The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells

Abstract

Aims/hypothesis: Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure.

Methods: To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells.

Results: Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP(+) ratio and activity of the pyruvate-malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion.

Conclusions/interpretation: Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Figure 1

PC siRNA decreases PC protein content, insulin secretion, cell proliferation and protein content in 48 h treated INS-1 cells. A, PC activity and PC protein content. The insert picture shows a representative Western blot (total 3) of PC protein (~130kDa) and actin in control and PC siRNA treated cells. The numbers under the protein bands indicate the data of protein band quantification (n=3). ROD means relative optical density after normalization to actin. B, glucose (2.8, 5.5 and 16.7 mmol/l) stimulated insulin secretion. C, KCl (50 mmol/l) stimulated insulin secretion. D, [³H]Thymidine incorporation. E, total protein content per well. Control means scrambled siRNA treatment. Data are mean ± S.E.M., n=4. * P<0.05, ** P<0.01 vs. controls.

Figure 2

Inhibition of INS-1 cell PC with siRNA treatment (48 h) does not change glucose utilization (A) and glucose oxidation (B), but does up-regulate active PDH activity (C) in INS-1 cells. Control means scrambled siRNA. Data are the mean ± S.E.M. n=4.

Figure 3

PC siRNA treatment (48 h) in dispersed primary islet cells reduces PC activity (A) and leads to a reduction in glucose (16.7 mmol/l) stimulated insulin secretion (B) and malate release from isolated mitochondria (C). Control means scrambled siRNA. Data are the mean ± S.E.M. n=4.

Figure 4

Stable overexpression of PC in INS-1 cells up-regulates insulin secretion and cell proliferation. A, PC activity and protein content. Western blot of PC protein (~130kDa) and actin shown on the top is a representative experiment (total 3). The numbers under the protein bands indicate the data of protein band quantification (n=3). ROD means relative optical density after normalization to actin. B, glucose (2.8 and 16.7 mmol/l) stimulated insulin secretion; C, [³H]Thymidine incorporation; and D, protein content per well. PC OVE indicates PC over-expressing cells. Control cells were treated with the vector that does not contain PC gene. Data are mean ± S.E.M, n=4. * P<0.05 vs. controls.

Figure 5

Reduction of active PDH (aPDH) activity by overexpression (over-exp) of PDK4 (A) does not significantly change PC activity (B) or glucose (2.8 and 16.7 mmol/l) stimulated insulin secretion (C) in INS-1 cells. Control cells were treated with the vector that does not contain PDK4 gene. Data are mean ± S.E.M. n=4.