Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver

Abstract

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEPCK protein content in the control of gluconeogenesis was unclear. In this study, metabolic control of liver gluconeogenesis was quantified in groups of mice with varying PEPCK protein content. Surprisingly, livers with a 90% reduction in PEPCK content showed only a approximately 40% reduction in gluconeogenic flux, indicating a lower than expected capacity for PEPCK protein content to control gluconeogenesis. However, PEPCK flux correlated tightly with TCA cycle activity, suggesting that under some conditions in mice, PEPCK expression must coordinate with hepatic energy metabolism to control gluconeogenesis.

Figure 1. Effect of PEPCK Protein Content on ²H and ¹³C Tracer Incorporation into Glucose

(A) Western blots for PEPCK and tubulin protein content. Relative PEPCK content was determined from the PEPCK/tubulin ratio normalized to 100% for the control strain (pck^lox/lox). Mean ± SEM is reported. (B) The carbons used for gluconeogenesis originate in the TCA cycle. Glucose cannot become ²H enriched at position H6s or labeled with ¹³C in any position without PEPCK activity. NMR can be used to measure these enrichments and combined with total glucose production to determine flux through the TCA cycle and gluconeogenesis. (C) Decreasing the PEPCK protein content in mice causes only small changes in the ²H enrichment of H6s (left box) or the C2 ¹³C multiplets (right box). This indicates significant rates of gluconeogenesis despite low levels of PEPCK protein content. Only glucose from the PEPCK null livers had dramatically reduced ²H or ¹³C enrichment in these positions, indicating that those livers produced glucose from glycerol rather than the TCA cycle (Burgess et al., 2004). ¹³C multiplets are labeled Q (quartet), D12 (doublet 12), D34 (doublet 34), and S (singlet). The areas of these multiplets represent the isotopomer distribution of ¹³C in glucose and are used to determine fluxes associated with the TCA cycle (Jones et al., 1997).

Figure 2. Influence of PEPCK Protein Content and TCA Cycle Flux on PEPCK Flux in Isolated Livers

(A) PEPCK flux versus liver PEPCK protein content. The data indicate that large changes in PEPCK content are required to modulate even small changes in flux through PEPCK. Metabolic control analysis of these data provided a control coefficient of 0.18, indicating that under these conditions, PEPCK content is not rate limiting for gluconeogenesis. (B) Plot of PEPCK flux versus TCA cycle flux (relative to the average of pck^lox/lox) for all 27 livers studied. There was a strong correlation (p < 0.001) between TCA cycle flux and PEPCK flux. These data directly link energy produced in the hepatic TCA cycle to the energy-consuming process of gluconeogenesis. (C) Under the conditions studied here, hepatic PEPCK protein content has only a weak influence on the rate of PEPCK flux and gluconeogenesis, while the rate of energy produced by the hepatic TCA cycle is strongly correlated with the rate of hepatic PEPCK flux (and gluconeogenesis), providing a potential mechanism by which gluconeogenesis and the energy required for this process are coordinated.