Mitochondrial mechanism of hyperglycemia-induced apoptosis in primary mouse hepatocytes with steatosis

AIM: To investigate the role of high glucose in primary hepatocytes of mice fed with a high fat diet.METHODS: Male C57BL/6J mice were fed a high fat (45% of calories) diet ad libitum for 6 weeks to induce hepatic steatosis. Primary hepatocytes were isolated from the mouse liver by the 2 step collagenase perfusion method. The cells were incubated in low glucose (5 mmol/L), low glucose plus mannitol (30 mmol/L), or high glucose (35 mmol/L) DMEM medium for 12 h. The cell viability, apoptosis, mitochondrial membrane potential, and caspase enzymatic activities were measured. Furthermore, proteins related to the stress-sensitive signaling pathway of regulating high glucose-induced apoptosis in primary hepatocytes were determined by Western blotting.RESULTS: Incubation with 35 mmol/L glucose resulted in a significant decrease in cell viability and an increase in apoptosis, whereas mannitol had no significant effect on the cell viability or apoptosis. A progressive depolarization of the mitochondria, an increase in cytosol cytochrome C and a dramatic decrease in mitochondrial cytochrome C in high-glucose stressed hepatocytes were observed. The enzymatic activities of caspase-9 and caspase-3, but not caspase-8, were significantly increased in high glucose-stressed hepatocytes (P<0.05). High glucose treatment suppressed the expression of Bcl-2 and Bcl-xL, while it increased the expression of the pro-apoptotic factor Bax.CONCLUSION: High glucose stress reduces mitochondrial membrane potential, initiates mitochondria-mediated apoptotic pathways and promotes apoptosis of hepatocytes with steatosis. This may be an important pathological mechanism of hyperglycemia-induced progression of nonalcoholic fatty liver disease.