Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.     

Metabolism of long-chain polyunsaturated fatty acids in isolated cardiac myocytes

The uptake and integrated intracellular metabolism of (n - 6) and (n - 3) polyunsaturated fatty acids was studied in isolated rat cardiac myocytes and in the perfused heart. Labeled linolenic acid (18:3(n - 3)) uptake and its subsequent metabolism into carbon dioxide as well as acylation into lipids was nonsaturable over a substrate range of 0.02 to 0.4 mM. [1-14C]Linoleic acid (18:2(n - 6)), dihomo-gamma-linolenic acid (20:3(n - 6)) and arachidonic acid (20:4(n - 6)) were transported into myocytes at rates similar to those for linolenic acid. Conversely both [1-14C]-gamma-linolenic acid (18:3(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were taken up at a slower rate. Oxidation of 18:3(n - 6) was 4-5-fold greater when compared with C18-C20 polyunsaturated fatty acids. When myocytes were incubated with labeled 18:2(n - 6), 18:3(n - 6), 18:3(n - 3), 20:4(n - 6) or 20:5(n - 3), it was not possible to detect any desaturation or chain-elongation products. Identical results were obtained when hearts were perfused with 1-14C-labeled linoleic acid.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Partitioning of polyunsaturated fatty acid oxidation between mitochondria and peroxisomes in isolated rat hepatocytes studied by HPLC separation of oxidation products

The extent of mitochondrial and peroxisomal contribution to beta-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO(2), while peroxisomal beta-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO(2) were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly beta-oxidized only one cycle in peroxisomes.     

Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts

Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form.     

Stimulation of fatty acid oxidation in myocytes by phosphodiesterase inhibitors and adenosine analogues.

The effect of various phosphodiesterase inhibitors, and adenosine analogues on palmitate oxidation, were studied in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a concentration of 250 microM, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. The specific A1-agonist, N6-cyclopentyladenosine, increased palmitate oxidation by 56%, at a concentration of 250 microM. Moreover, the nucleoside transport inhibitor, S-(P-Nitrobenzyl-)6-thioinosine, increased palmitate oxidation by 40%, at a concentration of 100 microM. These data suggest that the stimulation of palmitate oxidation by enoximone and adenosine analogues may be mediated via the inhibition of the uptake and/or the oxidation of glucose in myocytes.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.     

Novel inhibitors of fatty acid oxidation as potential metabolic modulators

We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles, benzoxazoles and introducing small alkyl substituents into the piperazine ring resulted in analogues with enhanced inhibitory activity against 1-(14)[C]-palmitoyl-CoA oxidation in isolated rat heart mitochondria (6, IC(50)=70 nM; 25, IC(50)=23 nM).     

Apparent in vivo retroconversion of dietary arachidonic to linoleic acid in essential fatty acid-deficient rats

Essential fatty acid-deficient rats were fed ethyl [U-14C]arachidonate (308 dpm/nmol) and when a decrease in the transepidermal water loss was seen, the epidermal sphingolipids, acylglucosylceramide and acylceramide were isolated. [14C]Linoleic acid (approx. 130 dpm/nmol) was present in both lipid classes, while the substrate was only detected in the former. These results intimate that in vivo retroconversion of arachidonic to linoleic acid can be induced in the rat.     

Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase

In an attempt to clarify why the brain oxidizes fatty acids poorly or not at all, the activities of beta-oxidation enzymes present in rat brain and rat heart mitochondria were measured and compared with each other. Although the apparent Km values and chain-length specificities of the brain and heart enzymes are similar, the specific activities of all but one brain enzyme are between 4 and 50% of those observed in heart mitochondria. The exception is 3-ketoacyl-CoA thiolase (EC 2.3.1.16) whose specific activity in brain mitochondria is 125 times lower than in heart mitochondria. The partially purified brain 3-ketoacyl-CoA thiolase was shown to be catalytically and immunologically identical with the heart enzyme. The low rate of fatty acid oxidation in brain mitochondria, estimated on the basis of palmitoylcarnitine-supported respiration and [1-14C]palmitoylcarnitine degradation to be less than 0.5 nmol/min/mg of protein, may be the consequence of the low activity of 3-ketoacyl-CoA thiolase. Inhibition of [1-14C]palmitoylcarnitine oxidation by 4-bromocrotonic acid proves the observed oxidation of fatty acids in brain to be dependent on 3-ketoacyl-CoA thiolase and thus to occur via beta-oxidation. Since the reactions catalyzed by carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA synthetase (EC 6.2.1.3) do not seem to restrict fatty acid oxidation in brain, it is concluded that the oxidation of fatty acids in rat brain is limited by the activity of the mitochondrial 3-keto-acyl-CoA thiolase.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system.

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart

Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [(14)C]glucose and 0.4 mM [(3)H]palmitate (low-fat/low-glucose buffer) or 20 mM [(14)C]glucose and 1.2 mM [(3)H]palmitate (high-fat/high-glucose buffer) +/-100 microU/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 +/- 85 to 1,150 +/- 147 nmol x g dry wt(-1) x min(-1) (P < 0. 05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P < 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P < 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low-fat/low-glucose buffer (9 +/- 1 nmol x g dry wt(-1) x min(-1)) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl-CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts.     

14CO2 production is no adequate measure of [14C]fatty acid oxidation

Palmitate oxidation was comparatively assayed in various cell-free and cellular systems by 14CO2 production and by the sum of 14CO2 and 14C-labeled acid-soluble products. The 14CO2 production rate was dependent on incubation time and amount of tissue in contrast to the total oxidation rate. The 14CO2 contribution to the oxidation rate of [1-14C]palmitate varied with homogenates from 1% with rat liver to 28% with rat kidney and amounted to only 2-4% with human muscles. With cellular systems the 14CO2 contribution varied between 20% in human fibroblasts and 70% in rat muscles and myocytes. Addition of cofactors increased the oxidation rate, but decreased the 14CO2 contribution. Various conditions appeared also to influence to a different extent the 14CO2 production and the total oxidation rate with rat tissue homogenates and with rat muscle mitochondria. Incorporation of radioactivity from [1-14C]palmitate into protein was not detectable in cell-free systems and only 2-3% of the sum of 14CO2 and 14C-labeled acid-soluble products in cellular systems. Assay of 14CO2 and 14C-labeled acid-soluble products is a much more accurate and sensitive estimation of fatty acid oxidation than assay of only 14CO2.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.