The role of endoplasmic reticulum stress on insulin-resistance induced by glucosamine in the L6 skeletal muscle cells

The endoplasmic reticulum (ER) is responsible for the quality control of proteins. When the productive folding process in the ER is perturbed by environmental stress conditions, eukaryotic cells activate the unfolded protein response (UPR). Recently, several studies reported that UPR might play an important role in Type 2 diabetes. Indeed, ER stress is involved both in pancreatic beta cell dysfunction and in peripheral insulin resistance. Hyperglycaemia causes insulin resistance and this process appears to be linked to UPR activation. Even glucosamine, generated by the activation of the hexosamine pathway during hyperglycaemia, causes insulin resistance and disturbances similar to glucose toxicity in muscle cells. Thus, in the present work we sought to evaluate the possible role of ER stress on the insulin-resistance induced by glucosamine in skeletal muscle cells. L6 skeletal muscle cells were incubated either with 10 mM glucosamine (GLC) or with 0.5 μM thapsigargin (THA), a well known ER stressing agent, in the presence or in absence of insulin (100nM). Upon either GLC or THA treatment, the mRNA levels of the two ER stress markers BiP and CHOP were increased up to 7- and 5-fold, respectively. Moreover, eIF2alpha inhibition was evident as early as 15 min after GLC treatment. We then evaluated the effect of GLC and THA on the insulin signalling. Both GLC and THA blunted insulin induced activation of Akt and phosphorylation of GSK3β. Furthermore, GLC and THA induced a 70% decrease of both GLUT4 mRNA and protein levels, and a 60% decrease of the mRNA levels of the transcription factor MEF2A. The decrease of GLUT4 expression was paralleled by a reduced capability of L6 cells to uptake the glucose analogue 2-deoxy-D-[3H]-glucose in response to insulin. Interestingly, the use of the chemical chaperon 4-Phenyl Butyric Acid (PBA) prevents the induction of ER stress by GLC and THA and their effects on Akt and glucose uptake inhibition. In conclusion, we show for the first time that glucosamine is able to activate the UPR in skeletal muscle cells, and it may play a role in insulin resistance through inhibition of Akt activity and Glut4 expression.