Palmitate-induced Slc2a4/GLUT4 downregulation in L6 muscle cells: evidence of inflammatory and endoplasmic reticulum stress involvement.

BACKGROUND: Obesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by Slc2a4 gene, in L6 skeletal muscle cells. METHODS: L6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed. RESULTS: Chronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, P < 0.01) and its mRNA (18%, P < 0.01) expression. Only acute palmitate treatment increased GRP78 (28%, P < 0.05), PERK (98%, P < 0.01), eIF-2A (35%, P < 0.01), IRE1a (60%, P < 0.05) and TRAF2 (23%, P < 0.05) protein content, and PERK phosphorylation (106%, P < 0.001), but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content (~ 30%, P < 0.05) and NFKB binding activity to Slc2a4 gene promoter (~ 45%, P < 0.05). CONCLUSION: Different pathways are activated in acute and chronic palmitate induced-repression of Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.

Reduced Slc2a4/GLUT4 expression in subcutaneous adipose tissue of monosodium glutamate obese mice is recovered after atorvastatin treatment.

BACKGROUND: Decreased expression of glucose transporter protein GLUT4, encoded by the solute carrier 2A4 (Slc2a4) gene, is involved in obesity-induced insulin resistance. Local tissue inflammation, by nuclear factor-κB (NFκB)-mediated pathway, has been related to Slc2a4 repression; a mechanism that could be modulated by statins. Using a model of obesity with insulin resistance, this study investigated whether (1) inflammatory markers and Slc2a4 expression are altered; (2) atorvastatin has beneficial effects on inflammation and Slc2a4 expression; and (3) inhibitor of NFκB (IKK)/NFκB pathway is involved in subcutaneous adipose tissue (SAT). FINDINGS: Obese mice showed insulin resistance, decreased expression of Slc2a4 mRNA (66%, P < 0.01) and GLUT4 protein (30%, P < 0.05), and increased expression of interleukin 6 (Il6) mRNA (44%, P < 0.05) in SAT. Obese mice treated with atorvastatin had enhanced in vivo insulin sensitivity, besides increased Slc2a4/GLUT4 expression and reduced Il6 expression in SAT. No alterations of tumor necrosis factor-α, interleukin 1ß and adiponectin expression or IKKα/ß activity in SAT of obese mice or obese mice treated with atorvastatin were observed. CONCLUSIONS: Atorvastatin has beneficial effect upon glycemic homeostasis, which may be related to its positive impact on Il6 and Slc2a4/GLUT4 expression in SAT.

Oleic and linoleic fatty acids downregulate Slc2a4/GLUT4 expression via NFKB and SREBP1 in skeletal muscle cells.

Oleic (OA) and linoleic (LA) fatty acids may be important regulators of Slc2a4 gene (GLUT4 protein) in skeletal muscle, thus participating in insulin resistance. We investigated the effect of OA and LA on the Slc2a4/GLUT4 expression in L6 muscle cells; as well as potential transcriptional regulators. OA and LA (50-400 µM) decreased the Slc2a4/GLUT4 expression in a dose-dependent way (maximum of ~50%, P < 0.001). OA and LA did not alter the Slc2a4-binding activity of oxysterols-receptor-LXR-alpha and peroxisome-proliferator-activated-receptor-gamma; but decreased the Slc2a4-binding activity of the sterol-regulatory-element-binding-protein-1 (SREBP1) enhancer (50%, P < 0.001), and increased (~30%, P < 0.001) the nuclear proteins binding into the Slc2a4-nuclear-factor-NF-kappa-B-binding site (repressor), and the phosphorylation of the inhibitors of nuclear-factor-kappa-B-kinase alpha/beta (150-300%, P < 0.001). In sum, OA and LA are potent inhibitors of the Slc2a4/GLUT4 expression in muscle cells; an effect involving reduced SREBP1 and increased NFKB transcriptional activity. These regulations may participate in the fatty acid-related pathophysiology of insulin resistance.

Proinsulin C-peptide prevents type-1 diabetes-induced decrease of renal Na+-K+-ATPase alpha1-subunit in rats.

AIMS/HYPOTHESIS: C-peptide reduces renal damage in diabetic patients and experimental animal models. In vitro studies suggest that the renal effects of C-peptide may, in part, be explained by stimulation of Na(+)/K(+)-ATPase activity. However, the responses of Na(+)/K(+)-ATPase expression in the kidney of diabetic animals to C-peptide administration remain unclear. The aim of this study was to clarify the responses. METHODS: Type 1 diabetic rats were produced by injecting streptozotocin (STZ), and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. The mRNA expression and immunohistochemical localization of Na(+)/K(+)-ATPase alpha1-, alpha2- and beta3-subunits were investigated in the kidney of these rats. RESULTS: Na(+)/K(+)-ATPase alpha1-subunit was abundantly expressed in the medullary collecting ducts of control animals, but the expression was markedly decreased in the diabetic state with concomitant decrease in its mRNA expression. Similar decreases were observed in the insulin-treated diabetic rats, whereas in the C-peptide-treated diabetic rats, there was no reduction in the alpha1-expression. The beta3-subunit was expressed in podocytes and parietal cells in the glomeruli, vascular endothelial cells, and cortical collecting ducts, but lesser signals were observed in the proximal and distal tubules. However, the beta3-subunit did not appear to be affected by the diabetic state. CONCLUSIONS: Diabetes selectively reduced Na(+)/K(+)-ATPase alpha1-subunit expression and abundance. Chronic administration of C-peptide prevented this decrease. This implies a role for C-peptide in the long-term regulation of Na(+)/K(+)-ATPase function.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue.

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 microL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced approximately 20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was approximately 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid ( approximately 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections.

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells.

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK gene transcription.