Inducible nitric oxide synthase modulates lipolysis in adipocytes

The role of inducible nitric oxide synthase (iNOS) in the modulation of adipocyte lipolysis was investigated. Treatment of white and brown adipose cell lines and mouse adipose explants with a mixture of tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide (LPS) doubled the lipolytic rate, and this was associated with marked induction of iNOS expression and nitric oxide (NO) production. iNOS inhibition by 1400W, aminoguanidine, or L-NIL pretreatment further increased the cytokine/LPS-mediated lipolysis by 30% (P < 0.05) in cultured adipocytes and in adipose explants. However, this potentiating effect of iNOS inhibition was abolished in adipose explants isolated from iNOS knockout mice. Pharmacological inhibitors of adenylyl cyclase or protein kinase A reduced cytokine/LPS-induced lipolysis and also blunted the potentiating effect of iNOS inhibition on the lipolytic rate. Furthermore, addition of the antioxidants l-cystine and l-glutathione to cytokine/LPS-stimulated adipocytes mimicked the lipolytic effect of iNOS inhibition. In conclusion, inhibition of iNOS activity in adipocytes potentiates cytokine/LPS-induced lipolysis. This effect was fully reversed by adenylyl cyclase and protein kinase A inhibitors but was mimicked by cellular antioxidants. These data suggest that iNOS-mediated NO production counteracts cytokine/LPS-mediated lipolysis in adipocytes and that this feedback mechanism involves an oxidative process upstream of cAMP production in the signaling pathway.     

Genetic control of ATGL-mediated lipolysis modulates adipose triglyceride stores in leptin-deficient mice

Dissecting the genetics of complex traits such as obesity allows the identification of causal genes for disease. Here, we show that the BALB/c mouse strain carries genetic variants that confer resistance to obesity induced by leptin-deficiency or a high-fat diet (HFD). We set out to identify the physiological and genetic bases underlying this phenotype. When compared with C57BL6/J ob/ob mice (B6), BALB/c ob/ob mice exhibited decreased food intake, increased thermogenic capacity, and improved fat catabolism, each of which can potentially modify obesity. Interestingly, analysis of F1 ob/ob (progeny of B6 ob/+ × BALB/c ob+) mice revealed that obesity resistance in BALB/c ob/ob mice principally relied upon improved fat mobilization. This was mechanistically explained by increased adipose triglyceride lipase (ATGL) content in adipocytes, along with increased lipolysis and fatty acid oxidation. We conducted a genome-wide scan and defined a quantitative trait locus (QTL) on chromosome 2. BALB/c alleles on chromosome 2 not only associated with the obesity resistance phenotype but also supported increased ATGL content in adipose tissue. In summary, our study provides evidence that leptin-independent control of adipocyte lipolysis rates directly modifies the balance of macronutrient handling and is sufficient to regulate fat mass in the absence of alterations in food intake and energy expenditure.-Marcelin, G., S-M. Liu, X. Li, G. J. Schwartz, and S. Chua.     

Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis

The majority of eukaryotic cells synthesize neutral lipids and package them into cytosolic lipid droplets. In vertebrates, triacylglycerol-rich lipid droplets of adipocytes provide a major energy storage depot for the body, whereas cholesteryl ester-rich droplets of many other cells provide building materials for local membrane synthesis and repair. These lipid droplets are coated with one or more of five members of the perilipin family of proteins: adipophilin, TIP47, OXPAT/MLDP, S3-12, and perilipin. Members of this family share varying levels of sequence similarity, lipid droplet association, and functions in stabilizing lipid droplets. The most highly studied member of the family, perilipin, is the most abundant protein on the surfaces of adipocyte lipid droplets, and the major substrate for cAMP-dependent protein kinase [protein kinase A (PKA)] in lipolytically stimulated adipocytes. Perilipin serves important functions in the regulation of basal and hormonally stimulated lipolysis. Under basal conditions, perilipin restricts the access of cytosolic lipases to lipid droplets and thus promotes triacylglycerol storage. In times of energy deficit, perilipin is phosphorylated by PKA and facilitates maximal lipolysis by hormone-sensitive lipase and adipose triglyceride lipase. A model is discussed whereby perilipin serves as a dynamic scaffold to coordinate the access of enzymes to the lipid droplet in a manner that is responsive to the metabolic status of the adipocyte.     

Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity

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.     

Effects of lipoic acid on lipolysis in 3T3-L1 adipocytes

Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser⁵⁶³ and at Ser⁶⁶⁰, which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E₂ released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E₂.     

Isolation and Properties of Alginate Lyase from the Mid-Gut Gland of Wreath Shell Turbo cornutus

Alginate lyase [EC 4.2.2.3] has been purified from mid-gut gland of wreath shell. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. A molecular weight of 32,000 was estimated by SDS-polyacrylamide gel electrophoresis. Absorption spectra of the reaction products indicated that the enzyme had lyase activity. The enzyme was most active at a pH range of about 8.8 to 9.2 and most stable at pH 5 to 6. Phosphate showed strong stabilizing and enhancing effects on the enzyme activity. Divalent cations behaved differently toward alginic acid and propylene glycol alginate, suggesting requirements for free carboxyl groups and a single glycosidic chain in the enzyme action.     

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.     

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS²³⁹S²⁴⁰, we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno­blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.     

Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation

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The role of inflamed adipose tissue in the insulin resistance

In this article, we discuss inflammation associated with adipose tissue dysfunction as a potential link with obesity-related insulin resistance, and how obesity-related inflammatory components, such as immune cells, cytokines/chemokines and adipocytokines, induce obesity-related pathologies.     

Fate of fat: The role of adipose triglyceride lipase in lipolysis

Lipolysis, the coordinated catabolism of triacylglycerol (TG) stored in cellular lipid droplets, provides fatty acids, di-, and monoglycerides. These products are important energy substrates, precursors for other lipids, or lipid signaling molecules. Following their discovery by Hollenberg, C.H., Raben, M.S., and Astwood, E.B.(1961) and Vaughan, M., Berger, J.E., and Steinberg, D. (1964), hormone-sensitive lipase (HSL) and monoacylglycerol lipase stayed in the focus of research for three decades. Within the last decade, however, it became evident that the lipolytic pathway is incompletely understood. Studies on the regulation of lipolysis and the characterization of HSL-deficient mice indicated that additional previously unrecognized factors that contribute to fat catabolism must exist. This led to the discovery of the perilipin, adipophilin, Tip47 (PAT) family of lipid droplet binding proteins and the identification of a novel TG hydrolase named adipose triglyceride lipase (ATGL). This review focuses on the importance of ATGL as TG lipase within the “lipolytic machinery” and the current knowledge of molecular mechanisms that regulate ATGL activity.     

Shrinking and development of lipid droplets in adipocytes during catecholamine-induced lipolysis

Time-lapse observation of adipocytes during catecholamine-induced lipolysis clearly shows that shrinking of existing lipid droplets (LDs) occurs in some adipocytes and that small LDs are newly developed in almost all cells. Immunofluorescence imaging reveals that activation and localization of hormone-sensitive lipase (HSL) on the surface of LDs, which are required for conferring maximal lipolysis, are necessary for the shrinking of the LDs. However, not all adipocytes in which phosphorylated HSL is localized on LDs exhibit shrinking of LDs. The simultaneous shrinking and development of LDs yield apparent fragmentation and dispersion of LDs in adipocytes stimulated with catecholamine.     

Prostacyclin: A major prostaglandin in the regulation of adipose tissue development

Prostaglandins (PGs) belong to the group lipid mediators and can act as local hormones. They contain 20 carbon atoms, including a 5-carbon ring, and are biosynthesized from membrane phospholipid derived arachidonic acid through the arachidonate cyclooxygenase (COX) pathway with the help of various terminal synthase enzymes. Prostacyclin (prostaglandin I₂) is one of the major prostanoids produced with the help of prostacyclin synthase (prostaglandin I₂ synthase) enzyme and rapidly hydrolyzed into 6-keto-PGF_1α in biological fluids. Obesity indicates an excess of body adiposity, which is globally considered as one of the major health disasters responsible for developing complex pathological situations in the human body. Adipose tissues can produce various PGs, and thus, the level and the molecular activity of these endogenously synthesized PGs are considered critical for the development of obesity. In this regard, the involvement of prostacyclin in adipogenesis has been studied in the last few decades. The current review, along with the background of other related PGs, presents the several molecular aspects of endogenous prostaglandin I₂ in adipose tissue development. Especially, the regulation of life cycle of adipocytes, impact on terminal differentiation, activity through prostacyclin receptor (IP), autocrine-paracrine manner, thermogenic adipose tissue remodeling and some future experimental aspects of prostacyclin have been focused upon in this study. This discussion might assist to develop new drug molecules acting on the signaling pathways of prostacyclin and devise therapeutic strategies for treating obesity.     

内质网应激响应在脂肪组织中的代谢调节作用

在真核细胞中,内质网是蛋白合成、加工及质量监控的关键细胞器,也是Ca2+储存及脂质合成的重要场所.细胞通过未折叠蛋白响应(unfolded protein response,UPR)感应外界不同刺激引发的内质网应激,在维持细胞功能稳态中发挥至关重要的作用.在哺乳动物中,三个位于内质网的跨膜蛋白——肌醇依赖酶la(ino...     

Cytoplasmic receptor-interacting protein 140 (RIP140) interacts with perilipin to regulate lipolysis

Receptor-interacting protein 140 (RIP140) is abundantly expressed in mature adipocyte and modulates gene expression involved in lipid and glucose metabolism. Protein kinase C epsilon and protein arginine methyltransferase 1 can sequentially stimulate RIP140 phosphorylation and then methylation, thereby promoting its export to the cytoplasm. Here we report a lipid signal triggering cytoplasmic accumulation of RIP140, and a new functional role for cytoplasmic RIP140 in adipocyte to regulate lipolysis. Increased lipid content, particularly an elevation in diacylglycerol levels, promotes RIP140 cytoplasmic accumulation and increased association with lipid droplets (LDs) by its direct interaction with perilipin. By interacting with RIP140, perilipin more efficiently recruits hormone-sensitive lipase (HSL) to LDs and enhances adipose triglyceride lipase (ATGL) forming complex with CGI-58, an activator of ATGL. Consequentially, HSL can more readily access its substrates, and ATGL is activated, ultimately enhancing lipolysis. In adipocytes, blocking cytoplasmic RIP140 accumulation reduces basal and isoproterenol-stimulated lipolysis and the pro-inflammatory potential of their conditioned media (i.e. activating NF-κB and inflammatory genes in macrophages). These results show that in adipocytes with high lipid contents, RIP140 increasingly accumulates in the cytoplasm and enhances triglyceride catabolism by directly interacting with perilipin. The study suggests that reducing nuclear export of RIP140 might be a useful means of controlling adipocyte lipolysis.     

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.