Genetic Inactivation of Pyruvate Dehydrogenase Kinases Improves Hepatic Insulin Resistance Induced Diabetes

Pyruvate dehydrogenase kinases (PDK1-4) play a critical role in the inhibition of the mitochondrial pyruvate dehydrogenase complex especially when blood glucose levels are low and pyruvate can be conserved for gluconeogenesis. Under diabetic conditions, the Pdk genes, particularly Pdk4, are often induced, and the elevation of the Pdk4 gene expression has been implicated in the increased gluconeogenesis in the liver and the decreased glucose utilization in the peripheral tissues. However, there is no direct evidence yet to show to what extent that the dysregulation of hepatic Pdk genes attributes to hyperglycemia and insulin resistance in vivo. To address this question, we crossed Pdk2 or Pdk4 null mice with a diabetic model that is deficient in hepatic insulin receptor substrates 1 and 2 (Irs1/2). Metabolic analyses reveal that deletion of the Pdk4 gene had better improvement in hyperglycemia and glucose tolerance than knockout of the Pdk2 gene whereas the Pdk2 gene deletion showed better insulin tolerance as compared to the Pdk4 gene inactivation on the Irs1/2 knockout genetic background. To examine the specific hepatic effects of Pdks on diabetes, we also knocked down the Pdk2 or Pdk4 gene using specific shRNAs. The data also indicate that the Pdk4 gene knockdown led to better glucose tolerance than the Pdk2 gene knockdown. In conclusion, our data suggest that hepatic Pdk4 may be critically involved in the pathogenesis of diabetes

Hepatic Pdk4 knockdown moderately improves glucose tolerance in IrsLDKO mice.

<p>A, Gene knockdown efficiency was analyzed by real-time PCR in IrsLDKO livers transduced with shRNA adenoviruses against GFP (shGFP), Pdk2 (shPdk2), or Pdk4 (shPdk4). B, Glucose tolerance tests were performed in shRNA adenoviruses infected IrsLDKO mice. C, Area under curve analysis (AUC) was performed for the above glucose tolerance test data. Data are presented as means ± SEM, n = 4–5. *, <i>P</i><0.05 relative to corresponding controls.</p

Knockout of the <i>Pdk</i> genes in wild-type and IrsLDKO mice.

<p>A, Control wild-type and IrsLDKO mice (n = 3) were fasted overnight for 16 hours and half of them were fed for 4 hours immediately after the fasting. <i>Pdks</i> gene expression in the liver was analyzed by real-time PCR and data were normalized to an internal control gene — Ppia. B, Western blot analysis of liver lysates from control and knockout mice. C, Body weight measurements in control and knockout mice (n = 6–20). D, Serum triglycerides (TG) were measured in overnight fasted control and knockout mice (n = 6–8). E, Liver TG analysis in control and knockout mice (n = 6–8). Pdk2KO, Pdk2 knockout; Pdk4KO, Pdk4 knockout; IrsLDKO, Irs1/2 liver-specific double knockout. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Pdk2 or Pdk4 knockdown has no significant effect on insulin tolerance in IrsLDKO mice.

<p>A, Insulin tolerance tests were performed on IrsLDKO mice (n = 4–5) that were injected with shGFP, shPdk2, and shPdk4 adenoviruses. B, Area under curve was analyzed for the above ITT data. C–F, Akt phosphorylation was analyzed in the liver and skeletal muscle of mice injected with shGFP, shPdk2, and shPdk4 adenoviruses. Western blot signals were also quantified using the Quantity One software. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Inactivation of Pdks improves insulin sensitivity in IrsLDKO mice.

<p>A, Insulin tolerance tests (ITT) were performed in age-matched control and knockout mice (n = 8–20). B, Fasting plasma insulin was analyzed in age-matched control and knockout mice (n = 5–9). C, HOMA-IR (homeostatic model assessment-insulin resistance) was analyzed using fasting glucose and insulin data. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Ablation of Pdks improves glucose tolerance in IrsLDKO mice.

<p>A, Glucose tolerance tests (GTT) were performed in age-matched control and knockout mice (n = 8–12). B and C, Expression of gluconeogenic genes <i>Pck1</i> and <i>G6pc</i> was analyzed in the liver of overnight fasted control and knockout mice (n = 3). Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Deletion of the <i>Pdk4</i> gene improves hyperglycemia in IrsLDKO mice.

<p>A, Blood glucose was measured in overnight fasted control and knockout mice. B, Blood glucose was measured in <i>ad libitum</i> fed control and knockout mice. Data are presented as means ± SEM, n = 8–23. *, <i>P</i><0.05 relative to corresponding controls.</p