Glucosamine-induced insulin-resistance in L6 skeletal muscle cells is associated with endoplasmic reticulum stress

Background and Aims: Endoplasmic reticulum (ER) stress might play an important role in the pathogenesis of insulin-resistance and Type 2 diabetes. Hyperglycaemia causes insulin resistance and this process appears to be linked to ER stress. Glucosamine, generated by hexosamine pathway during hyperglycaemia, also causes insulin resistance and disturbances similar to glucose toxicity. Here, we sought to evaluate the possible role of ER stress on the insulin-resistance induced by glucosamine in skeletal muscle cells. Materials and Methods: L6 skeletal muscle cells were incubated either with 10 mM glucosamine (GlcNac) or with 0.5 μM thapsigargin (Thap), a well known ER stressing agent, in the presence or in absence of insulin (100nM). Results: Upon 24 hours of GlcNac treatment, L6 cells showed a 6-fold increase of the ER stress marker BiP/GRP78 mRNA levels (p < 0.001) and a reduced uptake of the glucose analogue 2-deoxy-D-[3H]-glucose (2DG) in response to insulin (p < 0.01). These effects were paralleled by a 70% decrease of both mRNA and protein levels of the insulin-sensitive glucose transporter GLUT4. GLUT4 regulation was, very likely, at the transcriptional level, as demonstrated by parallel mRNA downregulation of two transcription factors known to regulate GLUT4 expression, such as MEF2A and PGC1. Time course experiments showed that GLUT4 expression was reduced by 30% after 4 hours (p < 0.001), and by 70% after 8 hours (p < 0.001) following GlcNac treatment. MEF2A mRNA showed a similar kinetic. Furthermore, immunofluorescence confocal imaging experiments showed that GlcNac, besides its effect on GLUT4 transcription, impaired also the insulin induced GLUT4 translocation onto the plasma membrane. The use of the chemical chaperon 4-Phenyl Butyric Acid (PBA), known to alleviate ER stress conditions, prevented both BiP/GRP78 induction and GLUT4 downregulation. As for GLUT4, also MEF2A and PGC1 mRNA levels did not show any appreciable variation when L6 cells were treated with GlcNac in the presence of PBA. Furthermore, PBA treatment restored insulin-induced 2DG uptake to levels comparable to those of L6 control cells. Interestingly, GlcNac caused a 60% increase of both mRNA and protein levels of the glucose transporter GLUT1 (p < 0.01), suggesting that GLUT1 gene could be considered an early target of ER stress. Indeed, bioinformatics researches revealed the presence of four ERSE (ER stress Responsive Element) consensus sequence for ER stress activated transcription factors in the proximal region of the rat GLUT1 promoter sequence. Conclusions: Glucosamine-induced ER stress may play a role in insulin-resistance of L6 skeletal muscle cells through the modulation of the expression and localization of the glucose transporters GLUT1 and GLUT4.