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.

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.

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.

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.

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.

Regulation of hepatic glutaminase in the streptozotocin-induced diabetic rat.

The liver of diabetic animals removes increased quantities of glutamine. We therefore examined factors that affect hepatic glutaminase activity in hepatocytes and mitochondria. Glutamine use, through glutaminase, was measured in isolated rat hepatocytes by monitoring the production of 14CO2 from [1-(14)C]glutamine. Hepatocytes from streptozotocin-induced diabetic rats use glutamine more rapidly than do hepatocytes from normal or insulin-maintained diabetic rats. Glutamine use in all of these hepatocytes was stimulated by glucagon and epinephrine. Glutaminase activity, assayed in broken mitochondrial membranes, was increased approximately 2.5-fold in diabetic rats. The sensitivity of glutaminase, measured in intact liver mitochondria, to phosphate was markedly left-shifted in mitochondria from diabetic rats compared with those from controls. In fact, glutaminase was increased 10-fold at 2.5 mmol/l phosphate compared with controls. This increased sensitivity of glutaminase to physiological concentrations of phosphate is characteristic of its hormonal activation. Therefore, activation of glutaminase plays a major role in diabetes and is as important as increases in its total enzyme amount in determining the increased glutamine uptake in diabetes.

Characterization of homocysteine metabolism in the rat kidney.

Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine beta-synthase and cystathionine gamma-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine beta-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine gamma-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.

A mass isotopomer study of urea and glutamine synthesis from 15N-labeled ammonia in the perfused rat liver.

This study examines the incorporation of 15N from 15NH4Cl into urea and glutamine, predicts the pattern of isotopomers produced as a function of the 15N enrichment of the relevant precursor pools, and presents a means of determining the isotopic enrichment of these pools. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM 15NH4Cl, and the isotopomers of urea and of glutamine produced were determined by gas chromatography-mass spectrometry methodology. Three different nitrogen mass isotopomers of urea were found, containing no, one, or two atoms of 15N. Four glutamine isotopomers were found, containing no 15N, one atom of 15N in either the amino or amide position, or two 15N atoms. A mathematical relationship was deduced that predicts that the relative proportions of the urea isotopomers depends not only on the relative enrichment of 15N in the two precursor pools of urea nitrogen (mitochondrial ammonia and cytoplasmic aspartate) but on their absolute enrichment. This relationship was validated in experiments in which the isotopic enrichment of the substrate, 15NH4Cl, was varied. The proportions of the urea isotopomers produced can be predicted if one knows the 15N enrichment in the two precursor pools. We found that when the 15N enrichment of citrulline and aspartate in the perfusate were used as proxies for that in the mitochondrial ammonia and cytoplasmic aspartate pools we could accurately predict the relative proportion of the three isotopomers. The production of the four nitrogen isotopomers of glutamine could be used to determine the 15N enrichment in the two precursor pools of glutamine nitrogen, the cytoplasmic ammonia and glutamate pools of the perivenous hepatocytes.