The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: Has it been overlooked?

Background

Plasma glucose levels are tightly regulated within a narrow physiologic range. Insulin-mediated glucose uptake by tissues must be balanced by the appearance of glucose from nutritional sources, glycogen stores, or gluconeogenesis. In this regard, a common pathway regulating both glucose clearance and appearance has not been described. The metabolism of glucose to produce ATP is generally considered to be the primary stimulus for insulin release from beta-cells. Similarly, gluconeogenesis from phosphoenolpyruvate (PEP) is believed to be the primarily pathway via the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). These models cannot adequately explain the regulation of insulin secretion or gluconeogenesis.

Scope of review

A metabolic sensing pathway involving mitochondrial GTP (mtGTP) and PEP synthesis by the mitochondrial isoform of PEPCK (PEPCK-M) is associated with glucose-stimulated insulin secretion from pancreatic beta-cells. Here we examine whether there is evidence for a similar mtGTP-dependent pathway involved in gluconeogenesis. In both islets and the liver, mtGTP is produced at the substrate level by the enzyme succinyl CoA synthetase (SCS-GTP) with a rate proportional to the TCA cycle. In the beta-cell PEPCK-M then hydrolyzes mtGTP in the production of PEP that, unlike mtGTP, can escape the mitochondria to generate a signal for insulin release. Similarly, PEPCK-M and mtGTP might also provide a significant source of PEP in gluconeogenic tissues for the production of glucose. This review will focus on the possibility that PEPCK-M, as a sensor for TCA cycle flux, is a key mechanism to regulate both insulin secretion and gluconeogenesis suggesting conservation of this biochemical mechanism in regulating multiple aspects of glucose homeostasis. Moreover, we propose that this mechanism may be important for regulating insulin secretion and gluconeogenesis compared to canonical nutrient sensing pathways.

Major conclusions

PEPCK-M, initially believed to be absent in islets, carries a substantial metabolic flux in beta-cells. This flux is intimately involved with the coupling of glucose-stimulated insulin secretion. PEPCK-M activity may have been similarly underestimated in glucose producing tissues and could potentially be an unappreciated but important source of gluconeogenesis.

General significance

The generation of PEP via PEPCK-M may occur via a metabolic sensing pathway important for regulating both insulin secretion and gluconeogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Endotoxin induced hyperlactatemia and hypoglycemia is linked to decreased mitochondrial phosphoenolpyruvate carboxykinase

AimsPhosphoenolpyruvate carboxykinase (PEPCK) is the rate limiting enzyme for gluconeogenesis, and plays a key role in recycling lactate for glucose production. It is synthesized as two separate isoforms; cytosolic (PEPCK-C, gene code; PCK1) and mitochondrial (PEPCK-M, gene code; PCK2). Previous studies of gluconeogenesis in endotoxemia have focused solely on PCK1. We investigated the relative roles of the two isoforms in hepatic and renal gluconeogenesis in a rat model of endotoxic shock, and in cultured hepatocytes.Main methodsRats were administered lipopolysaccharide (6 mg/kg; LPS) for 6 h. Cultured cells were incubated with lactate (5 mM) with or without tumor necrosis factor alpha (1 – 10 ng/ml). Rat liver and kidney samples as well as cultured cells were subjected to subcellular fractionation to produce mitochondrial and cytosolic fractions for PEPCK activity assay. PCK1 and PCK2 mRNA levels were measured using quantitative RT-PCR.Key findingsIn rat endotoxemia, hepatic PCK2 mRNA and PEPCK-M enzyme activity decreased by 53% and 38%, compared to sham controls. Hepatic PCK1 mRNA levels increased by 44%, but PEPCK-C enzyme activity remained unchanged. The changes in hepatic PEPCK-M coincided with a marked hypoglycemia and hyperlactatemia as well as elevated plasma interleukin 1 beta (IL1beta). Incubation of cultured hepatocytes with TNF-alpha inhibited lactate-induced increases in glucose production, PCK2 mRNA levels and PEPCK-M enzyme activity but had no effect on PCK1 mRNA levels or PEPCK-C activity.SignificanceThese results indicate that decreases in hepatic PEPCK-M play a key role in the manifestation of hyperlactatemia and hypoglycemia in endotoxemia.     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Carbohydrate metabolism of the perfused rat liver

1. The rates of gluconeogenesis from most substrates tested in the perfused livers of well-fed rats were about half of those obtained in the livers of starved rats. There was no difference for glycerol. 2. A diet low in carbohydrate increased the rates of gluconeogenesis from some substrates but not from all. In general the effects of a low-carbohydrate diet on rat liver are less marked than those on rat kidney cortex. 3. Glycogen was deposited in the livers of starved rats when the perfusion medium contained about 10mm-glucose. The shedding of glucose from the glycogen stores by the well-fed liver was greatly diminished by 10mm-glucose and stopped by 13.3mm-glucose. Livers of well-fed rats that were depleted of their glycogen stores by treatment with phlorrhizin and glucagon synthesized glycogen from glucose. 4. When two gluconeogenic substrates were added to the perfusion medium additive effects occurred only when glycerol was one of the substrates. Lactate and glycerol gave more than additive effects owing to an increased rate of glucose formation from glycerol. 5. Pyruvate also accelerated the conversion of glycerol into glucose, and the accelerating effect of lactate can be attributed to a rapid formation of pyruvate from lactate. 6. Butyrate and oleate at 2mm, which alone are not gluconeogenic, increased the rate of gluconeogenesis from lactate. 7. The acceleration of gluconeogenesis from lactate by glucagon was also found when gluconeogenesis from lactate was stimulated by butyrate and oleate. This finding is not compatible with the view that the primary action of glucagon in promoting gluconeogenesis is an acceleration of lipolysis. 8. The rate of gluconeogenesis from pyruvate at 10mm was only 70% of that at 5mm. This ;inhibition' was abolished by oleate or glucagon.     

C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1-10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.     

Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes

The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca(2+) being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca(2+) so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.     

Gluconeogenesis in chick embryo isolated hepatocytes

1. The effectiveness of gluconeogenic precursors in hepatocytes isolated from 18 day old chick embryos is:Lactate much much greater than pyruvate greater than alanine = glutamine greater than glycerol and other amino acids. This result is qualitatively and quantitatively similar to hepatocytes isolated after hatching. 2. In the presence of endogenous glycogenolysis, conversion of [U-14C]lactate to glucose was used to estimate gluconeogenic flux and its control by hormones. 3. Glucagon failed to stimulate lactate gluconeogenesis although simultaneously increasing glycogenolysis. Insulin had no effects on gluconeogenesis.     

Effects of epinephrine on the net hepatic uptake of lactate, pyruvate, and glycerol in sheep

Epinephrine caused hyperglycemia in part by increasing gluconeogenesis. However, the mechanism of its gluconeogenic effects has not been studied in ruminants. This study was undertaken to examine the effect of epinephrine on the net hepatic uptake of selected glucose precursors in sheep. The major abdominal blood vessels of the sheep were catheterized in normal and alloxan diabetic sheep. Glucose production, metabolic clearance of glucose, and the hepatic removal of certain glucose precursors were determined before, during, and after epinephrine infusion. Epinephrine increased the hepatic glucose output, the concentrations of lactate and glycerol in plasma, and the net hepatic uptake and fractional hepatic extraction of lactate and glycerol. These effects were independent of changes in the concentrations of insulin and glucagon in plasma. These results show that epinephrine directly stimulates hepatic gluconeogenesis in sheep.     

Evidence that L‐glutamine is better than L‐alanine as gluconeogenic substrate in perfused liver of weaned fasted rats submitted to short‐term insulin‐induced hypoglycaemia

Gluconeogenesis in livers from overnight fasted weaned rats submitted to short‐term insulin‐induced hypoglycaemia (IIH) was investigated. For this purpose, a condition of hyperinsulinemia/hypoglycaemia was obtained with an intraperitoneal (ip) injection of regular insulin (1.0 U kg^?1). Control group (COG group) received ip saline. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. The livers from IIH and COG rats were perfused with L‐alanine (5 mM), L‐lactate (2 mM), L‐glutamine (10 mM) or glycerol (2 mM). Hepatic glucose, L‐lactate and pyruvate production from L‐alanine was not affected by IIH. In agreement with this result, the hepatic ability in producing glucose from L‐lactate or glycerol remained unchanged (IIH group vs. COG group). However, livers from IIH rats showed higher glucose production from L‐glutamine than livers from COG rats and, in the IIH rats, the production of glucose from L‐glutamine was higher than that from L‐alanine. The higher glucose production in livers from the IIH group, when compared with the COG group was due to its entrance further on gluconeogenic pathway. Taken together, the results suggest that L‐glutamine is better than L‐alanine, as gluconeogenic substrate in livers of hypoglyceaemic weaned rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone

Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-¹³C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-¹³C]glucose, [2-¹³C]glycerol 3-phosphate, [2-¹³C]phosphoenolpyruvate (PEP), [2-¹³C]pyruvate, [2-¹³C]alanine, and [2-¹³C]lactate. [2-¹³C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-¹³C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Ca2(+)-fatty acid interaction in the control of hepatic gluconeogenesis

Calcium depletion induced by perfusing livers with calcium-free buffer did not alter the rates of basal glucose production from pyruvate or from increasing concentrations of lactate. However, calcium deficiency selectively prevented the fatty acid-induced stimulation of gluconeogenesis from lactate. This effect is not related to the higher NAD redox potential consistently observed in Ca2(+)-deficient livers. On the other hand, octanoate was capable of inducing dose-dependent changes in the [pyruvate]0.5 in calcium-depleted livers perfused with lactate, ruling out that low cellular calcium content could perturb the mitochondrial transport of pyruvate. The observation that the effect of calcium deficiency can be overcome by supraphysiological concentrations of pyruvate supports the proposal that stimulation of the maximal capacity of the gluconeogenic pathway by fatty acid relies largely on the tricarboxylic acid cycle activity, restricted in calcium deficiency conditions.     

Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the β-cells of the islets. An erratum to this article can be found at     

Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes

Dietary iron deficiency in rats results in increased blood glucose turnover and recycling. We measured the rates of glucose production in isolated hepatocytes from iron-sufficient (Fe+) and iron-deficient (Fe-) rats to assess the intrinsic capacity of the Fe- liver to carry out gluconeogenesis. Low-iron and control diets were given to 21-day-old female rats. After 4-5 wk, hemoglobin concentrations averaged 4.1 g/dl in the Fe- and 14.3 g/dl in the Fe+ animals. In the hepatocytes from Fe- rats, there was a 35% decrease in the rate of glucose production from 1 mM pyruvate + 10 mM lactate, a 48% decrease from 0.1 mM pyruvate + 1 mM lactate, a 39% decrease from 1 mM alanine, and a 48% decrease from 1 mM glycerol. The addition of 5 microM norepinephrine or 0.5 microM glucagon to the incubation media produced stimulatory effects on hepatocytes from both Fe- and Fe+ rats, resulting in the maintenance of an average difference of 38% in the rates of gluconeogenesis between the two groups. Studies on isolated liver mitochondria and cytosol revealed alpha-glycerophosphate-cytochrome c reductase and phospho(enol)pyruvate carboxykinase activities to be decreased by 27% in Fe- rats. We conclude that because severe dietary iron deficiency decreases gluconeogenesis in isolated rat hepatocytes, the increased gluconeogenesis demonstrated by Fe- rats in vivo is attributable to increased availability of gluconeogenic substrates and upregulation of the pathway.     

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.