A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction.

OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. METHODS: We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. RESULTS: Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. CONCLUSION: These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).

Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate.

2-Bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a nonselective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.

Metabolome response to glucose in the ß-cell line INS-1 832/13.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 ß-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.