Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids and fatty acid hydroxyoctadecadienoic acids in white adipose tissue depots and in adipocytes cocultured with macrophages. In lipopolysaccharide (LPS)-induced coculture systems, we confirm that adipose triglyceride lipase is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized fatty acid hydroxyoctadecadienoic acids show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of proinflammatory hydroxy fatty acids into FAHFAs may dampen inflammation. Indeed, we demonstrate that proinflammatory lipids, such as hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4), can be converted by cells in culture to weakly antiinflammatory FAHFAs.

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids (FAHSAs) and fatty acid hydroxyoctadecadienoic acids (FAHODEs) in white adipose tissue depots and in adipocytes co-cultured with macrophages. In LPS-induced co-culture systems, we confirm that adipose triglyceride lipase (ATGL) is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized FAHODEs show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of pro-inflammatory hydroxy fatty acids into FAHFAs may modulate inflammation. We test this concept by showing the pro-inflammatory lipids—hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4)—are converted into FAHFAs in cell culture, and that two LTB4-derived FAHFAs have are modestly anti- not pro-inflammatory. Further research is needed to establish whether these increased FAFHA levels have a role in inflammation or are simply markers of inflammation, but the discovery of significant increases in FAHFA upon acute inflammation advances our knowledge of FAHFAs.

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by β-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGLAdpKO). Here, we find that upon activation of β-adrenergic receptors in vivo, ATGLAdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGLfl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGLAdpKO mice was indeed from the adipocytes. ATGLAdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGLAdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

Fatty acid binding protein 4 (FABP4) is linked with the pathogenesis of metabolic diseases, including diabetes and cardiovascular disease in both mice and humans. It has also been demonstrated that the levels of hormonal FABP4 are strongly associated with obesity, and secretion is stimulated under conditions of fasting and lipolysis both in vivo and in vitro . Here, we utilized adipocyte-specific deficiency of adipose triglyceride lipase (ATGL) in a mouse model (ATGLAdpKO) to evaluate the regulation of FABP4 secretion by lipolytic signals in the absence of actual lipolysis in vivo . Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of ATGL, and from adipose tissue explants from ATGLAdpKO mice. Unexpectedly, upon activation beta-adrenergic receptors, ATGLAdpKO mice exhibited significantly higher levels of circulating FABP4 as compared to ATGLfl/fl controls in vivo , with no corresponding increase in non-esterified free fatty acids or glycerol, confirming the lack of lipolysis. We also generated an additional model with adipocyte-specific deletion of FABP4 in the background of ATGLAdpKO mice (ATGL/FABP4AdpKO or DKO) to evaluate the cellular source of circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the elevated FABP4 hormone levels in the ATGLAdpKO mice were indeed from the adipocytes. ATGLAdpKO mice did not exhibit an increase in insulin secretion upon stimulation of lipolysis, but had a normal insulin response to glucose injection along with increased FABP4 secretion, suggesting the elevated FABP4 secretion is not due to lack of insulin. Inhibition of sympathetic signaling during lipolysis using hexamethonium significantly reduced FABP4 secretion in ATGLAdpKO mice compared to controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se , is not required for stimulated in vivo FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.### Competing Interest StatementG.S.H. is in the Scientific Advisory Board of Crescenta Biosciences and holds equity. The Hotamisligil lab holds intellectual property related to hormonal FABP4 and its therapeutic targeting. Other authors have no conflicts of interest to declare.

Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

AbstractFatty acid binding protein 4 (FABP4) is linked with the pathogenesis of metabolic diseases, including diabetes and cardiovascular disease in both mice and humans. It has also been demonstrated that the levels of hormonal FABP4 are strongly associated with obesity, and secretion is stimulated under conditions of fasting and lipolysis bothin vivoandin vitro. Here, we utilized adipocyte-specific deficiency of adipose triglyceride lipase (ATGL) in a mouse model (ATGLAdpKO) to evaluate the regulation of FABP4 secretion by lipolytic signals in the absence of actual lipolysisin vivo. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of ATGL, and from adipose tissue explants from ATGLAdpKOmice. Unexpectedly, upon activation beta-adrenergic receptors, ATGLAdpKOmice exhibited significantly higher levels of circulating FABP4 as compared to ATGLfl/flcontrolsin vivo, with no corresponding increase in non-esterified free fatty acids or glycerol, confirming the lack of lipolysis. We also generated an additional model with adipocyte-specific deletion of FABP4 in the background of ATGLAdpKOmice (ATGL/FABP4AdpKOor DKO) to evaluate the cellular source of circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the elevated FABP4 hormone levels in the ATGLAdpKOmice were indeed from the adipocytes. ATGLAdpKOmice did not exhibit an increase in insulin secretion upon stimulation of lipolysis, but had a normal insulin response to glucose injection along with increased FABP4 secretion, suggesting the elevated FABP4 secretion is not due to lack of insulin. Inhibition of sympathetic signaling during lipolysis using hexamethonium significantly reduced FABP4 secretion in ATGLAdpKOmice compared to controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL,per se, is not required for stimulatedin vivoFABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS?based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9(th) carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo. Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

Androgen‐induced gene 1 (AIG1) and Androgen‐dependent TFPI‐regulating protein (ADTRP) are atypical transmembrane hydrolases that rely on a catalytic threonine for their enzymatic activity. In vitro characterization of AIG1 and ADTRP in lysates and cells identified fatty acid ester of hydroxy fatty acids (FAHFAs) as their putative substrates. Here, we generate ADTRP knockout (Adtrp‐KO), AIG1 knockout (Aig1‐KO), and ADTRP/AIG1 double deficient (DKO) mice using CRISPR‐Cas9 technology to test whether these enzymes regulate FAHFAs in vivo. AIG1, ADTRP, or a deficiency in both enzymes leads to decreased FAHFA hydrolytic activity in tissue lysates. Quantitative measurement of FAHFA levels in several tissues revealed increased FAHFA levels in brown adipose tissue (BAT), subcutaneous adipose tissue (SQWAT), and perigonadal WAT (PGWAT) of Adtrp‐KO mice consistent with the loss of FAHFA degrading activity. Furthermore, contribution by AIG1 was modest and only observed in the kidney and BAT of DKO mice. Lipidomics of tissues from knockout and wild type control mice detected no significant changes in other lipid classes to indicate that these enzymes are specific for FAHFA substrates. Furthermore, we developed a potent and selective, dual AIG1/ADTRP inhibitor to enable pharmacological interrogation of these enzymes in vivo. Chemical inhibition of AIG1 and ADTRP raised FAHFA levels demonstrating acute regulation of FAHFAs. In aggregate, the results establish AIG1 and ADTRP as the only endogenous FAHFA hydrolases known, and describe resources (mice, inhibitors) needed to elucidate the biochemical and physiological role of these exciting enzymes.Support or Funding InformationThis research was supported by the NIH (DK106210, DK114785, DA033760), The Leona M. and Harry B. Helmsley Charitable Trust (grant #2012‐PG‐MED002 to A.S.), NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), a NIH F32 postdoctoral fellowship, DK111159 (M.EE.), a Hewitt Foundation for Medical Research Fellowship (W.H.P.), Mass Spectrometry Core of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NIH 1S10OD021815‐01 and the Helmsley Center for Genomic Medicine, and Transgenic Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195.

BACKGROUND:Chronic ER stress and dysfunction is a hallmark of obesity and a critical contributor to metaflammation, abnormal hormone action and altered substrate metabolism in metabolic tissues, such as liver and adipocytes. Lack of STAMP2 in lean mice induces inflammation and insulin resistance on a regular diet, and it is dysregulated in the adipose tissue of obese mice and humans. We hypothesized that the regulation of STAMP2 is disrupted by ER stress.METHODS:3T3-L1 and MEF adipocytes were treated with ER stress inducers thapsigargin and tunicamycin, and inflammation inducer TNFα. The treatments effect on STAMP2 expression and enzymatic function was assessed. In addition, 3T3-L1 adipocytes and HEK cells were utilized for Stamp2 promoter activity investigation performed with luciferase and ChIP assays.RESULTS:ER stress significantly reduced both STAMP2 mRNA and protein expression in cultured adipocytes whereas TNFα had the opposite effect. Concomitant with loss of STAMP2 expression during ER stress, intracellular localization of STAMP2 was altered and total iron reductase activity was reduced. Stamp2 promoter analysis by reporter assays and chromatin immunoprecipitation, showed that induction of ER stress disrupts C/EBPα-mediated STAMP2 expression.CONCLUSION:These data suggest a clear link between ER stress and quantitative and functional STAMP2-deficiency.

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.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous lipids with antidiabetic and anti-inflammatory activities. Interest in these lipids is due to their unique biological activites and the observation that insulin-resistant people have lower palmitic acid esters of hydroxystearic acid (PAHSA) levels, suggesting that a FAHFA deficiency may contribute to metabolic disease. Rigorous testing of this hypothesis will require the measurement of many clinical samples; however, current analytical workflows are too slow to enable samples to be analyzed quickly. Here we describe the development of a significantly faster workflow to measure FAHFAs that optimizes the fractionation and chromatography of these lipids. We can measure FAHFAs in 30 min with this new protocol versus 90 min using the older protocol with comparable performance in regioisomer detection and quantitation. We also discovered through this optimization that oleic acid esters of hydroxystearic acids (OAHSAs), another family of FAHFAs, have a much lower background signal than PAHSAs, which makes them easier to measure. Our faster workflow was able to quantify changes in PAHSAs and OAHSAs in mouse tissues and human plasma, highlighting the potential of this protocol for basic and clinical applications.

Lipids encompass a wide variety of molecules such as fatty acids, sterols, phospholipids, and triglycerides. These molecules represent a highly efficient energy resource and can act as structural elements of membranes or as signaling molecules that regulate metabolic homeostasis through many mechanisms. Cells possess an integrated set of response systems to adapt to stresses such as those imposed by nutrient fluctuations during feeding-fasting cycles. While lipids are pivotal for these homeostatic processes, they can also contribute to detrimental metabolic outcomes. When metabolic stress becomes chronic and adaptive mechanisms are overwhelmed, as occurs during prolonged nutrient excess or obesity, lipid influx can exceed the adipose tissue storage capacity and result in accumulation of harmful lipid species at ectopic sites such as liver and muscle. As lipid metabolism and immune responses are highly integrated, accumulation of harmful lipids or generation of signaling intermediates can interfere with immune regulation in multiple tissues, causing a vicious cycle of immune-metabolic dysregulation. In this review, we summarize the role of lipotoxicity in metaflammation at the molecular and tissue level, describe the significance of anti-inflammatory lipids in metabolic homeostasis, and discuss the potential of therapeutic approaches targeting pathways at the intersection of lipid metabolism and immune function.

Adipocyte fatty acid binding protein 4, aP2, contributes to the pathogenesis of several common diseases including type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Although the biological functions of aP2 have classically been attributed to its intracellular action, recent studies demonstrated that aP2 acts as an adipokine to regulate systemic metabolism. However, the mechanism and regulation of aP2 secretion remain unknown. Here, we demonstrate a specific role for lipase activity in aP2 secretion from adipocytes in vitro and ex vivo. Our results show that chemical inhibition of lipase activity, genetic deficiency of adipose triglyceride lipase and, to a lesser extent, hormone-sensitive lipase blocked aP2 secretion from adipocytes. Increased lipolysis and lipid availability also contributed to aP2 release as determined in perilipin1-deficient adipose tissue explants ex vivo and upon treatment with lipids in vivo and in vitro. In addition, we identify a nonclassical route for aP2 secretion in exosome-like vesicles and show that aP2 is recruited to this pathway upon stimulation of lipolysis. Given the effect of circulating aP2 on glucose metabolism, these data support that targeting aP2 or the lipolysis-dependent secretory pathway may present novel mechanistic and translational opportunities in metabolic disease.

Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes.