Aims: Increased oxidative stress and mitochondrial dysfunction in obese adipocytes contribute to adipokine dysregulation, inflammation, and insulin resistance. Results: Through an advanced proteomic analysis, we found that peroxiredoxin 3 (Prx3), a thioredoxin-dependent mitochondrial peroxidase, is highly expressed in 3T3-L1 adipocytes compared to preadipocytes. Interestingly, in obese db/db mice and human subjects, adipose Prx3 levels were significantly decreased, indicating its association with obesity. We therefore employed Prx3 knockout (KO) mice and transfected 3T3-L1 cells to examine the role of endogenous Prx3 in adipocyte metabolism. Prx3 KO mice had increased fat mass compared to wild-type due to adipocyte hypertrophy. Increased adipogenic transcription factors and lipogenic gene expression during differentiation of adipose tissue-derived stem cells from Prx3-deficient mice confirmed that these adipocytes are likely to accumulate fat. Mitochondrial protein carbonylation in Prx3 KO adipose tissue and mitochondrial superoxide level in Prx3 knockdown 3T3-L1 cells were increased showing aberrant regulation of oxidative stress. Proteomic analysis and gene expression analysis of Prx3 KO mice adipocytes also showed defect in mitochondria biogenesis along with enzymes involved in glucose/lipid metabolism and oxidative phosphorylation. In addition, expression level of adiponectin was downregulated and plasminogen activator inhibitor-1 was upregulated in Prx3 KO adipocytes. Impaired glucose tolerance and insulin resistance further implied metabolic dysregulation in Prx3 KO mice. Innovation and Conclusion: These data suggest that endogenous Prx3 may play an essential role in maintaining normal characteristics of adipocytes and that defect in Prx3 alters mitochondrial redox state and function, and adipokine expression in adipocytes leading to metabolic alteration. Antioxid. Redox Signal. 16,[229][230][231][232][233][234][235][236][237][238][239][240][241][242][243]

Background/Aims: Plasminogen activator inhibitor (PAI)-1 is increasingly recognized as a profibrotic factor but the mechanisms are not entirely clear. The present study examined the profibrotic mechanism of PAI-1 focusing on its effect on transforming growth factor (TGF)-β1 in experimental diabetes. Methods: PAI-1 knockout (KO) mesangial cells cultured under high glucose (HG) in addition to streptozotocin-induced diabetic PAI-1 KO mice were used. Results: PAI-1 deficiency did not affect plasma glucose significantly but reduced the fractional mesangial area, fibronectin and collagen I expression in the renal cortex after 20 weeks of diabetes as well as in HG-stimulated mesangial cells along with suppression of TGF-β1 mRNA expression. PAI-1 deficiency also reduced HG-induced βig-h3, a TGF-β1-induced gene product, mRNA expression. All these losses-of-function in PAI-1 KO mesangial cells were effectively gained by recombinant PAI-1. Recombinant PAI-1-induced fibronectin and collagen I expression was abrogated by TGF-β1 receptor inhibitor or anti-TGF-β antibody suggesting that the effect of PAI-1 was mediated by TGF-β1. In a similar context, recombinant PAI-1 stimulated TGF-β1 promoter activity to the same extent as TGF-β1 itself. Conclusion: Since TGF-β1 is well known to stimulate the PAI-1 promoter, we suggest that TGF-β1 and PAI-1 together constitute a positive feedback loop in the development of renal fibrosis in diabetes.

) is a potent chemokine that plays an important role in the recruitment of macrophages. Although previous studies have demonstrated the importance of MCP-1 in the pathogenesis of diabetic nephropathy (DN) in terms of inflammation, the role of MCP-1 and its receptor (C-C chemokine receptor 2; CCR2) in extracellular matrix (ECM) accumulation under diabetic conditions has been largely unexplored. This study was undertaken to investigate the functional role of the MCP-1/CCR2 system in high glucoseinduced ECM (fibronectin and type IV collagen) protein expression in cultured mesangial cells (MCs). Mouse MCs were exposed to medium containing 5.6 mM glucose (NG), NGϩ24.4 mM mannitol (NGϩM), or 30 mM glucose (HG) with or without mutant MCP-1 (mMCP-1), CCR2 small interfering (si)RNA, or CCR2 inhibitor (RS102895). To examine the relationship between MCP-1 and transforming growth factor (TGF)-␤1, MCs were also treated with TGF-␤1 (2 ng/ml) with or without mMCP-1 or CCR2 siRNA. Transient transfection was performed with Lipofectamine 2000 for 24 h. Cell viability was determined by an MTT assay, mouse and human MCP-1 and TGF-␤1 levels by ELISA, and CCR2 and ECM protein expression by Western blotting. Transfections of mMCP-1 and CCR2 siRNA increased human MCP-1 levels and inhibited CCR2 expression, respectively. HG-induced ECM protein expression and TGF-␤1 levels were significantly attenuated by mMCP-1, CCR2 siRNA, and RS102895 (P Ͻ 0.05). MCP-1 directly increased ECM protein expression, and this increase was inhibited by an anti-TGF-␤1 antibody. In addition, TGF-␤1-induced ECM protein expression was significantly abrogated by the inhibition of the MCP-1/CCR2 system (P Ͻ 0.05). These results suggest that an interaction between the MCP-1/CCR2 system and TGF-␤1 may contribute to ECM accumulation in DN. diabetic nephropathy; monocyte chemoattractant protein-1; transforming growth factor-␤1; extracellular matrix MONOCYTES/MACROPHAGES ARE the principle inflammatory cells found in the diabetic kidney (6, 9, 29). These cells are extravasculated from the bloodstream through a process mediated by chemokines secreted from resident glomerular cells. Chemokines are a family of chemotactic cytokines that induce the migration of various cell types, and to date Ͼ40 chemokines have been identified (31). Among them, monocyte chemoattractant protein (MCP)-1 is the most extensively studied chemokine. In the kidney, MCP-1 is expressed in mesangial cells (MCs) and tubular epithelial cells (22,26) and is known to be involved in the pathogenesis of various renal diseases, including diabetic nephropathy. Previous studies have demonstrated that plasma MCP-1 levels are increased in type 1 diabetes with microalbuminuria (4) and that urinary levels of MCP-1 are also increased in accordance with the extent of albuminuria (1,20). In addition, it has been reported that glomerular MCP-1 expression is increased in experimental diabetic rats and that this increase is associated with the number of infiltrated monocytes in the glomeruli (5, 6). Moreover, an angio...

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Diabetes is associated with significantly accelerated rates of atherosclerosis, key features of which include the presence of excessive macrophage-derived foam cells in the subendothelial space. We examined the hypothesis that enhanced monocyte-vascular smooth muscle cell (VSMC) interactions leading to subendothelial monocyte retention and differentiation to macrophages under diabetic conditions may be underlying mechanisms. Human aortic VSMCs (HVSMCs) treated with diabetic stimuli high glucose (HG) or S100B, a ligand of the receptor for advanced glycation end products, exhibited significantly increased binding of THP-1 monocytic cells. Diabetic stimuli increased the expression of the adhesive chemokine fractalkine (FKN) in HVSMCs. Pretreatment of HVSMCs with FKN or monocyte chemoattractant protein-1 (MCP-1) neutralizing antibodies significantly inhibited monocyte-VSMC binding, whereas monocytes treated with FKN showed enhanced binding to VSMC. Mouse aortic VSMCs (MVSMCs) derived from type 2 diabetic db/db mice exhibited significantly increased FKN levels and binding to mouse WEHI78/24 monocytic cells relative to nondiabetic control db/+ cells. The enhanced monocyte binding in db/db cells was abolished by both FKN and MCP-1 antibodies. Endothelium-denuded aortas from db/db mice and streptozotocin-induced diabetic mice also exhibited enhanced FKN expression and monocyte binding, relative to respective controls. Coculture with HVSMCs increased CD36 expression in THP-1 cells, and this was significantly augmented by treatment of HVSMCs with S100B or HG. CD36 mRNA and protein levels were also significantly increased in WEHI78/24 cells after coincubation with db/db MVSMCs relative to control MVSMCs. These results demonstrate that diabetic conditions may accelerate atherosclerosis by inducing key chemokines in the vasculature that promote VSMC-monocyte interactions, subendothelial monocyte retention, and differentiation.