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Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.

Complete lecithin cholesterol acyltransferase (LCAT) deficiency uniformly results in a profound HDL deficiency. We recently reported unexpected enhanced insulin sensitivity in LCAT knock-out mice in the LDL receptor knock-out background (Ldlr(-/-)×Lcat(-/-); double knock-out (DKO)), when compared with their Ldlr(-/-)×Lcat(+/+) (single knock-out (SKO)) controls. Here, we report that LCAT-deficient mice (DKO and Lcat(-/-)) are protected against high fat high sucrose (HFHS) diet-induced obesity without hypophagia in a gender-specific manner compared with their respective (SKO and WT) controls. The metabolic phenotypes are more pronounced in the females. Changes in endoplasmic reticulum stress were examined as a possible mechanism for the metabolic protection. The female DKO mice developed attenuated HFHS-induced endoplasmic reticulum stress as evidenced by a lack of increase in mRNA levels of the hepatic unfolded protein response (UPR) markers Grp78 and CHOP compared with SKO controls. The DKO female mice were also protected against diet-induced insulin resistance. In white adipose tissue of chow-fed DKO mice, we also observed a reduction in UPR, gene markers for adipogenesis, and markers for activation of Wnt signaling. In skeletal muscles of female DKO mice, we observed an unexpected increase in UCP1 in association with increase in phospho-AMPKα, PGC1α, and UCP3 expressions. This increase in UCP1 was associated with ectopic islands of brown adipocytes between skeletal muscle fibers. Our findings suggest that LCAT deficiency confers gender-specific protection against diet-induced obesity and insulin resistance at least in part through regulation in UPR, white adipose tissue adipogenesis, and brown adipocyte partitioning.

Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Here, we analyze adipocytes exposed to hyperglycemia both in vitro as well as in vivo. Comparing cells differentiated at 4 mm to cells differentiated at 25 mm glucose (the standard differentiation protocol) reveals severe insulin resistance in cells exposed to 25 mm glucose. A global assessment of transcriptional changes shows an up-regulation of a number of mitochondrial proteins. Exposure to hyperglycemia is associated with a significant induction of reactive oxygen species (ROS), both in vitro as well as in vivo in adipocytes isolated from streptozotocin-treated hyperglycemic mice. Furthermore, hyperglycemia for a few hours in a clamped setting will trigger the induction of a pro-inflammatory response in adipose tissue from rats that can effectively be reduced by co-infusion of N-acetylcysteine (NAC). ROS levels in 3T3-L1 adipocytes can be reduced significantly with pharmacological agents that lower the mitochondrial membrane potential, or by overexpression of uncoupling protein 1 or superoxide dismutase. In parallel with ROS, interleukin-6 secretion from adipocytes is significantly reduced. On the other hand, treatments that lead to a hyperpolarization of the mitochondrial membrane, such as overexpression of the mitochondrial dicarboxylate carrier result in increased ROS formation and decreased insulin sensitivity, even under normoglycemic conditions. Combined, these results highlight the importance ROS production in adipocytes and the associated insulin resistance and inflammatory response.

Increased flux through the hexosamine biosynthesis pathway (HBP) has been shown to stimulate the expression of a number of genes. We previously demonstrated in glomerular mesangial and endothelial cells that both high glucose concentrations and glucosamine activated the plasminogen activator inhibitor-1 (PAI-1) gene promoter through the transcription factor, Sp1; and that the glutamine:fructose-6-phosphate amidotransferase inhibitor, 6-diazo-5-oxonorleucine, inhibited the effect of high glucose, but not that of glucosamine. Here, we examined the role of protein kinase C (PKC) isoforms in the regulation of the PAI-1 promoter and Sp1 transcriptional activity by the HBP. In transient transfections, exposure to 2 mm glucosamine or 20 mm glucose for 4 days increased the activities of a PAI-1 promoter-luciferase reporter gene as well as the Sp1 transcriptional activation domain fused to the GAL4 DNA-binding domain cotransfected with a GAL4 promoter-luciferase reporter. Cotransfected dominant negative PKC-betaI and -delta completely blocked the induction of PAI-1 promoter transcription by both sugars, whereas only dominant negative PKC-betaI interfered with Sp1-GAL4 activation. Both glucosamine and high glucose stimulated the in vitro kinase activity of immunoprecipitated PKC-betaI and -delta. Furthermore, 6-diazo-5-oxonorleucine suppressed high glucose-induced PKC kinase activity and Sp1-GAL4 transcriptional activation. These findings demonstrate a requirement for the PKC-betaI and -delta signal transduction pathways in HBP-induced transcription.

A fructose-fed hamster model of insulin resistance was previously documented to exhibit marked hepatic very low density lipoprotein (VLDL) overproduction. Here, we investigated whether VLDL overproduction was associated with down-regulation of hepatic insulin signaling and insulin resistance. Hepatocytes isolated from fructose-fed hamsters exhibited significantly reduced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2. Phosphatidylinositol 3-kinase activity as well as insulin-stimulated Akt-Ser473 and Akt-Thr308 phosphorylation were also significantly reduced with fructose feeding. Interestingly, the protein mass and activity of protein-tyrosine phosphatase-1B (PTP-1B) were significantly higher in fructose-fed hamster hepatocytes. Chronic ex vivo exposure of control hamster hepatocytes to high insulin also appeared to attenuate insulin signaling and increase PTP-1B. Elevation in PTP-1B coincided with marked suppression of ER-60, a cysteine protease postulated to play a role in intracellular apoB degradation, and an increase in the synthesis and secretion of apoB. Sodium orthovanadate, a general phosphatase inhibitor, partially restored insulin receptor phosphorylation and significantly reduced apoB secretion. In summary, we hypothesize that fructose feeding induces hepatic insulin resistance at least in part via an increase in expression of PTP-1B. Induction of hepatic insulin resistance may then contribute to reduced apoB degradation and enhanced VLDL particle assembly and secretion.