Fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous bioactive lipids known for their anti-inflammatory and anti-diabetic properties. Despite their therapeutic potential, little is known about the sex-specific variations in FAHFA metabolism. This study investigated the role of sex and Androgen Dependent TFPI Regulating Protein (ADTRP), a FAHFA hydrolase. Additionally, tissue-specific differences in FAHFA levels, focusing on the perigonadal white adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), plasma, and liver, were evaluated using metabolomics and lipidomics. We found that female mice exhibited higher FAHFA levels in pgWAT, scWAT, and BAT compared to males. FAHFA levels were inversely related to testosterone and Adtrp mRNA, which showed significantly lower expression in females compared with males in pgWAT and scWAT. However, no significant differences between the sexes were observed in plasma and liver FAHFA levels. Adtrp deletion had minimal impact on both sexes\' metabolome and lipidome of pgWAT. However, we discovered higher endogenous levels of triacylglycerol estolides containing FAHFAs, a FAHFA metabolic reservoir, in the pgWAT of female mice. These findings suggest that sex-dependent differences in FAHFA levels occur primarily in specific WAT depots and may modulate local insulin sensitivity in adipocytes, and the role of ADTRP is limited to adipose depots. However, further investigations are warranted to fully comprehend the underlying mechanisms and implications of sex-dependent regulation of human FAHFA metabolism.

Mitochondrial fatty acid oxidation serves as an essential process for cellular survival, differentiation, proliferation, and energy metabolism. Numerous studies have utilized etomoxir (ETO) for the irreversible inhibition of carnitine palmitoylcarnitine transferase 1 (CPT1) which catalyzes the rate-limiting step for mitochondrial long-chain fatty acid β-oxidation to examine the bioenergetic roles of mitochondrial fatty acid metabolism in many tissues in multiple diverse disease states. Herein, we demonstrate that intact mitochondria robustly metabolize etomoxir to etomoxir-carnitine (ETO-carnitine) prior to nearly complete etomoxir-mediated inhibition of CPT1. The novel pharmaco-metabolite, ETO-carnitine, was conclusively identified by accurate mass, fragmentation patterns, and isotopic fine structure. On the basis of these data, ETO-carnitine was successfully differentiated from isobaric structures (e.g., 3-hydroxy-C18:0 carnitine and 3-hydroxy-C18:1 carnitine). Mechanistically, generation of ETO-carnitine from mitochondria required exogenous Mg2+, ATP or ADP, CoASH, and L-carnitine indicating that thioesterification by long-chain acyl-CoA synthetase to form ETO-CoA precedes its conversion to ETO-carnitine by CPT1. CPT1-dependent generation of ETO-carnitine was substantiated by an orthogonal approach using ST1326 (a CPT1 inhibitor) which effectively inhibits mitochondrial ETO-carnitine production. Surprisingly, purified ETO-carnitine potently inhibited calcium-independent PLA2γ and PLA2β as well as mitochondrial respiration independent of CPT1. Robust production and release of ETO-carnitine from HepG2 cells incubated in the presence of ETO was also demonstrated. Collectively, this study identifies the chemical mechanism for the biosynthesis of a novel pharmaco-metabolite of etomoxir, ETO-carnitine, that is generated by CPT1 in mitochondria and likely impacts multiple downstream (non-CPT1 related) enzymes and processes in multiple subcellular compartments.

All-trans retinoic acid (atRA), a metabolite of vitamin A, reduces hepatic lipid accumulation in liver steatosis model animals. Lipophagy, a new lipolysis pathway, degrades a lipid droplet (LD) via autophagy in adipose tissue and the liver. We recently found that atRA induces lipophagy in adipocytes. However, it remains unclear whether atRA induces lipophagy in hepatocytes. In this study, we investigated the effects of atRA on lipophagy in Hepa1c1c7 cells and the liver of mice fed a high-fat diet (HFD). First, we confirmed that atRA induced autophagy in Hepa1c1c7 cells by Western blotting and the GFP-LC3-mCherry probe. Next, we evaluated the lipolysis in fatty Hepa1c1c7 cells treated with the knockdown of Atg5, an essential gene in autophagy induction. Atg5-knockdown partly suppressed the atRA-induced lipolysis in fatty Hepa1c1c7 cells. We also found that atRA reduced the protein, but not mRNA, expression of Rubicon, a negative regulator of autophagy, in Hepa1c1c7 cells and the liver of HFD-fed mice. Rubicon-knockdown partly inhibited the atRA-induced lipolysis in fatty Hepa1c1c7 cells. In addition, atRA reduced hepatic Rubicon expression in young mice, but the effect of atRA on it diminished in aged mice. Finally, we investigated the mechanism underlying reduced Rubicon protein expression by atRA in hepatocytes. A protein synthesis inhibitor, but not proteasome or lysosomal inhibitors, significantly blocked the reduction of Rubicon protein expression by atRA in Hepa1c1c7 cells. These results suggest that atRA may promote lipophagy in fatty hepatocytes by reducing hepatic Rubicon expression via inhibiting protein synthesis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) versus 38 (18-58) μmol/min; simple steatosis: 62 (46-78) versus 45 (25-65) μmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P <0.05 time-effect) in both groups. Insulin-mediated glucose disposal was similar in the two groups (P = 0.54). Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from visceral adipose tissue lipolysis delivered to the liver. These results demonstrate that the splanchnic balances of FFA and VLDL-TG do not differ between obese men with NASH and those with simple steatosis.

Angiopoietin-like protein (ANGPTL) complexes 3/8 and 4/8 are established inhibitors of LPL and novel therapeutic targets for dyslipidemia. However, the effects of regular exercise on ANGPTL3/8 and ANGPTL4/8 are unknown. We characterized ANGPTL3/8 and ANGPTL4/8 and their relationship with in vivo measurements of lipase activities and cardiometabolic traits before and after a 5-month endurance exercise training intervention in 642 adults from the HERITAGE (HEalth, RIsk factors, exercise Training And GEnetics) Family Study. At baseline, higher levels of both ANGPTL3/8 and ANGPTL4/8 were associated with a worse lipid, lipoprotein, and cardiometabolic profile, with only ANGPTL3/8 associated with postheparin LPL and HL activities. ANGPTL3/8 significantly decreased with exercise training, which corresponded with increases in LPL activity and decreases in HL activity, plasma triglycerides, apoB, visceral fat, and fasting insulin (all P < 5.1 × 10-4). Exercise-induced changes in ANGPTL4/8 were directly correlated to concomitant changes in total cholesterol, LDL-C, apoB, and HDL-triglycerides and inversely related to change in insulin sensitivity index (all P < 7.0 × 10-4). In conclusion, exercise-induced decreases in ANGPTL3/8 and ANGPTL4/8 were related to concomitant improvements in lipase activity, lipid profile, and cardiometabolic risk factors. These findings reveal the ANGPTL3-4-8 model as a potential molecular mechanism contributing to adaptations in lipid metabolism in response to exercise training.

Intracellular lipolysis-the enzymatic breakdown of lipid droplet-associated triacylglycerol (TAG)-depends on the cooperative action of several hydrolytic enzymes and regulatory proteins, together designated as lipolysome. Adipose triglyceride lipase (ATGL) acts as a major cellular TAG hydrolase and core effector of the lipolysome in many peripheral tissues. Neurons initiate lipolysis independently of ATGL via DDHD domain-containing 2 (DDHD2), a multifunctional lipid hydrolase whose dysfunction causes neuronal TAG deposition and hereditary spastic paraplegia. Whether and how DDHD2 cooperates with other lipolytic enzymes is currently unknown. In this study, we further investigated the enzymatic properties and functions of DDHD2 in neuroblastoma cells and primary neurons. We found that DDHD2 hydrolyzes multiple acylglycerols in vitro and substantially contributes to neutral lipid hydrolase activities of neuroblastoma cells and brain tissue. Substrate promiscuity of DDHD2 allowed its engagement at different steps of the lipolytic cascade: In neuroblastoma cells, DDHD2 functioned exclusively downstream of ATGL in the hydrolysis of sn-1,3-diacylglycerol (DAG) isomers but was dispensable for TAG hydrolysis and lipid droplet homeostasis. In primary cortical neurons, DDHD2 exhibited lipolytic control over both, DAG and TAG, and complemented ATGL-dependent TAG hydrolysis. We conclude that neuronal cells use noncanonical configurations of the lipolysome and engage DDHD2 as dual TAG/DAG hydrolase in cooperation with ATGL.

Angiopoietin-like proteins, ANGPTL3, ANGPTL4, and ANGPTL8, are involved in regulating plasma lipids. In vitro and animal-based studies point to LPL and endothelial lipase (EL, LIPG) as key targets of ANGPTLs. To examine the ANGPTL mechanisms for plasma lipid modulation in humans, we pursued a genetic mimicry analysis of enhancing or suppressing variants in the LPL, LIPG, lipase C hepatic type (LIPC), ANGPTL3, ANGPTL4, and ANGPTL8 genes using data on 248 metabolic parameters derived from over 110,000 nonfasted individuals in the UK Biobank and validated in over 13,000 overnight fasted individuals from 11 other European populations. ANGPTL4 suppression was highly concordant with LPL enhancement but not HL or EL, suggesting ANGPTL4 impacts plasma metabolic parameters exclusively via LPL. The LPL-independent effects of ANGPTL3 suppression on plasma metabolic parameters showed a striking inverse resemblance with EL suppression, suggesting ANGPTL3 not only targets LPL but also targets EL. Investigation of the impact of the ANGPTL3-ANGPTL8 complex on plasma metabolite traits via the ANGPTL8 R59W substitution as an instrumental variable showed a much higher concordance between R59W and EL activity than between R59W and LPL activity, suggesting the R59W substitution more strongly affects EL inhibition than LPL inhibition. Meanwhile, when using a rare and deleterious protein-truncating ANGPTL8 variant as an instrumental variable, the ANGPTL3-ANGPTL8 complex was very LPL specific. In conclusion, our analysis provides strong human genetic evidence that the ANGPTL3-ANGPTL8 complex regulates plasma metabolic parameters, which is achieved by impacting LPL and EL. By contrast, ANGPTL4 influences plasma metabolic parameters exclusively via LPL.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

White adipose tissue (WAT) possesses the endocannabinoid system (ECS) machinery and produces the two major endocannabinoids (ECs), arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). Accumulating evidence indicates that WAT cannabinoid 1 receptors (CB1R) are involved in the regulation of fat storage, tissue remodeling and secretory functions but their role in controlling lipid mobilization is unclear. In the present study, we used different strategies to acutely increase ECS activity in WAT and tested the consequences on glycerol production as a marker of lipolysis. Treating lean mice or rat WAT explants with JLZ195, which inhibits ECs degrading enzymes, induced an increase in 2-AG tissue contents that was associated with a CB1R-dependent decrease in lipolysis. Direct treatment of rat WAT explants with AEA also inhibited glycerol production while mechanistic studies revealed it could result from the stimulation of Akt-signaling pathway. Interestingly, AEA treatment decreased lipolysis both in visceral and subcutaneous WAT collected on lean subjects suggesting that ECS also reduces fat store mobilization in Human. In obese mice, WAT content and secretion rate of ECs were higher than in control while glycerol production was reduced suggesting that over-produced ECs may inhibit lipolysis activating local CB1R. Strikingly, our data also reveal that acute CB1R blockade with Rimonabant did not modify lipolysis in vitro in obese mice and human explants nor in vivo in obese mice. Taken together, these data provide physiological evidence that activation of ECS in WAT, by limiting fat mobilization, may participate in the progressive tissue remodeling that could finally lead to organ dysfunction. The present findings also indicate that acute CB1R blockade is inefficient in regulating lipolysis in obese WAT and raise the possibility of an alteration of CB1R signaling in conditions of obesity.

Adaptive thermogenesis is highly dependent on uncoupling protein 1 (UCP1), a protein expressed by thermogenic adipocytes present in brown adipose tissue (BAT) and white adipose tissue (WAT). Thermogenic capacity of human and mouse BAT can be measured by positron emission tomography-computed tomography quantifying the uptake of 18F-fluodeoxyglucose or lipid tracers. BAT activation is typically studied in response to cold exposure or treatment with β-3-adrenergic receptor agonists such as CL316,243 (CL). Currently, it is unknown whether cold-stimulated uptake of glucose or lipid tracers is a good surrogate marker of UCP1-mediated thermogenesis. In metabolic studies using radiolabeled tracers, we found that glucose uptake is increased in mildly cold-activated BAT of Ucp1 -/- versus WT mice kept at subthermoneutral temperature. Conversely, lower glucose disposal was detected after full thermogenic activation achieved by sustained cold exposure or CL treatment. In contrast, uptake of lipoprotein-derived fatty acids into chronically activated thermogenic adipose tissues was substantially increased in UCP1-deficient mice. This effect is linked to higher sympathetic tone in adipose tissues of Ucp1 -/- mice, as indicated by elevated levels of thermogenic genes in BAT and WAT. Thus, glucose and lipoprotein handling does not necessarily reflect UCP1-dependent thermogenic activity, but especially lipid uptake rather mirrors sympathetic activation of adipose tissues.