Oncogenic Ras expression increases cellular formate production.

One-carbon units, critical intermediates for cell growth, may be produced by a variety of means, one of which is via the production of formate. Excessive formate accumulation, known as formate overflow and a characteristic of oxidative cancer, has been observed in cancer cells. However, the basis for this high rate of formate production is unknown. We examined the effect of elevated expression of oncogenic Ras (RasV12), on formate production in NIH-3T3 cells (mouse fibroblasts) cultured with either labelled 13C-serine or 13C-glycine. Formate accumulation by the fibroblasts transformed by RasV12 was increased two-threefold over those by vector control (Babe) cells. The production of formate exceeded the rate of utilization in both cell types. 13C-formate was produced almost exclusively from the #3 carbon of 13C-serine. Virtually no labelled formate was produced from either the #2 carbon of serine or the #2 carbon of glycine. The increased formate production by RasV12 cells was associated with increased mRNA abundances for enzymes of formate production in both the mitochondria and the cytosol. Thus, we find the oncogenic RasV12 significantly increases formate overflow and may be one way for tumor cells to produce one-carbon units required for enhanced proliferation of these cells and/or for other processes which have not been identified.

Homocysteine metabolism in diabetes.

An increase in the plasma level of Hcy (homocysteine), an intermediate in the catabolism of methionine, has been identified as a risk factor for many diseases including CVD (cardiovascular disease). CVD is the major cause of death in patients with diabetes mellitus. Therefore the study of Hcy metabolism in diabetes mellitus has been a major focus of current research. Studies conducted in our laboratory were able to show that in both Type 1 and Type 2 diabetes with no renal complications, the plasma Hcy levels were lower than in controls. In Type 1 diabetes, increased activities of the trans-sulfuration enzymes were the major cause for the reduction in plasma Hcy. In Type 2 diabetes, BHMT (betaine:homocysteine methyltransferase) was also observed to play a major role in the increased catabolism of Hcy in addition to the trans-sulfuration enzymes. We were also able to demonstrate the direct effect of insulin and the counter-regulatory hormones on the regulation of cystathionine beta-synthase and BHMT, which accounts for the changes in the activities of these two enzymes seen in diabetes mellitus.

Threonine metabolism in isolated rat hepatocytes.

The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO(2). Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in alpha-ketobutyrate, which is oxidized in the mitochondria to CO(2). Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mM L-[1-(14)C]threonine, and (14)CO(2) production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, alpha-cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial alpha-keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that approximately 65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10(-7) M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.

Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate.

S-adenosylmethionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates.

Hyperglucagonemia in rats results in decreased plasma homocysteine and increased flux through the transsulfuration pathway in liver.

An elevated plasma level of homocysteine is a risk factor for the development of cardiovascular disease. The purpose of this study was to investigate the effect of glucagon on homocysteine metabolism in the rat. Male Sprague-Dawley rats were treated with 4 mg/kg/day (3 injections per day) glucagon for 2 days while control rats received vehicle injections. Glucagon treatment resulted in a 30% decrease in total plasma homocysteine and increased hepatic activities of glycine N-methyltransferase, cystathionine beta-synthase, and cystathionine gamma-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine beta-synthase was accompanied by a 2-fold increase in its mRNA level. Hepatocytes prepared from glucagon-injected rats exported less homocysteine, when incubated with methionine, than did hepatocytes of saline-treated rats. Flux through cystathionine beta-synthase was increased 5-fold in hepatocytes isolated from glucagon-treated rats as determined by production of (14)CO(2) and alpha-[1-(14)C]ketobutyrate from l-[1-(14)C]methionine. Methionine transport was elevated 2-fold in hepatocytes isolated from glucagon-treated rats resulting in increased hepatic methionine levels. Hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine, allosteric activators of cystathionine beta-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway.

Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.

Amino acids, then and now--a reflection on Sir Hans Krebs' contribution to nitrogen metabolism.

H. A. Krebs made an enormous contribution to our knowledge of amino acid metabolism, beginning with his studies on proteolysis in the early 1930s, progressing through his work on urea synthesis to an extensive series of papers on deamination and, then, to work on gluconeogenesis from amino acids. This paper addresses three of Krebs' early contributions-urea synthesis, glutamine metabolism, and D-amino acid oxidase-and relates them to our modern understanding of amino acid metabolism.

Characterization of homocysteine metabolism in the rat liver.

Recent evidence suggests that an increased plasma concentration of the sulphur amino acid homocysteine is a risk factor for the development of vascular disease. The tissue(s) responsible for homocysteine production and export to the plasma are not well known. However, given the central role of the liver in amino acid metabolism, we developed a rat primary hepatocyte model in which homocysteine (and cysteine) production and export were examined. The dependence of homocysteine export from incubated hepatocytes on methionine concentration fitted well to a rectangular hyperbola, with half-maximal homocysteine export achieved at methionine concentrations of approx. 0.44 mM. Hepatocytes incubated with 1 mM methionine and 1 mM serine (a substrate for the transulphuration pathway of homocysteine removal) produced and exported significantly less homocysteine (25-40%) compared with cells incubated with 1 mM methionine alone. The effects of dietary protein on homocysteine metabolism were also examined. Rats fed a 60% protein diet had a significantly increased total plasma homocysteine level compared with rats fed a 20% protein diet. In vitro effects of dietary protein were examined using hepatocytes isolated from animals maintained on these diets. When incubated with 1 mM methionine, hepatocytes from rats fed the high protein diet exported significantly more homocysteine compared with hepatocytes from rats fed the normal protein diet. Inclusion of serine significantly lowered homocysteine export in the normal protein group, but the effect was more marked in the high protein group. In vivo effects of serine were also examined. Rats fed a high protein diet enriched with serine had significantly lower total plasma homocysteine (25-30%) compared with controls. These data indicate a significant role for the liver in the regulation of plasma homocysteine levels.

Plasma homocysteine is decreased in the hypothyroid rat.

Recent clinical studies have indicated that plasma homocysteine was significantly increased in hypothyroid patients. Since hyperhomocysteinemia is an independent risk factor for cardiovascular disease we investigated homocysteine metabolism in hypothyroid rats. Hypothyroidism was induced in one study by addition of propylthiouracil (PTU) to the drinking water for 2 weeks. In a second study, thyroidectomized and sham-operated rats were used with thyroid hormone replacement via mini-osmotic pumps. Unlike the human hypothyroid patients, both groups of hypothyroid rats exhibited decreased total plasma homocysteine (30% in PTU rats, 50% in thyroidectomized rats) versus their respective controls. Thyroid replacement normalised homocysteine levels in the thyroidectomized rat. Increased activities of the hepatic trans-sulfuration enzymes were found in both models of hypothyroidism. These results provide a possible explanation for the decreased plasma homocysteine concentrations. The hypothyroid rat cannot be used as a model to study homocysteine metabolism in hypothyroid patients.

Folate and vitamin B12 status of women in Newfoundland at their first prenatal visit.

BACKGROUND: Newfoundland has one of the highest rates of neural tube defects in North America. Given the association between low maternal folic acid levels and neural tube defects, a cross-sectional study was conducted to obtain base-line data on the folate and vitamin B12 status of a sample of women in Newfoundland who were pregnant. METHODS: Blood samples were collected between August 1996 and July 1997 from 1424 pregnant women in Newfoundland during the first prenatal visit (at approximately 16 weeks' gestation); this represented approximately 25% of the women in Newfoundland who were pregnant during this period. The samples were analysed for serum folate, vitamin B12, red blood cell folate and homocysteine. RESULTS: Median values for serum folate, red blood cell folate and serum vitamin B12 were 25 nmol/L, 650 nmol/L and 180 pmol/L, respectively. On the basis of the interpretive criteria used for red blood cell folate status, 157 (11.0%) of the 1424 women were deficient (< 340 nmol/L) and a further 180 (12.6%) were classified as indeterminate (340-420 nmol/L). Serum homocysteine levels, measured in subsets of the red blood cell folate status groups, supported the inadequate folate status. Serum vitamin B12 levels of 621 (43.6%) women were classified as deficient or marginal; however, the validity of the interpretive criteria for pregnant women is questionable. INTERPRETATION: A large proportion of pregnant women surveyed in Newfoundland in 1997 had low red blood cell folate levels.

Glutamate, at the interface between amino acid and carbohydrate metabolism.

The liver is the major site of gluconeogenesis, the major organ of amino acid catabolism and the only organ with a complete urea cycle. These metabolic capabilities are related, and these relationships are best exemplified by an examination of the disposal of the daily protein load. Adults, ingesting a typical Western diet, will consume approximately 100 g protein/d; the great bulk of this is metabolized by the liver. Although textbooks suggest that these amino acids are oxidized in the liver, total oxidation cannot occur within the confines of hepatic oxygen uptake and ATP homeostasis. Rather, most amino acids are oxidized only partially in the liver, with the bulk of their carbon skeleton being converted to glucose. The nitrogen is converted to urea and, to a lesser extent, to glutamine. The integration of the urea cycle with gluconeogenesis ensures that the bulk of the reducing power (NADH) required in the cytosol for gluconeogenesis can be provided by ancillary reactions of the urea cycle. Glutamate is at the center of these metabolic events for three reasons. First, through the well-described transdeamination system involving aminotransferases and glutamate dehydrogenase, glutamate plays a key catalytic role in the removal of alpha-amino nitrogen from amino acids. Second, the "glutamate family" of amino acids (arginine, ornithine, proline, histidine and glutamine) require the conversion of these amino acids to glutamate for their metabolic disposal. Third, glutamate serves as substrate for the synthesis of N-acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, a key regulatory enzyme in the urea cycle.

Catabolism of arginine and ornithine in the perfused rat liver: effect of dietary protein and of glucagon.

The rates of oxidation of arginine and ornithine that occurred through a reaction pathway involving the enzyme ornithine aminotransferase (EC 2.6.1.13) were determined using (14)C-labeled amino acids in the isolated nonrecirculating perfused rat liver. At physiological concentrations of these amino acids, their catabolism is subject to chronic regulation by the level of protein consumed in the diet. (14)CO(2) production from [U-(14)C]ornithine (0.1 mM) and from [U-(14)C]arginine (0.2 mM) was increased about fourfold in livers from rats fed 60% casein diets for 3-4 days. The catabolism of arginine in the perfused rat liver, but not that of ornithine, is subject to acute regulation by glucagon (10(-7) M), which stimulated arginine catabolism by approximately 40%. Dibutyryl cAMP (0.1 mM) activated arginine catabolism to a similar extent. In retrograde perfusions, glucagon caused a twofold increase in the rate of arginine catabolism, suggesting an effect of glucagon on arginase in the perivenous cells.

Studies of hepatic glutamine metabolism in the perfused rat liver with (15)N-labeled glutamine.

This study examines the role of glucagon and insulin in the incorporation of (15)N derived from (15)N-labeled glutamine into aspartate, citrulline and, thereby, [(15)N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM NH(4)Cl and either 2-(15)N- or 5-(15)N-labeled glutamine (1 mM). The isotopic enrichment of the two nitrogenous precursor pools (ammonia and aspartate) involved in urea synthesis as well as the production of [(15)N]urea isotopomers were determined using gas chromatography-mass spectrometry. This information was used to examine the hypothesis that 5-N of glutamine is directly channeled to carbamyl phosphate (CP) synthesis. The results indicate that the predominant metabolic fate of [2-(15)N] and [5-(15)N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of (15)N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form U(m+1) and U(m+2) (urea containing one or two atoms of (15)N). However, insulin had little effect compared with control. The [5-(15)N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-(15)N]glutamine was predominantly incorporated via aspartate. This is evident from the relative enrichments of aspartate and of citrulline generated from each substrate. Furthermore, the data indicate that the (15)NH(3) that was generated in the mitochondria by either PDG (from 5-(15)N) or glutamate dehydrogenase (from 2-(15)N) enjoys the same partition between incorporation into CP or exit from the mitochondria. Thus, there is no evidence for preferential access for ammonia that arises by the action of PDG to carbamyl-phosphate synthetase. To the contrary, we provide strong evidence that such ammonia is metabolized without any such metabolic channeling. The glucagon-induced increase in [(15)N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [(15)N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis of N-acetylglutamate (an obligatory activator of CP). The current study may provide the theoretical and methodological foundations for in vivo investigations of the relationship between the hepatic urea cycle enzyme activities, the flux of (15)N-labeled glutamine into the urea cycle, and the production of urea isotopomers.

Regulation of homocysteine metabolism.

We have used a combination of in vivo and in vitro techniques to measure factors regulating homocysteine metabolism and the plasma concentration of this atherogenic amino acid. The germane findings include: 1. Homocysteine metabolism in rat kidney proceeds predominantly through the transsulfuration pathway, whose enzymes are enriched within the proximal cells of kidney tubules. Furthermore, the rat kidney possesses significant reserve capacity to handle both acute and chronic elevations in plasma homocysteine concentrations. 2. Plasma homocysteine concentrations are lower in diabetic rats. Insulin administration corrects this perturbation. Therefore, insulin and/or one of its counter-regulatory hormones affects homocysteine metabolism, possibly through an increased flux in the hepatic transsulfuration pathway. In support of these data, glucagon administration to rats produced similar results. Further support was provided by studies with isolated rat hepatocytes, from which homocysteine export was reduced when incubated in the presence of glucagon.

Comments on metabolic needs for glucose and the role of gluconeogenesis.

The metabolism of carbohydrates is largely determined by their chemical properties. Glucose may have been selected, over the other aldohexoses, because of its low propensity for glycation of proteins. That carbohydrate is stored in polymeric form (glycogen) is dictated by osmotic pressure considerations. That stored fat is about eight times more calorically dense than glycogen, when attendant water is factored in, accounts for the predominance of fat as a storage form of calories and, also, for the fact that ingested carbohydrate is oxidized promptly (that is, fuel of the fed state) rather than being extensively stored. That stored glycogen is accompanied by so much water accounts for the fact that the brain only has very small glycogen stores. Carbohydrate has two important advantages, over fat, as a metabolic fuel; it is the only fuel that can produce ATP in the absence of oxygen, and more ATP is produced per O2 consumed when glucose is oxidized, compared with when fat is oxidized. These advantages probably determine the preference of many cell types for carbohydrate. In addition to its use as a metabolic fuel, glucose plays other important roles such as provision of NADPH via the pentose phosphate pathway, and as a source material for the synthesis of other key carbohydrates, for example, ribose and deoxyribose for nucleic acid synthesis and substrates for the synthesis of glycoproteins, glycolipids and glycosaminoglycans. It can also play a key role in anaplerosis. Although it is widely acknowledged that gluconeogenesis plays a crucial role in starvation it is now apparent that prandial gluconeogenesis occurs, both in the metabolic disposal of dietary amino acids and in the synthesis of glycogen by the indirect pathway. Although there is, strictly speaking, no dietary requirement for carbohydrate it is evident that glucose is a universal fuel for probably all cells in the body and carbohydrate is the cheapest source of calories and the major source of dietary fibre. These observations, together with the fact that glucose is the preferred metabolic fuel for the brain, permit us to recommend appreciable quantities of carbohydrate in all prudent diets.

Effects of streptozotocin-induced diabetes and of insulin treatment on homocysteine metabolism in the rat.

An elevation in the concentration of total plasma homocysteine is known to be an independent risk factor for the development of vascular disease. Alterations in homocysteine metabolism have also been observed clinically in diabetic patients. Patients with either type 1 or type 2 diabetes who have signs of renal dysfunction tend to exhibit elevated total plasma homocysteine levels, whereas type 1 diabetic patients who have no clinical signs of renal dysfunction have lower than normal plasma homocysteine levels. The purpose of this study was to investigate homocysteine metabolism in a type 1 diabetic animal model and to examine whether insulin plays a role in its regulation. Diabetes was induced by intravenous administration of 100 mg/kg streptozotocin to Sprague-Dawley rats. We observed a 30% reduction in plasma homocysteine in the untreated diabetic rat. This decrease in homocysteine was prevented when diabetic rats received insulin. Transsulfuration and remethylation enzymes were measured in both the liver and the kidney. We observed an increase in the activities of the hepatic transsulfuration enzymes (cystathionine beta-synthase and cystathionine gamma-lyase) in the untreated diabetic rat. Insulin treatment normalized the activities of these enzymes. The renal activities of these enzymes were unchanged. These results suggest that insulin is involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway, which is involved in the catabolism of homocysteine.

Renal uptake and excretion of homocysteine in rats with acute hyperhomocysteinemia.

BACKGROUND: Elevated plasma total homocysteine, an independent risk factor for cardiovascular disease, is commonly observed in renal patients. We have previously shown that the kidney is a major site for the removal of plasma homocysteine in the rat. The present investigation was performed to further characterize the capacity of the kidney to handle acute elevations in plasma homocysteine concentrations. METHODS: Acute hyperhomocysteinemic conditions (4- to 7-fold > controls) in rats were produced by either a primed-continuous infusion of L-homocysteine or exposure to 80:20% nitrous oxide:oxygen, which results in the inhibition of methionine synthase. RESULTS: At physiological homocysteine concentrations, approximately 15% of the arterial plasma homocysteine was removed on passage through the kidney. Renal homocysteine uptake was approximately 85% of the filtered load. The urinary excretion of homocysteine was negligible (<2%). During acute hyperhomocysteinemia produced by the infusion of L-homocysteine, renal homocysteine uptake was increased fourfold and was equivalent to 50% of the infused dose, while urinary excretion remained negligible. Renal homocysteine uptake during nitrous oxide-induced hyperhomocysteinemia increased threefold, with urinary excretion remaining negligible. CONCLUSIONS: These results provide strong evidence that the kidney has a significant capacity for metabolizing acute elevations in plasma homocysteine, and support a very limited role for the re-methylation pathway in renal homocysteine metabolism.

Hepatic zonation of the catabolism of arginine and ornithine in the perfused rat liver.

The metabolism of 14C-labelled arginine and ornithine was studied in the isolated, nonrecirculating, perfused rat liver. The catabolism of these amino acids required ornithine aminotransferase since treatment of rats with gabaculine, an inhibitor of this enzyme, decreased substantially the production of 14CO2 from the 14C-labelled amino acids. In the liver, ornithine aminotransferase is restricted to a small population of hepatocytes proximal to the terminal hepatic vein [Kuo, F.C., Hwu, W.L., Valle, D. and Darnell Jr., J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9468-9472], i.e. the perivenous subpopulation of hepatocytes. Catabolism of arginine requires arginase to convert arginine to ornithine which can then be catabolized through ornithine aminotransferase. The presence of arginase activity in the perivenous hepatocytes was demonstrated by experiments in which livers were perfused with [14C]arginine in both antegrade and retrograde directions. Identical rates of 14CO2 production were obtained in these experiments, a result which could only occur if the process of arginine catabolism through ornithine aminotransferase can be carried out in its entirety in the perivenous cells.

Cell signalling and the hormonal stimulation of the hepatic glycine cleavage enzyme system by glucagon.

The glycine cleavage enzyme system (GCS) is found in mitochondria. In liver it is activated by glucagon and other hormones but it is not known how the hormonal signal is transmitted to the mitochondria. We found that the cell-permeant protein phosphatase inhibitor okadaic acid stimulated flux through GCS and could induce a significant increase in the sensitivity of GCS and of glycogenolysis to glucagon. Half-maximal stimulation of GCS by glucagon occurred at 3.2+/-0.6 nM, whereas it was fully activated at 0.3 nM in the presence of 1 microM okadaic acid. The protein kinase A agonist adenosine-3',5'-cyclic monophosphorothioate, Sp isomer (10 microM) stimulated the GCS flux by approx. 100%. This stimulation was inhibited by the protein kinase A antagonist 8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS). Although Rp-8-Br-cAMPS significantly inhibited glucagon-stimulated glycogenolysis it had no effect on the glucagon-stimulated GCS flux. These results indicate that a cytoplasmic phosphorylated protein is involved in transmitting glucagon's effect to the mitochondria. However, protein kinase A does not have a necessary role in transmitting glucagon's signal. We also examined the role of protein kinase C because angiotensin II also stimulated flux through GCS. However, the phorbol ester PMA had no effect on either GCS or on glycogenolysis.