OBJECTIVE-We sought to determine the role of adipocyte death in obesity-induced adipose tissue (AT) inflammation and obesity complications.RESEARCH DESIGN AND METHODS-Male C57BL/6 mice were fed a high-fat diet for 20 weeks to induce obesity. Every 4 weeks, insulin resistance was assessed by intraperitoneal insulin tolerance tests, and epididymal (eAT) and inguinal subcutaneous AT (iAT) and livers were harvested for histological, immunohistochemical, and gene expression analyses.RESULTS-Frequency of adipocyte death in eAT increased from Ͻ0.1% at baseline to 16% at week 12, coincident with increases in 1) depot weight; 2) AT macrophages (ATM⌽s) expressing F4/80 and CD11c; 3) mRNA for tumor necrosis factor (TNF)-␣, monocyte chemotactic protein (MCP)-1, and interleukin (IL)-10; and 4) insulin resistance. ATM⌽s in crown-like structures surrounding dead adipocytes expressed TNF-␣ and IL-6 proteins. Adipocyte number began to decline at week 12. At week 16, adipocyte death reached ϳ80%, coincident with maximal expression of CD11c and inflammatory genes, loss (40%) of eAT mass, widespread collagen deposition, and accelerated hepatic macrosteatosis. By week 20, adipocyte number was restored with small adipocytes, coincident with reduced adipocyte death (fourfold), CD11c and MCP-1 gene expression (twofold), and insulin resistance (35%). eAT weight did not increase at week 20 and was inversely correlated with liver weight after week 12 (r ϭ Ϫ0. 85, P Ͻ 0.001). In iAT, adipocyte death was first detected at week 12 and remained Յ3%.CONCLUSIONS-These results implicate depot-selective adipocyte death and M⌽-mediated AT remodeling in inflammatory and metabolic complications of murine obesity. Diabetes
Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517.Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis. Triglyceride (TAG)3 and other neutral lipids are stored in adipocyte lipid droplets (LDs) and, in response to energy demand, are hydrolyzed by lipases (lipolysis) to generate fatty acids (FAs) as fuel for peripheral tissues (1-5). Tight regulation of adipocyte lipolysis in response to the inhibitory actions of insulin and the stimulatory actions of lipolytic hormones such as catecholamines maintains whole body energy homeostasis and metabolic health (6 -8). Catecholamines bind to -adrenergic receptors on adipocytes, resulting in up-regulation of adenyl cyclase, activation of cAMP-dependent protein kinase A (PKA), and increased lipolytic rate (9). The ability of PKA to stimulate adipocyte lipolysis is mediated in large part by the LD-associated phosphoprotein perilipin (Peri) (1, 10, 11). Peri A (the predominant perilipin isoform in adipocytes) is the most prevalent PKA substrate in adipocytes. In the absence of hormonal stimulation (i.e. basal state), Peri A functions to sequester lipases from stored neutral lipid, thereby maintaining a low rate of constitutive lipolysis. After phosphorylation by PKA, Peri A facilitates lipase accessibility to lipid stores, thereby promoting lipolysis (12)(13)(14)(15)(16)(17)(18)(19)(20). The mechanism(s) by which Peri A phosphorylation facilitates TAG/lipase interaction in adipocytes is not elucidated.Previous studies of Pe...
Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr 221 and Tyr 570 as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr 221 , Tyr 570 lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2 Y570F ) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr 570 does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr 570 in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr 570 -dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.Type I cytokines mediate a plethora of physiologic processes, ranging from hematopoietic and immune functions (such as those mediated by erythropoietin [EPO] and the interleukins [ILs]) to growth and neuroendocrine responses (such as those mediated by growth hormone and leptin) (12,14,16,23). These actions are mediated by the activation of cytokine receptor proteins found on the surface of target cells. Cytokine receptors each contain an extracellular domain that recognizes its specific cytokine ligand, a single transmembrane domain, and an intracellular domain that, although devoid of enzymatic activity, transmits intracellular signals by means of an associated Jak family tyrosine kinase. Ligand binding activates the associated intracellular Jak kinase, resulting in the tyrosine phosphorylation of the Jak kinase and the intracellular domain of the cytokine receptor. These tyrosine phosphorylation events mediate the recruitment of downstream signaling molecules that contain phosphotyrosine-binding SH2 domains (such as STAT proteins) (12, 16); tyrosine phosphorylation may also mediate other regulatory events during cytokine signaling (8, 31).The Jak kinase family contains four members: Jak1 to Jak3 and Tyk2 (12, 16). Of these, Jak1, Jak2, and Tyk2 are ubiquitously expressed, while Jak3 is found predominantly in immune and hematopoietic tissues. Jak kinases are composed of four conserved domains. The NH 2 -terminal FERM domain is required for interaction with cytokine receptors (24, 30), while the adjacent SH2-like fold has no known function. The COOH-terminal portion of Jak kinases contains a kinase-like JH2 domain that is devoid of enzymatic activity but that inhibits the activity of the COOH-terminal JH1 tyrosine kinase domain (10,19,22,28,29).Our laboratory studies s...
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