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Summary: Our study identifies a transcriptional role of cell death-inducing DNA fragmentation factor-like effector A (CIDEA), a lipid-droplet-associated protein, whereby it regulates human adipocyte britening/beiging with consequences for the regulation of energy expenditure. The comprehensive transcriptome analysis revealed CIDEA's control over thermogenic function in brite/beige human adipocytes. In the absence of CIDEA, achieved by the modified dual-RNA-based CRISPR-Cas9nD10A system, adipocytes lost their britening capability, which was recovered upon CIDEA re-expression. Uncoupling protein 1 (UCP1), the most upregulated gene in brite human adipocytes, was suppressed in CIDEA knockout (KO) primary human adipocytes. Mechanistically, during induced britening, CIDEA shuttled from lipid droplets to the nucleus via an unusual nuclear bipartite signal in a concentration-dependent manner. In the nucleus, it specifically inhibited LXRα repression of UCP1 enhancer activity and strengthened PPARγ binding to UCP1 enhancer, hence driving UCP1 transcription. Overall, our study defines the role of CIDEA in increasing thermogenesis in human adipocytes. : Biological Sciences; Molecular Biology; Cell Biology; Transcriptomics Subject Areas: Biological Sciences, Molecular Biology, Cell Biology, Transcriptomics

Treatment with PPARγ agonists in vivo improves human adipocyte metabolism, but the cellular mechanisms and possible depot differences in responsiveness to their effects are poorly understood. To examine the ex vivo metabolic effects of rosiglitazone (Rosi), we cultured explants of human visceral (omental) and abdominal subcutaneous adipose tissues for 7 days. Rosi increased mRNA levels of transcriptional regulators of brite/beige adipocytes (PGC1α, PRDM16), triglyceride synthesis (GPAT3, DGAT1), and lipolysis (ATGL) similarly in adipose tissues from both depots. In parallel, Rosi increased key modulators of FA oxidation (UCP1, FABP3, PLIN5 protein), rates of FA oxidation, and protein levels of electron transport complexes, suggesting an enhanced respiratory capacity as confirmed in newly differentiated adipocytes. Rosi led to the formation of small lipid droplets (SLDs) around the adipocyte central lipid droplet; each SLD was decorated with redistributed mitochondria that colocalized with PLIN5. SLD maintenance required lipolysis and FA reesterification. Rosi thus coordinated a structural and metabolic remodeling in adipocytes from both visceral and subcutaneous depots that enhanced oxidative capacity. Selective targeting of these cellular mechanisms to improve adipocyte FA handling may provide a new approach to treat metabolic complications of obesity and diabetes.

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). Keywords: Adipocytes, Fatty acid metabolism, Obesity, Arachidonic acid, Polyunsaturated fatty acid

Objective: Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT. Methods: The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice). Results: Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved. Conclusions: Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance. Author Video: Author Video Watch what authors say about their articles Keywords: Adipose tissue, Obesity, Insulin, AKT, Ubiquitin, GPS2

The glucocorticoid-induced leucine zipper (GILZ), a primary target of glucocorticoids, is expressed in human adipocytes, but its importance in adipocyte function is unknown. Because TNFα is increased in obese adipose tissue and antagonizes a number of glucocorticoid actions, we investigated the interplay of these pathways. GILZ knockdown increased and GILZ overexpression decreased interleukin-6 (IL-6) and leptin mRNA and protein secretion. GILZ knockdown increased the magnitude of the glucocorticoid effect on leptin secretion, but did not affect the glucocorticoid suppression of IL-6. Although GILZ silencing decreased adiponectin mRNA levels, it did not affect the amount of adiponectin secreted. GILZ negatively modulated pro-inflammatory signaling pathways, blocking basal and TNFα-stimulated (1 h) p65 nuclear factor κB nuclear translocation and transcriptional activity by binding to p65 in the cytoplasm. GILZ silencing increased basal ERK1/2 and JNK phosphorylation, and decreased MAPK phosphatase-1 protein levels. Longer term TNFα (4 h or 24 h) treatment decreased GILZ expression in human adipocytes. Furthermore, adipose tissue GILZ mRNA levels were reduced in proportion to the degree of obesity and expression of inflammatory markers. Overall, these results suggest that GILZ antagonizes the pro-inflammatory effects of TNFα in human adipocytes, and its downregulation in obesity may contribute to adipose inflammation and dysregulated adipokine production, and thereby systemic metabolism.

Objective: Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx). Methods: Adrx knockdown and overexpressing 3T3-L1 murine adipocyte cell lines and a knockout mouse model were used to assess the influence of Adrx on secreted proteins as well as the redox state of ER resident chaperones. The metabolic phenotypes of Adrx null mice were characterized and compared to WT mice. The correlation of Adrx levels BMI, adiponectin levels, and other inflammatory markers from adipose tissue of human subjects was also studied. Results: Adiporedoxin functions via a CXXC active site, and is upstream of protein disulfide isomerase whose direct function is disulfide bond formation, and ultimately protein secretion. Over and under expression of Adrx in vitro enhances and reduces, respectively, the secretion of the disulfide-bonded proteins including adiponectin and collagen isoforms. On a chow diet, Adrx null mice have normal body weights, and glucose tolerance, are moderately hyperinsulinemic, have reduced levels of circulating adiponectin and are virtually free of adipocyte fibrosis resulting in a complex phenotype tending towards insulin resistance. Adrx protein levels in human adipose tissue correlate positively with adiponectin levels and negatively with the inflammatory marker phospho-Jun kinase. Conclusion: These data support the notion that Adrx plays a critical role in adipocyte biology and in the regulation of mouse and human metabolism via its modulation of adipocyte protein secretion.

The mechanisms of the increased serum leptin in response to feeding are poorly understood. Therefore, we used metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover in adipose tissue from 14 h-starved compared with fed 12–14 week old rats. Starvation decreased serum leptin (−47 ± 7%), adipose tissue leptin content (−32 ± 5%), and leptin secretion during 3 h of incubation (−65 ± 12%). Starvation did not affect leptin mRNA levels but decreased rates of leptin biosynthesis by tissue fragments, as determined by [35S]methionine/cysteine incorporation into immunoprecipitable leptin. Insulin in vitro did not acutely increase leptin biosynthesis or rates of 125I-leptin degradation. Pulse-chase studies showed that in adipose tissue from fed but not starved rats, insulin accelerated the secretion of [35S]leptin by ∼2-fold after 30 and 60 min of chase. Degradation of newly synthesized leptin was slower in adipose tissue of starved than fed rats (half-lives of 50 and 150 min, respectively). Inhibitor experiments showed that both lysosomes and proteosomes contributed to leptin degradation. In conclusion, feeding compared with starvation influences leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion. The insulin-stimulated release of leptin from a preformed intracellular leptin pool may contribute to increases in serum leptin levels after meals.