Rate Of Pyruvate Oxidation Research Articles

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

Use of a five-compartment closed model to describe the effects of ethanol inhalation on the transport and elimination of injected pyruvate in the rat.

A five-compartment closed model was established using the system of differential equations. The completely analytical solution of a five-compartment closed model was found using the Laplace transform. 2-[14C]Pyruvate kinetics were studied in rats without and with inhalation of vaporized ethanol, and were modelled by the five-compartment closed model, i.e. injected site, blood, eliminated 14CO2 in air, eliminated 14C in urine and faeces. The kinetic parameters were estimated using the analytical solution of the five-compartment closed model to fit eliminated 14CO2, and 14C in urine and faeces simultaneously. The compartmental analysis showed that the inhalation of vaporized ethanol can increase 2-[14C]pyruvate trans-membrane, trans-tissue processes and oxidation rate. The model developed is useful for general pharmacokinetic and toxicokinetic analysis as well as for studies on ethanol.

Plasticity of fish muscle mitochondria with thermal acclimation

Short-horned sculpin Myoxocephalus scorpius were acclimated to 5 and 15 °C to evaluate the impact of thermal acclimation upon maximal rates of substrate oxidation by mitochondria and upon the thermal sensitivity of their ADP affinity. Cold acclimation virtually doubled maximal rates of pyruvate oxidation at all experimental temperatures (2.5, 7.5, 12.5 and 20 °C). Rates of palmitoyl carnitine oxidation were also enhanced by cold acclimation, but to a lesser degree. At their respective acclimation temperatures, the mitochondria attained similar rates of pyruvate oxidation. For warm-acclimated sculpin, the Q10 values for mitochondrial pyruvate and palmitoyl carnitine oxidation were higher between 2.5 and 7.5 °C than between 7.5 and 12.5 °C or between 12.5 and 20 °C. In contrast, for cold-acclimated fish, the Q10 values did not differ over these thermal ranges. The Arrhenius activation energy for pyruvate oxidation was reduced by cold acclimation (from 70 to 55 kJ mol-1), whereas that for palmitoyl carnitine oxidation was unchanged (approximately 75 kJ mol-1). Cold acclimation did not alter the ADP affinity of mitochondria at low temperatures but markedly increased the apparent Km for ADP (Km,app) at 12.5 and 20 °C. At the acclimation temperatures, mitochondrial ADP Km,app values did not differ. The loss of ADP affinity at higher temperatures may represent a cost of the enhanced maximal oxidative capacity achieved during cold acclimation.

Deficiency of the Voltage-Dependent Anion Channel: A Novel Cause of Mitochondriopathy

A patient with a deficient voltage-dependent anion channel (VDAC) is reported, presenting clinically with psychomotor retardation and minor dysmorphic features. Biochemical studies on muscle mitochondria showed impaired rates of pyruvate oxidation and ATP production; however, no specific deficient activity of one of the mitochondrial enzymes was involved. Western blotting experiments indicated an almost complete VDAC deficiency in skeletal muscle. The only moderately decreased VDAC content in the patient's fibroblasts might indicate that VDAC is expressed in a tissue-specific manner. The deficiency is likely caused by a mutation in the HVDAC1 gene or by a distributed posttranslational modification. This is the first described deficiency of a component of the outer mitochondrial membrane associated with the pyruvate oxidation pathway. Defects in this membrane should be considered as a possible cause of otherwise unexplained mitochondrial disorders.

In VivoMetabolic Response of Glucose to Dichloroacetate in Humans

Hyperglycemia is common in severely ill patients and is related principally to an increase in glucose production. Dichloroacetate (DCA), which is known to increase the rate of pyruvate oxidation, has been shown to lower plasma glucose concentrations in normal fasting subjects and in diabetics and thus may be efficacious in treating stress induced hyperglycemia. However, the mechanism by which DCA lowers the plasma glucose concentration in humans has not been elucidated. To examine the humanin vivometabolic alterations induced by DCA, six fasting volunteers were infused with 6,6-D2-glucose and indirect calorimetry was performed prior to and following DCA administration. Glucose, lactate, and alanine net balance across the leg were also quantitated. Following DCA administration, plasma glucose concentrations decreased by 9% due to a proportional decrease in the rate of glucose production (P< 0.05). DCA had no affect on glucose clearance or leg net balance; however, the rate of glucose oxidation increased by 24% from baseline (P< 0.05). This increase in glucose oxidation without a compensatory change in peripheral glucose consumption suggests an improved efficiency in peripheral glucose utilization induced by DCA. Plasma concentrations of lactate and alanine were also lowered by DCA (56% for lactate, 66% for alanine,P< 0.05) without a significant alteration in leg net balance. These results suggest that DCA may decrease gluconeogenesis by limiting the availability of the precursor substrates lactate and alanine. Thus dichloroacetate may be an appropriate alternative to insulin in correcting mild elevations in plasma glucose concentrations. Furthermore, DCA may be especially effective in severely ill patients where hyperglycemia is largely due to increases in gluconeogenesis.

CHANGES IN GROWTH, PO(2) AND PH AFTER EXPOSURE TO OXAMATE - STUDIES OF 2 HUMAN TUMOR-CELL LINES GROWING AS MULTICELLULAR SPHEROIDS

The basic metabolism and pO(2) and pH gradients in spheroids were characterized and, in some cases, changed by the addition of the pyruvate analogue oxamate. Two human tumour spheroid types, colon adenocarcinoma HT29 and malignant glioma U118MG, were applied as models. Microelectrode measurements in HT29 spheroids showed steep pO(2) gradients with large differences between surface and center, Delta pO(2), and low central pO(2) values. The HT29 spheroids had rather flat pH gradients. The U118MG spheroids had less steep pO(2) gradients but steeper pH gradients. Determinations of 1-C-14 and 2-C-14 pyruvate oxidation rates, for characterization of the oxidative glucose breakdown as well as of lactate dehydrogenase kinetics, showed consistent results with the microelectrode measurements in that there was high oxidative metabolism in the HT29 spheroids whereas the U118MG spheroids relied more on glycolysis. Western blot investigations of the LDH isoenzyme composition showed different isoenzyme patterns in the two spheroid types with a lack of LDH1 in U118MG spheroids. Addition of 40 mM of oxamate gave decreased 1-C-14- and 2-C-14-pyruvate oxidation rates in the HT29 cells and inhibition of LDH activity in the U118MG cells. Oxamate increased the central pO(2) values in HT29 spheroids and the central pH values in U118MG spheroids. One example of experimental therapy was applied and oxamate acted as a radiation sensitizer in the U118MG system and as a radioprotective substance in the HT29 system. This has to be analysed in more detail.

Skeletal muscle pyruvate dehydrogenase activity during maximal exercise in humans.

The regulation of the active form of pyruvate dehydrogenase (PDHa) and related metabolic events were examined in human skeletal muscle during repeated bouts of maximum exercise. Seven subjects completed three consecutive 30-s bouts of maximum isokinetic cycling, separated by 4 min of recovery. Biopsies of the vastus lateralis were taken before and immediately after each bout. PDHa increased from 0.45 +/- 0.15 to 2.96 +/- 0.38, 1.10 +/- 0.11 to 2.91 +/- 0.11, and 1.28 +/- 0.18 to 2.82 +/- 0.32 mmol.min-1.kg wet wt-1 during bouts 1, 2, and 3, respectively. Glycolytic flux was 13-fold greater than PDHa in bouts 1 and 2 and 4-fold greater during bout 3. This discrepancy between the rate of pyruvate production and oxidation resulted in substantial lactate accumulation to 89.5 +/- 11.6 in bout 1, 130.8 +/- 13.8 in bout 2, and 106.6 +/- 10.1 mmol/kg dry wt in bout 3. These events coincided with an increase in the mitochondrial oxidation state, as reflected by a fall in mitochondrial NADH/NAD, indicating that muscle lactate production during exercise was not an O2-dependent process in our subjects. During exercise the primary factor regulating PDHa transformation was probably intracellular Ca2+. In contrast, the primary regulatory factors causing greater PDHa during recovery were lower ATP/ADP and NADH/NAD and increased concentrations of pyruvate and H+. Greater PDHa during recovery facilitated continued oxidation of the lactate load between exercise bouts.

Interaction of decreased arterial PO2 and exercise on carbohydrate metabolism in the dog

To determine the mechanism by which low arterial PO2 (PaO2) affects muscle carbohydrate (CHO) metabolism during exercise, dogs inhaled gas consisting of 0.21 (NO; n = 6) or 0.11 (LO; n = 6) inspired oxygen fraction (FIO2) during rest and 150 min of moderate treadmill exercise. Limb arteriovenous difference and isotopic ([3H]- and [14C]glucose) methods were used to assess muscle carbohydrate metabolism: PaO2 was reduced by approximately 50% in LO vs. NO, but limb O2 uptake was similar. Glucose disappearance was increased during rest (13 +/- 2 vs. 19 +/- 1 mumol.kg-1.min-1) and exercise (23 +/- 4 vs. 36 +/- 6 mumol.kg-1.min-1 at 150 min) in LO vs. NO, but arterial glucose was unchanged because hepatic glucose production was increased similarly. Limb glucose and pyruvate oxidation (derived from vein [14C]lactate specific activity) rates were elevated about twofold during rest and exercise in LO vs. NO. Estimated limb glycogenolysis increased at rest (21 +/- 9 vs. 96 +/- 23 mumol/min) and during exercise (70 +/- 21 vs. 184 +/- 41 mumol/min at 150 min) in LO vs. NO. The %CO2 and %lactate from glucose in LO were about twofold the values in NO in rest and exercise. The %CO2 from pyruvate was greater and free fatty acid levels were lower, suggesting reduced fat metabolism in LO. Arterial lactate and pyruvate levels were elevated during rest and the initial 30 min of exercise, even though net limb outputs were no greater. Lactate-to-pyruvate ratios and pH were similar in LO and NO during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse Ubiquitous Mitochondrial Creatine Kinase: Gene Organization and Consequences from Inactivation in Mouse Embryonic Stem Cells

Individual members of the creatine kinase isoenzyme family (CK; EC 2.7.3.2), which play a prominent role in energy homeostasis, are encoded by four separate nuclear genes. We have isolated and characterized the complete mouse UbCKmit gene, the product of which is ubiquitously expressed and is located in the intermembrane space of mitochondria. Transcription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features with genes encoding housekeeping proteins. These include a high G/C content, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 recognition sequences. In addition, a binding site for HIP1, hormone-responsive elements, and three Mt-motifs, known as boxes shared between nuclear genes encoding mitochondrial proteins, were identified. To study the functional role of the UbCKmit protein, we have inactivated both UbCKmit alleles in mouse embryonic stem (ES) cells. UbCKmit-deficient cells, obtained by consecutive rounds of gene targeting using homologous recombination and drug selection-driven gene conversion events, show no obvious growth disadvantage or abnormal differentiation potential. Activities of mitochondrial cytochrome c oxidase and citrate synthase, as well as the rate of pyruvate oxidation, showed values equal to wild-type cells, indicating a normal aerobic metabolism. Mitochondria of in vivo differentiated knock-out cells were structurally intact, as demonstrated by electron microscopy. Approaches to study the role of the UbCKmit gene further are discussed.

Metabolic specialization of mitochondria from scallop phasic muscles

In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol O2 min-1 mg-1 mitochondrial protein at 25°C, while malate and glutamine were oxidized at ∼ 75% and proline at ∼ 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent Km, app=0.013 mM) than at pH 7.0 (Km, app=0.026 mM) while the affinity for ADP (Km, app=0.015 mM) and that for glutamate (Km, app=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.

13C-nuclear magnetic resonance spectroscopy studies of hepatic glucose metabolism in normal subjects and subjects with insulin-dependent diabetes mellitus.

To determine the effect of insulin-dependent diabetes mellitus (IDDM) on rates and pathways of hepatic glycogen synthesis, as well as flux through hepatic pyruvate dehydrogenase, we used 13C-nuclear magnetic resonance spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units of hepatic glycogen, in combination with acetaminophen to sample the hepatic UDP-glucose pool and phenylacetate to sample the hepatic glutamine pool, during a hyperglycemic-hyperinsulinemic clamp using [1-13C]-glucose. Five subjects with poorly controlled IDDM and six age-weight-matched control subjects were clamped at a mean plasma glucose concentration of approximately 9 mM and mean plasma insulin concentrations approximately 400 pM for 5 h. Rates of hepatic glycogen synthesis were similar in both groups (approximately 0.43 +/- 0.09 mumol/ml liver min). However, flux through the indirect pathway of glycogen synthesis (3 carbon units-->-->glycogen) was increased by approximately 50% (P < 0.05), whereas the relative contribution of pyruvate oxidation to TCA cycle flux was decreased by approximately 30% (P < 0.05) in the IDDM subjects compared to the control subjects. These studies demonstrate that patients with poorly controlled insulin-dependent diabetes mellitus have augmented hepatic gluconeogenesis and relative decreased rates of hepatic pyruvate oxidation. These abnormalities are not immediately reversed by normalizing intraportal concentrations of glucose, insulin, and glucagon and may contribute to postprandial hyperglycemia.

ARE MITOCHONDRIA SUBJECT TO EVOLUTIONARY TEMPERATURE ADAPTATION?

Thermal tolerance and the respiratory properties of isolated red muscle mitochondria were investigated in Oreochromis alcalicus grahami from the alkaline hot-springs, Lake Magadi, Kenya. Populations of O. a. grahami were resident in pools at 42.8 &deg;C and migrated into water reaching temperatures of 44.8 &deg;C for short periods. The maximum respiration rates of mitochondria with pyruvate as substrate were 217 and 284 natom O mg-1 mitochondrial protein min-1 at 37 &deg;C and 42 &deg;C, respectively (Q10=1.71). Fatty acyl carnitines (chain lengths C8, C12 and C16), malate and glutamate were oxidised at 70&shy;80 % of the rate for pyruvate. In order to assess evolutionary temperature adaptation of maximum mitochondrial oxidative capacities, the rates of pyruvate and palmitoyl carnitine utilisation in red muscle mitochondria were measured from species living at other temperatures: Notothenia coriiceps from Antarctica (-1.5 to +1 &deg;C); summer-caught Myoxocephalus scorpius from the North Sea (10&shy;15 &deg;C); and Oreochromis andersoni from African lakes and rivers (22&shy;30 &deg;C). State 3 respiration rates had Q10 values in the range 1.8&shy;2.7. At the lower lethal temperature of O. andersoni (12.5 &deg;C), isolated mitochondria utilised pyruvate at a similar rate to mitochondria from N. coriiceps at 2.5 &deg;C (30 natom O mg-1 mitochondrial protein min-1). Rates of pyruvate oxidation by mitochondria from M. scorpius and N. coriiceps were similar and were higher at a given temperature than for O. andersoni. At their normal body temperature (-1.2 &deg;C), mitochondria from the Antarctic fish oxidised pyruvate at 5.5 % and palmitoyl-dl-carnitine at 8.8 % of the rates of mitochondria from the hot-spring species at 42 &deg;C. The results indicate only modest evolutionary adjustments in the maximal rates of mitochondrial respiration in fish living at different temperatures.

Alteration of Pyruvate Metabolism in African Trypanosomes During Differentiation from Bloodstream into Insect Forms

In the presence of glucose and ample oxygen, insect form African trypanosomes release pyruvate more than 100-fold more slowly than do bloodstream forms. This rate decrease could not be accounted for simply by an increased mitochondrial pyruvate oxidation rate as inhibiting mitochondrial respiration increases pyruvate efflux to rates only 2-3% of that observed for bloodstream form trypanosomes. Alternatively, decreased pyruvate efflux from insect form trypanosomes could not be accounted for by decreased pyruvate transporter activity, which, surprisingly, was nearly as high in insect form trypanosomes as reported by us earlier for bloodstream forms (J. P. Barnard, B. Reynafarje, and P. L. Pedersen (1993) J. Biol. Chem. 268, 3654-3661). Rather, the low pyruvate efflux rate appears to be due primarily to reduced levels of the enzyme pyruvate kinase, which, in contrast to conclusions of an earlier study, is readily detected in insect form trypanosomes in the absence of added activators at an activity level about 4% of that found in bloodstream forms. Insect form pyruvate kinase seems to be located in the cytosol and exhibits kinetic profiles and constants nearly identical to those reported by us earlier for the bloodstream form enzyme (J. P. Barnard, and P. L. Pedersen (1988) Mol. Biochem. Parasitol. 31, 141-148). It is suggested that the reduced levels of pyruvate kinase, and hence the reduced pyruvate efflux rates, in insect form trypanosomes result from down regulation of the gene encoding the cytosolic enzyme.

Mitochondrial pyruvate transport in working guinea-pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 μM-1.2 mM α-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, α-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO 2 production from [1- 14C]pyruvate or [U- 14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 μmol/min per g dry mass at S 0.5 ≈ 120 μ M , which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of ≈ 32 μ mol/min per g dry mass at 37° C . Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 μM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O 2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H + symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

Effects of pH and Temperature on the Kinetics of Pyruvate Oxidation by Muscle Mitochondria from Rainbow Trout (Oncorhynchus mykiss)

We measured the kinetics of pyruvate oxidation by mitochondria of rainbow trout (Oncorhynchus mykiss) red muscle and the kinetics of pyruvate decarboxylation by partially purified pyruvate dehydrogenase (PDH) from the same tissue. The measurements were done at three temperatures (8°, 15°, and 22°C) and at two pH conditions, a stable pH and an adjusted pH, that is, a pH that mimics the cellular pH adjustments with body temperature (ΔpH/ΔT= -0.03°C⁻¹). At constant pH, a change in temperature from 15° to 8°C decreased the apparent Km of mitochondria for pyruvate. However, when pH covaried with temperature, the apparent Km did not change. The Q10 for the Vmax of pyruvate oxidation was 2.5 between 8° and 15°C and 0.89 between 15° and 22°C (adjusted pH). Temperature significantly affected the pyruvate kinetics of PDH when pH was maintained constant (7.8) but not when pH covaried with temperature. Thus, pH regulation would minimize the functional impact of changes in temperature for both PDH and mitochondria. Comparison of the pyruvate kinetics suggests that PDH partially determines the mitochondrial affinity for pyruvate as well as its sensitivity to temperature and pH. Between 8° and 15°C, the Q10 of the Vmax of PDH was lower than that of intact mitochondria, suggesting that PDH did not limit the maximal rates of mitochondrial pyruvate oxidation. The thermal sensitivity of mitochondrialpyruvate oxidation was lower than that of the aerobic scope of the trout.

Postnatal development of pyruvate oxidation in quadriceps muscle of the rat.

In order to evaluate the age dependency of enzymes involved in the energy-generating system, skeletal muscle specimens from rats of different ages were investigated for several mitochondrial enzymes. [1-14C]pyruvate (+/- ADP) oxidation rates and pyruvate dehydrogenase complex (PDHC) activity increased significantly from low early values during the neonatal period to nearly adult values at the end of the suckling period. Other enzymes of the pyruvate oxidation route such as citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC detected a clear increase in the intensity of the bands of the alpha subunits of E1 (pyruvate dehydrogenase) and E2 (dihydrolipoyl transacetylase) within the first 3 weeks of life. The ratio between the individual PDHC proteins indicated that E1 alpha, the regulatory subunit of the multienzyme complex, is the most rapidly increasing protein with age.

147 POSTNATAL DEVELOPMENT OF PYRUVATE OXIDATION IN SKELETAL MUSCLE OF THE RAT

Age dependency of enzymes involved in the energy generating system was investigated in 600 g supernatants of quadriceps muscle from rats of different ages. (14-C) pyruvate oxidation rates (± ADP) increased a significantly from low early values in the neonatal period (321 ± 113 nmol/h/mg prot.) to nearly adult values at the end of the suckling period (859 ± 199 nmol/h/mg prot.) (p < 0.01). Pyruvate dehydrogenase complex (PDHC), citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC indicated that E1α, the regulatory subunit of the multi-enzyme complex, is ihe most rapidly increasing protein with age. This study demonstrates a clear increase in mitochondrial oxidative capacity in rat muscle up until weaning which is in accordance with the known differententiation pattern of skeletal muscle fibers.

Inhibition of hepatic propionyl-CoA synthetase activity by organic acids: Reversal of propionate inhibition of pyruvate metabolism

Intracellular accumulation of propionyl-CoA is associated with impairment of important hepatic metabolic pathways. Since propionate absorbed from the intestine can be converted to propionylCoA in the liver, inhibition of propionyl-CoA synthesis from propionate and CoA may provide a strategy for decreasing toxicity from plasma propionate. Therefore, inhibition of propionyl-CoA formation by several organic acids was investigated. In isolated, solubilized mitochondria, octanoate, butyrate, salicylate and p-nitrobenzoate inhibited propionyl-CoA synthesis. Octanoate was the most potent inhibitor of propionyl-CoA synthetase activity and had a K i of 58 μM. In isolated hepatocytes, octanoate inhibited propionate oxidation in a concentration-dependent manner. Consistent with previous studies, propionate (1.0 mM) inhibited the rates of 14CO 2 formation from [1- 14C]pyruvate (10 mM) to 55% of the control values in the hepatocyte system. Octanoate (0.8 mM) had no effect on [1- 14C]pyruvate oxidation under control conditions, but increased 14CO 2 formation from pyruvate to 88% of the control values in the presence of 1.0 mM propionate. Reversal of propionate inhibition of pyruvate oxidation by octanoate was associated with a 44% decrease in hepatocyte propionyl-CoA content. In contrast, while pyruvate oxidation rates were decreased to 53% of control rates in the presence of 10 mM propionylcarnitine, octanoate stimulated pyruvate oxidation under these conditions only to 67% of control levels. In conclusion, mitochondrial propionyl-CoA synthetase activity and hepatocyte propionylCoA accumulation can be inhibited by octanoate with consequent decreased propionate oxidation and toxicity in intact hepatocytes. The reversal by octanoate of propionate's inhibition of cellular metabolism may be useful in reducing tissue toxicity from circulating propionate.

Separation of enzymatically distinct populations of trout hepatocytes

Two different approaches were chosen to investigate the heterogeneity of rainbow trout (Oncorhynchus mykiss) hepatocytes. (i) Hepatocytes were isolated following selective liver infusion with a short pulse of digitonin from either the portal or the hepatic vein. Cell isolates, presumably enriched in cells from either "periportal" or "perivenous" liver regions, were then compared with cells obtained from the nondigitonized part of the same liver (control). No significant differences between hepatocytes were found in the activities of six key metabolic enzymes or in the rate of pyruvate oxidation. All cell isolates were responsive to glucagon, glucagon-like peptide, and insulin (all at 10 nM). If the liver was digitonized through the hepatic vein, resulting cell isolates revealed higher rates of gluconeogenesis from pyruvate, and gluconeogenic flux was more responsive to glucagon (at 10 nM) than controls. The experimental evidence for metabolic zonation in trout liver is weak. (ii) Enzymatically different subpopulations of hepatocytes were successfully separated by Percoll gradient centrifugation after isolation of cells by the conventional collagenase perfusion technique. Two prominent subpopulations of hepatocytes differed in the activities of glycogen phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, glutamate dehydrogenase, phosphoenolpyruvate carboxykinase, and alanine and aspartate aminotransferases, but not in their content of glycogen. However, the relative distribution of enzymes did not follow the mammalian pattern of perivenous or periportal hepatocytes. These data support cellular heterogeneity in the trout liver, with gluconeogenesis and oxidative metabolism dominating in some hepatocytes, whereas others display a relatively larger scope for glycogen breakdown, glycolysis, and amino acid metabolism.

Regional differences of substrate oxidation capacity in rat hearts: Effects of extra load and endurance training

Male rats, aged 17 weeks at the end of experiments, were divided into four groups. Two groups lived in normal cage conditions with or without extra load (20% of the body weight) and two groups were trained by running with or without extra load for 8 weeks. Oxidation rates of succinate, glutamate + malate, palmitoylcarnitine, and pyruvate, and the activities of lactate dehydrogenase, citrate synthase, isocitrate dehydrogenase and cytochrome oxidase were measured in homogenates of the right ventricle and in those of the subendocardial and subepicardial layers of the left ventricle. Oxidation rates of succinate and palmitoylcarnitine tended to be higher in the subendocardium than in the subepicardium of sedentary control animals (p less than 0.1 and p less than 0.05, respectively). Transmural differences of succinate and palmitoylcarnitine oxidation rates were even more clear after running training (p less than 0.01 and p less than 0.05, respectively), after carrying extra load (p less than 0.001 and p less than 0.001, respectively) and after training carrying extra load (p less than 0.001 and p less than 0.05, respectively). Training also enhanced pyruvate oxidation rate in the subendocardium. Oxidation rates of all substrates were lower in the right ventricle than in the left ventricle. In control animals there were no regional differences in the myocardial enzyme activities and the training- or extra-load-induced changes were modest compared with the changes in the oxidation rates. The most significant change was the training-induced enhancement in the lactate dehydrogenase activity of the subendocardium (p less than 0.001 vs subepicardium). These results show greater subendocardial than subepicardial oxidation rates of certain substrates in the normal heart. These results also suggest that the myocardium adapts to increased work by increasing the subendocardial oxidation rate of some but not all substrates, indicating further that there may be qualitative mitochondrial differences in the different regions of the heart.