Effect of starvation and exercise on actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Starvation does not change the actual activity per g of tissue of the branched-chain 2-oxo acid dehydrogenase in skeletal muscles, but affects the total activity to a different extent, depending on the muscle type. The activity state (proportion of the enzyme present in the active state) does not change in diaphragm and decreases in quadriceps muscle. Liver and kidney show an increase of both activities, without a change of the activity state. In heart and brain no changes were observed. Related to organ wet weights, the actual activity present in the whole-body muscle mass decreases on starvation, whereas the activities present in liver and kidney do not change, or increase slightly. Exercise (treadmill-running) of untrained rats for 15 and 60 min causes a small increase of the actual activity and the activity state of the branched-chain 2-oxo acid dehydrogenase complex in heart and skeletal muscle. Exercise for 1 h, furthermore, increased the actual and the total activity in liver and kidney, without a change of the activity state. In brain no changes were observed. The actual activity per g of tissue in skeletal muscle was less than 2% of that in liver and kidney, both before and after exercise and starvation. Our data indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and to a smaller extent in kidney and skeletal muscle in fed, starved and exercised rats.     

Postnatal development of the actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues

Actual and total activities of the branched-chain 2-oxo acid dehydrogenase complex were determined in homogenates of quadriceps muscle, heart, liver, kidney and brain from rats of 0-70 days age. All rat tissues except quadriceps muscle showed a marked increase of total activity between 0 and 21 days, heart and kidney also after weaning. The actual activity rose after birth in liver, kidney and brain and after weaning in liver, kidney and heart. The activity state was always about 100% in liver and varied between 40-60% in kidney and brain, 10-23% in heart and 6-12% in quadriceps muscle. The actual activities measured indicate, that the degradation of branched-chain 2-oxo acids mainly takes place in the liver of the newborn, suckling and young-adult rat.     

Effects of diet and of alloxan-diabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues.

The total activities (sum of active and inactive forms) of branched-chain 2-oxo acid dehydrogenase complex in tissues of normal rats fed on a standard diet were (unit/g wet wt.): liver, 0.82; kidney, 0.77; heart, 0.57; hindlimb skeletal muscles, 0.034. Total activity was decreased in liver by 9%- or 0%-casein diets and by 48 h starvation, but not by alloxan-diabetes. Total activities were unchanged in kidney and heart. The amount of active form of the complex (in unit/g wet wt. and as % of total) in tissues of normal rats fed on standard diet was: liver, 0.45, 55%; kidney, 0.55, 71%; heart, 0.03, 5%; skeletal muscle less than 0.007, less than 20% (below lower limit of assay). The concentration of the active form of the complex was decreased in liver and kidney, but not in heart, by low-protein diets, 48 h starvation and alloxan-diabetes. In heart muscle alloxan-diabetes increased the concentration of active complex. The concentration of activator protein (which activates phosphorylated complex without dephosphorylation) in liver and kidney was decreased by 70-90% by low-protein diets and 48 h starvation. Alloxan-diabetes decreased activator protein in liver, but not in kidney. Evidence is given that in tissues of rats fed on a normal diet approx. 70% of whole-body active branched chain complex is in the liver and that the major change in activity occasioned by low-protein diets is also in the liver.     

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.     

The effect of starvation on branched-chain 2-oxo acid oxidation in rat muscle.

Oxidative-decarboxylation rates of branched-chain amino acids in rat hemidiaphragm and of branched-chain 2-oxo acids in hemidiaphragm, soleus muscle and heart slices of 110-120 g rats were increased considerably by 3-4 days of starvation, when they were calculated from the specific radioactivity in the medium. When the supply from endogenous protein degradation to the oxidation-precursor pool was severely limited by transaminase inhibitors, oxidative-decarboxylation rates of branched-chain 2-oxo acids rose significantly. Since this apparent increase was relatively larger in preparations from fed rats than from 3-days-starved rats, the differences in oxidation rates with nutritional state became less or even not significant. With rat heart the smaller dilution of the oxidation precursor pool after starvation is in accordance with the reported decrease in protein breakdown. Since protein degradation increases with starvation in skeletal muscles, we suggest that the amino acid pool arising from protein degradation is more segregated from the oxidation precursor pool in muscles from starved than from fed rats. We conclude that starvation increases branched-chain amino acid and 2-oxo acid oxidation in skeletal and cardiac muscle considerably less than has been suggested by previous studies.     

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial beta-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.     

Mitochondrial 2-oxoacid dehydrogenase complexes of animal tissues

The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation. In addition, phosphorylated branched-chain complex is reactivated, apparently without dephosphorylation, by a protein or protein-associated factor present in liver and kidney mitochondria but not in heart or skeletal muscle mitochondria. Interconversion of the branched-chain complex may adjust the degradation of branched-chain amino acids in different tissues in response to supply. Phosphorylation is inhibited by branched-chain ketoacids, ADP and TPP. The pyruvate dehydrogenase complex is almost totally inactivated (99%) by starvation or diabetes, the kinase reactions being accelerated by products of fatty acid oxidation and by a protein or protein-associated factor induced by starvation or diabetes. There are three sites of phosphorylation, but only sites 1 and 2 are inactivating. Site 1 phosphorylation accounts for 98% of inactivation except during dephosphorylation when its contribution falls to 93%. Sites 2 and 3 are only fully phosphorylated when the complex is fully inactivated (starvation, diabetes). Phosphorylation of sites 2 and 3 inhibits reactivation by phosphatase. The phosphatase reaction is activated by Ca2+ (which may mediate effects of muscle work) and possibly by uncharacterized factors mediating insulin action in adipocytes.     

Evidence for the presence of a prostaglandin E 2 -9-keto reductase in rat organs

Antibodies directed toward PGF_2α which cross react with PGE₂ only slightly were used to detect conversion of PGE₂ to PGF_2α by homogenates of several rat tissues. This conversion by rat heart homogenates was demonstrated to be reversible, lost after trypsin digestion, and inhibited by several sulfhydryl blocking agents. The activity of the rat heart homogenate was precipitable by ammonium sulphate, was not dialyzable, and was 50% destroyed when the homogenate was incubated at 50° for 5 min. In the rat, the heart had the highest activity, followed by the kidney, brain, and liver. Negligible activity was found in smooth muscle, skeletal muscle, and whole blood of rat.     

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.     

Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.     

Interaction of various metabolites and agents with branched-chain 2-oxo acid oxidation in rat and human muscle in vitro

The interaction of various metabolites and agents with the 14CO2 production from 0.1 mM [1-14C]-labelled 2-oxoisocaproate (KIC) and 2-oxoisovalerate (KIV) was studied in rat and human heart and skeletal muscle preparations. Glucose and carnitine had no effect in any of the studied systems; palmitate gave a small increase of KIC oxidation only in soleus muscle. With rat hemidiaphragms a considerable decrease was found in the presence of high concentrations of a competitive branched-chain 2-oxo acid and of pyruvate, and in the presence of ketone bodies. A considerable increase was found in the presence of the branched-chain 2-oxo acid dehydrogenase kinase inhibitor 2-chloroisocaproate and the transminase inhibitor amino-oxyacetate. 2-Oxoglutarate increased and clofibric acid decreased only KIC oxidation. Divergent effects were given by intermediates of the degradation route of KIC and KIV and by monocarboxylate translocator inhibitors. The observed interactions are discussed and related to regulatory mechanisms which are known to affect the branched-chain 2-oxo acid dehydrogenase complex.     

Tissue metabolism and enzyme activities in the rodent Heterocephalus glaber, a poor temperature regulator.

1. Tissue oxygen uptake and enzyme activities were investigated in the naked mole rat, Heterocephalus glaber, a mammal notable for its low body temperature and metabolism and poor temperature regulating ability. 2. Q10 for O2 uptake of Heterocephalus crude liver homogenates ranged from 1.91 for the temperature interval 25-30 degrees C to 1.76 within the range 30-38 degrees C, values similar to those reported for typical homoiotherms. 3. Km pyruvate of lactate dehydrogenase in heart muscle had the same temperature dependence in the mole rat and mouse. 4. O2 uptake and cytochrome oxidase activity of skeletal muscle were higher for mole rat than mouse. The reverse was true for heart muscle. Brain and liver O2 uptake showed similar values for both species, while kidney O2 uptake was highest in the mouse. 5. Pyruvate kinase activity in heart and skeletal muscle was higher in mouse than mole rat, suggesting a greater reliance on glycolysis in the former. 6. Na+, K+ -ATPase activity of liver and kidney was 60% higher in mouse than mole rat, while brain was 30% higher in mouse. 7. The results indicate that the effects of temperature on tissue metabolism in the mole rat conform to those in typical homoiotherms. The low body temperature and O2 uptake in the mole rat find no expression in the tissue respiratory capacity.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.     

Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.     

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.     

Changes in drug-metabolizing activities in the livers of suckling rats as a result of treatment of the lactating mothers with phenobarbitone and chlorpromazine

1. The activities in rat tissues of 3-oxo acid CoA-transferase (the first enzyme involved in acetoacetate utilization) were found to be highest in kidney and heart. In submaxillary and adrenal glands the activities were about one-quarter of those in kidney and heart. In brain it was about one-tenth and was less in lung, spleen, skeletal muscle and epididymal fat. No activity was detectable in liver. 2. The activities of acetoacetyl-CoA thiolase were found roughly to parallel those of the transferase except for liver and adrenal glands. The high activity in the latter two tissues may be explained by additional roles of thiolase, namely, the production of acetyl-CoA from fatty acids. 3. The activities of the two enzymes in tissues of mouse, gerbil, golden hamster, guinea pig and sheep were similar to those of rat tissues. The notable exception was the low activity of the transferase and thiolase in sheep heart and brain. 4. The activities of the transferase in rat tissues did not change appreciably in starvation, alloxan-diabetes or on fat-feeding, where the rates of ketone-body utilization are increased. Thiolase activity increased in kidney and heart on fat-feeding. 5. The activity of 3-hydroxybutyrate dehydrogenase did not change in rat brain during starvation. 6. The factors controlling the rate of ketone-body utilization are discussed. It is concluded that the activities of the relevant enzymes in the adult rat do not control the variations in the rate of ketone-body utilization that occur in starvation or alloxan-diabetes. The controlling factor in these situations is the concentration of the ketone bodies in plasma and tissues.     

The effect of SH-groups of cysteine on the rate of oxygen uptake by some homogenates of the rabbit tissues

The effect of SH-groups of cysteine on the rate of oxygen uptake by some homogenates of the rabbit tissues. Acta Physiol. Pol., 1977, 28 (6): 541-551. In the present experiments the effect of SH-groups of cysteine on the respiration rate of homogenates of kidney, liver, brain, myocardium and skeletal muscle was investigated in the rabbit. Using the Warburg's method of respirometry it was found that cysteine added to the incubation medium modifies oxygen uptake by the above-mentioned tissue homogenates and that this reaction depends both on the kind of tissue and cysteine concentration in the medium. Addition of cysteine to the incubation medium in the concentration of 0.1 mg/ml exerted only slight, insignificant influence on the tissue respiration but in the concentration of 0.4 mg/ml it increased the respiration rate in homogenates of kidney (by 30%), liver (by 55%) and skeletal muscle (by 59%). Cysteine added in the concentration of 0.8 mg/ml increased the respiration rate of all the examined tissue homogenates. The strongest effect of cysteine in this concentration was found in the liver and skeletal muscle homogenates (an increase in O2 uptake by 88% and 89% respectively) and the lowest in the myocardium (by 53%). Under control conditions (without cysteine) kidney homogenates had the highest oxygen consumption and skeletal muscle ones the lowest.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.