A role for mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) in the regulation of hepatic gluconeogenesis

Abstract

Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.

Keywords: Diabetes; GTPase; Gluconeogenesis; Glyceroneogenesis; Intermediary Metabolism; Metabolic Regulation; Metabolic Tracers; Metabolism; Mitochondrial Metabolism; Phosphoenolpyruvate Carboxykinase.

FIGURE 1.

Oxygen consumption and gluconeogenesis from hepatocytes. A, PEPCK-M mRNA levels from hepatocytes isolated from Con^ASO (white bars) and PCK2^ASO (black bars). B and C, PEPCK-M and PEPCK-C protein expression quantified by Western blotting and normalized to actin. Knockdown levels in hepatocytes are indicative of PEPCK-M levels at the time of the in vivo and in vitro studies. D, oxygen consumption rates in hepatocytes incubated in the presence of either 10 mm lactate or pyruvate alone or uncoupled by 100 μm dinitrophenol (DNP) (n = 5/5) in isolated primary hepatocytes Con^ASO (white bars) and PCK2^ASO (black bars) rats (n = 6/6). E, glucose production rates from hepatocytes treated with the indicated concentration of substrate. The percentage of decrease from Con^ASO is indicated in parentheses for each substrate (n = 6/6). F and G, PEPCK-M and PEPCK-C mRNA levels from isolated hepatocytes acutely transfected (n = 6/6) with control (white bars) or two different siRNAs (PCK2-A and PCK2-B, black bars). H and I, glucose production from hepatocytes incubated with 9 mm lactate and 1 mm pyruvate (H) or 10 mm glutamine or alanine with siRNA PCK-2A (I). Data are presented as mean ± S.E. t test: *, p < 0.05; ***, p < 0.001.

FIGURE 2.

Fed and fasted glucose homeostasis. Rodents were allowed to eat ad lib overnight, and then food was withdrawn at 6 a.m. (Fed) before rodents underwent a 36-h fast (Fasted). A, PEPCK-M mRNA in indicated tissue Con^ASO (white bars) and PCK2^ASO (black bars) rats determined by quantitative PCR. Results are normalized to the control liver mRNA to demonstrate relative abundance of message, and significance is between CON^ASO and PCK2^ASO. WAT, white adipose tissue; BAT, brown adipose tissue. B and C, initial weights (B) and weight loss during and white adipose mass after the 36-h fast (C). E–H, glucose, insulin, glucagon, and corticosterone were measured. Data are shown as mean ± S.E. for n = 14/12 rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

FIGURE 3.

Basal turnover studies from overnight fasted rats. A and B, CON^ASO (white bars) and PCK2^ASO (black bars) rats were given tracer infusions of labeled (A) lactate and (B) glycerol (n = 7/8) for basal turnover calculations. C, the individual differences between glycerol and lactate R_a are indicative of a substantial switch in gluconeogenic precursor preference. Data are presented as mean ± S.E., **, p < 0.01; ***, p < 0.001.