Chronic high glucose lowers pyruvate dehydrogenase activity in islets through enhanced production of long chain acyl-CoA: prevention of impaired glucose oxidation by enhanced pyruvate recycling through the malate-pyruvate shuttle

In islet beta-cells, the high expression of pyruvate carboxylase and the functional importance of the downstream anaplerosis pathways result in a unique characteristic whereby high glucose and fatty acids both increase production of a key fatty acid metabolite, long chain acyl-CoA, for signaling and enzyme regulation in beta-cells. We showed previously in islets that pyruvate dehydrogenase (PDH) activity is lowered by excess fatty acids (the so-called Randle effect). We have now investigated PDH activity and pyruvate metabolism in islets after 48-h culture at 16.7 mmol/liter glucose. Active PDH V(max) was lowered 65% by 48 h of high glucose, and this effect was markedly attenuated by co-culture with triacsin C, which inhibits acyl-CoA synthase. Despite the large reduction in PDH activity, glucose oxidation was twice normal. The reason was continued metabolism of pyruvate through pyruvate carboxylase (V(max), 83% of control) and diversion of flux through the pyruvate-malate shuttle. The result was a 3-fold increase of the pyruvate concentration that overcame the lowered PDH activity by mass action as shown by glucose oxidation measured with [6-(14)C]glucose being twice normal. In addition, glucose-induced insulin secretion was 3-fold increased after 48 h of high glucose, and this effect was totally blocked by co-culture with triacsin C. These results show that a unique feature of islet beta-cells is not only fatty acids but also excess glucose that impairs PDH activity. Also, a specialized trait of beta-cells is a long chain acyl-CoA-mediated defense mechanism that prevents a reduction in glucose oxidation and consequently in insulin secretion.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.     

Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Interleukin-1 beta inhibits glucokinase activity in clonal HIT-T15 beta-cells

Interleukin-1 beta (IL-1 beta) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. In the present study we have investigated the effects of IL-1 beta on glucose metabolism in clonal HIT-T15 beta cells. In the short-term (1 h), 25 U/ml IL-1 beta significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL-1 beta inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6-phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL-1 beta may be due to impaired glucokinase activity.     

Leptin regulates energy metabolism in MCF-7 breast cancer cells

Obesity is known to be a poorer prognosis factor for breast cancer in postmenopausal women. Among the diverse endocrine factors associated to obesity, leptin has received special attention since it promotes breast cancer cell growth and invasiveness, processes which force cells to adapt their metabolism to satisfy the increased demands of energy and biosynthetic intermediates. Taking this into account, our aim was to explore the effects of leptin in the metabolism of MCF-7 breast cancer cells. Polarographic analysis revealed that leptin increased oxygen consumption rate and cellular ATP levels were more dependent on mitochondrial oxidative metabolism in leptin-treated cells compared to the more glycolytic control cells. Experiments with selective inhibitors of glycolysis (2-DG), fatty acid oxidation (etomoxir) or aminoacid deprivation showed that ATP levels were more reliant on fatty acid oxidation. In agreement, levels of key proteins involved in lipid catabolism (FAT/CD36, CPT1, PPARα) and phosphorylation of the energy sensor AMPK were increased by leptin. Regarding glucose, cellular uptake was not affected by leptin, but lactate release was deeply repressed. Analysis of pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH) and pyruvate carboxylase (PC) together with the pentose-phosphate pathway enzyme glucose-6 phoshate dehydrogenase (G6PDH) revealed that leptin favors the use of glucose for biosynthesis. These results point towards a role of leptin in metabolic reprogramming, consisting of an enhanced use of glucose for biosynthesis and lipids for energy production. This metabolic adaptations induced by leptin may provide benefits for MCF-7 growth and give support to the reverse Warburg effect described in breast cancer.     

Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.     

Glucose oxidation modulates anoikis and tumor metastasis

Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.     

Effect of nutritional status on insulin sensitivity in vivo and tissue enzyme activities in the rat.

The hyperinsulinaemic-glucose-clamp technique, in combination with measurement of glucose turnover in conscious unrestrained rats, was used to assess the effects of nutritional status on insulin sensitivity in vivo and glucose metabolism. Liver, heart and quadriceps skeletal-muscle glycogen content and activities of pyruvate dehydrogenase (PDH) and glycogen synthase were measured both basally and at the end of a 2.5 h glucose clamp (insulin 85 munits/h) in rats 6, 24 and 48 h after food withdrawal. Clamp glucose requirement and glucose turnover were unchanged by fasting. Activation of glycogen synthase and glycogen deposition in liver and skeletal muscle during the clamps were also not impaired in rats after a prolonged fast. By contrast with skeletal muscle, activation of cardiac-muscle glycogen synthase and glycogen deposition during the clamps were markedly impaired by 24 h of fasting and were undetectable at 48 h. Skeletal-muscle PDH activity fell with more prolonged fasting (6 h, 15.3 +/- 3.4%; 24 h, 4.7 +/- 0.7%; 48 h, 4.3 +/- 0.6% active; P less than 0.005), but at 24 and 48 h was stimulated by the clamp to values unchanged by the duration of fasting. Stimulation of cardiac PDH activity by the clamp was, however, impaired in rats fasted for 24 or 48 h. Basal hepatic PDH did not change significantly with fasting (6 h, 5.3 +/- 1.1%; 24 h, 4.6 +/- 0.7%; 48 h, 3.9 +/- 0.5%), and, although it could be partly restored at 24 h, very little stimulation occurred at 48 h. Hepatic pyruvate kinase and acetyl-CoA carboxylase activity were both stimulated by the clamps, and this was not impaired with more prolonged fasting. During the glucose clamps, blood concentrations of lactate, pyruvate and alanine were increased to a greater extent in rats fasted for 24 and 48 h than in rats studied 6 h after food withdrawal. The findings suggest that, although sensitivity to insulin of whole-body glucose disposal is unchanged with fasting, there may be qualitative differences in the metabolism of glucose.     

Inhibition of glucose oxidation by vasoactive intestinal peptide in isolated rat enterocytes

Glucose metabolism and its hormonal regulation have been investigated in isolated enterocytes from rat small intestine. About 70% of the glucose consumed by the cells was transformed into lactate, 5% into pyruvate, and 4% into alanine. The remaining 20% was oxidized. Among several tested gastrointestinal peptides and hormones, only vasoactive intestinal peptide (VIP) was found to affect the metabolic fate of glucose. VIP (10(-7) M) induced a 40% inhibition of glucose oxidation without significant modification of either glucose uptake or production of lactate, pyruvate, and alanine. This acute inhibition was dose-dependent (Ki = 3.10(-11) M) and appeared to be dependent on the stimulation of cAMP production (K0.5 = 3.10(-9) M) since dibutyryl-cAMP and forskolin reproduced all the effects of VIP. Similar inhibition of cell respiration by VIP was observed when pyruvate, fructose, and dihydroxyacetone were used as substrates, while the oxidation of glutamine, ketone bodies, and octanoate was unaffected, suggesting that the peptide acts on pyruvate metabolism. The suppression of VIP effects by dichloroacetate (5 mM) and pyruvate (10 mM) and the significant decrease (18%) of the activity of the pyruvate dehydrogenase complex after incubation of the cells with the neuropeptide, support the hypothesis that the effects of VIP on glucose oxidation may occur through an inhibition of the pyruvate dehydrogenase complex. The total suppression of the inhibitory effects of VIP by sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of long-chain fatty acid oxidation, suggests that VIP did not affect the pyruvate dehydrogenase directly, but more probably acted through modifications of fatty acid oxidation.     

Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids.

During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44+/-1% peak oxygen consumption (mean+/-SE) until exhaustion (exhaustion at 3 h 23 min+/-11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P<0.05). PDH activity peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced (approximately 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P<0.05) was also associated with decreasing PDH activity (P<0.05) and increased PDH kinase 4 mRNA (P<0.05) during exercise and recovery. At 1 h of exercise, pyruvate production was greatest and was closely linked to glutamate, which was the predominant amino acid taken up during exercise and recovery. Alanine and glutamine were also associated with pyruvate metabolism, and they comprised approximately 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism in early exercise.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Enhanced rat beta-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway

Islet beta-cell proliferation is a very important component of beta-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of beta-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of beta-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only approximately 1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.     

Carvacrol partially reverses symptoms of diabetes in STZ-induced diabetic rats

Little is known about the protective effects of carvacrol on the symptoms of streptozotocin induced diabetes in rats. Hence, this present study was designed to evaluate the protective effect of the strong antioxidant, carvacrol, on the symptoms of streptozotocin induced diabetes in rats. Carvacrol at the doses of 25 and 50 mg/kg body weight were orally administered to diabetic rats for a period of 7 days after the onset of diabetes. Food-water intake and body weight changes were daily recorded. Biochemical parameters such as serum glucose, insulin, total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were measured. Although treatment of diabetic rats with oral administration of carvacrol resulted in a slight reduction in serum glucose level and significant reduction in serum total cholesterol, alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in comparison with diabetic control rats, there were no significant differences in serum insulin levels, food-water intake values and body weight changes. Despite the inadequacy of carvacrol on diabetes treatments, it was determined to have at least a partially protective role on liver enzymes.     

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.     

Rat brain slices oxidize glucose at high rates: a (13)C NMR study

Since glucose is the main cerebral substrate, we have characterized the metabolism of various ¹³C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO₂) and determined absolute fluxes through pathways of glucose metabolism. After 60 min of incubation, lactate and CO₂ were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO₂. High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.     

The insulin signal and its effects on the pyruvate dehydrogenase complex in circulating lymphocytes of obese children.

1. Studies have shown that in circulating lymphocytes pyruvate dehydrogenase (PDH) is responsive to insulin. 2. To improve existing knowledge on how insulin influences PDH behaviour, situations in which cell responsiveness to insulin is impaired could be of interest. 3. PDH behaviour in circulating lymphocytes from obese children, with high plasma insulin levels and normal glucose tolerance, was examined. 4. Masking and unmasking processes of insulin receptors on the plasma membrane appear to modulate the enzyme response to insulin.     

Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets

Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK.     

Lipogenesis in rat and guinea-pig isolated epididymal fat-cells

Fat-cells were prepared from rat and guinea-pig epididymal adipose tissue and compared on the basis of the intracellular distributions and activities of enzymes and with respect to their utilization of various U-(14)C-labelled substrates for lipogenesis. 1. Compared with the rat, guinea-pig extramitochondrial enzyme activities differed in that aconitate hydratase, alanine aminotransferase, ATP-citrate lyase, lactate dehydrogenase, NAD-malate dehydrogenase, NADP-malate dehydrogenase and phosphoenolpyruvate carboxykinase activities were appreciably lower, whereas aspartate aminotransferase, glucose 6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase activities were appreciably higher. Mitochondrial activities of citrate synthase, NADP-isocitrate dehydrogenase and pyruvate carboxylase were appreciably lower, whereas mitochondrial activities of aspartate aminotransferase, glutamate dehydrogenase, NAD-malate dehydrogenase and phosphoenolpyruvate carboxykinase were higher in the guinea pig compared with the rat. 2. In general guinea-pig fat-cells incorporated acetate and lactate into fatty acids more readily than rat fat-cells, whereas rat fat-cells incorporated glucose and pyruvate more readily than guinea-pig fat-cells. 3. Acetate stimulated the incorporation of glucose into fatty acids in rat fat-cells, but had no appreciable effect upon this process in guinea-pig fat-cells. Acetate greatly decreased the incorporation of lactate into fatty acids in cells from both species. 4. Lactate/pyruvate ratios produced by incubation of guinea-pig cells with glucose+insulin were very low compared with those found with rat cells under the same conditions. 5. With glucose (+insulin) or with glucose+acetate (+insulin) as substrates guinea-pig cells produced enough NADPH by the hexose monophosphate pathway to satisfy the NADPH requirements of lipogenesis. In rat fat-cells under the same conditions, hexose monophosphate-pathway NADPH provision was not sufficient to meet the requirements of lipogenesis. 6. These results are discussed, particularly in relationship to the disposition of cytosolic reducing equivalents in the cells.     

The conversion of alanine into glutamine in guinea-pig renal cortex. Essential role of pyruvate carboxylase.

1. The metabolism of L-alanine was studied in isolated guinea-pig kidney-cortex tubules. 2. In contrast with previous conclusions of Krebs [(1935) Biochem. J. 29, 1951-1969], glutamine was found to be the main carbon and nitrogenous product of the metabolism of alanine (at 1 and 5 mM). Glutamate and ammonia were only minor products. 3. At neither concentration of alanine was there accumulation of glucose, glycogen, pyruvate, lactate, aspartate or tricarboxylic acid-cycle intermediates. 4. Carbon-balance calculations and the release of 14CO2 from [U-14C]alanine indicate that oxidation of the alanine carbon skeleton occurred at both substrate concentrations. 5. A pathway involving alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, pyruvate carboxylase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of alanine into glutamine. 6. Strong evidence for this pathway was obtained by: (i) suppressing alanine removal by amino-oxyacetate, and inhibitor of transaminases, (ii) measuring the release of 14CO2 from [1-14C]alanine, (iii) the use of L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from alanine, and (iv) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from alanine. 7. In this pathway, the central role of pyruvate carboxylase, which explains the discrepancy between our results and those of Krebs (1935), was also demonstrated.