Palmitic Acid Esters Of Hydroxy Stearic Acids Research Articles

The chemical and biological characteristics of fatty acid esters of hydroxyl fatty acids.

With the continuous advancements in detection methods and the exploration of unknown substances, an increasing number of bioactive compounds are being discovered. Fatty acid esters of hydroxyl fatty acids (FAHFAs), a class of endogenous lipids found in 2014, exhibit various physiological activities, such as improving glucose tolerance and insulin sensitivity, stimulating insulin secretion, and demonstrating broad anti-inflammatory effects. Moreover, some FAHFAs are closely linked to intestinal health and can serve as potential biomarkers for gut health. Various FAHFAs have been observed in food, including palmitic acid esters of hydroxy stearic acids (PAHSA), oleic acid esters of hydroxy stearic acids (OAHSA), linoleic acid esters of hydroxy linoleic acid (LAHLA). As a type of lipid regularly consumed in the daily diet, it is highly important to ascertain the types and quantities of FAHFAs present in the diet. This article, based on existing research, provides a review of the analysis methods for FAHFAs, particularly focusing on the separation of chiral isomers. It also summarizes the sources and contents of dietary FAHFAs, emphasizing their bioavailability and impact on the gut. Understanding the beneficial effects of these lipids in the diet can serve as a valuable reference for the development of specific functional foods.

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.

1832-P: Palmitic Acid Esters of Hydroxy Stearic Acids Reduce Endogenous Glucose Production through GPR43 Activation

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<0.05). This effect was absent in GPR43 KO mice (KO: vehicle= 25.3±4.2 vs. PAHSA= 23.4±2.7 mg/kg/min). We also examined whether GPR43 activation is required for the direct effect of 9-PAHSA on WAT lipolysis. Treatment with the β-adrenergic agonist isoproterenol increased FFA release from WAT explants by 3.8-fold, and 9-PAHSA inhibited isoproterenol-induced lipolysis by 21% which was similar to the effects of insulin and 4-CMTB. In WAT explants from GPR43 KO mice, 9-PAHSA suppression of isoproterenol-induced FFA release was abolished while insulin still suppressed lipolysis. 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

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 β 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 β 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 β cell proliferation in both NOD mice and MIN6 cells and increased the number of β cells in NOD mice. PAHSAs attenuated cytokine-induced apoptotic and necrotic β cell death and increased β 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 β cell survival and function.

Hybrid lipids, peptides, and lymphocytes: new era in type 1 diabetes research.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing β cells in islets of Langerhans. Many genetic and immunological insights into autoimmune disease pathogenesis were initially uncovered in the context of T1D and facilitated by preclinical studies using the nonobese diabetic (NOD) mouse model. Recently, the study of T1D has led to the discovery of fatty acid esters of hydroxyl fatty acids (FAHFAs), which are naturally occurring hybrid peptides that modulate inflammation and diabetes pathogenesis, and a hybrid lymphocyte that expresses both B and T cell receptors. Palmitic acid esters of hydroxy stearic acids (PAHSAs) are the most extensively studied FAHFA. In this issue of the JCI, Syed et al. have shown that PAHSAs both attenuate autoimmune responses and promote β cell survival in NOD mice. Given the lack of effective T1D therapies and the paucity of known side effects of PAHSAs, this lipid may have therapeutic potential for individuals at risk for or newly diagnosed with T1D.

361-OR: ER Stress Attenuation Is Involved in PAHSA-Induced Reduction of T1D in Nonobese Diabetic (NOD) Mice

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<0.002). Since GLP-1 receptor (GLP-1R) antagonism causes β-cell death and G-protein coupled receptor GPR40 plays a vital role in β-cell function, we assessed if PAHSA beneficial effects on β-cell survival involve these pathways. In vitro, GLP-1R antagonism lowered PAHSA protective effects on β-cell viability by 53% and proliferation by 44% (p<0.005) under cytokine stress but GPR40 antagonism had no effect. 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.)

1846-P: The Insulin-Sensitizing Effects of Palmitic Acid Esters of Hydroxy Stearic Acids Involve Intertissue Communication

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.)

De novo Lipogenesis in Adipocytes Results in the Production of Structurally Novel Signaling Lipids with Beneficial Metabolic and Anti‐inflammatory Effects

Increased de novo lipogenesis in adipose tissue is associated with enhanced insulin sensitivity in humans and rodents. Mice overexpressing the Glut4 glucose transporter in adipose tissue have elevated adipose tissue lipogenesis and increased glucose tolerance in spite of obesity and elevated circulating fatty acids. Untargeted lipidomic analysis of adipose tissue from these mice revealed marked elevation of a structurally novel class of lipids, branched Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs). More than 25 FAHFA family members, which are distinguished by different acyl chain composition, have been identified in mammalian tissues and plants. Levels of some family members such as Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs) are highly regulated in altered nutritional and metabolic states. PAHSA levels are reduced in serum and subcutaneous adipose tissue of insulin‐resistant humans and serum PAHSA levels correlate very strongly with insulin sensitivity in humans. Chronic PAHSA treatment improves glucose tolerance and insulin sensitivity in insulin‐resistant mice. PAHSAs also have anti‐inflammatory effects in states such as obesity, colitis and auto‐immune Type 1 diabetes in mice. Many effects of PAHSA are mediated through G‐protein coupled receptors. Thus, PAHSAs exert multiple beneficial metabolic and anti‐inflammatory effects in diabetes and immune‐mediated diseases. The pathways that regulate these lipids provide new opportunities for disease prevention and treatment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Palmitic Acid Esters of Hydroxy Stearic Acids Are Hepatic Insulin Sensitizers in Chow and High-Fat Diet (HFD)–Fed Mice

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.

Absence of Carbohydrate Response Element Binding Protein in Adipocytes Causes Systemic Insulin Resistance and Impairs Glucose Transport

Lower adipose-ChREBP and de novo lipogenesis (DNL) are associated with insulin resistance in humans. Here, we generated adipose-specific ChREBP knockout (AdChREBP KO) mice with negligible sucrose-induced DNL in adipose tissue (AT). Chow-fed AdChREBP KO mice are insulin resistant with impaired insulin action in the liver, muscle, andAT and increased AT inflammation. HFD-fed AdChREBP KO mice are also more insulin resistantthan controls. Surprisingly, adipocytes lacking ChREBP display a cell-autonomous reduction in insulin-stimulated glucose transport that is mediated by impaired Glut4 translocation and exocytosis, not lower Glut4 levels. AdChREBP KO mice have lower levels of palmitic acid esters of hydroxy stearic acids (PAHSAs) in serum, and AT. 9-PAHSA supplementation completely rescues their insulin resistance and AT inflammation. 9-PAHSA also normalizes impaired glucose transport and Glut4 exocytosis in ChREBP KO adipocytes. Thus, lossof adipose-ChREBP is sufficient to cause insulin resistance, potentially by regulating AT glucose transport and flux through specific lipogenic pathways.

Branched Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) Protect against Colitis by Regulating Gut Innate and Adaptive Immune Responses

We recently discovered a structurally novel class of endogenous lipids, branched palmitic acid esters of hydroxy stearic acids (PAHSAs), with beneficial metabolic and anti-inflammatory effects. We tested whether PAHSAs protect against colitis, which is a chronic inflammatory disease driven predominantly by defects in the innate mucosal barrier and adaptive immune system. There is an unmet clinical need for safe and well tolerated oral therapeutics with direct anti-inflammatory effects. Wild-type mice were pretreated orally with vehicle or 5-PAHSA (10 mg/kg) and 9-PAHSA (5 mg/kg) once daily for 3 days, followed by 10 days of either 0% or 2% dextran sulfate sodium water with continued vehicle or PAHSA treatment. The colon was collected for histopathology, gene expression, and flow cytometry. Intestinal crypt fractions were prepared for ex vivo bactericidal assays. Bone marrow-derived dendritic cells pretreated with vehicle or PAHSA and splenic CD4+ T cells from syngeneic mice were co-cultured to assess antigen presentation and T cell activation in response to LPS. PAHSA treatment prevented weight loss, improved colitis scores (stool consistency, hematochezia, and mouse appearance), and augmented intestinal crypt Paneth cell bactericidal potency via a mechanism that may involve GPR120. In vitro, PAHSAs attenuated dendritic cell activation and subsequent T cell proliferation and Th1 polarization. The anti-inflammatory effects of PAHSAs in vivo resulted in reduced colonic T cell activation and pro-inflammatory cytokine and chemokine expression. These anti-inflammatory effects appear to be partially GPR120-dependent. We conclude that PAHSA treatment regulates innate and adaptive immune responses to prevent mucosal damage and protect against colitis. Thus, PAHSAs may be a novel treatment for colitis and related inflammation-driven diseases.

GLUT4 Expression in Adipocytes Regulates De Novo Lipogenesis and Levels of a Novel Class of Lipids With Antidiabetic and Anti-inflammatory Effects.

Adipose tissue (AT) regulates systemic insulin sensitivity through multiple mechanisms, and alterations in de novo lipogenesis appear to contribute. Mice overexpressing GLUT4 in adipocytes (AG4OX) have elevated AT lipogenesis and enhanced glucose tolerance despite being obese and having elevated circulating fatty acids. Lipidomic analysis of AT identified a structurally unique class of lipids, branched fatty acid esters of hydroxy–fatty acids (FAHFAs), which were elevated in AT and serum of AG4OX mice. Palmitic acid esters of hydroxy–stearic acids (PAHSAs) are among the most upregulated FAHFA families in AG4OX mice. Eight PAHSA isomers are present in mouse and human tissues. PAHSA levels are reduced in insulin resistant people, and levels correlate highly with insulin sensitivity. PAHSAs have beneficial metabolic effects. Treatment of obese mice with PAHSAs lowers glycemia and improves glucose tolerance while stimulating glucagon-like peptide 1 and insulin secretion. PAHSAs also reduce inflammatory cytokine production from immune cells and ameliorate adipose inflammation in obesity. PAHSA isomer concentrations are altered in physiological and pathophysiological conditions in a tissue- and isomer-specific manner. The mechanisms most likely involve changes in PAHSA biosynthesis, degradation, and secretion. The discovery of PAHSAs reveals the existence of previously unknown endogenous lipids and biochemical pathways involved in metabolism and inflammation, two fundamental physiological processes.

A LC-MS-based workflow for measurement of branched fatty acid esters of hydroxy fatty acids.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous mammalian lipids with antidiabetic and anti-inflammatory effects. We previously identified 16 different FAHFA families, such as branched palmitic acid esters of hydroxy stearic acids (PAHSAs); each family consists of multiple isomers in which the branched ester is at different positions (e.g., 5- and 9-PAHSA). We anticipate increased need for PAHSA measurements as markers of metabolic and inflammatory health. In this protocol, we provide a detailed description of the extraction of FAHFAs from human or mouse tissues, their enrichment by solid-phase extraction and subsequent analysis by LC-MS. For a sample size of 6-12, the time frame is 2-3 d.