Palmitic Acid Hydroxy Stearic Acids Activate GPR40 Which is Involved in Their Beneficial Effects on Glucose Homeostasis. Ismail Syed1,5, Jennifer Lee1,5, Pedro M. Moraes-Vieira1,$, Cynthia J. Donaldson2, Alexandra Sontheimer1, Pratik Aryal1, Kerry Wellenstein,1 Matthew J Kolar2, Andrew T Nelson3, Dionicio Siegel3, Jacek Mokrosinski4, I. Sadaf Farooqi4, Juan Juan Zhao1, Mark M. Yore1, Odile D. Peroni1, Alan Saghatelian2, Barbara B. Kahn1*. Affiliations: 1 Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215. 2 Clayton Foundation Laboratories for Peptide Biology, Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N Torrey Pines Road, La Jolla, CA 92037. 3 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA, 92093. 4 University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, CB2 0QQ, United Kingdom. $ Current address: University of Campinas, Campinas-SP 13083-970, Brazil. 5 these authors contributed equally. * Correspondence: Dr. Barbara B. Kahn Division of Endocrinology, Diabetes and Metabolism Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Center for Life Sciences, 7th floor 330 Brookline Ave, Boston, MA 02215 Phone: 617-735-3324, Fax: 617-735-3323 Email: bkahn@bidmc.harvard.edu Summary Branched Palmitic Acid Esters of Hydroxy Stearic Acids (PAHSAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. PAHSA levels are reduced in serum and adipose tissue of insulin-resistant people and high-fat-diet (HFD)-fed mice. Here, we investigated whether PAHSAs enhance insulin sensitivity and which receptors mediate their effects. Chronic PAHSA administration in chow-fed and HFD-fed mice raises serum and tissue PAHSA levels ~1.4 to 3 fold. This improves insulin sensitivity, glucose tolerance and adipose tissue inflammation without altering food intake or body weight. Chronic PAHSA administration in chow-fed mice, but not in HFD-fed mice, also augments insulin and glucagon-like peptide (GLP-1) secretion. PAHSAs are partial agonists for GPR40, increasing Ca2+ flux but not intracellular cAMP levels. Blocking GPR40 reverses the improvement in glucose tolerance and insulin sensitivity in PAHSA-treated chow-fed and HFD-fed mice and directly inhibits PAHSA augmentation of glucose-stimulated insulin secretion (GSIS) in human islets. In contrast, blocking the GLP-1 receptor in PAHSA-treated mice reduces PAHSA effects only on glucose tolerance and not on insulin sensitivity. Thus, PAHSAs activate GPR40 which plays a major role in mediating their beneficial metabolic effects. Introduction Type 2 Diabetes (T2D) is a global epidemic (International Diabetes Federation Atlas 2013) characterized by hyperglycemia due to impaired pancreatic islet function and insulin resistance in peripheral tissues. Despite advances in understanding the molecular mechanisms contributing to T2D and the development of new treatment modalities, the medical management of T2D remains inadequate (Madsbad et al., 2011; Aroda et al., 2012; Pratley et al., 2010). At present, many available treatment strategies including analogues of the incretin, GLP-1, and DPP-lV inhibitors work primarily by increasing insulin secretion. Other effective agents promote glucose excretion (Campbell & Drucker 2013; Meier 2012). But there is still a need for effective and safe agents that enhance insulin sensitivity to improve diabetic treatment and prevent complications. We recently discovered a structurally novel class of endogenously synthesized lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), with beneficial metabolic and anti-inflammatory effects (Yore et al., 2014). More than 16 FAHFA family members have been identified (Yore et al., 2014; Zhang et al., 2016; Yan et al., 2015). Levels of one of the FAHFA family members, palmitic acid hydroxy stearic acid (PAHSAs), are markedly lower in serum and adipose tissue of insulin-resistant humans. PAHSA levels correlate strongly with insulin sensitivity as measured by euglycemic clamps in humans (Yore et al., 2014). Acute oral treatment with 5- or 9-PAHSA isomers in chow and high-fat diet (HFD)-fed mice improves glucose tolerance and augments insulin and GLP-1 secretion in vivo. In vitro, PAHSAs directly enhance GLP-1 secretion from enteroendocrine cells and glucose-stimulated insulin secretion from human pancreatic islets (Yore et al., 2014). Furthermore, PAHSAs have anti-inflammatory effects including decreasing adipose inflammation in HFD mice and attenuating LPS-induced dendritic cell activation and cytokine production (Yore et al., 2014). Although we reported that a single dose of PAHSAs acutely improves glucose tolerance, whether PAHSAs have effects on insulin sensitivity in vivo has not been investigated. Therefore, the first aim of this study was to determine whether PAHSA treatment enhances insulin sensitivity in vivo, and the second aim was to determine whether the beneficial effects of PAHSAs are sustained with chronic treatment. The receptors responsible for PAHSA effects on insulin secretion and insulin action in vivo have not been identified. The effect of PAHSAs to enhance insulin-stimulated glucose transport in adipocytes is mediated by the G-protein coupled receptor, GPR120 (Yore et al., 2014), but PAHSAs are likely to activate other GPCRs because of the diversity of their actions in multiple tissues. Major pharmaceutical companies have had high-priority programs screening for activators of GPR120 and GPR40 to treat T2D. Molecules that activate more than one of these GPCRs may be more effective for T2D treatment than agonists for single receptors. Recent research has focused on identifying key agonists and receptors mediating nutrient-induced GLP-1 and insulin secretion. The long-chain fatty acid receptor, GPR40 is the most abundant GPCR expressed in islet β-cells and is also expressed on intestinal L-cells where it contributes to GLP-1 release along with GPR120 (Itoh et al., 2003; Edfalk et al., 2008). GPR40 activation augments glucose-stimulated insulin secretβ-cells and is also expressed on intestinal L-cells where it contributes to GLP-1 release along with GPR120 (Itoh et al., 2003; Edfalk et al., 2008). GPR40 activation augments glucose-stimulated insulin secretion (GSIS) and improves glycemia in rodent T2D models (Itoh et al., 2003; Steneberg et al., 2005; Latour et al., 2007), and the GPR40 a(Leifke et al., 2012; Burant et al., 2012). Because PAHSAs acutely augment GSIS directly from human islets (Yore et al., 2014), the third aim of this study was to determine whether PAHSAs activate GPR40 and whether this contributes to their beneficial effects in vivo. Here we show that PAHSAs directly activate GPR40 which is important for PAHSA effects on glucose homeostasis in both chow and HFD-fed mice. Since PAHSAs augment GLP-1 secretion in insulin-resistant mice (Yore et al., 2014), we also investigated the role of the GLP-1R pathway in the beneficial effects of PAHSAs. While GLP-1 which is secreted in response to intestinal luminal nutrients, potentiates insulin secretion and suppresses glucagon release (Host 2007), its beneficial metabolic actions are not limited to the endocrine pancreas (Ayala et al., 2009; Villanueva-Peñacarrillo et al., 2011; Christensen et al., 2015). In this study, we show that the GLP-1 receptor contributes to the beneficial effects of PAHSAs on insulin secretion and glucose tolerance but not on insulin sensitivity in chow-fed mice. In addition, the PAHSA-stimulated augmentation of GSIS in chow-fed mice is directly mediated by GPR40 and is independent of GLP1 secretion. Thus, in chow-fed mice PAHSAs improve glucose tolerance and insulin sensitivity, and these effects are sustained for 5 months. In HFD-fed mice, glucose tolerance and insulin sensitivity are also improved but the effects are more modest. Furthermore, this study shows that PAHSAs activate GPR40 and this contributes to their beneficial metabolic effects in vivo. Results and Discussion Chronic PAHSA treatment improves insulin sensitivity and glucose tolerance, and reduces adipose tissue inflammation in chow-fed mice without altering food intake or adiposity Up to 18 weeks of treatment with 5- and 9-PAHSA by subcutaneous mini pump in chow-fed male C57bl6 mice had no effect on body weight, fat mass (Figure 1A), food intake, lean mass, serum triglycerides or free fatty acid levels (Supplementary Figure 1A and 1B). Serum 5- and 9-PAHSA levels increased 2-fold with 2 months of treatment compared to vehicle-treated mice (Figure 1B). After 5 months of treatment, only 9-PAHSA levels were elevated in serum in the treatment group (Figure 1B). However, 5- and 9-PAHSA levels were increased 1.5- to 3-fold in perigonadal (PG) and subcutaneous (SC) white adipose tissue and brown adipose tissue. In liver 9-PAHSA levels were elevated 2.5-fold in treated mice, and 5-PAHSA was easily detectable even though this isomer was not found in liver of vehicle-treated mice as expected (Yore et al., 2014) (Figure 1B). 5- and 9-PAHSA levels were not increased in the pancreas or brain with chronic PAHSA treatment (Figure 1B). 5- and 9-PAHSA treatment improved insulin sensitivity as early as 13 days of treatment and these effects were sustained for at least 5 months (Figure 1C). Glucose tolerance (Figure 1D) was also improved similar to the effects reported with a single dose (Yore et al. 2014). Loss of first phase insulin response to glucose is one of the major and early impairments of islet β-cell function in T2D (Luzi and DeFronzo 1989). Restoration of this response is associated with significant improvement in postprandial glucose excursion (Bruce et al., 1988). Here we show that, chronic 5- and 9-PAHSA treatment enhances insulin secretion in response to glucose by 40% over vehicle-treated mice (Figure 1E, left panel). GLP-1 levels tend to be reduced at baseline in PAHSA-treated mice, and PAHSA treatment enhanced glucose-stimulated GLP-1 secretion 225% over vehicle (Figure 1E, right panel). Therefore, not only does an acute oral dose of PAHSAs improve first-phase insulin and GLP-1 responses to glucose (Yore et al., 2014), but this effect is maintained over 5 months without tachyphylaxis or islet β-cell exhaustion. To confirm that the beneficial effects of PAHSAs on metabolic parameters are distinct from effects of ordinary fatty acids, we performed similar studies with palmitate since PAHSAs are made up. To confirm that the beneficial effects of PAHSAs on metabolic parameters are distinct from effects of ordinaralmitate since PAHSAs are made up of palmitate and hydroxystearic acid. Palmitate treatment at the same dose used for PAHSAs does not have any favorable effects on glucose tolerance, insulin tolerance, or glucose-stimulated insulin or GLP-1 secretion (Figure 1F-1I). In addition, at the same concentration as PAHSAs (20µM), palmitate did not augment GSIS in cultured islets (Supplementary Figure 1C). This was not because of lower palmitate uptake into cells since lipid uptake as measured by the ratio of PAHSA or palmitate in the low density microsomes, high density microsomes, and cytosol fractions compared to the plasma membranes, appeared greater for palmitate than for PAHSAs (Supplemental Figure 1D). We next investigated whether chronic PAHSA treatment reduces adipose tissue (AT) Inflammation. PAHSA treatment reduced the total number of AT CD11c+ (pro-inflammatory) macrophages with no effect on AT CD206+ (anti-inflammatory) macrophages (Figure 1J; Gating strategy in Supplementary Figure 1E). The total number of AT macrophages tended to be reduced with PAHSA treatment (Figure 1J) with no change in monocytes or neutrophils (Supplementary Figures 1F). Moreover, PAHSA treatment reduced the number of macrophages positive for IL-1β or TNF-α (Figure 1J). Together, these data indicate that chronic PAHSA treatment reduces AT Inflammation which could contribute to enhanced glucose-insulin homeostasis in chow-fed mice. PAβ or TNF-α (Figure 1J). Together, these data indicate that chronic PAHSA treatment reduces AT Inflammation which could contribute to enhanced glucose-insulin homeostasis in chow-fed mice. PAHSAs activate GPR40 and this plays a role in their effects on insulin se Since free fatty acids can induce insulin and GLP-1 secretion through GPR40, we investigated the role of GPR40 in mediating PAHSA effects. 9-PAHSA potentiated GSIS in isolated human islets, and pharmacological inhibition of GPR40 using a GPR40 antagonist, GW1100, completely blocked this effect (Figure 2A). Furthermore, knocking down GPR40 in pancreatic β-cells (MIN6 cells) using a combination of three different GPR40 siRNAs completely reversed 9-PAHSA-potentiated GSIS without altering insulin pancreatic β-cells (MIN6 cells) using a combination of three different GPR40 siRNAs completely reversed 9-PAHSA-potentiated GSIS without altering insulin secretion at low glucose (Figure 2B, GPR40 knockdown efficiency in Supplementary Figure 2A). Thus, both pharmacologic and genetic approaches indicat To determine whether PAHSAs directly activate GPR40, we transfected HEK293 cells with GPR40 and SRE-luc. 9-PAHSA activates GPR40 in a dose-dependent manner (Figure 2C). 5-PAHSA also activates GPR40 (Supplementary Figure 2B). 9-PAHSA-induced GPR40 activation was attenuated by a GPR40 antagonist, in a dose-dependent manner (Figure 2D). We next determined whether PAHSAs are full or partial GPR40 agonists. 9-PAHSA increased Ca2+ flux similar to the positive control Linoleic acid, and the GPR40 antagonist, GW1100 attenuated this effect (Figure 2E). 9-PAHSA had no effect on intracellular cAMP levels (Figure 2F). Thus, PAHSAs are partial, not full, agonists for GPR40. We also tested whether GPR40 mediates the effects of PAHSAs on GLP-1 secretion directly in STC-1 enteroendocrine cells. Both 5- and 9-PAHSAs augmented GLP-1 secretion in STC-1 cells by 1.5- to 2-fold, but GPR40 antagonism did not reduce this (Supplementary Figure 2C). This suggests that the direct effects of PAHSAs to augment GLP-1 secretion in enteroendocrine cells are independent of GPR40. Inhibition of GPR40 reverses the beneficial effects of 5- and 9-PAHSA on both glucose tolerance and insulin sensitivity To determine whether the GSIS effects of PAHSAs are mediated by GPR40 in vivo, vehicle- and 5- and 9-PAHSA-treated chow-fed mice were injected with the GPR40 antagonist, DC260126. A single dose of DC260126 attenuated the beneficial effects of PAHSAs on insulin sensitivity and glucose tolerance (Figure 2G-2H, compare to Figure 1C-1D), but had no effect in the vehicle-treated mice(Supplementary Figure 2D-2F). There was no effect of GPR40 inhibition on glycemia at baseline or 5 minutes post glucose administration (Figure 2I and Supplementary Figure 2G). However, GPR40 inhibition lowered baseline insulinemia (30 min after DC260126) and completely blocked glucose-stimulated insulin secretion (GSIS) in PAHSA-treated mice (Figure 2I), but had no effect in vehicle-treated mice (Supplementary Figure 2G). In PAHSA- and vehicle-treated mice, GPR40 inhibition did not affect glucose-stimulated GLP-1 secretion (Figure 2I and Supplementary Figure 2G). Thus, PAHSAs stimulate insulin secretion through GPR40 and these effects are independent of GLP-1 secretion. We also determined whether the effects of chronic PAHSA treatment in vivo are sustained when pancreatic islets are removed and studied ex vivo. Compared to islets isolated from vehicle-treated mice, in islets from PAHSA-treated mice ex vivo there was no further augmentation of GSIS (Supplementary Figure 2H). This suggests the continuous presence of PAHSAs is required to trigger the GPR40 activity. Blockade of the GLP-1 receptor reverses the beneficial effects of chronic PAHSA treatment on glucose tolerance but not insulin sensitivity To determine whether PAHSA-induced improvements in glucose tolerance and insulin sensitivity are mediated through enhanced GLP-1 secretion, we injected vehicle- and 5- and 9-PAHSA-treated mice with the GLP-1 receptor antagonist, Exendin (9-39) (Figure 3A). A single dose of Exendin (9-39) completely reversed PAHSA-induced improvement in glucose tolerance in chow-fed mice (Figure 3B; Supplementary Figure 3A) but not insulin sensitivity (Figure 3C). In PAHSA-treated mice, GLP-1 receptor blockade increased glycemia at baseline (30 minutes after Exendin treatment) (Figures 3B-3D) and at 5 minutes post glucose administration (Figure 3D). In addition, Exendin (9-39) treatment attenuated PAHSA-mediated increase in GSIS. Exendin (9-39) increased baseline but attenuated glucose-stimulated GLP-1 secretion (Figure 3D). In vehicle-treated mice, Exendin (9-39) had no effect on baseline glucose (30 minutes after Exendin treatment) or on glucose tolerance except at 45 min after glucose administration (Supplemental Figure 3B). There was also no effect on insulin sensitivity or on glycemia at baseline or at 5 minutes post glucose administration (Supplementary Figure 3C-3D). Furthermore, GLP-1R antagonism in vehicle-treated mice increased glucose-stimulated insulin and GLP-1 secretion (Supplementary Figure 3D). This increase in GLP-1 secretion may be a response to GLP-1 receptor blockade and mostly likely stimulates insulin secretion. As expected, PAHSAs do not directly activate GLP-1R (Supplementary Figure 3E) which is activated by peptides (GLP-1) not lipids. Thus these experiments indicate that GLP-1 action is necessary for the full effects of PAHSAs on glucose tolerance but not insulin sensitivity, and this results from PAHSA-mediated increase in GLP-1 secretion. These observations are in accordance with a study in T2D people, in which GLP-1 improves glycemic control by increasing insulin secretion and inhibiting glucagon secretion, without improving insulin sensitivity (Vella et al., 2000). Chronic PAHSA treatment improves insulin sensitivity and glucose tolerance, and reduces adipose tissue inflammation in HFD-fed mice without altering food intake or adiposity. 15-18 weeks of 9-PAHSA treatment via subcutaneous mini pumps in HFD-fed male C57bl6 mice did not alter body weight, fat mass, lean mass, food intake, or serum triglycerides or free fatty acid levels (Figure 4A, Supplementary Figure 4A-4B). 9-PAHSA levels are elevated ~2 fold in serum and liver (Figure 4B) and ~33-42% in PG and SQ WAT compared to vehicle-treated HFD mice. 9-PAHSA levels are restored to chow-fed mouse levels in serum and PG WAT but not completely restored in SQ WAT (Figure 4B and Figure 1B). Chronic PAHSA treatment in HFD-fed mice improved insulin sensitivity (Figure 4C and Supplementary Figure 4C) and glucose tolerance (Figure 4D) compared to vehicle-treated HFD-fed mice and these effects were sustained for at least 4 months. We found that 9-PAHSA treatment (12 mg/kg per day) was more effective than a split dose of 5- and 9-PAHSA (6 mg/kg of each) in HFD mice (data not shown) although the combination treatment consistently lowered glycemia 5 hours after food removal (Supplementary Figure 4D). Further studies showed 9-PAHSA lowered glycemia at 5 minutes post-glucose in HFD mice compared to vehicle-treated HFD mice with similar insulin levels (Figure 4E), supporting enhanced insulin sensitivity. Also in support of this, chronic 9-PAHSA treatment in HFD-fed mice prevented the increase in islet mass seen in vehicle-treated HFD-fed mice (Figure 4F). This again suggests that the same insulin levels are more efficient in lowering glucose in PAHSA-treated HFD mice and expansion of islet mass is not necessary. In contrast to PAHSA treatment in chow-fed mice (Figure 1E), chronic PAHSA treatment in HFD-fed mice did not enhance glucose-stimulated insulin or GLP-1 secretion compared to vehicle-treated HFD mice (Figure 4E) but similar insulin levels lowered glucose more in PAHSA-treated HFD mice. Thus, PAHSA beneficial effects on glucose homeostasis in HFD-fed mice are independent of changes in glucose-stimulated insulin or GLP-1 secretion and appear to involve increased insulin sensitivity. AT TNF-α mRNA levels are lower in PAHSA-treated HFD-fed mice compare TNF-α mRNA levels are lower in PAHSA-treated HFD-fed mice compared to vehicle-treated HFD-fed mice (Figure 4G), indicating that reduced AT Inflammation could contribute to the enhanced glucose-insulin homeostasis in HFD-fed mice. Overall, the PAHSA-mediated beneficial effects in HFD-fed mice are more moderate than effects in chow-feSC AT. Since PAHSA beneficial effects on glucose homeostasis in chow-fed mice are in part mediated by GPR40 (Figure 2G and 2H), we studied the role of GPR40 in the PAHSA effects in HFD-fed mice. The GPR40 antagonist, DC260126, reverses the beneficial effects of PAHSAs on insulin tolerance test and glucose tolerance test in HFD-fed mice (Figure 4H and 4I), and had no effect in the vehicle-treated HFD mice (Supplementary Figure 4E and 4F). This effect of GPR40 inhibition in PAHSA-treated HFD mice is mainly due to the reversal of the effects of PAHSAs to lower glycemia at time “0” min of the OGTT and ITT (Figure 4H and 4I, Supplementary Figure 4G and 4H). These data suggest that GPR40 mediates at least some of the beneficial effects of PAHSAs in HFD-fed mice similar to the effects in chow-fed mice. Prior to this study, we investigated only the acute PAHSA effects on glucose homeostasis, primarily with a single dose (Yore et al., 2014). Furthermore, we did not report data on insulin sensitivity. A major advance of this study is demonstrating that PAHSAs enhance insulin sensitivity. This is important since a major “unmet medical need” is safe insulin-sensitizing agents. None of the anti-diabetic drugs on the market are primarily insulin-sensitizers except the thiazolidinediones which have adverse effects that have markedly curtailed their use. Safe insulin-sensitizing agents could be used both to prevent and treat insulin resistance, T2D, and cardiovascular complications. Since PAHSAs correlate highly with insulin sensitivity in humans, and are reduced in serum and SC AT of insulin-resistant people and HFD-fed mice, we designed the current study to restore circulating and tissue PAHSA levels within the physiologic range (Yore et al., 2014). Although we achieved this in chow-fed mice and in serum and PG WAT of HFD-fed mice, we did not fully restore levels in SC AT of HFD-fed mice. This could contribute to the more modest effects on insulin sensitivity and glucose tolerance in HFD mice. The effects in chow-fed mice are also greater with a mixed background, which is more relevant to human disease, than inbred C57bl6 mice. Future studies will investigate the effects of PAHSA treatment in HFD mice on a mixed genetic background. Another major advance is that we identified a new receptor that is directly activated by PAHSAs, GPR40. GPR40 blockade in chow-fed mice attenuates PAHSA-mediated improvements in glucose tolerance, insulin sensitivity, and insulin secretion but not glucose-stimulated GLP-1 secretion. The fact that PAHSAs have direct effects on insulin secretion independent of GLP-1 is supported by our data showing they stimulate GSIS in isolated human islets (Figure 2A; Yore et al., 2014). In HFD-fed mice, GPR40 blockade attenuates PAHSA-mediated beneficial effects on glucose tolerance and systemic insulin sensitivity even though chronic PAHSA treatment does not potentiate GSIS. The GLP1 receptor plays a role in some but not all PAHSA effects in chow-fed mice since GLP-1R blockade attenuates PAHSA-mediated improvement only in glucose tolerance and not in insulin sensitivity. These observations do not eliminate a role for GPR120 which could mediate the PAHSA effects on GLP1 secretion as well as glucose uptake (Yore et al 2014) and which has some overlapping biologic effects with GPR40. This study expands our understanding of the biology of PAHSAs by demonstrating sustained beneficial effects of chronic treatment without tachyphylaxis or islet exhaustion. In fact, PAHSAs appear to enhance islet function in HFD-fed mice since expansion of islet mass which we observe in vehicle-treated HFD-fed mice, is prevented with PAHSA treatment. Yet the islets produce the same amount of insulin and lower glycemia more in PAHSA-treated HFD mice than vehicle-treated HFD mice. PAHSA-mediated beneficial effects are achieved with subcutaneous delivery via mini pumps, indicating that the oral route is not necessary for PAHSA effects on glucose-homeostasis. These sustained effects of PAHSAs are mediated, at least in part, by GPR40. Because PAHSAs can activate multiple GPCRs which are known to have beneficial metabolic effects, and PAHSA levels are reduced in insulin-resistant people, restoring PAHSA levels has high therapeutic potential to prevent and/or treat T2D. EXPERIMENTAL PROCEDURES Animal studies and measurement of metabolic parameters Chow-fed (Purina Lab diet, 5008) male C57Bl/6J wild-type mice (Jackson Laboratories) and in-house bred mixed background mice (C57Bl/6J and FVB) were randomized to treatment group based on body weight at 8 weeks of age and implanted with subcutaneous mini pumps (Alzet, Model 2006). Similarly, randomized male C57Bl/6J wild-type mice were placed on HFD (Research Diets, Td93075) at 6 weeks of age. After 8 weeks on HFD, insulin resistance was confirmed by an insulin tolerance test. Mini pumps were implanted after 9 weeks on HFD. All mice were singly housed in ventilated cages with ad libitum access to food and water. All animals were kept on a 14 hr light, 10 hr dark schedule at an ambient temperature between 72-74˚F. Body weight and food intake were measured weekly and body composition was measured by MInsulin tolerance test and oral glucose tolerance test were performed as previously described (Moraes-Vieira et al. 2014) following 5-hour food removal. All animal care and use procedures were in strict accordance with, and approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center, Boston, MA. Experimental treatments, materials and chemical reagents Mini pumps filled with either vehicle (50% PEG-400, 0.5% Tween-80, 49.5% distilled water) or 5- and 9-PAHSAs (Chow: 2mg/kg of each PAHSA or 2mg/kg of palmitate; HFD: 6mg/kg of each PAHSA or 12mg/kg of 9-PAHSA) were inserted subcutaneously at 8-weeks of age in chow-fed and 15 weeks of age in HFD-fed mice. Minipumps were changed every 42 days. Insulin and D-glucose (Sigma Aldrich, St. Louis, MO) were used for tolerance tests. For in vivo studies, the GLP-1R antagonist, Ex(9-39) (5µg/mouse) and the GPR40 antagonist, DC260126 (5mg/kg; Tocris Bioscience) was administered intraperitoneally. The GPR40 antagonist, GW1100 (10µM, Cayman Chemicals) was used for in vitro studies. All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. ELISA kits were obtained from Crystal Chem (Mouse Insulin), Millipore (Mouse GLP-1) and Alpco Diagnostics (Human Insulin). Organic solvents for LC-MS were purchased from Honeywell Burdick & Jackson. PAHSA extraction and measurements from serum and tissues Lipid extraction and PAHSA measurements were performed as described (Zhang et al., 2016). Anti-inflammatory effects of 5- and 9-PAHSAs in vivo Perigonadal AT stromal vascular fraction cells were harvested and surface markers were stained with monoclonal antibodies (Biolegend) for multicolor flow cytometry. For intracellular cytokine measurements, cells were stained as previously described (Moraes-Vieira et al., 2014). GPR40 Reporter assay HEK293 cells transfected with GPR40 and SRE-luc 44-235 were treated with DMSO control, 5-PAHSA (100µM), or 9-PAHSA (0-100µM). Transfected cells were treated with GW1100 (0-10µM) in combination with DMSO or 9-PAHSA. Glucose-stimulated insulin secretion (GSIS) in human islets Human islets from normal donor were obtained from BADERC Core facility, MGH, Bosotn. GSIS was assessed as previously outlined (Kowluru et al., 2010). Briefly, human islets were cultured overnight in human islet media and stimulated with either 2.5mM or 20mM glucose in presence or absence of DMSO, 9-PAHSA (20µM), and GW1100 (10µM) for 45 mins. At the end, media was collected and insulin was quantitated by ELISA (Alpco Diagnostics). Cell uptake study INS-1 cells were treated with 20µM double labelled PAHSA and 13C-palmitate for 1 hour. At the end of incubation, cells were collected in homogenization buffer with protease inhibitor and homogenized. Plasma membrane, high density microsome, low density microsome and cytosolic fractions were obtained as described by Evans et al., 2009. Protein was quantified using BCA reagent and purity of subcellular fractions was determined by Western blot. Double labelled PAHSA and 13C-Palmitate in different fractions were measured as previously described (Zhang et al., 2016). Determination of β-as previously described (Zhang et al., 2016). Determination of β-cell mass Pancreata from mice treated chronically with vehicle or 9-PAHSA by minipumps were collected and fixed in 10% formalin. From each pancreas, 2-3 duplicate sections were imaged to determine islet β-cell mass as previously described (Iglesias et. al., 2012). Data Analysis All values are means ± SEM. Differences between groups were assessed using Student’s t tests and/or ANOVA with Tukey post-test for multiple comparisons where appropriate. All statistical analysi Author Contributions I.S. and J.L. conceived of, designed, performed, and interpreted experiments and made figures. P.M.M.-V. designed, performed and interpreted the immunology experiments and made the figures. C.J.D., A.S. and B.B.K. designed and performed the receptor activation experiments. M.J.K. performed the uptake studies. A.T.N., D.S. and A.S. designed and performed chemical synthesis of the lipids. A. Sontheimer, P.A., O.D.P., K.W., and J.J.Z., assisted with animal studies. J.M. and I.S.F. contributed to GPR40 signalling assays. M.M.Y designed and performed GLP-1 assay in vitro. B.B.K. and A.S. conceived of, designed and supervised the experimental plan and interpreted experiments. B.B.K., I.S., and J.L. wrote the manuscript. A.S. and P.M.M.V. edited the manuscript. Acknowledgements We thank Dr. Ji Lei, the director of BADERC Pancreatic Islet Core for providing human islets; and Dr. Douglas Hanahan for the STC1 cells. We thank Dr. Susan Bonner-Weir from the Joslin Diabetes Center for her expertise and conversations related to islet biology analyses. Supported by grants from the NIH R01 DK43051 and P30 DK57521 (B.B.K.); R01 DK106210 (B.B.K. and A.S.); a grant from the JPB foundation (B.B.K.). T32DK07516 (B.B.K. and J.L.). I.S.F. is supported by the Wellcome Trust and Bernard Wolfe Endowment. B.B.K., A.S., I.S. P.M.M.-V. and M.M.Y. are inventors on a patent related to the fatty acid hydroxy-fatty acids. References Aroda VR, Henry R, Han J, Huang W, DeYoung MB, Darsow T, Hoogwerf BJ. (2012). Efficacy of GLP-1 receptor agonists and DPP-4 inhibitors: meta-analysis and systematic review. Clin Ther. 34, 1247–1258. Ayala JE, Bracy DP, James FD, Julien BM, Wasserman DH, Drucker DJ (2009). The glucagon-like peptide-1 receptor regulates endogenous glucose production and muscle glucose uptake independent of its incretin action. Endocrinology. 150(3), 1155-1164. Brownlie R, Mayers RM, Pierce JA, Marley AE, Smith DM (2008). The long-chain fatty acid receptor, GPR40, and glucolipotoxicity: investigations using GPR40-knockout mice. Biochemical Society Transactions 36(5), 950-954. Burant CF, Viswanathan P, Marcinak J, Cao C, Vakilynejad M, Xie B, Leifke E (2012). TAK-875 versus placebo or glimepiride in type 2 diabetes mellitus: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet. 379, 1403–1411. Calabria AC, Li C, Gallagher PR, Stanley CA, De León DD (2012). GLP-1 receptor antagonist exendin-(9-39) elevates fasting blood glucose levels in congenital hyperinsulinism owing to inactivating mutations in the ATP-sensitive K+ channel. Diabetes. 61(10), 2585-2591. Campbell, J.E. & Drucker, D.J (2013). Pharmacology physiology and mechanisms of incretin hormone action. Cell Metab. 17, 819–837. Christensen LW, Kuhre RE, Janus C, Svendsen B, Holst JJ (2015). Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine. Physiol Rep. 3(9), e12551. Edfalk S, Steneberg P, Edlund H (2008). Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion. Diabetes. 57(9), 2280-22877. Evans SA, Doblado M, Chi MM, Corbett JA, Moley KH (2009). Facilitative Glucose Transporter 9 Expression Affects Glucose Sensing in Pancreatic β-Cells. Endocrinology. 150(12), 5302–5310. Holst JJ (2007). The ph-Cells. Endocrinology. 150(12), 5302–5310. Holst JJ (2007). The physiology of glucagon-like peptide 1. Physiol Rev. 87, 1409–1439. International Diabetes Federation (2013). IDF Diabetes Atlas (6th Edn.), International Diabetes Federation, Brussels, Belgium. Iglesias J, Barg S, Vallois D, Lahiri S, Roger C, Yessoufou A, Pradevand S, McDonald A, Bonal C, Reimann F, Gribble F, Debril M, Metzger D, Chambon P, Herrera P, Rutter GA, Prentki M, Thorens B, and Wahli W (2012). PPARβ/δ affects pancreatic β-cell mass and insulin se. J Clin Invest. 122(11), 4105–4117. Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H, Tanaka H, Maruyama M, Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura F, Noguchi Y, Shinohara T, Hinuma S, Fujisawa Y, Fujino M (2003). Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature. 422(6928), 173-176. Latour MG, Alquier T, Oseid E, Tremblay C, Jetton TL, Luo J, Lin DC, Poitout V (2007). GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo. Diabetes. 56, 1087–1094. Leifke E, Naik H, Wu J, Viswanathan P, Demanno D, Kipnes M, Vakilynejad M (2012). A multiple ascending-dose study to evaluate safety, pharmacokinetics, and pharmacodynamics of a novel GPR40 agonist, TAK-875, in subjects with type 2 diabetes. Clin Pharmacol Ther. 92, 29–39. Ma Y, Kind T, Vaniya A, Gennity I, Fahrmann JF, Fiehn O (2015). An in silico MS/MS library for automatic annotation of novel FAHFA lipids. J Cheminform. 7, 53. Madsbad S, Kielgast U, Asmar M, Deacon CF, Torekov SS, Holst JJ (2011). An overview of once-weekly glucagon-like peptide-1 receptor agonists-available efficacy and safety data and perspectives for the future. Diabetes Obes Metab. 13, 394–407. Meier, J.J (2012). GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 728–742. Pratley RE, Nauck M, Bailey T, Montanya E, Cuddihy R, Filetti S, Thomsen AB, Søndergaard RE, Davies M; 1860-LIRA-DPP-4 Study Group (2010). Liraglutide versus sitagliptin for patients with type 2 diabetes who did not have adequate glycaemic control with metformin: a 26-week, randomized, parallel-group, open label trial. Lancet. 375, 1447–1456. Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD, Edlund H (2005). The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metab. 1, 245–258. Vella A, Shah P, Basu R, Basu A, Holst JJ, Rizza RA (2000). Effect of glucagon-like peptide 1(7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes. Diabetes. 49(4), 611-617. Villanueva-Peñacarrillo ML, Martín-Duce A, Ramos-Álvarez I, Gutiérrez-Rojas I, Moreno P, Nuche-Berenguer B, Acitores A, Sancho V, Valverde I, González N (2011). Characteristic of GLP-1 effects on glucose metabolism in human skeletal muscle from obese patients. Regul Pept. 168(1-3), 39-44. Yore MM*, Syed I*, Moraes-Vieira PM, Zhang T, Herman MA, Homan EA, Patel RT, Lee J, Chen S, Peroni OD, Dhaneshwar AS, Hammarstedt A, Smith U, McGraw TE, Saghatelian A, Kahn BB (2014). Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects. Cell. 159(2), 318-332. Zhang T, Chen S, Syed I, Ståhlman M, Kolar MJ, Homan EA, Chu Q, Smith U, Borén J, Kahn BB, Saghatelian A (2016). A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids. Nature Protocols. 11(4), 747-763. Figure Legends Figure 1: Chronic PAHSA treatment improves insulin sensitivity and glucose tolerance, and reduces adipose tissue inflammation without altering food intake or adiposity. (A) Body weight and fat mass of C57bl6 male chow-fed mice treated with 5- and 9-PAHSAs (2mg/kg body weight per day of each) via mini pumps. n=16/group. (B) 5- and 9-PAHSA levels in sera at 2 and 5 months, and tissues at 5 months of 5-and 9-PAHSA treatment. n=5-6/group. (C) insulin tolerance tests (ITT) and (D) oral glucose tolerance tests (OGTT) 5-hours after food removal in vehicle- and PAHSA-treated mice. n=8-11/group. For A-D, *p<0.05 vs. vehicle. (E) Serum insulin and GLP-1 levels 5 min postglucose challenge in vehicle- and PAHSA-treated mice. n=14-16/group. *p<0.05 vs. baseline within same treatment, #p<0.05 vs. vehicle at same time point, $p=0.08 vs. vehicle at same time point. (F) ITT and (G) OGTT 5-hours after food removal in vehicle-, Palmitate- and PAHSA-treated outbred mice. n=7-9/group. (H-I) Serum insulin and GLP-1 levels 5 min postglucose challenge in vehicle-, Palmitate and PAHSA-treated outbred mice. n=7-9/group. *p<0.05 vs. baseline within same treatment, #p<0.05 vs. vehicle at same time point. (J) Number of adipose tissue (AT) CD11c+, CD206+, total number of AT macrophages (ATM), and macrophages expressing IL-1β and TNFα from PG WAT were measured by flow cytometry. n=4-5/group. *p<0.05 vs. vehicle; †p<0.08 vs. vehicle. Data are means±SEM. Figure 2: PAHSAs directly activate GPR40, and GPR40 antagonism reverses PAHSA-mediated improvements in glucose IL-1β and TNFα from PG WAT were measured by flow cytometry. n=4-5/group. *p<0.05 vs. vehicle; †p<0.08 vs. vehicle. Data are means±SEM. Figure 2: PAHSAs directly activate GPR40, and GPR40 antagonism reverses PAHSA-mediated improvements in glucose tolerance