leupeptins and Insulin-Resistance

An early insulin regimen ameliorates glucotoxicity but also lipotoxicity in type 2 diabetes; however, the underlying mechanism remains elusive. In the present study, we investigated the role of mitochondria in lipid regulation following early insulin administration in insulin-resistant skeletal muscle cells. Male C57BL/6 mice, fed a high-fat diet (HFD) for 8 weeks, were treated with insulin for 3 weeks, and L6 myotubes cultured with palmitate (PA) for 24 h were incubated with insulin for another 12 h. The results showed that insulin facilitated systemic glucose disposal and attenuated muscular triglyceride accumulation in vivo. Recovery of AMP-activated protein kinase (AMPK) phosphorylation, inhibition of sterol-regulated element binding protein-1c (SREBP-1c) and increased carnitine palmitoyltransferase‑1B (CPT1B) expression were observed after insulin administration. Moreover, increased ATP concentration and cellular energy charge elicited by over-nutrition were suppressed by insulin. Despite maintaining respiratory complex activities, insulin restored muscular uncoupling protein 3 (UCP3) protein expression in vitro and in vivo. By contrast, knockdown of UCP3 abrogated insulin-induced restoration of AMPK phosphorylation in vitro. Importantly, the PA-induced decrease in UCP3 was blocked by the proteasome inhibitor MG132, and insulin reduced UCP3 ubiquitination, thereby prohibiting its degradation. Our findings, focusing on energy balance, provide a mechanistic understanding of the promising effect of early insulin initiation on lipotoxicity. Insulin, by recovering UCP3 activity, alleviated energy surfeit and potentiated AMPK-mediated lipid homeostasis in skeletal muscle cells following exposure to PA and in gastrocnemius of mice fed HFD.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Carnitine O-Palmitoyltransferase; Diabetes Mellitus, Type 2; Diet, High-Fat; Glucose; Insulin; Insulin Resistance; Leupeptins; Lipid Metabolism; Male; Mice; Muscle, Skeletal; Phosphorylation; Protein Kinases; Sterol Regulatory Element Binding Protein 1; Triglycerides; Uncoupling Protein 3

Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in murine skeletal muscle cell line C

Topics: Ammonium Chloride; Animals; Blotting, Western; Cell Differentiation; Cell Line; Co-Repressor Proteins; Glucose; Insulin; Insulin Resistance; Leupeptins; Lysosomes; Mice; Models, Biological; Muscle, Skeletal; Phosphorylation; Proteolysis; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering

Obesity-associated insulin resistance is a major pathogenesis of type 2 diabetes mellitus and is characterized by defects in insulin signaling. High concentrations of plasma free fatty acids (FFAs) are involved in the etiology of obesity-associated insulin resistance. However, the detailed mechanism by which FFAs contribute to the development of insulin resistance is not yet fully understood. We investigated the molecular basis of insulin resistance elicited by FFAs using the human hepatocyte cell line HepG2. Among major human FFAs, palmitate markedly inhibited insulin-stimulated phosphorylation of key insulin signaling molecules such as insulin receptor, insulin receptor substrate-1, and Akt, indicating that palmitate is the principal inducer of insulin resistance. We revealed that palmitate facilitates ubiquitination of the key insulin signaling molecules, and subsequently elicits their proteasomal degradation. Furthermore, we demonstrated that inhibition of ubiquitination by the ubiquitin-activating enzyme E1 inhibitor PYR41 significantly prevents palmitate-inducible insulin resistance but not by the proteasome inhibitor MG132, implying that ubiquitinated signaling molecules may be dysfunctional. In conclusion, inhibition of ubiquitination of the key insulin signaling molecules may be a potential strategy for preventing and treating obesity-associated insulin resistance.

Topics: Benzoates; Fatty Acids, Nonesterified; Furans; Gene Expression Regulation; Hep G2 Cells; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Leupeptins; Palmitic Acid; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptor, Insulin; Signal Transduction; Ubiquitin-Activating Enzymes; Ubiquitination

Although well-established, the underlying mechanisms involved in obesity-related plasma adiponectin decline remain elusive. Oxidative stress is associated with obesity and insulin resistance and considered to contribute to the progression toward obesity-related metabolic disorders. In this study, we investigated the effects of 4-hydroxynonenal (4-HNE), the most abundant lipid peroxidation end product, on adiponectin production and its potential implication in obesity-related adiponectin decrease. Long-term high-fat diet feeding led to obesity in mouse, accompanied by decreased plasma adiponectin and increased adipose tissue 4-HNE content. Exposure of adipocytes to exogenous 4-HNE resulted in decreased adiponectin secretion in a dose-dependent manner, which was consistent with significantly decreased intracellular adiponectin protein abundance. In contrast, adiponectin gene expression was significantly elevated by 4-HNE treatment, which was concomitant with increased peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. The effect was abolished by T0070907, a PPAR-γ antagonist, suggesting that PPAR-γ activation plays a critical role in this process. To gain insight into mechanisms involved in adiponectin protein decrease, we examined the effects of 4-HNE on adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that 4-HNE exposure accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that 4-HNE exposure increased ubiquitinated adiponectin protein levels. These data altogether indicated that 4-HNE enhanced adiponectin protein degradation via ubiquitin-proteasome system. Finally, we demonstrated that supplementation of HF diet with betaine, an antioxidant and methyl donor, alleviated high-fat-induced adipose tissue 4-HNE increase and attenuated plasma adiponectin decline. Taken together, our findings suggest that the lipid peroxidation product 4-HNE can differentially regulates adiponectin gene expression and protein abundance and may play a mechanistic role in obesity-related plasma adiponectin decline.

Topics: 3T3-L1 Cells; Adiponectin; Adipose Tissue; Aldehydes; Animals; Benzamides; Diet, High-Fat; Dose-Response Relationship, Drug; Gene Expression; Insulin Resistance; Leupeptins; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Obesity; Oxidative Stress; PPAR gamma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Pyridines; Ubiquitin; Up-Regulation

Endoplasmic reticulum (ER) stress is associated with obesity-induced insulin resistance, yet the underlying mechanisms remain to be fully elucidated. Here we show that ER stress-induced insulin receptor (IR) down-regulation may play a critical role in obesity-induced insulin resistance. The expression levels of IR are negatively associated with the ER stress marker C/EBP homologous protein (CHOP) in insulin target tissues of db/db mice and mice fed a high-fat diet. Significant IR down-regulation was also observed in fat tissue of obese human subjects and in 3T3-L1 adipocytes treated with ER stress inducers. ER stress had little effect on IR tyrosine phosphorylation per se but greatly reduced IR downstream signaling. The ER stress-induced reduction in IR cellular levels was greatly alleviated by the autophagy inhibitor 3-methyladenine but not by the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Inhibition of autophagy prevented IR degradation but did not rescue IR downstream signaling, consistent with an adaptive role of autophagy in response to ER stress-induced insulin resistance. Finally, chemical chaperone treatment protects cells from ER stress-induced IR degradation in vitro and obesity-induced down-regulation of IR and insulin action in vivo. Our results uncover a new mechanism underlying obesity-induced insulin resistance and shed light on potential targets for the prevention and treatment of obesity-induced insulin resistance and type 2 diabetes.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Autophagy; Disease Models, Animal; Down-Regulation; Endoplasmic Reticulum; Humans; Insulin Resistance; Leupeptins; Mice; Mice, Inbred Strains; Obesity; Phosphorylation; Receptor, Insulin; Taurochenodeoxycholic Acid; Tyrosine

In the current study, we examined the mechanisms that regulate hepatic apolipoprotein B (apoB)-containing lipoprotein secretion in Psammomys obesus, a good animal model for the investigation of insulin resistance and diabetes.. When fed chow ad libitum, 22% maintained normoglycemia and normoinsulinemia (group A), 33% exhibited normoglycemia and appreciable hyperinsulinemia (group B), and 45% developed overt diabetes (group C). Body weight gain, plasma free fatty acid elevation, hypertriglyceridemia, and hypercholesterolemia characterized groups B and C. Triton WR-1339 injection, at fasting, resulted in higher plasma VLDL-triglyceride and VLDL-apoB accumulation in groups B and C, suggesting increased VLDL production by the liver. Pulse-chase labeling experiments in cultured hepatocytes with [35S]methionine revealed reduced intracellular degradation and enhanced secretion of newly synthesized apoB in groups B and C. Concomitant with the raised triglyceride and cholesterol contents in the livers of groups B and C, there was an increase in lipogenesis and in the activity of microsomal triglyceride transfer protein, monoacylglycerol acyltransferase, and diacylglycerol transferase. Pretreatment of hepatocytes with proteasomal inhibitors eliminated the differences in apoB secretion among groups A, B, and C.. Our data indicate that both insulin resistance and diabetes triggered the intracellular machinery involved in VLDL assembly and secretion.

Topics: Acetylcysteine; Animals; Apolipoproteins B; Cells, Cultured; Cysteine Endopeptidases; Diabetes Mellitus; Disease Models, Animal; Gerbillinae; Hepatocytes; Hypercholesterolemia; Hyperinsulinism; Hypertriglyceridemia; Insulin; Insulin Resistance; Leupeptins; Lipoproteins, VLDL; Liver; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex