Early experiments on isolated hepatocytes treated with pharmacological inhibitors of PEPCK activity yielded conflicting results concerning the role of PEPCK in control of gluconeogenesis. Rognstad (1979)
concluded that PEPCK is the rate-controlling enzyme for gluconeogenesis based upon inhibition of hepatocyte glucose production from lactate in the presence of the PEPCK inhibitor mercaptopicolinate. This conclusion was later disputed by Groen et al. (1982)
and others, who used both inhibitor methods and elasticities to show that PEPCK was only weakly controlling in isolated hepatocytes (Argaud et al., 1991
; Groen et al., 1986
; Rigoulet et al., 1987
). These groups concluded that, depending on hormone exposure, pyruvate carboxylase and/or pyruvate kinase were more important enzymes in controlling the rate of gluconeogenesis (Groen et al., 1986
). More recently, similar results were obtained by a combination of GC/MS-based metabolomic and mass isotopomer analysis in perfused rat liver treated with mercaptopicolinate (Yang et al., 2006
When PEPCK flux was measured as a function of PEPCK protein content in the intact isolated mouse liver, the results suggested only a weak relationship between PEPCK protein content and flux through the enzyme. One important consideration is whether gradations in PEPCK protein levels in the present study are representative of typical physiological levels in response to hormonemediated PEPCK expression. Presumably, PEPCK protein content in 24 hr-fasted control mice represents the highest normal hepatic PEPCK content. The 90% reduction of PEPCK found in the pcklox + neo/del
mouse is sub-stantially more dramatic than the 50% reduction found after hyperinsulinemic clamp in mice (Sun et al., 2002
), yet hyperinsulinemic clamp dramatically suppresses gluconeogenesis, while PEPCK deficiency alone only decreases it by 40%. This suggests that other factors such as peripheral substrate supply or insulin-mediated effects on other gluconeogenic enzymes and/or hepatic energy metabolism must coordinate with PEPCK expression to attenuate gluconeogenesis.
It remains unclear whether compensatory mechanisms in hepatic metabolism due to chronic PEPCK deficiency play a role in the apparent low control strength of PEPCK over hepatic gluconeogenesis or whether PEPCK might even have a different control strength in vivo. These questions linger mainly because the low control strength for PEPCK over its own flux is difficult to rationalize in light of the exquisite hormonal control of PEPCK expression and the fact that several interventions of PEPCK expression have demonstrated remarkable effects on systemic glucose metabolism in mice. A 7-fold overexpression of PEPCK results in hyperglycemia (Valera et al., 1994
), while a 2-fold overexpression results in insulin resistance (Sun et al., 2002
). The complexity of transcriptional regulation of gluconeogenesis was nicely illustrated in the latter study, where a point mutation in PEPCK expression also resulted in overexpression of glucose-6-phosphatase and underexpression of glucokinase and GLUT2, all conditions that naturally contribute to increased gluconeogenic flux. The opposite was illustrated by Perales and coworkers (Gomez-Valades et al., 2006
), who used PEPCK RNA silencing to suppress PEPCK expression in diabetic mice, resulting in correction of hyperglycemia. Interestingly, they also noted that inhibiting PEPCK expression in diabetic mice decreased circulating fatty acids and increased circulating ketones, suggesting an interaction between PEPCK expression and hepatic energy metabolism. On the other hand, a variety of studies in mice demonstrate important modulations in hepatic glucose metabolism even in the absence of altered PEPCK expression (Burgess et al., 2006
; Kersten et al., 1999
) or altered PEPCK expression without significant effect on gluconeogenesis (Xu et al., 2006
). This disconnection might occur because other factors, such as hepatic energy metabolism (Pryor et al., 1987
), can also project substantial control over the rate of gluconeogenesis. The present data indicate that changes in PEPCK content alone may be insufficient to modulate gluconeogenesis; thus, taken in combination with prior knowledge regarding the exquisite regulation of this enzyme, it seems likely that PEPCK expression must coordinate with other mechanisms to regulate gluconeogenesis.
The finding that PEPCK flux is intimately linked to hepatic energy metabolism as well as gluconeogenesis fits with earlier observations that flux through PEPCK is necessary for normal energy generation in the hepatic TCA cycle (Burgess et al., 2004
; Hakimi et al., 2005
). In the complete absence of hepatic PEPCK, livers accumulate triglycerides (She et al., 2000
) secondary to decreased fat oxidation in the TCA cycle (Burgess et al., 2004
; Hakimi et al., 2005
). Moreover, when hepatic TCA cycle activity is limited by decreased TCA cycle and electron transport chain enzyme expression, PEPCK flux and gluconeogenesis are also impaired (Burgess et al., 2006
). Together, these data suggest a bidirectional feedback between cataplerosis and energy generated in the hepatic TCA cycle (), a mechanism which may allow appropriate coordination of energy production and anabolism, specifically gluconeogenesis. This coordination may be facilitated by PC flux since this enzyme is under allosteric control by acetyl-CoA, whose levels are responsive to the energy state of the hepatocyte via β-oxidation and oxidation in the TCA cycle. The ostensibly separate pathways of hepatic energy production and gluconeogenesis are in fact linked transcriptionally by a number of coactivators, such as PGC-1 (Lin et al., 2005
), TORC (Koo et al., 2005
), and the forkhead family of transcription factors (Wolfrum et al., 2004
; Zhang et al., 2006
), that act as “master regulators” controlling the expression of multiple enzymes of gluconeogenesis and fatty acid oxidation in parallel. In this way, the liver is well equipped to coordinate the energy-consuming pathway of gluconeogenesis with the energy-producing pathway of fat oxidation. Results presented here suggest that one mechanism by which the metabolic fluxes of gluconeogenesis and fat oxidation interact involves oxidative flux through the TCA cycle, leading to stimulation of anaplerosis, cataplerotic flux through PEPCK, and ultimately gluconeogenesis ().
In summary, contrary to the widely held view that PEPCK is the primary control point for gluconeogenesis, the present results show that hepatic PEPCK content alone only weakly influences gluconeogenesis. In light of the robust response of PEPCK expression to physiology, these data suggest a broader role for PEPCK, perhaps in integrating hepatic energy metabolism and gluconeogenesis. In agreement with this role, we observed that PEPCK flux correlates strongly with energy generated in the hepatic TCA cycle. We hypothesize that in mice, hormone-regulated PEPCK expression is generally controlled in parallel with hepatic energy production, and these two factors cooperate (perhaps with other factors as well) to determine the rate of gluconeogenesis. Finally, these findings demonstrate that metabolic flux is subject to regulation by the entire metabolic network and that changes in the expression, content, or activity of individual enzymes do not always predictably affect flux through the intact pathway.