α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway.
ABSTRACT Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.
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ABSTRACT: Aims/hypothesis The aim of the study was to address the importance of mitochondrial function in insulin resistance and type 2 diabetes, and also to identify effective agents for ameliorating insulin resistance in type 2 diabetes. We examined the effect of two mitochondrial nutrients, R-α-lipoic acid (LA) and acetyl-l-carnitine (ALC), as well as their combined effect, on mitochondrial biogenesis in 3T3-L1 adipocytes. Methods Mitochondrial mass and oxygen consumption were determined in 3T3-L1 adipocytes cultured in the presence of LA and/or ALC for 24 h. Mitochondrial DNA and mRNA from peroxisome proliferator-activated receptor gamma and alpha (Pparg and Ppara) and carnitine palmitoyl transferase 1a (Cpt1a), as well as several transcription factors involved in mitochondrial biogenesis, were evaluated by real-time PCR or electrophoretic mobility shift (EMSA) assay. Mitochondrial complexes proteins were measured by western blot and fatty acid oxidation was measured by quantifying CO2 production from [1-14C]palmitate. Results Treatments with the combination of LA and ALC at concentrations of 0.1, 1 and 10 μmol/l for 24 h significantly increased mitochondrial mass, expression of mitochondrial DNA, mitochondrial complexes, oxygen consumption and fatty acid oxidation in 3T3L1 adipocytes. These changes were accompanied by an increase in expression of Pparg, Ppara and Cpt1a mRNA, as well as increased expression of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (Ppargc1a), mitochondrial transcription factor A (Tfam) and nuclear respiratory factors 1 and 2 (Nrf1 and Nrf2). However, the treatments with LA or ALC alone at the same concentrations showed little effect on mitochondrial function and biogenesis. Conclusions/interpretation We conclude that the combination of LA and ALC may act as PPARG/A dual ligands to complementarily promote mitochondrial synthesis and adipocyte metabolism.Diabetologia 01/2008; 51(1):165-174. · 6.49 Impact Factor
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ABSTRACT: Induction of stress kinases leading to serine hyperphosphorylation of IRS1 may link oxidative stress to insulin resistance. The aim of this study was to investigate the roles of the phosphorylated serine residues Ser307 and Ser632, two sites implicated in the inhibition of IRS1 function in insulin signalling. Fao hepatoma cells were exposed to an H(2)O(2)-generating system, and antibodies against the two phosphorylated serine residues were used for immunoprecipitation, immunoblot and immunofluorescence analyses. Exposure to approximately 50 mumol/l H(2)O(2) for 2 h resulted in IRS1 phosphorylation on both Ser307 and Ser632, concomitant with activation of inhibitor kappa kinase beta (IKKbeta) and c-Jun kinase (JNK). Immunoprecipitation studies revealed that the maximum overlap between phospho (p) Ser307-IRS1 and pSer632-IRS1 was 20%, and confocal microscopy suggested distinct localisations of IRS1 molecules phosphorylated on either site. Although pSer307-IRS1 showed decreased insulin-induced tyrosine phosphorylation and interaction with phosphatidylinositol 3-kinase (PI3K) in response to insulin, pSer632-IRS1 molecules were normally tyrosine-phosphorylated and exhibited typical associated PI3K activity. Salicylic acid and SP600125 partially inhibited IKKbeta and JNK, respectively, which indicated distinct roles for these two kinases in the phosphorylation of IRS1 at the two serine sites. Protection against oxidation-mediated impairment in insulin-induced phosphorylation of protein kinase B/Akt and in glycogen synthesis was achieved only by combining salicylic acid and SP600125. These results suggest that pSer307-IRS1 and pSer632-IRS1 may define two minimally overlapping pools of IRS1 in response to oxidative stress, contributing differentially to insulin resistance. A combination of stress kinase inhibitors is required to protect against insulin resistance and IRS1 hyperphosphorylation induced by oxidative stress.Diabetologia 11/2006; 49(10):2463-73. · 6.49 Impact Factor
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ABSTRACT: OBJECTIVE The inability of pancreatic beta-cells to appropriately respond to glucose and secrete insulin are primary defects associated with beta-cell failure in type 2 diabetes. Mitochondrial dysfunction has been implicated as a key factor in the development of type 2 diabetes; however, a link between mitochondrial dysfunction and defective insulin secretion is unclear. RESEARCH DESIGN AND METHODS We investigated the changes in islet mitochondrial function and morphology during progression from insulin resistance (3 weeks old), immediately before hyperglycemia (5 weeks old), and after diabetes onset (10 weeks old) in transgenic MKR mice compared with controls. The molecular and protein changes at 10 weeks were determined using microarray and iTRAQ proteomic screens. RESULTS At 3 weeks, MKR mice were hyperinsulinemic but normoglycemic and beta-cells showed negligible mitochondrial or morphological changes. At 5 weeks, MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (DeltaPsi(m)), reduced mitochondrial Ca(2+) uptake, slightly enlarged mitochondria, and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic and hyperinsulinemic and beta-cells contained swollen mitochondria with disordered cristae. beta-Cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of DeltaPsi(m), impaired Ca(2+)-signaling, and reduced glucose-stimulated ATP/ADP and insulin release. Furthermore, decreased cytochrome c oxidase-dependent oxygen consumption and signs of oxidative stress were observed in diabetic islets. Protein profiling of diabetic islets revealed that 36 mitochondrial proteins were differentially expressed, including inner membrane proteins of the electron transport chain. CONCLUSIONS We provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance-induced beta-cell failure.Diabetes 11/2009; 59(2):448-59. · 7.90 Impact Factor