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Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in [beta]-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic [beta]-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/[TxNIP.sup.-/-]) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and [beta]-cell mass than control mice (C3H). Hcb-19/[TxNIP.sup.-/-] did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although [beta]-cell mass remained higher in Hcb-19/[TxNIP.sup.-/-] mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1[[beta], interferon-[gamma], and tumor necrosis factor-[alpha]. Furthermore, when corrected for insulin content, isolated pancreatic islets from [TxNIP.sup.-/-] mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of [beta]-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in [beta]-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced [beta]-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions. thioredoxin interacting protein; pancreatic [beta]-cell; apoptosis; insulin secretion

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-[[beta].sub.1] is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC)-[zeta] activation. We investigated the role of TGF-[[beta].sub.1] in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-[[beta].sub.1] stimulated PKC-[zeta] kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-[[beta].sub.1] antibodies. Neutralizing TGF-[[beta].sub.1] antibodies, or a TGF-[beta] receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-[zeta] activation by high glucose. TGF-[[beta].sub.1] also stimulated cellular membrane translocation of PKC-[alpha], -[[beta].sub.1], -[delta], and -[epsilon], similar to high glucose. High glucose and TGF-[[beta].sub.1] enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-[[beta].sub.1] antibodies, LY364947, or a specific PKC-[zeta] pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-[zeta] in normal glucose caused upregulation of [p22.sup.phox], a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-[[beta].sub.1] stimulation of PKC isozymes including PI3 kinase activation of PKC-[zeta] and consequent generation of ROS by NADPH oxidase. reactive oxygen species; transforming growth factor-[[beta].sub.1]; PI3 kinase

Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against [p22.sup.phox], a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-[[beta].sub.1] with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-[zeta] with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-[beta], or PKC-[zeta] inhibition. In normal glucose (5.6 mM), overexpression of [p22.sup.phox] or constitutively active PKC-[zeta] enhanced VEGF expression. Hypoxia inducible factor-1[alpha] protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-[[beta].sub.1] and -[zeta], play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells. NADPH oxidase; vascular endothelial growth factor; [p22.sup.phox]; protein kinase C-[beta]; protein kinase C-[zeta]

Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-[zeta]. Am J Physiol Renal Physiol 282: F151-F163, 2002. First published August 15, 2001; 10.1152/ajprenal.00055.2001.--In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (ML[C.sub.20]) phosphorylation, or [Ca.sup.2+] signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 [+ or -] 135 vs. 3,952 [+ or -] 225 (SE) [micro][m.sup.2] in NG, P < 0.01, n = 31] with no contractile response to 0.1 [micro]M ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced [Ca.sup.2+] signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated ML[C.sub.20] showed that HG increased mono- and decreased unphosphorylated ML[C.sub.20] (42 [+ or -] 16 and 49 [+ or -] 15 vs. 13 [+ or -] 3 and 80 [+ or -] 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-[delta] and -[epsilon] from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-[zeta] translocation. In HG, membrane accumulation of PKC-[zeta] was observed. Membrane PKC-[zeta] activity measured by immunoprecipitation and [sup.32]P phosphorylation of PKC-[epsilon] pseudosubstrate peptide was 190 [+ or -] 18% of NG (P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal [Ca.sup.2+] signaling and normal ML[C.sub.20] phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-[zeta]. endothelin-1; calcium signaling; myosin light chain phosphorylation; protein kinase C-[zeta]

A study of the role of Ca2+, cytoprotective amino acid glycine and pH on the accumulation of unesterified fatty acids by intact proximal tubules during antimycin-, hypoxia- or antimycin and ionomycin-induced injury reveals that Ca2+-independent pathways regulate most of the fatty acid accumulation during proximal tubule cell injury. Fatty acid accumulation is suppressed by decreasing the pH to 6.9. While glycine prevents the occurrence of lethal membrane damage due to the accumulation of high levels of unesterified fatty acids, it does not influence the degree of accumulation under Ca2+-rich or Ca2+-restricted conditions.

James, Leighton R., I. George Fantus, Howard Goldberg, Hao Ly, and James W. Scholey. Overexpression of GFAT activates PAI-1 promoter in mesangial cells. Am J Physiol Renal Physiol 279: F718-F727, 2000.--Effects of hyperglycemia on glomerular cells may be mediated by glucose entry into the hexosamine pathway, and mesangial cell (MC) expression of the hexosamine pathway rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) is increased in diabetic glomerulosclerosis. We hypothesized that GFAT activity would be an important determinant of gene expression in glomerular MC. When overexpressed in primary MC, GFAT produced a two- to threefold increase in the activity of plasminogen activator inhibitor-1 (PAI-1) promoter. There was a 1.4-fold increase in PAI-1 promoter activity in cells exposed to high glucose (20 mM), whereas in MC overexpressing GFAT, exposure to high glucose caused a 3.5- to 4-fold increase in promoter activity. PAI-1 promoter activation was dependent on GFAT enzyme activity because o-diazoacetyly-L-serine and 6-diazo-5-oxonorleucine, inhibitors of GFAT enzyme activity, abrogated the activation of PAI-1 promoter in MC overexpressing GFAT. Glucosamine, which is downstream of GFAT in the hexosamine pathway, produced a 2.5-fold increase in the PAI-1 promoter activity. In addition to increasing the mRNA levels for transforming growth factor-[Beta]1 (TGF-[Beta]1), GFAT overexpression also increased mRNA levels for the TGF-[Beta] type I and type II receptors. TGF-[Beta]-neutralizing antibody did not normalize PAI-1 promoter activity in MC exposed to glucosamine or those overexpressing GFAT. We conclude that GFAT expression and activity are important determinants of gene expression in MC and that flux through the hexosamine pathway activates expression of genes implicated in vascular injury pathways. glutamine:fructose-6-phosphate amidotransferase; plasminogen activator inhibitor-1; diabetic nephropathy; gene expression

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-β1 is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -ζ activation. We investigated the role of TGF-β1 in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-β1 stimulated PKC-ζ kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-β1 antibodies. Neutralizing TGF-β1 antibodies, or a TGF-β receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-ζ activation by high glucose. TGF-β1 also stimulated cellular membrane translocation of PKC-α, -β1, -δ, and -ϵ, similar to high glucose. High glucose and TGF-β1 enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-β1 antibodies, LY364947, or a specific PKC-ζ pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-ζ in normal glucose caused upregulation of p22phox, a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-β1 stimulation of PKC isozymes including PI3 kinase activation of PKC-ζ and consequent generation of ROS by NADPH oxidase. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEOF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22phox, a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-β1 with the specific pharmacological inhibitor LY-33353 1 or inhibition of PKC-ζ with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEOF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-β1, or PKC-ζ inhibition. In normal glucose (5.6 mM), overexpression of p22phox or constitutively active PKC-ζ enhanced VEGF expression. Hypoxia inducible factor-1α protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-β1 and -ζ, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells. [ABSTRACT FROM AUTHOR]

Examines the effect of high glucose on mesangial cell calcium signaling, myosin light chain phosphorylation and protein kinase C isozyme activity. Mediation of cytoskeletal dysfunction and hypocontractility; Effects of high glucose and chronic phorbol ester on mesangial cell planar area; Confocal fluorescence image analysis.