Browsing by Subject "Adipocyte"

Now showing 1 - 4 of 4
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    Adipocyte protein carbonylation and oxidative stress in obesity-linked mitochondrial dysfunction and insulin resistance.
    (2011-09) Curtis, Jessica Marie
    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. The antioxidant enzyme glutathione S-transferase A4 (GSTA4) catalyzes a major detoxification pathway for such reactive lipids but its expression was selectively down regulated in the obese, insulin resistant adipocyte resulting in increased protein carbonylation. The effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. Mitochondrial functions in adipocytes of lean or obese GSTA4 null mice were significantly compromised compared to wild type controls and were accompanied by an increase in superoxide anion. Silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration and altered glucose transport and lipolysis. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4- silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1 alpha subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production and a reduction in membrane potential without changes in mitochondrial number, area or density. These results suggest protein carbonylation plays a major instigating role in mitochondrial dysfunction and may be a linked to the development of insulin resistance in the adipocyte.
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    Distinct functions of autophagy kinases ULK1 and ULK2 in adipogenesis and adipocyte metabolism
    (2011-11) Ro, Seung-Hyun
    Autophagy, the catabolic process through which intracellular constituents are degraded in the lysosome under nutrient starvation or stress, has gained growing attention in the field of diabetes and obesity (Goldman S 2010; Ost A 2010; Beau I 2011; Kovsan J 2011). Despite the fundamental cellular function of autophagy in maintaining cellular energy homeostasis and survival under nutrient– or energy– deprived conditions and stress, the role of adipose autophagy in metabolism and metabolic diseases remains largely unknown. The goal of my study has been to better understand the function of autophagy in adipogenesis and in the regulation of adipocyte metabolism. My study has been focused on defining the role of ULK1 (Unc–51 like kinase 1, mammalian homolog of Atg1, hATG1) and its homologue ULK2 in the regulation of adipogenesis, metabolism and mitochondrial functions in adipocytes. ULK1 and ULK2 are key regulators of autophagy induction in mammalian cells (Kundu M 2009; Chang YY 2009; Ganley IG 2009; Hosokawa N 2009; Jung CH 2009). Knockdown of ULK1 or ULK2 inhibited autophagy in 3T3–L1 adipocytes, suggesting that they play important roles in autophagy in adipocytes. The knockdown experiment also revealed that ULK1 and ULK2 share key functions in lipolysis, mitochondrial respiration and protection of cells against oxidative stress. Despite these shared functions, their knockdown had different or even opposing effects on several metabolic parameters. Knockdown of ULK1 raised PPAR–γ level, facilitated differentiation of 3T3–L1 cells, increased the levels of GLUT4, insulin receptorβ(IRβ) and insulin receptor substrate–1 (IRS–1), and insulin–stimulated glucose uptake, and reduced fatty acid oxidation. By contrast, knockdown of ULK2 had opposite or no significant effects on these parameters. Through knocking down both ULK1 and ULK2, we found that ULK2 has a dominant effect over ULK1 in the regulation of adipogenesis. These results demonstrate that ULK1 and ULK2 have distinct functions in the regulation of adipogenesis and adipocyte metabolism, and that ULK2–dependent autophagy appears to be important for adipogenesis.
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    Functional role of receptor-interacting protein 140 (RIP140) in adipocyte dysfunctions and inflammatory response in macrophages.
    (2012-03) Ho, Ping-Chih
    The prevalence of metabolic diseases in modern society, including Type II diabetes mellitus (T2DM), hypertension and cardiovascular diseases, is a major burden on health care systems. Among these diseases, T2DM and its associated complications contribute to the progression of other metabolic diseases such as fatty liver diseases and atherosclerosis. Understanding the initiation and progression of T2DM is critical for developing treatments for T2DM and its associated metabolic disorders. Adipocyte dysfunctions and chronic inflammation have been shown recently to play essential roles in the progression of T2DM. Normally, adipocytes can store energy as triglycerides, fine-tune other metabolic tissues¡¦ lipid and glucose metabolism, and secreted cytokines (adipokines) to modulate immune response. In T2DM or obesity, adipocytes become dysfunctional, with increased lipolysis, an altered adipokine profile, and decreased insulin sensitivity and glucose uptake ability. These changes affect not only the adipocytes themself but also systemic glucose and lipid metabolism. In obese patients and in the high-fat diet (HFD)-fed mouse model, increased inflammatory response in macrophages also contributes to adipocyte dysfunction. The escalated inflammatory response plays pathophysiological roles in various metabolic disorders, including atherosclerosis and arthritis, and increases the incidence of septic shock. However, the underlying mechanisms for initiation of adipocyte dysfunctions and escalation of inflammatory response remain unclear. Receptor-interacting protein 140 (RIP140) is a co-regulator for various transcription factors and nuclear receptors and is expressed mainly in macrophages and metabolic tissues, including adipocytes, hepatocytes and muscle cells. RIP140 affects the progression of T2DM through its nuclear activity as shown by the resistance of knockout mice to diet-induced diabetes and its associated metabolic disorders. In my studies, I found that when I used HFD feeding to induce T2DM, RIP140 could accumulate within the cytoplasm of adipocytes. I further demonstrated that cytoplasmic RIP140 not only interacted with AS160 to impede GLUT4 vesicle trafficking and adiponectin vesicle secretion, but also formed a complex with perilipin A to enhance lipolysis. These findings suggest that HFD feeding can alter RIP140¡¦s cellular distribution, which leads to adipocyte dysfunctions including higher lipolysis, lower glucose uptake, and reduction in adiponectin secretion. I also showed that HFD feeding promoted cytoplasmic accumulation of RIP140 in adipocytes through a PKCϵ-dependent signaling pathway by enhancing intracellular lipid content (as an intrinsic stimulus) and circulating endothelin-1 (as an extrinsic stimulus). Most importantly, administration of a selective ET-1 receptor anatagonist, ambrisentan, reduced HFD-induced cytoplasmic accumulation of RIP140 in adipocytes and further ameliorate hepatic steatosis and insulin sensitivity in vivo. These findings reveal the novel roles of cytoplasmic RIP140 in adipocyte dysfunctions and provide evidence for cytoplasmic RIP140 as a promising target for treatment of T2DM. Recently, RIP140 has also been shown to affect proinflammatory cytokine production by functioning as co-activator for NF-fÛB in macrophages. I showed that HFD feeding up-regulated RIP140 expression by promoting intracellular cholesterol level which led to increased proinflammatory potential in macrophages. In this study, intracellular cholesterol level regulates RIP140 expression by decreasing microRNA-33a, which targeted RIP140 via a conserved region in 3¡¦-UTR of RIP140 mRNA. I further discovered that TLR ligands could trigger RIP140 degradation to resolve inflammation. This RIP140 degradation was modulated by RelA-recruited SCF E3 ligase and Syk-mediated phosphorylation on RIP140. My studies in macrophages demonstrate that RIP140 in macrophages can be modulated by a HFD to affect the systemic inflammatory response and further suggest that defects in RIP140 degradation may cause non-resolving inflammation which is involved in septic shock and various metabolic disorders. Taken together, my studies provide evidence for the novel functions of RIP140 in adipocyte dysfunction and inflammatory response in macrophages and determine the mechanisms by which HFD affect RIP140¡¦s distribution and expression in adipcoytes and macrophages. These findings contribute to our understanding of how HFD causes adipocyte dysfunctions and increase inflammatory response.
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    The role of renin in the adipose tissue renin-angiotensin system.
    (2009-07) Fowler, Jason Dean
    The renin angiotensin system (RAS) has been implicated in a variety of adipose tissue functions including tissue growth, differentiation, metabolism, and inflammation. While expression of all components necessary for a locally derived adipose tissue RAS have been demonstrated within adipose tissue, independence of local adipose RAS component concentrations from corresponding plasma RAS fluctuations has not been addressed. To analyze this, we varied in vivo rat plasma concentrations of two RAS components, renin and angiotensinogen (AGT), to determine the influence of their plasma concentrations on adipose and cardiac tissue levels in both perfused (plasma removed) and nonperfused samples. Variation of plasma RAS components was accomplished by 4 treatment groups: Normal, DOCA-salt, Bilateral nephrectomy, and Losartan. Adipose and cardiac tissue AGT concentrations correlated positively with plasma values. Perfusion of adipose tissue decreased AGT concentrations by 11.1% indicating that adipose tissue AGT was in equilibrium with plasma. Cardiac tissue renin levels positively correlated with plasma renin concentration for all treatments. In contrast, adipose tissue renin levels did not correlate with plasma renin, with the exception of extremely high plasma renin concentrations achieved in the Losartan treated group. These results suggest that adipose tissue may control its own local renin concentration independently of plasma renin as a potential mechanism for maintaining a functional local adipose RAS. Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were up regulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA while TNF alpha treatment decreased renin mRNA 4-fold. IL-6, insulin, and angiotensin II (Ang II) were without effect. In contrast, forskolin and TNF alpha each increased renin protein secretion by 12- and 7-fold, respectively. Although both forskolin and TNFalpha induce lipolysis in adipocytes, fatty acids, prostaglandin E2 or lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate mechanism(s) by which forskolin and/or TNF alpha are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin induced renin release while having no effect on TNF alpha regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels while H89 had no effect. Neither inhibitor had any influence on TNFalpha regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNF alpha-mediated down regulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes.