Oxidative phosphorylation. The effect of anions on the inhibition by triethyltin of various mitochondrial functions, and the relationship between this inhibition and binding of triethyltin

1. The binding of triethyltin to rat liver mitochondria is unaffected by the nature of the predominant anion in the incubation medium. 2. With chloride, bromide or iodide as the predominant anion, ATP synthesis linked to the oxidation of pyruvate or succinate and ATP hydrolysis stimulated by 2,4-dinitrophenol are much more sensitive to triethyltin than they are when nitrate or isethionate is the predominant anion. 3. When nitrate or isethionate is the predominant anion, oxygen uptake stimulated by 2,4-dinitrophenol is not inhibited by triethyltin. 4. In the presence of nitrate or isethionate anions, inhibition of ATP synthesis is directly related to the binding of triethyltin to mitochondria. 5. The relationship of the above effects to the anion–hydroxide ion exchange mediated by triethyltin and the relevance of this to published arrangements for coupling of electron transport to ATP synthesis are discussed.     

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.     

Oxidative phosphorylation. Halide-dependent and halide-independent effects of triorganotin and trioganolead compounds on mitochondrial functions.

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.     

The influence of adenine nucleotides and oxidizable substrates on triethyltin-mediated chloride uptake by rat liver mitochondria in potassium chloride media.

In a 100 mM-KCl medium, pH 6.8, containing ATP increasing concentrations of triethyltin cause an uptake of Cl- into mitochondria with a maximum at 1 muM. This can be inhibited by atractylate or oligomycin, but is virtually unaffected by the presence of rotenone. When the medium contains substrate (pyruvate, beta-hydroxybutyrate or succinate), both in the presence and absence of adenine nucleotides, Cl- uptake is greater with a maximum at 1-10 muM-triethyltin. If substrate oxidation is blocked by respiratory-chain inhibitors the Cl- uptake mediated by triethyltin is inhibited except in the media containing ATP, when the characteristics of Cl- uptake similar to that found in the medium containing ATP alone are observed. Under all conditions tested Cl- uptake is decreased by the presence of 2,4-dinitrophenol. It is concluded that energy from either the oxidation of substrate or the hydrolysis of ATP is associated with the generation of sufficient OH- to enable the triethyltin-mediated Cl-/OH- exchange to occur under the metabolic conditions relevant to this action of triethyltin.     

Studies on brain-cortex slices: The influence of various inhibitors on the retention of potassium ions and amino acids with glucose or pyruvate as substrate

1. The rate of appearance of (14)CO(2) from [6-(14)C]glucose and [3-(14)C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30mum-2,4-dinitrophenol increases the output of (14)CO(2) from [6-(14)C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0.1m-potassium chloride. The stimulating action of these two agents on the output of (14)CO(2) from [3-(14)C]pyruvate is much less than on that from [6-(14)C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1.3mum) inhibited the oxidation of [6-(14)C]glucose more than 70%, but did not inhibit the oxidation of[3-(14)C]pyruvate. [3-(14)C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH(2) produced during glycolysis.     

Oxidative phosphorylation. The specific binding of trimethyltin and triethyltin to rat liver mitochondria

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.     

THE ACTION OF TRIETHYLTIN ON THE RESPIRATION OF RAT BRAIN CORTEX SLICES

Glucose oxidation by rat brain cortex slices is inhibited by low concentrations of triethyltin and this sensitive inhibition is shown to be chloride-dependent. Pyruvate oxidation is only inhibited at high concentrations of triethyltin whether chloride is present or not. In contrast, the stimulation of both glucose and pyruvate oxidation observed with low concentrations of triethyltin prior to inhibition is chloride-dependent. The results are discussed in relation to the chloride-hydroxide exchange reaction known to be mediated across the inner mitochondrial membrane by triethyltin and other organo-metals.     

The influence of pH on the inhibition of oxidative phosphorylation and electron transport by triethyltin

The ability of triethyltin to inhibit oxidative phosphorylation and electron transport in tightly coupled rat liver mitochondria is very dependent on the pH and the ionic constitution of the assay medium.

1. 1. In an assay medium containing Cl⁻ at an alkaline pH, above 7.1, triethyltin inhibited both the ADP stimulated rate of oxygen uptake and the dinitrophenol-induced ATPase (EC 3.6.1.3) but had no effect on the dinitrophenol-stimulated rate of oxygen uptake. If the pH was reduced to below 6.9 the pattern of inhibition changed and both the ADP and dinitrophenol-stimulated rates of oxygen uptake were inhibited by triethyltin.

2. 2. In the absence of Cl⁻ in the medium triethyltin inhibited both the ADP-stimulated rate of oxygen uptake and dinitrophenol-induced ATPase and had no effect on the dinitrophenol-stimulated rate of oxygen uptake at either pH 7.4 or 6.6.

3. 3. In either the presence or absence of Cl⁻ the ability of triethyltin to inhibit ATP synthesis appears to markedly decrease as the pH is lowered from 7.4 to 6.6.

4. 4. The significance of these observations is discussed in relation to the operation of a Cl⁻/OH⁻ antiport in the coupling membrane.

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Studies on brain-cortex slices: Differences in the oxidation of 14C-labelled glucose and pyruvate revealed by the action of triethyltin and other toxic agents

1. The rate of appearance of ¹⁴CO₂ from [6-¹⁴C]glucose and [3-¹⁴C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30μm-2,4-dinitrophenol increases the output of ¹⁴CO₂ from [6-¹⁴C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0·1m-potassium chloride. The stimulating action of these two agents on the output of ¹⁴CO₂ from [3-¹⁴C]pyruvate is much less than on that from [6-¹⁴C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1·3μm) inhibited the oxidation of [6-¹⁴C]glucose more than 70%, but did not inhibit the oxidation of[3-¹⁴C]pyruvate. [3-¹⁴C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH₂ produced during glycolysis.     

Effect of atractyloside on glucose and pyruvate metabolism in rat diaphragm muscle

1. In the presence of 1.2mm-atractyloside oxygen uptake by rat diaphragm muscle incubated with 5.6mm-glucose decreases, as well as glycogen synthesis and carbon dioxide production. Lactate formation from glucose increases, but that of phosphoglycerate diminishes fivefold. 2. When pyruvate is used as substrate, atractyloside decreases oxygen uptake. 3. The specific radioactivity of the (14)CO(2) (mumoles of (14)CO(2)/mumole of oxygen), calculated at concentrations of [1-(14)C]pyruvate between 0.091mm and 91mm, lies between 3.1x10(-4) and 5.7x10(-1). Atractyloside increases the specific radioactivity of the (14)CO(2) with the lowest concentrations of substrate and has no effect when the substrate concentration is 91mm. 4. No appreciable effect of atractyloside on the anaerobic production of (14)CO(2) from [1-(14)C]pyruvate at various incubation times and various concentrations is found. 5. It is suggested that atractyloside induces anaerobic conditions in the tissue. Further, it produces a rise in the pyruvate concentration and an ATP deficiency in the cell. Consequently it stimulates pyruvate dismutation, and glycolysis, to which phosphorylation is linked at the substrate level.     

THE BINDING OF TRIETHYLTIN TO RAT BRAIN MYELIN

—A high affinity binding site for triethyltin was found in rat brain myelin with an affinity of approx 6·6 × 10⁵m ⁻¹ at pH 7·5. Competitive binding studies showed that triethyl-lcad had about the same affinity and trimethyltin 30 times lower affinity than triethyltin. Hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide did not prevent triethyltin binding to rat brain myelin. Since triethyltin, hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide all produce similar oedematous lesions in the brain of rats, whereas triethyl-lead and trimethyltin do not, it is concluded that the high affinity triethyltin binding site either is not involved or is not the only factor in oedema production.     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Glucocorticoid action on rat thymic lymphocytes. Experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions.

Inhibition of glucose uptake has been proposed as a primary cause of many of the subsequent inhibitory effects of glucocorticoids. This hypothesis has been tested in experiments where adenosine is substituted for glucose. Like glucose, adenosine maximally supports glycolytic and oxidative ATP generation, and by its use the hormonal inhibition of glucose uptake is circumvented. With adenosine, inhibition by cortisol is seen at at least one other metabolic site, respiratory ATP synthesis. This action can be observed by hormone-induced increases in levels of lactate, pyruvate, and AMP that accompany a lowering of ATP. Evidence for this metabolic action is also seen when cells are provided with a limiting amount of glucose; despite inhibition of glucose uptake, a cortisol-induced increase in lactate accompanies the reduction in levels of ATP. Decreased respiratory ATP synthesis is also suggested by a hormonal reduction in the metabolism of labeled exogenous pyruvate to 14CO2. Several experimental approaches suggest that inhibition of oxidative ATP production, rather than of glucose uptake, is the event most responsible for glucocorticoid-induced changes in the balance of adenine nucleotides, which in turn contribute to effects on protein synthesis and uridine uptake. First, the characteristic inhibitory cortisol effects on adenine nucleotides and protein synthesis are undiminished when adenosine is substituted for glucose. Second, in adenosine-supported cells the onset of the hormone-induced increase in levels of lactate corresponds closely to the appearance of measurable reductions in ATP. In contrast, when cells are supported by glucose, the hormonal inhibition of glucose uptake is maximal by 30 to 35 min, nearly an hour before effects on levels of ATP are detectable. Third, when cells are made strongly dependent upon glucose for ATP production by deprivation of exogenous substrate and cortisol is added at 90 min, a characteristic inhibition of the uptake of glucose added 40 min later is seen; nevertheless, this is insufficient to prevent added glucose from immediately and fully restoring ATP, rates of protein synthesis, and uridine uptake. Inhibitory effects on ATP, protein synthesis, and uridine do appear after an additional hour or so, a time commensurate with the development of an inhibition of oxidative metabolism. Fourth, limiting added glucose can reduce uptake more than cortisol, without reducing levels of ATP.     

Respiratory inhibition by copper in Tetrahymena pyriformis GL

An excess of copper incorporated into Tetrahymena cells was mainly distributed in mitochondria, and inhibited oxygen uptake of Tetrahymena cells. The inhibition of oxygen uptake was clearly to copper uptake in mitochondria. Succinate was most favorable as a substrate stimulating oxygen uptake in mitochondria, and oxygen uptake was most strongly inhibited by copper (0.1 mM) in the presence of succinate among various substrates. The copper incorporated into mitochondria was in the fraction with the inner membranes. Succinate dehydrogenase (SDH) was inhibited at the lowest copper concentration (0.1 mM) among respiratory related enzymes. The redox potential of respiratory components was raised by copper. These results suggest that respiratory inhibition of Tetrahymena cells by copper may be mainly cause by inhibition of SDH as a FAD-protein and oxidation of electron carriers. At higher copper concentrations, MDH, cytochrome c reductase, and ATP synthesis were also inhibited. Growth inhibition may be due to these effects of copper in mitochondria. Mercury affected both oxygen uptake and SDH more strongly than copper. Zinc (0.1 mM) also affected oxygen uptake in mitochondria and a little in whole cells, however, it did not inhibit SDH. Cobalt, manganese, and nickel affected both oxygen uptake and SDH only a little at the same concentration (0.1 mM) as copper.     

Use of β-Methylene-D,L-Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of reconstituted cholesterol 7 alpha-hydroxylase system from rat and rabbit liver microsomes.

1. Pyruvate carboxylase is present in brown adipose tissue mitochondria. 2. In isolated mitochondria, pyruvate, bicarbonate and ATP, the substrates for pyruvate carboxylase, are able to replace added malate in supplying a condensing partner for acetyl-CoA formed from beta-oxidation of fatty acids. 3. In brown adipocytes, pyruvate and CO2 increase the rate of norepinephrine-stimulated respiration synergistically. 4. The norepinephrine-stimulated respiration in brown adipocytes is diminished when pyruvate transport into the mitochondria is inhibited. 5. Pyruvate carboxylation increases the intramitochondrial level of citric acid cycle intermediates, as shown by titrations of malonate inhibition of respiration. 6. Pyruvate carboxylation can continuously supply the mitochondria with citric acid cycle intermediates, as evidenced by its ability to maintain respiration when oxoglutarate conversion to glutamate is stimulated. 7. Pyruvate carboxylation is necessary for maximal oxygen consumption even when drainage of the citric acid cycle for amino acid synthesis is eliminated. 8. Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.     

Mechanism for the oleate stimulation of gluconeogenesis from dihydroxyacetone by hepatocytes from fasted rats

Oleate stimulates glucose production and concomitantly decreases lactate and pyruvate production by rat hepatocyte suspensions incubated with dihydroxyacetone as substrate. The actions of oleate could be blocked by D-(+)dodecanoylcarnitine, which inhibits transport of the fatty acid into the mitochondria and the subsequent oxidation. beta-Hydroxybutyrate, but not acetoacetate, also stimulated glucose synthesis and inhibited lactate and pyruvate production. Furthermore, both beta-hydroxybutyrate and oleate stimulated oxygen consumption to the same extent. This suggests that oleate stimulates glucose production by the provision of energy subsequent to mitochondrial beta-oxidation of the fatty acids. The content of ATP itself did not appear to be responsible for the effects of oleate. Crossover analysis of the gluconeogenic intermediates implicated a site of oleate action between fructose 1,6-bisphosphate and fructose 6-phosphate, suggesting phosphofructokinase and/or fructose-bisphosphatase as possible regulatory sites. Coupled with the finding that intracellular citrate accumulates upon addition of oleate or beta-hydroxybutyrate, but not acetoacetate, the results suggest that citrate inhibition of phosphofructokinase accounts for the redirection of carbon flow from lactate and pyruvate formation and towards that of glucose.     

Impaired oxidative phosphorylation in skeletal muscle of intrauterine growth-retarded rats

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in later life. We have developed a model of uteroplacental insufficiency, a common cause of intrauterine growth retardation, in the rat. Early in life, the animals are insulin resistant and by 6 mo of age they develop diabetes. Glycogen content and insulin-stimulated 2-deoxyglucose uptake were significantly decreased in muscle from IUGR rats. IUGR muscle mitochondria exhibited significantly decreased rates of state 3 oxygen consumption with pyruvate, glutamate, alpha-ketoglutarate, and succinate. Decreased pyruvate oxidation in IUGR mitochondria was associated with decreased ATP production, decreased pyruvate dehydrogenase activity, and increased expression of pyruvate dehydrogenase kinase 4. Such a defect in IUGR mitochondria leads to a chronic reduction in the supply of ATP available from oxidative phosphorylation. Impaired ATP synthesis in muscle compromises energy-dependent GLUT4 recruitment to the cell surface, glucose transport, and glycogen synthesis, which contribute to insulin resistance and hyperglycemia of type 2 diabetes.