016 Banting Medal for Scientific Achievement Lecture.

My scientific career has focused on understanding the mechanisms underlying insulin resistance with the goal of developing new strategies to prevent and treat type 2 diabetes. My early studies focused on understanding how insulin promotes glucose transport into adipocytes, a classic model of highly insulin-responsive target cells. When we found changes in adipocyte glucose transport in altered metabolic states, we were highly motivated to understand the consequences of this on whole-body glucose homeostasis. In the late 1980s, when GLUT4, the major insulin-regulated glucose transporter, was identified, my lab observed that it was downregulated in adipocytes but not in skeletal muscle in insulin-resistant states, such as obesity and type 2 diabetes, in humans and rodents. We investigated the role of GLUT4 in adipose tissue and muscle in whole-body insulin sensitivity, making tissue-specific GLUT4-overexpressing and GLUT4 knockout mice. These studies led to the discovery that adipocytes, and specifically glucose transport into adipocytes, regulate whole-body glucose homeostasis. As adipocytes take up relatively little glucose, we investigated the underlying mechanisms. In the 1990s, we performed DNA microarrays on adipose tissue from adipose-specific GLUT4-overexpressing and GLUT4 knockout mice to find reciprocally regulated genes, and we identified several molecules that were not previously known to regulate systemic insulin sensitivity and/or energy balance. More recently, with Alan Saghatelian's lab, we discovered a novel class of lipids with antidiabetes and anti-inflammatory effects. We also investigated the effects of the adipose-secreted hormone, leptin, on insulin sensitivity. We found that the AMP-activated protein kinase (AMPK) pathway mediates leptin's effects on fatty acid oxidation in muscle and also plays a role in leptin's anorexigenic effects in the hypothalamus. These studies transformed AMPK from a "fuel gauge" that regulates energy supply at the cellular level to a sensing and signaling pathway that regulates organismal energy balance. Overall, these studies have expanded our understanding of the multifaceted role of adipose tissue in metabolic health and how adipose dysfunction increases the risk for type 2 diabetes.

Adipose tissue dysfunction is associated with low levels of the novel Palmitic Acid Hydroxystearic Acids.

Adipose tissue dysfunction is considered an important contributor to systemic insulin resistance and Type 2 diabetes (T2D). Recently, a novel family of endogenous lipids, palmitic acid hydroxy stearic acids (PAHSAs), was discovered. These have anti-diabetic and anti-inflammatory effects in mice and are reduced in serum and adipose tissue of insulin resistant humans. In the present study, we investigate if adipose tissue dysfunction is associated with reduced PAHSA levels in human subjects and if PAHSAs influence adipocyte differentiation. Our results show that low expression of adipocyte GLUT4 and adipocyte hypertrophy, markers of adipose tissue dysfunction, are associated with reduced expression of key enzymes for de novo lipogenesis and adipose tissue levels of PAHSAs in human subjects. We also show that GLUT4 is not only a marker of adipose tissue dysfunction, but may be causally related to the observed impairments. PAHSAs may also act locally in the adipose tissue to improve adipogenesis through a mechanism bypassing direct activation of peroxisome proliferator-activated receptor (PPARγ). The discovery of PAHSAs and our current results provide novel insights into positive effects of lipid species in adipose tissue and mechanisms by which dysfunctional adipose tissue is associated with insulin resistance and risk of developing T2D.