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The gut microbiome contributes to host health and has therapeutic potential to treat metabolic disease. A newer class of lipids, PAHSAs, have antidiabetic effects in high-fat diet (HFD)-fed mice. We aimed to determine whether the gut microbiome contributes to the beneficial metabolic effects of PAHSAs and if these effects are transmissible by Fecal Microbiota Transplantation (FMT) in mice. In HFD-fed germ-free (GF) mice, PAHSA treatment for 35 days did not improve glucose homeostasis vs. vehicle-treated mice, in contrast to previous data in conventional HFD mice. Feces from conventional Chow fed mice with enhanced insulin sensitivity after PAHSA treatment for 21 days were used for oral FMT in HFD-GF mice. HFD-GF mice treated with feces from PAHSA-treated chow mice gained less weight, had reduced glycemia 5-hrs after food removal, and were more glucose tolerant and insulin sensitive than HFD-GF-mice that received feces from vehicle-treated Chow mice. Metagenome sequencing and metabolomics analysis performed on cecal contents from conventional Chow-fed PAHSA- and Vehicle-treated mice showed that 26 fatty acids (out of 600 metabolites) and several microbial species and their metabolic pathways correlated most highly with PAHSA-mediated insulin sensitivity. Also, Bacteroides thetaiotaomicron (Bt), a gut microbe associated with leanness in humans, and cecal C34:2 Phosphatidylethanolamine (PE), an abundant phospholipid class that is reduced in obese humans, were most strongly associated with the beneficial PAHSA effects. Initial studies with Bt treatment in male chow mice show improved glucose tolerance and lower glycemia 5-hrs after food removal vs. controls. Thus, the gut microbiota is necessary for the effects of PAHSAs to improve glucose homeostasis in HFD-fed mice. These effects can be conferred by FMT. PAHSA-associated microbes could provide novel gut-based therapeutic strategies to treat obesity and insulin resistance. Disclosure J. Lee: None. A. Rahnavard: None. K. Wellenstein: None. A.T. Nelson: None. C.B. Clish: None. D. Siegel: None. B. Kahn: Advisory Panel; Self; Harrington Discovery Institute, Janssen Pharmaceuticals, Inc., National Institute of Diabetes and Digestive and Kidney Diseases. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1K01DK114162-01A1, DK1NADK-057521-01 to J.L.); JPB Foundation (to B.K.)

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with anti-inflammatory and antidiabetic effects, improve systemic and hepatic insulin sensitivity in insulin-resistant mice. PAHSAs augment insulin action on endogenous glucose production (EGP) through direct and indirect actions involving inter-tissue communication between liver and adipose tissue. The direct effect of PAHSAs to inhibit EGP is mediated through a cAMP dependent pathway involving Gα/i protein-coupled receptors. Here, we investigated which receptor mediates PAHSA effects on EGP. We found that 9-PAHSA activates GPR43 in a Ca+2 flux assay. 4-CMTB (0.4μM), a selective agonist-allosteric modulator of GPR43, reduced basal EGP by 23% and glucagon-stimulated EGP by 20% in primary hepatocytes ex vivo. Acute 9-PAHSA treatment (40μM) inhibited basal EGP to the same extent as 4-CMTB. 9-PAHSA suppression of basal EGP was abolished in primary hepatocytes from whole body GPR43 KO mice. Intravenous infusion of 9-PAHSA (9μg/hr) for 3 hrs decreased ambient EGP in chow-fed mice studied 5 hours after food removal (WT: vehicle= 24.2±1.9 vs. PAHSA= 17.2±0.7 mg/kg/min p Sum: 9-PAHSA activates GPR43, which is involved in its direct effects to reduce hepatic glucose production and WAT lipolysis. Conclusion: We have identified a new receptor that mediates some of the beneficial metabolic effects of PAHSAs. Disclosure A. Santoro: None. P. Zhou: None. Y. Zhu: None. O.D. Peroni: None. A.T. Nelson: None. D. Siegel: None. B. Kahn: Advisory Panel; Self; Harrington Discovery Institute, Janssen Pharmaceuticals, Inc., National Institute of Diabetes and Digestive and Kidney Diseases. Funding American Diabetes Association (1-18-PDF-134 to P.Z.); National Institutes of Health (DK43051, DK57521, DK106210, DK112622); JPB Foundation; American Heart Association

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with anti-inflammatory and antidiabetic effects, improve glucose tolerance and insulin sensitivity in insulin-resistant mice. We found that PAHSA treatment enhances insulin action to suppress endogenous glucose production (EGP) in chow and HFD mice and promotes glucose uptake in glycolytic muscle and heart in HFD-fed mice. We aimed to determine the mechanisms by which PAHSAs enhance hepatic insulin sensitivity. We examined both direct effects in hepatocytes and indirect effects resulting from augmentation of insulin-mediated suppression of WAT lipolysis. Here, we show that PAHSAs inhibit lipolysis in WAT explants to the same extent as insulin. PAHSAs also enhance the anti-lipolytic effect of insulin in vivo, as indicated by a 30% reduction in FFAs during the clamp in PAHSA-treated mice under conditions with no suppression in Vehicle treated mice. To determine whether lowering FFAs is necessary for PAHSA effects on insulin sensitivity, we infused intralipid during a hyperinsulinemic-euglycemic clamp. In chow-fed mice infused with intralipid, PAHSAs still have a partial effect to lower EGP (Intralipid: vehicle 12±0.8 vs. PAHSA 7±1.7 mg/kg/minute), whereas in HFD-fed mice, preventing PAHSA-induced FFA reduction blocks PAHSA effects on hepatic insulin sensitivity (EGP Intralipid: vehicle 13±0.6 vs. PAHSA 12±1.7 mg/kg/minute). Our mechanistic studies show that PAHSAs inhibit basal EGP and reduce glucagon stimulated EGP directly in isolated hepatocytes. This is mediated by a cAMP-dependent pathway involving Gα/i protein-coupled receptors. Hepatic phospho-CREB is also reduced by PAHSAs in vivo. Sum: PAHSAs enhance hepatic insulin sensitivity through direct and indirect actions involving inter-tissue communication between adipose tissue and liver. These data reveal new mechanisms underlying the beneficial effects of PAHSAs, which will clarify their roles in healthy and disease states. Disclosure P. Zhou: None. A. Santoro: None. O.D. Peroni: None. A.T. Nelson: None. A. Saghatelian: None. D. Siegel: None. B. Kahn: Advisory Panel; Self; Alterna Therapeutics, Inc., American Diabetes Association, Harrington Discovery Institute, Janssen Research & Development. Consultant; Self; Ironwood Pharmaceuticals, Inc. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases. Funding American Diabetes Association (1-18-PDF-134 to P.Z.); National Institutes of Health (R01DK43051 to B.K.), (R01DK106210 to B.K., A.S.); JPB Foundation (to B.K.); American Heart Association (to A.T.N.)

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. We found that PAHSAs protect against T1D by attenuating autoimmune responses and by direct effects on islets. We aimed to determine the mechanisms by which PAHSAs directly promote β-cell survival. Daily oral gavage of PAHSAs in NOD mice starting at 4 or 13 weeks of age delays T1D onset and reduces T1D incidence from 82-90% of mice with vehicle to 35-40% of mice receiving PAHSAs. PAHSA treatment reduces leukocyte infiltration into islets by 50% and reduces T cell activation. PAHSA-treated mice had greater β-cell area relative to total islet area (vehicle 60±7 vs. PAHSA 77±1). PAHSAs enhance β-cell proliferation in NOD mice 5 fold vs. vehicle. PAHSA-treated NOD mice pancreata have lower ER stress markers, CHOP and XBP-1 (vehicle 68±12 vs. PAHSA 42±3), and higher insulin (vehicle 12±2 vs. PAHSA 17±1) immunostaining intensities per β-cell area. PAHSAs attenuate thapsigargin-induced PARP cleavage by 55% in human islets, and JNK1/2 phosphorylation by 40% in MIN6 cells. Also, PAHSAs attenuate apoptotic and necrotic β-cell death under cytokine stress and increase β-cell viability by 34% and proliferation by 31% (p Conclusion: In addition to attenuating immune responses, PAHSAs delay the onset and reduce T1D incidence in NOD mice through direct effects. PAHSAs directly reduce cytokine-induced β-cell death and increase cell proliferation in autoimmune diabetes through mechanisms involving ER stress reduction. This appears to be mediated in part by GLP-1R and not by GPR40. Disclosure I. Syed: None. M. Fernandez Rubin de Celis: None. J.F. Mohan: None. P.M. Moraes-Vieria: None. A. Vijayakumar: None. A.T. Nelson: None. D. Siegel: None. A. Saghatelian: None. D. Mathis: None. B. Kahn: Advisory Panel; Self; Alterna Therapeutics, Inc., American Diabetes Association, Harrington Discovery Institute, Janssen Research & Development. Consultant; Self; Ironwood Pharmaceuticals, Inc. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases. Funding National Institutes of Health (R01DK43051, P30DK57521 to B.K.), (R01DK106210 to B.K., A.S.), (K01DK118041 to I.S.); JPB Foundation (to B.K., D.M.); Deutsche Forschungsgemeinschaft (to M.F.R.)

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with potent anti-inflammatory and antidiabetic effects, reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin resistant HFD-fed mice. We aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. We treated chow- and HFD-fed mice with 5-PAHSA (0.1 mg/day) and 9-PAHSA (0.4 mg/day) by SQ osmotic minipumps for 13 weeks. Serum 5-PAHSA levels increased 3.5 fold and 9-PAHSA 2.5 fold in chow-fed mice, while 5-PAHSA levels increased 17 fold and 9-PAHSA 6 fold in HFD-fed mice. PAHSAs had no effect on weight gain or fat mass. We performed hyperinsulinemic euglycemic clamps (2.5 mU/Kg/min insulin infusion rate). PAHSA treatment increased systemic insulin sensitivity in both chow- [glucose infusion rate (GIR): vehicle 13±2 vs. PAHSA 27±4 mg/kg/min] and HFD-fed mice (GIR: vehicle 2±0.5 vs. PAHSA 10±3 mg/kg/min). Endogenous glucose production (EGP) was suppressed 28% by insulin in vehicle-treated mice and 55% in PAHSA-treated mice on chow diet. Remarkably, while insulin failed to suppress EGP in vehicle-treated HFD-fed mice, PAHSA treatment led to a 37% reduction in EGP. PAHSAs also decreased glycemia during a pyruvate tolerance test, indicating that suppression of hepatic gluconeogenesis contributes to the EGP improvement. Mechanistic studies showed that PAHSAs inhibit basal and glucagon-stimulated EGP and reduce cAMP in isolated hepatocytes. This effect is blocked by pertussis toxin indicating it is mediated by Gα/i protein-coupled receptors. PAHSAs also reduce G6pase activity and phosphorylation of CREB in liver. Sum: PAHSAs are systemic insulin sensitizers and augment insulin action on EGP in vivo. In vitro, PAHSAs reduce EGP through a cAMP dependent pathway involving Gα/i protein-coupled receptors. Thus, PAHSAs could be effective antidiabetic agents and the pathways they engage could provide novel drug targets for type 2 diabetes. Disclosure A. Santoro: None. P. Zhou: None. O.D. Peroni: None. I. Syed: None. A.T. Nelson: None. D. Siegel: None. A. Saghatelian: None. B. Kahn: Advisory Panel; Self; Janssen Research & Development. Research Support; Self; Janssen Research & Development. Advisory Panel; Self; Alterna Biotech.