Pioglitazone Inhibits Pyruvate Metabolism in Hepatocytes

The thiazoldinedione compound pioglitazone (PIO) is an effective hypoglycemic agent commonly prescribed for the treatment of type 2 diabetes mellitus (T2DM), the actions of which have typically been assigned to transcriptional regulation through the peroxisome proliferator‐activated receptor gamma (PPAR‐γ). Growing evidence supports the presence of non‐PPAR‐γ related targets of PIO. For example, modulation of glucose metabolism has been demonstrated in human skeletal muscle myotubes through PIO‐mediated inhibition of the newly identified mitochondrial pyruvate carriers MPC1 and MPC2 (Divakuruni et al ,. 2013). However, the existence of PPAR‐γ sparing mechanisms of PIO in hepatocytes has not been established. Therefore, we explored the effects of pharmacologically‐relevant concentrations of PIO in cultured hepatocytes (H4IIE) on whole‐cell substrate oxidation ( 14 CO 2 capture), mitochondrial ATP production (luciferin‐luciferase assay) and pyruvate/lactate‐driven glucose production, as well as any impact upon the expression of key regulatory gluconeogenic genes. We also assessed the dependence of these effects, if any, on MPC1 and MPC2 using siRNA transfection by electroporation. Four hour PIO treatment (10 μM) suppressed pyruvate oxidation by 29% (P<0.01), but had no impact upon palmitate oxidation. Consistent with this, a dose‐dependent inhibition of mitochondrial ATP production from 500 μM pyruvate/2 mM malate was observed with PIO at 1 μM (27%; p=0.07), 5 μM (36%; P<0.05) and 10 μM (61%; P<0.01) concentrations, but did not influence ATP production from glutamate, succinate or palmitoylcarnitine substrates. Glucose production from pyruvate/lactate was 53% lower following overnight treatment with 10 μM PIO (P<0.01) and this occurred in the absence of any changes in the expression of phosphoenolpyruvate carboxykinase, fructose 1,6‐bisphosphatase, glucose 6‐phosphatase or pyruvate carboxylase. Treatment with the specific inhibitor of mitochondrial pyruvate import UK50995 (5 – 10 μM) similarly inhibited pyruvate oxidation (−85%), pyruvate‐driven mitochondrial ATP production (−66%) and glucose production (−71%). Silencing of MPC1 or MPC2 led to a ~20% reduction in pyruvate oxidation, but only MPC1 silencing significantly impaired glucose production (−48%). The inhibition of pyruvate oxidation by PIO was preserved in cells silenced for MPC1 (P<0.001) and MPC2 (P<0.05), as was the suppression of glucose production in cells silenced for MPC1 (P<0.05). Together, these data demonstrate a role for PIO in the downregulation of liver mitochondrial metabolism that is specific to pyruvate and not dependent on the expression of MPC1 or MPC2. Moreover, these experiments indicate the existence of a novel PPAR‐γ‐independent mechanism through which PIO may suppress hepatic glucose production and hence ameliorate hyperglycemia in T2D patients.