Accumulation Of Long-chain acyl-CoA Research Articles

Suppression of RCAN1 alleviated lipid accumulation and mitochondrial fission in diabetic cardiomyopathy

BackgroundAlthough metabolic disturbance is a characteristic of diabetic cardiomyopathy (DbCM), the detailed pathogenesis of DbCM remains unknown. MethodsWe used a heart transplantation (HTx) cohort to explore the effect of diabetes mellitus on heart failure (HF) progression dependent of myocardium. Microscopic and ultramicroscopic pathology were used to depict the pathological features of human myocardium of DbCM. We performed targeted metabolomics to characterize the metabolic phenotype of human DbCM. Transcriptomics data were analyzed and weighted gene co-expression network analysis was performed to explore the potential upstream regulator for metabolic remodeling of DbCM. In vivo and in vitro experiments were further conducted to demonstrate the therapeutic effects and molecular mechanisms. ResultsDbCM promoted the progression of HF and increased death or HF-rehospitalization after HTx. Lipid accumulation and mitochondrial fission were the obvious pathological features of DbCM myocardium. The concentrations of C14:0-CoA and C16:1-CoA were significantly increased in the myocardium, and they were positively correlated with the accelerated HF progression and RCAN1 expression in DbCM patients. Knockdown of RCAN1 improved cardiac dysfunction, lipid accumulation, and mitochondrial fission in db/db mice. In vitro studies showed that RCAN1 knockdown improved mitochondrial dysfunction in DbCM cardiomyocytes via the RCAN1-p-Drp1 Ser616 axis. ConclusionsDiabetes is associated with faster progression of HF and causes poor prognosis after HTx, accompanied by metabolic remodeling in the myocardium. Accumulation of long chain acyl-CoA in the myocardium is the metabolic hallmark of human DbCM and is associated with more rapid disease progression for DbCM patients. Upregulation of RCAN1 in the myocardium is associated with the metabolic signatures of DbCM and RCAN1 is a potential therapeutic target for DbCM.

Treatment with the 3-Ketoacyl-CoA Thiolase Inhibitor Trimetazidine Does Not Exacerbate Whole-Body Insulin Resistance in Obese Mice

There is a growing need to understand the underlying mechanisms involved in the progression of cardiovascular disease during obesity and diabetes. Although inhibition of fatty acid oxidation has been proposed as a novel approach to treat ischemic heart disease and heart failure, reduced muscle fatty acid oxidation rates may contribute to the development of obesity-associated insulin resistance. Our aim was to determine whether treatment with the antianginal agent trimetazidine, which inhibits fatty acid oxidation in the heart secondary to inhibition of 3-ketoacyl-CoA thiolase (3-KAT), may have off-target effects on glycemic control in obesity. We fed C57BL/6NCrl mice a high-fat diet (HFD) for 10 weeks before a 22-day treatment with the 3-KAT inhibitor trimetazidine (15 mg/kg per day). Insulin resistance was assessed via glucose/insulin tolerance testing, and lipid metabolite content was assessed in gastrocnemius muscle. Trimetazidine-treatment led to a mild shift in substrate preference toward carbohydrates as an oxidative fuel source in obese mice, evidenced by an increase in the respiratory exchange ratio. This shift in metabolism was accompanied by an accumulation of long-chain acyl-CoA and a trend to an increase in triacylglycerol content in gastrocnemius muscle, but did not exacerbate HFD-induced insulin resistance compared with control-treated mice. It is noteworthy that trimetazidine treatment reduced palmitate oxidation rates in the isolated working mouse heart and neonatal cardiomyocytes but not C2C12 skeletal myotubes. Our findings demonstrate that trimetazidine therapy does not adversely affect HFD-induced insulin resistance, suggesting that treatment with trimetazidine would not worsen glycemic control in obese patients with angina.

Inhibition of malonyl-CoA decarboxylase reduces the inflammatory response associated with insulin resistance

We previously showed that genetic inactivation of malonyl-CoA decarboxylase (MCD), which regulates fatty acid oxidation, protects mice against high-fat diet-induced insulin resistance. Development of insulin resistance has been associated with activation of the inflammatory response. Therefore, we hypothesized that the protective effect of MCD inhibition might be caused by a favorable effect on the inflammatory response. We examined if pharmacological inhibition of MCD protects neonatal cardiomyocytes and peritoneal macrophages against inflammatory-induced metabolic perturbations. Cardiomyocytes and macrophages were treated with LPS to induce an inflammatory response, in the presence or absence of an MCD inhibitor (CBM-301106, 10 μM). Inhibition of MCD attenuated the LPS-induced inflammatory response in cardiomyocytes and macrophages. MCD inhibition also prevented LPS impairment of insulin-stimulated glucose uptake in cardiomyocytes and increased phosphorylation of Akt. Additionally, inhibition of MCD strongly diminished LPS-induced activation of palmitate oxidation. We also found that treatment with an MCD inhibitor prevented LPS-induced collapse of total cellular antioxidant capacity. Interestingly, treatment with LPS or an MCD inhibitor did not alter intracellular triacylglycerol content. Furthermore, inhibition of MCD prevented LPS-induced increases in the level of ceramide in cardiomyocytes and macrophages while also ameliorating LPS-initiated decreases in PPAR binding. This suggests that the anti-inflammatory effect of MCD inhibition is mediated via accumulation of long-chain acyl-CoA, which in turn stimulates PPAR binding. Our results also demonstrate that pharmacological inhibition of MCD is a novel and promising approach to treat insulin resistance and its associated metabolic complications.

Organization and Function of the YsiA Regulon of Bacillus subtilis Involved in Fatty Acid Degradation

The organization and function of the Bacillus subtilis YsiA regulon involved in fatty acid degradation were investigated. Northern and primer extension analyses indicated that this regulon comprises five operons, i.e. lcfA-ysiA-B-etfB-A, ykuF-G, yhfL, yusM-L-K-J, and ywjF-acdA-rpoE. YusJ and AcdA, YsiB and YusL, and YusK presumably encode acyl-CoA dehydrogenases, 3-hydroxyl-CoA dehydrogenase/enoyl-CoA hydratase complexes, and acetyl-CoA C-acyltransferase, respectively, which are directly involved in the fatty acid beta-oxidation cycle. In addition, LcfA and YhfL are likely to encode long chain acyl-CoA ligases. On gel retardation and footprinting analyses involving the purified YsiA protein, we identified cis-sequences for YsiA binding (YsiA boxes) in the promoter regions upstream of ysiA, ykuF, yusL, yhfL, and ywjF, the equilibrium dissociation constants (K(d)) for YsiA binding being 20, 21, 37, 43, and 65 nm, respectively. YsiA binding was specifically inhibited by long chain acyl-CoAs with 14-20 carbon atoms, acyl-CoAs with 18 carbon atoms being more effective; out of long chain acyl-CoAs tested, monounsaturated oleoyl-CoA, and branched chain 12-metyltetradecanoyl-CoA were most effective. These in vitro findings were supported by the in vivo observation that the knock-out of acyl-CoA dehydrogenation through yusJ, etfA, or etfB disruption resulted in YsiA inactivation, probably because of the accumulation of long chain acyl-CoAs in the cells. Furthermore, the disruption of yusL, yusK, yusJ, etfA, etfB, or ykuG affected the utilization of palmitic acid, a representative long chain fatty acid. Based on this work, ysiA, ysiB, ykuF, ykuG, yhfL, yusM, yusL, yusK, yusJ, and ywjF can be renamed fadR, fadB, fadH, fadG, lcfB, fadM, fadN, fadA, fadE, and fadF.

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells

Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-κB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATP total/ADP total ratio by 26 ± 5% (mean ± SEM) and 15 ± 2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34 ± 5%, while oleate stimulated it by 12 ± 2%. α-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by α-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B 1. Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-κB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.

Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24 ± 6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N ε-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Δ ψ), ADP/O ratio, and the content of coenzyme Q 9, cytochromes b and a + a 3. Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C = 0.14) and phosphorylation fluxes ( C = 0.15), extra- ( C = 0.23) and intramitochondrial ( C = − 0.56) ATP/ADP ratios, and Δ ψ ( C = − 0.11). We conclude that although HFD induces accumulation of LCAC and N ε-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.

Mitochondrial Glycerol-3-phosphate Acyltransferase-1 Is Essential inLiver for the Metabolism of ExcessAcyl-CoAs

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis.

Kir6.2 polymorphisms sensitize beta-cell ATP-sensitive potassium channels to activation by acyl CoAs: a possible cellular mechanism for increased susceptibility to type 2 diabetes?

The commonly occurring E23K and I337V Kir6.2 polymorphisms in the ATP-sensitive potassium (KATP) channel are more frequent in Caucasian type 2 diabetic populations. However, the underlying cellular mechanisms contributing to the pathogenesis of type 2 diabetes remain uncharacterized. Chronic elevation of plasma free fatty acids observed in obese and type 2 diabetic subjects leads to cytosolic accumulation of long-chain acyl CoAs (LC-CoAs) in pancreatic beta-cells. We postulated that the documented stimulatory effects of LC-CoAs on KATP channels might be enhanced in polymorphic KATP channels. Patch-clamp experiments were performed on inside-out patches containing recombinant KATP channels (Kir6.2/SUR1) to record macroscopic currents. KATP channels containing Kir6.2 (E23K/I337V) showed significantly increased activity in response to physiological palmitoyl-CoA concentrations (100-1,000 nmol/l) compared with wild-type KATP channels. At physiological intracellular ATP concentrations (mmol/l), E23K/I337V polymorphic KATP channels demonstrated significantly enhanced activity in response to palmitoyl-CoA. The observed increase in KATP channel activity may result in multiple defects in glucose homeostasis, including impaired insulin and glucagon-like peptide-1 secretion and increased glucagon release. In summary, these results suggest that the E23K/I337V polymorphism may have a diabetogenic effect via increased KATP channel activity in response to endogenous levels of LC-CoAs in tissues involved in the maintenance of glucose homeostasis.

Overexpression of a Modified Human Malonyl-CoA Decarboxylase Blocks the Glucose-induced Increase in Malonyl-CoA Level but Has No Impact on Insulin Secretion in INS-1-derived (832/13) β-Cells

The long-chain acyl-CoA (LC-CoA) model of glucose-stimulated insulin secretion (GSIS) holds that secretion is linked to a glucose-induced increase in malonyl-CoA level and accumulation of LC-CoA in the cytosol. We have previously tested the validity of this proposal by overexpressing goose malonyl-CoA decarboxylase (MCD) in INS-1 cells, but these studies have been criticized due to: 1) the small insulin secretion response (2-4-fold) of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K(+) (K(ATP)) channel-independent pathway of GSIS in INS-1 cells, which has been implicated as the site at which lipids regulate insulin granule exocytosis; and 3) deletion of the N-terminal mitochondrial targeting sequence, but not the C-terminal peroxisomal targeting sequence in the goose MCD construct, raising the possibility that a significant fraction of the overexpressed enzyme was localized to peroxisomes. To address these outstanding concerns, INS-1-derived 832/13 cells, which exhibit robust K(ATP) channel-dependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lacking both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD Delta 5), resulting in large increases in cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD Delta 5 completely blocked the glucose-induced rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fatty acid oxidation. However, MCD overexpression had no effect on K(ATP) channel-dependent or -independent GSIS in 832/13 cells. Furthermore, combined treatment of 832/13 cells with AdCMV-MCD Delta 5 and triacsin C, an inhibitor of long chain acyl-CoA synthetase that reduces LC-CoA levels, did not impair GSIS. These findings extend our previous observations and are not consistent with the LC-CoA hypothesis as originally set forth.

Muscle lipid accumulation and protein kinase C activation in the insulin-resistant chronically glucose-infused rat.

Chronic glucose infusion results in hyperinsulinemia and causes lipid accumulation and insulin resistance in rat muscle. To examine possible mechanisms for the insulin resistance, alterations in malonyl-CoA and long-chain acyl-CoA (LCA-CoA) concentration and the distribution of protein kinase C (PKC) isozymes, putative links between muscle lipids and insulin resistance, were determined. Cannulated rats were infused with glucose (40 mg. kg(-1). min(-1)) for 1 or 4 days. This increased red quadriceps muscle LCA-CoA content (sum of 6 species) by 1.3-fold at 1 day and 1.4-fold at 4 days vs. saline-infused controls (both P < 0.001 vs. control). The concentration of malonyl-CoA was also increased (1.7-fold at 1 day, P < 0.01, and 2.2-fold at 4 days, P < 0.001 vs. control), suggesting an even greater increase in cytosolic LCA-CoA. The ratio of membrane to cytosolic PKC-epsilon was increased twofold in the red gastrocnemius after both 1 and 4 days, suggesting chronic activation. No changes were observed for PKC-alpha, -delta, and -theta. We conclude that LCA-CoAs accumulate in muscle during chronic glucose infusion, consistent with a malonyl-CoA-induced inhibition of fatty acid oxidation (reverse glucose-fatty acid cycle). Accumulation of LCA-CoAs could play a role in the generation of muscle insulin resistance by glucose oversupply, either directly or via chronic activation of PKC-epsilon.

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.

Comparision of the effects of orally administered linoleic acid, and its hydroperoxides and secondary autoxidation products on hepatic lipid metabolism in rats.

Linoleic acid, and its hydroperoxides and secondary autoxidation products were orally administered to rats (400mg/rat). Their effects on hepatic lipid metabolism were examined. Linoleic acid reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase. It decreased the CoASH level and caused the accumulation of long-chain acyl-CoA. Hydroperoxides changed the compositions of unsaturated fatty acids in the hepatic lipids and lowered the content of neutral lipids. Secondary products stimulated carnitine palmitoyltransferase and decreased the content of neutral lipids. They reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase, and the levels of CoASH and acetyl-CoA. Thus, the effect of secondary products was apparently different from those of linoleic acid and its hydroperoxides.

Metabolic disturbances during acute lack of oxygen: a short overview.

Oxygen deficiency results in several alterations in lipid metabolism in cardiac muscle. The primary events appear to involve a rise in mitochondrial NADH/NAD secondary to the low oxygen supply. The more reduced state of mitochondrial adenine nucleotides in turn inhibits β-oxidation of long chain fatty acids resulting in the accumulation of long-chain acyl CoA and acylcarnitine derivatives. These compounds accumulate in both the mitochondrial matrix and the cytosolic compartments, but acyl CoA is largely matrix and acylcarnitine is largely cytosolic. The rise in cytosolic acyl CoA may directly stimulate conversion of fatty acids to Triacylglycerol.

Involvement of long-chain acyl CoA in the antagonistic effects of halothane and L-carnitine on mitochondrial energy-linked processes

Incubation of rat liver mitochondria in the presence of halothane induced a consistent impairment of mitochondrial oxidative phosphorylation without significantly affecting the steady-state of transmembrane electrical potential. These alterations of mitochondrial energy-linked processes were associated with a consistent accumulation of long-chain acyl CoA. Addition of L-carnitine partially prevented the effects of halothane on oxidative phosphorylation and completely abolished the halothane-induced long-chain acyl CoA accumulation. The possibility is discussed that the damaging action of halothane on mitochondrial functions might be partially ascribed to the noxious action of the excess of long-chain acyl CoA induced the anesthetic.

Lipid intermediates in chronically volume-overloaded rat hearts. Effect of diffuse ischemia.

Tissue contents of intermediates of fatty acid metabolism were determined in isolated volume-overloaded rat hearts, 3 months after creation of an aorto-caval fistula. In the absence of any modification of blood carnitine, tissue levels of total carnitine were reduced by 33% in overloaded hearts compared to normal hearts. Total tissue CoA was unchanged. Fifteen minutes of whole-heart ischemia (i.e. a 50% reduction in coronary flow) did not increase levels of long-chain acyl esters of CoA and carnitine of the overloaded myocardium, in the presence of glucose as the only exogenous substrate. This was associated with lower than normal levels of long-chain acyl carnitine under normoxic conditions. The addition of exogenous palmitate (1.5 mM) resulted in an ischemia-induced accumulation of long-chain acyl-CoA and acyl carnitine in the overloaded heart although to a smaller extent than in the normal heart under similar perfusion conditions.

Rate of pyridine nucleotide oxidation and cytochrome oxidase interaction with intracellular oxygen in hearts from rats with compensated volume overload.

Volume overload was created in rats by means of an aortocaval fistula. The animals were sacrificed after the fistula had been in place for 1 month, at which time most of the animals were in the compensated stage of cardiac hypertrophy. Their hearts were excised and used for in vitro perfusions. Their ability to oxidize an excess of reduced pyridine nucleotide (state 5 to state 3 transition) was tested fluorometrically during recovery from a short period of anoxic perfusion. In a parallel series of experiments, the kinetics of myoglobin reoxygenation and cytochromeaa3 reoxidation were studied by means of double wave length recordings of myoglobin (581–621 nm) and cytochromeaa3 (606–626 nm) absorption changes. The sequential analysis of a series of reflectance spectra was also used to assess the range of mean intracellular oxygen concentrations (myoglobin O2-saturation) compatible with unlimited respiration (full oxidation of cytochromeaa3). Normoxic and anoxic patterns of adenine nucleotide phosphorylation, as well as the patterns of coenzyme A esterification were determined biochemically in order to assess the state of respiratory control at the level of the initial span of the respiratory chain. The results are as follows: (1) hearts from animals with compensated volume overload reoxidized an anoxia induced excess of NADH more rapidly than those of controls. (2) The improved oxidation of reduced pyridine nucleotide was not related to changes in coronary perfusion, since coronary flow rate (ml/min gdw) as well as the rate of myoglobin reoxydation were unchanged. Total tissue phosphocreatine contents and total tissue ATP/ADP·Pi ratio were comparable in control and overloaded hearts, both under normoxic conditions, and after 2 min of anoxia. (3) On the other hand, anoxic patterns of coenzyme A esterification differed significantly: while in control hearts, there was an accumulation of long chain acyl-CoA during anoxia, no such change was observed in overloaded hearts. (4) During recovery from anoxia, the preanoxic reduction degree of cytochromeaa3 appeared at lower level of intracellular meanpO2 in overloaded hearts, as compared with controls. This was suggested by the degree of myoglobin O2-saturation that was simultaneously detected.

The inhibition of alpha-ketoglutarate oxidation by fatty acids in rat liver mitochondria.

The oxidation of a-ketoglutarate in rat liver mito- chondria incubated in State 3 and in the uncoupled state in the absence and presence of oligomycin was inhibited by the simultaneous oxidation of fatty acids. Palmitylcarnitine inhibited a-ketoglutarate oxidation by up to 80% and octanoate by up to 30%. Various possibilities for the mechanism of this inhi- bition were experimentally evaluated. The inhibition was not associated with the damage of mitochondrial membranes due to the detergent action of fatty acids or with a restriction of a-ketoglutarate translocation. Also, a severe depletion of intramitochondrial CoASH did not occur. a-Ketoglutarate dehydrogenase extracted from liver mitochondria which was stabilized with rabbit serum was inhibited by (listed from the highest to the lowest potency) palmityl-CoA, NADH, succinyl-CoA, GTP, and ATP. The intramitochondrial content of these inhibi- tory metabolites was examined in mitochondria oxidiz- ing a-ketoglutarate in the absence and presence of fatty acids. The addition of octanoate increased the content of only one inhibitor, NADH. The addition of palmityl- carnitine resulted in an accumulation of long chain acyl-CoA and the elevation of the succinyl-CoA/CoASH ratio while the NADH content increased less than in the case of octanoate. A definitive correlation between the rate of a-keto- glutarate oxidation and the succinyl-CoA/CoASH ratio was not found at similar NADH levels in various ener- getic states of mitochondria. The significant suppres- sion of the process by palmitylcarnitine was not nec- essarily associated with the high succinyl-CoA/CoASH ratio but it was correlated with the level of long chain acyl-CoA. Hence, the strong inhibition of the a-ketoglutarate oxidation in rat liver mitochondria by long chain fatty acids is postulated to be primarily due to a direct effect of long chain acyl-CoA on a-ketoglutarate dehydrogen- ase. The elevation of the succinyl-CoA/CoASH ratio and NADH level as well as the competition for CoASH have only secondary importance for this inhibition. The much weaker inhibition of the process by medium chain fatty acids is mediated exclusively by the increase of NADH content. It has been demonstrated that the tricarboxylic acid cycle is suppressed during rapid /? oxidation of fatty acids in the