The role of malate in exercise-induced enhancement of mitochondrial respiration

Liver mitochondria isolated from rats immediately after exercise oxidize substrates more rapidly than do mitochondria from resting animals. In both fed and fasted rats, a 1-h period of exercise resulted in increased concentrations of malate in their livers and in the mitochondria isolated therefrom. This increase occurred in both untrained and exercise-trained rats. Because mitochondrial malate is known to facilitate mitochondrial uptake of other carboxylic substrates, it seems likely that the increased mitochondrial malate is responsible for the increased rate of oxidation. Rats injected with small amounts of malate (4.6 mumol/100 g body wt) yielded liver mitochondria with increased malate concentration and increased rates of oxidation of citrate, alpha-ketoglutarate, and succinate. The beta adrenergic antagonist propranolol (0.25 mg/100 g body wt) and the alpha 1 antagonist prazosin (same dose) did not abolish the effect of exercise on mitochondrial malate concentration or substrate oxidation.     

The role of malate in hormone-induced enhancement of mitochondrial respiration

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.     

The role of malate in regulating the rate of mitochondrial respiration in vitro

Depletion of endogenous malate by preincubation of mitochondria at 30 degrees C in substrate-free media sharply decreases the rate of citrate oxidation and inhibits mitochondrial respiration in the presence of pyruvate and alpha-ketoglutarate. Addition of catalytic amounts of endogenous malate and its production via succinate oxidation promote rapid oxidation of citrate and pyruvate in the mitochondria and abolishes the lag period with alpha-ketoglutarate Malate increases the rate of membrane potential generation after addition of citrate, pyruvate or alpha-ketoglutarate to mitochondrial suspensions. Studies with controlled malate concentrations revealed that the changes in malate concentrations observed in the mitochondria in the presence of gluconeogenesis-inducing hormones may be due to the influence of these hormones on mitochondrial oxidation.     

Antioxidant enzyme systems in rat liver and skeletal muscle. Influences of selenium deficiency, chronic training, and acute exercise

The influences of selenium deficiency (Se-D), chronic training, and an acute bout of exercise on hepatic and skeletal muscle antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), as well as glutathione S-transferase (GST) and tissue lipid peroxidation, were investigated in post-weaning male Sprague-Dawley rats. Se-D per se depleted GPX in both liver and skeletal muscle but had no effect on SOD or catalase activity. One hour of treadmill running (20 m/min, 0% grade and 27 m/min, 15% grade for untrained and trained rats, respectively) significantly elevated hepatic catalase and cytosolic SOD activity; more prominent activations were found in the Se-D or untrained rats, whereas skeletal muscle antioxidant enzymes were little affected. Ten weeks of training (1 h/day, 5 days/week at 27 m/min, 15% grade) increased hepatic mitochondrial SOD by 23% (P less than 0.05) in Se-D rats. Both hepatic mitochondrial and cytosolic GPX were decreased by training whereas GPX was increased twofold in skeletal muscle mitochondria. Se-independent GPX was elevated by training only in the skeletal muscle mitochondria of Se-D rats. Lipid peroxidation (malondialdehyde formation) was increased by an acute bout of exercise in hepatic mitochondria of the untrained rats and in skeletal muscle mitochondria of the Se-D rats. These data indicate that antioxidant enzymes in liver and skeletal muscle are capable of adapting to selenium deficiency and exercise to minimize oxidative injury caused by free radicals.     

Stimulation of mitochondrial functions by glucagon treatment, starvation and by treatment of isolated mitochondria with glycogen-bound enzymes

Liver mitochondria isolated from rats starved overnight, or fed rats injected with glucagon, exhibited a similar increase of the respiration rate with succinate (by 30-40%) and glutamate plus malate (by 20-30%), as compared to mitochondria from control fed animals. The content of mitochondrial adenine nucleotides was elevated by 30-45% by glucagon treatment or starvation. Mitochondrial respiration and citrulline synthesis were stimulated by 30-40% when mitochondria isolated from fed rats were briefly preincubated with the extract from liver glycogen granules, ATP and MgCl2. This effect was abolished by heating the extract at 100 degrees C.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.     

Synthesis of phosphoenolpyruvate from propionate in sheep liver

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or alpha-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas alpha-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of alpha-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ;malic' enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate-dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate-malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.     

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.     

Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline

The respiratory control and rate of oxidation of exogenous NADH in vitro by liver mitochondria from vitamin E deficient rats were studied as a means of providing information concerning possible mitochondrial membrane alterations due to the deficiency.When mitochondria were aged at different temperatures for various periods of time, half-maximal inhibition of respiratory control occurred at lower temperatures and shorter aging periods in deficient mitochondria than in normal ones. Also, respiratory control was lost more rapidly in deficient mitochondria than in normal ones in the presence of either digitonin or low (hypotonic) concentrations of mannitol.Microsomes, both freshly prepared and boiled, dramatically lowered respiratory control and the effect was greater in the deficient mitochondria. Bovine serum albumin overcame the suppressed respiratory control, and exogenously added fatty acids mimiced the action of the microsomes.NADH oxidation by normal mitochondria proceeded slowly in isotonic media, while mitochondria of vitamin E deficient rats oxidized NADH much more rapidly. When mitochondria were subjected to ultrasonic disruption or incubated in hypotonic media, the rates of NADH oxidation by both types of mitochondria were similar.Respiratory decline associated with oxidation of β-hydroxybutyrate by the deficient mitochondria was decreased by including in the medium either a high concentration of NAD⁺, 0.5 mm oxalacetate, or 2 mm aspartate plus 1 mm α-ketoglutarate. This observation, plus the finding of similar activities of malate dehydrogenase and glutamic-oxalacetic transminase in normal and deficient livers, suggests that the action of each was due to an elevation of the mitochondrial NAD⁺/NADH ratio via a malate shuttle and cytoplasmic and mitochondrial glutamic-oxalacetate transaminase. It is postulated that the marked mitochondrial respiratory decline in the deficient rats is attributed to a limiting availability of NAD⁺ and a low ratio of NAD⁺ to NADH.     

Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism

In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P < 0.05) and decreased in PTU-treated (2.0 +/- 0. 3 vs. 5.2 +/- 1; P < 0.05) rats. Aralar1 mRNA levels were unchanged in T3-treated and somewhat decreased in PTU-treated (7.1 +/- 1.0 vs. 9.3 +/- 0.1, P < 0.05) rats. Citrin mRNA levels were decreased in T3-treated and unchanged in PTU-treated rats. EAAT1 protein levels (arbitrary units) in T3-treated cardiac mitochondria were increased compared with controls (8.9 +/- 0.4 vs. 5.9 +/- 0.6; P < 0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found between PTU-treated and control cardiac mitochondria. Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.     

Synthesis of N-acetyl-l-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport

1. The synthesis and efflux of N-acetyl-l-aspartate from brain mitochondria of rats of different ages has been studied. 2. Brain mitochondrial State 3 (+ADP) respiration rate, using 10mm-glutamate and 2.5mm-malate as substrates, increases during the suckling period and reaches approx. 50% of the adult value at 17 days after birth [adult State 3 respiration rate=160+/-7ng-atoms of O/min per mg of mitochondrial protein(mean+/-s.d.; n=3)]. 3. The influence of 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate on aspartate efflux from brain mitochondira from rats of different ages oxidizing glutamate and malate was studied. In all cases the aspartate efflux in State 3 was greater than in State 4, but, whereas the aspartate efflux in State 3 increased as the animals developed, that of State 4 showed only a small increase. However, the rate of aspartate efflux in the presence of pyruvate or 3-hydroxybutyrate as well as glutamate and malate was approx. 60-65% of that in the presence of glutamate and malate alone. 4. An inverse relationship between aspartate efflux and N-acetylaspartate efflux was observed with adult rat brain mitochondria oxidizing 10mm-glutamate and 2.5mm-malate in the presence of various pyruvate concentrations (0-5mm). 5. N-Acetylaspartate efflux by brain mitochondria of rats of different ages was studied in States 3 and 4, utilizing 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate as acetyl-CoA sources. A similar pattern of increase during development was seen in State 3 for N-acetylaspartate efflux as for aspartate efflux (see point 3 above). Also only very small increases in N-acetylaspartate efflux occurred during development in State 4.6. Rat brain mitochondria in the presence of iso-osmotic N-acetylaspartate showed some swelling which was markedly increased in the presence of malate. 7. It is concluded that N-acetylaspartate may be synthesized and exported from both neonatal and adult rat brain mitochondria. It is proposed that the N-acetylaspartate is transported by the dicarboxylic acid translocase and may be an additional mechanism for mitochondrial/cytosolic carbon transport to that of citrate.     

Influence of mitochondrial membrane fatty acid composition on proton leak and H2O2 production in liver

Mitochondrial membrane fatty acid composition has been proposed to play a role in determining mitochondrial proton leak rate. The purpose of this study was to determine if feeding rats diets with different fatty acid sources produces changes in liver proton leak and H(2)O(2) production. Six-month-old male FBNF(1) rats were fed diets with a primary fat source of either corn or fish oil for a 6-month period. As expected, diet manipulations produced substantial differences in mitochondrial fatty acid composition. These changes were most striking for 20:4n6 and 22:6n3. However, proton leak and phosphorylation kinetics as well as lipid and protein oxidative damage were not different (P > 0.10) between fish and corn oil groups. Metabolic control analysis, however, did show that control of both substrate oxidation and phosphorylation was shifted away from substrate oxidation reactions to increased control by phosphorylation reactions in fish versus corn oil groups. Increased mitochondrial H(2)O(2) production was observed in corn versus fish oil-fed rats when mitochondria were respiring on succinate alone or on either succinate or pyruvate/malate in the presence of antimycin A. These results show that mitochondrial H(2)O(2) production and the regulation of oxidative phosphorylation are altered in liver mitochondria from rats consuming diets with either fish or corn oil as the primary lipid source.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

Effect of training on H(2)O(2) release by mitochondria from rat skeletal muscle

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.     

Differential long-term effects of d-chloramphenicol on the biogenesis of mitochondria in normal and regenerating rat liver

1. Normal and partially hepatectomized rats (150g) were injected daily with d-chloramphenicol (20mg) for a period of 4 weeks, in order to investigate whether defective mitochondria could be induced in vivo in higher organisms as in yeast, and to measure the degree of inhibition of the mitochondrial function thus obtained. 2. The antibiotic did not affect growth and increased the amount of liver protein without changing the mitochondrial yield. 3. The respiration of isolated mitochondria from regenerated liver (regeneration completed) with succinate, α-oxo-glutarate, isocitrate and malate, was decreased in the chloramphenicol-treated rats, whereas in normal liver the antibiotic increased the mitochondrial oxygen consumption with succinate and did not significantly change the respiration with other substrates. 4. Mitochondrial cytochromes and respiratory enzymes were also decreased in amount in regenerated liver from the treated rats and enhanced in normal liver. 5. The protein specific radioactivities of most mitochondrial and microsomal subfractions, 30min after an injection of [¹⁴C]leucine, were decreased in regenerated liver under the action of chloramphenicol. Conversely, the incorporation of [¹⁴C]leucine into proteins of most subfractions in incubations of liver slices was enhanced in the case of normal rats treated with the antibiotic. 6. It is concluded that in regenerated liver chloramphenicol induces functionally defective mitochondria by inhibiting their biogenesis, whereas in normal liver the stimulation of respiration and protein synthesis is probably a secondary detoxication response.     

Induction of endogenous uncoupling protein 3 suppresses mitochondrial oxidant emission during fatty acid-supported respiration

Uncoupling protein 3 (UCP3) expression increases dramatically in skeletal muscle under metabolic states associated with elevated lipid metabolism, yet the function of UCP3 in a physiological context remains controversial. Here, in situ mitochondrial H(2)O(2) emission and respiration were measured in permeabilized fiber bundles prepared from both rat and mouse (wild-type) gastrocnemius muscle after a single bout of exercise plus 18 h of recovery (Ex/R) that induced a approximately 2-4-fold increase in UCP3 protein. Elevated uncoupling activity (i.e. GDP inhibitable) was evident in Ex/R fibers only upon the addition of palmitate (known activator of UCP3) or under substrate conditions eliciting substantial rates of H(2)O(2) production (i.e. respiration supported by succinate or palmitoyl-L-carnitine/malate but not pyruvate/malate), indicative of UCP3 activation by endogenous reactive oxygen species. In mice completely lacking UCP3 (ucp3(-/-)), Ex/R failed to induce uncoupling activity. Surprisingly, when UCP3 activity was inhibited by GDP (rats) or in the absence of UCP3 (ucp3(-/-)), H(2)O(2) emission was significantly (p < 0.05) higher in Ex/R versus non-exercised control fibers. Collectively, these findings demonstrate that the oxidant emitting potential of mitochondria is increased in skeletal muscle during recovery from exercise, possibly as a consequence of prolonged reliance on lipid metabolism and/or altered mitochondrial biochemistry/morphology and that induction of UCP3 in vivo mediates an increase in uncoupling activity that restores mitochondrial H(2)O(2) emission to non-exercised, control levels.     

The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells.

The possibility that hormones control hepatic gluconeogenesis via the regulation of the rate of mitochondrial pyruvate carboxylation was investigated with the use of suspensions of liver cells isolated from fasted rats. The mitochondria prepared from liver cells were judged in good condition as they exhibited satisfactory phosphorus-oxygen and respiratory control ratios and transported Ca2+ and K+ ions in an energy-dependent manner. Addition of glucagon, epinephrine, or cyclic adenosine 3':5'-monophosphate to liver cells caused a 50 to 80% increase in the rate of glucose synthesis from lactate. When mitochondria were isolated from the cells after treatment with these agonists, they displayed 2- to 3-fold increases in the rate of pyruvate carboxylation, pyruvate decarboxylation, and pyruvate uptake. These mitochondrial changes are similar to those obtained in hepatic mitochondria prepared from intact, hormone-treated rats. The mitochondrial responses were specific for agents that stimulated gluconeogenesis; no response occurred with 5'-AMP or cyclic adenosine 2':3'-monophosphate. In the cell suspensions, the dose response curves for the activation of mitochondrial pyruvate metabolism and for increased glucose synthesis from L-lactate were coincident with four different agonists. The mitochondrial changes resulting from stimulation with glucagon developed in 1 to 2 min after the rise in cyclic adenosine 3':5'-monophosphate and occurred at least as early as the increase in the rate of gluconeogenesis. When the intracellular level of cyclic adenosine 3':5'-monophosphate returned to basal values, the rates of mitochondrial pyruvate carboxylation and glucose synthesis also declined to control levels. It is concluded that the rate of mitochondrial pyruvate metabolisms can be increased by hormones and cyclic nucleotides and that control of mitochondrial pyruvate carboxylation is an important regulatory site of hepatic gluconeogenesis.