Myocardial function and energy substrate metabolism in the insulin-resistant JCR:LA corpulent rat.

Alterations in myocardial energy substrate utilization contribute to the development of cardiomyopathic changes in insulin-dependent and non-insulin-dependent diabetic rats. Energy substrate utilization and contractile function, however, have not been characterized in insulin-resistant diabetes. In this study, we studied these parameters in the insulin-resistant obese JCR:LA-cp rat homozygous for the corpulent gene (cp/cp). Homozygous (+/+) or heterozygous (+/cp) lean non-insulin-resistant rats were used as controls. Isolated working hearts from cp/cp and lean control rats were perfused with Krebs-Henseleit buffer containing either 11 mM [U-14C]glucose and 0.4 mM palmitate or 11 mM glucose and 0.4 mM [1-14C]palmitate. Unlike control hearts, hearts from cp/cp rats were found to require high doses of insulin and Ca2+ concentrations of less than or equal to 1.75 mM to maintain mechanical function. In the presence of 2,000 microU/ml insulin, contractile function from cp/cp rat hearts was not depressed in the presence of either 1.25 or 1.75 mM Ca2+. Steady-state glucose oxidation rates in hearts perfused with 1.25 mM Ca2+ and 2,000 microU/ml insulin were 811 +/- 86 (SE) and 612 +/- 51 nmol.min-1.g dry wt-1 in cp/cp and control rats, respectively. Palmitate oxidation was 307 +/- 47 and 307 +/- 47 nmol.min-1.g dry wt-1 in cp/cp and lean control hearts, respectively. Under these perfusion conditions, 40% of myocardial ATP production was derived from glucose, whereas 60% was derived from palmitate in both cp/cp and control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.     

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.     

Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

Metabolic effects of treadmill exercise training on the diabetic heart.

This study determined whether exercise training in rats would prevent the accumulation of lipids and depressed glucose utilization found in hearts from diabetic rats. Diabetes was induced by intravenous streptozotocin (60 mg/kg). Trained diabetic rats were run on a treadmill for 60 min, 27 m/min, 10% grade, 6 days/wk for 10 wk. Training of diabetic rats had no effect on glycemic control but decreased plasma lipids. In vivo myocardial long-chain acylcarnitine, acyl-CoA, and high-energy phosphate levels were similar in sedentary control, sedentary diabetic, and trained diabetic groups. The levels of myocardial triacylglycerol were similar in sedentary control and diabetic rats but decreased in trained diabetic rats. Hearts were perfused with buffer containing diabetic concentrations of glucose (22 mM) and palmitate (1.2 mM). D-[U-14C] glucose oxidation rates (14CO2 production) were depressed in hearts from sedentary diabetic rats relative to sedentary control rats. Hearts from trained diabetic rats exhibited increased glucose oxidation relative to those of sedentary diabetic rats, but this improvement was below that of the sedentary control rats. [9,10(-3)H]palmitate oxidation rates (3H2O production) were identical in all three groups. These findings suggest that exercise training resulted in a partial normalization of myocardial glucose utilization in diabetic rats.     

Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts

The objective of this study was to determine the contribution of myocardial triglycerides to overall ATP production in isolated working rat hearts. Endogenous lipid pools were initially prelabeled (pulsed) by perfusing hearts for 60 min with Krebs-Henseleit buffer containing 1.2 mM [1-14C]palmitate. During a subsequent 60-min period (chase), hearts were perfused with either no fat, low fat (0.4 mM [9,10-3H] palmitate), or high fat (1.2 mM [9,10-3H]palmitate). All buffers contained 11 mM glucose. During the "chase," 14CO2 production (a measure of endogenous fatty acid oxidation) and 3H2O production (a measure of exogenous fatty acid oxidation) were determined. Oxidative rates of endogenous fatty acids during the chase were 279 +/- 50, 88 +/- 14, and 88 +/- 8 nmol of [14C]palmitate oxidized per g dry weight.min in the no fat, low fat, and high fat groups, respectively, compared to exogenous palmitate oxidation rates of 0, 361 +/- 68, and 633 +/- 60 nmol of [3H]palmitate/g dry weight.min, in the no fat, low fat, and high fat groups, respectively. Endogenous [14C]palmitate oxidation rates were matched by loss of [14C]palmitate from endogenous myocardial triglycerides. Overall triglyceride content decreased during the no fat and low fat chase perfusion but did not change during the high fat chase. Loss of triglyceride [14C]palmitate during the high fat chase was matched by incorporation of exogenous [3H]palmitate in triglycerides. In a second series of perfusions, three groups of hearts were perfused under similar conditions, except that unlabeled palmitate was used during the "pulse" and that 11 mM [2-3H/U-14C]glucose and unlabeled palmitate was present during the chase. During the chase, both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured. Rates of glucose oxidation were inversely related to the fatty acid concentration in the perfusate (1257 +/- 158, 366 +/- 40, and 124 +/- 26 nmol of glucose oxidized per min.g dry weight in the no fat, low fat, and high fat groups, respectively), while rates of glycolysis were not significantly different between these groups. Calculation of overall ATP production from both oxidative and glycolytic sources determined that even in the presence of high concentrations of fatty acids, myocardial triglyceride turnover can provide over 11% of steady state ATP production in the aerobically perfused heart. In the absence of fatty acids, myocardial triglyceride fatty acids can become the major energy substrate of the heart.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

Phosphorylation of cardiac protein kinase B is regulated by palmitate

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 microU/ml insulin or 100 microU/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor C(2)-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation.     

Glucose and insulin improve cardiac efficiency and postischemic functional recovery in perfused hearts from type 2 diabetic (db/db) mice

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 microU/ml insulin (GI), or 33 mM glucose and 900 microU/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications.     

Differences in myocardial ischemic tolerance between 1- and 7-day-old rabbits.

Between 1 and 7 days of life, the newborn rabbit heart shifts from predominantly using carbohydrates to predominantly using fatty acids as an energy substrate. We therefore used isolated working hearts from 1- or 7-day-old rabbits to determine the effects of fatty acids on myocardial glucose use and the ability of hearts to recover following various periods of transient no-flow ischemia. One-day-old hearts were perfused via the inferior vena cava and ejected buffer through the cannulated aorta and pulmonary artery. Seven-day-old hearts were perfused via the left atrium and ejected buffer through the cannulated aorta. To measure glucose use, hearts were perfused with 11 mM [3H, 14C]glucose, 3% albumin, and 500 microU insulin/mL, in the presence or absence of 0.4 mM palmitate. In the absence of fatty acids, glycolytic rates were similar in 1- and 7-day-old hearts, whereas glucose oxidation rates were 5 times greater in 7-day-old hearts. Palmitate did not have any major effects on overall glucose use in 1-day-old hearts, but did markedly inhibit glycolysis and glucose oxidation in 7-day-old hearts. A series of hearts were also subjected to periods (25-60 min) of no-flow ischemia, followed by 30 min of aerobic reperfusion. In the absence of palmitate, 1-day-old hearts subjected to ischemic periods of up to 60 min recovered some degree of mechanical function during reperfusion, whereas 7-day-old rabbit hearts failed to recover if hearts were subjected to ischemic periods of 35 min or longer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of type 2 diabetic db/db mice with a novel PPARgamma agonist improves cardiac metabolism but not contractile function

Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-gamma that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward K+ currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.     

Estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of hearts from ovariectomized female rats

Women less than 50 years of age, the majority of whom are likely premenopausal and exposed to estrogen, are at greater risk of a poor short-term recovery after myocardial ischemia than men and older women. Since estrogen enhances non-cardiac lipid utilization and increased lipid utilization is associated with poor post-ischemic heart function, we determined the effect of estrogen replacement on post-ischemic myocardial function and fatty acid oxidation. Female Sprague-Dawley rats, either intact (n = 15) or ovariectomized and treated with 17beta-estradiol (0.1 mg x kg(-1) x day(-1), s.c., n = 14) or corn oil vehicle (n = 16) for 5 weeks, were compared. Function and fatty acid oxidation of isolated working hearts perfused with 1.2 mM [9,10-3H]palmitate, 5.5 mM glucose, 0.5 mM lactate, and 100 mU/L insulin were measured before and after global no-flow ischemia. Only 36% of hearts from estrogen-treated rats recovered after ischemia compared with 56% from vehicle-treated rats (p > 0.05, not significant), while 93% of hearts from intact rats recovered (p < 0.05). Relative to pre-ischemic values, post-ischemic function of estrogen-treated hearts (26.3 +/- 10.1%) was significantly lower than vehicle-treated hearts (53.4 +/- 11.8%, p < 0.05) and hearts from intact rats (81.9 +/- 7.0%, p < 0.05). Following ischemia, fatty acid oxidation was greater in estrogen-treated hearts than in the other groups. Thus, estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of isolated working hearts from ovariectomized female rats.     

Insulin fails to alter plasma LCFA metabolism in muscle perfused at similar glucose uptake

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 microM palmitate and [1-(14)C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 microU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 +/- 0.6 vs. 11.0 +/- 0.5 micromol x g(-1) x h(-1); P > 0.05). Total palmitate uptake and oxidation were not significantly different (P > 0.05) between the G (10.1 +/- 1.0 and 0.8 +/- 0.1 nmol x min(-1) x g(-1)) and I (10.2 +/- 0.6 and 1.1 +/- 0.2 nmol. min(-1) x g(-1)) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion (P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups (P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.     

Impact of lactate in the perfusate on function and metabolic parameters of isolated working rat heart

The goal of this study was to investigate the effect of 1 mM exogenous lactate on cardiac function, and some metabolic parameters, such as glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation, in isolated working rat hearts. Hearts from male Sprague-Dawley rats were isolated and perfused with 5 mM glucose, 1.2 mM palmitate, and 100 μU/ml insulin with or without 1 mM lactate. The rates of glycolysis, glucose, lactate, and fatty acid oxidation were determined by supplementing the buffer with radiolabeled substrates. Cardiac function was similar between lactate+ and lactate− hearts. Glycolysis was not affected by 1 mM lactate. The addition of lactate did not alter glucose oxidation rates. Interestingly, palmitate oxidation rates almost doubled when 1 mM lactate was present in the perfusate. This study suggests that subst rate supply to the heart is crucially important when evaluating the data from metabolic studies.     

Evidence of separate pathways for lactate uptake and release by the perfused rat heart

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.     

Glycolysis and glucose oxidation during reperfusion of ischemic hearts from diabetic rats

Stimulationg of glucose oxidation by dichloroacetate (DCA) treatment is beneficial during recovery of ischemic hearts from non-diabetic rats. We therfore determined whether DCA treatment of diabetic rat hearts (in which glucose use is extremely low), increases recovery of function of hearts reperfused following ischemia. Isolated working hearts from 6 week streptozotocindiabetic rats were perfused with 11 mM [2-³H/U-¹⁴C]glucose, 1.2 mM palmitate, 20 μU/ml insulin, and subjected to 30 min of no flow ischemia followed by 60 min reperfusion. Heart function (expressed as the product of heart rate and peak systolic pressure), prior to ischemia, was depressed in diabetic hearts compared to controls (HR × PSP × 10^?3 was 18.2 ± 1 and 24.3 ± 1 beats/mm Hg/min in diabetic and control hearts respectively) but recover to pre-ischemic levels following ischemia, whereas recovery of control of control hearts was significantly decreased (17.8 ± 1 and 11.9 ± 3 beats/mm Hg/min in diabetic and control hearts respectively). This enhanced recovery of diabetic rat hearts occurred even though glucose oxidation during reperfusion was significantly reduced as compared to controls (39 ± 6 and 208 ± 42 nmol/min/g dry wt, in diabetic and control hearts respectively). Glycolytic rate (³G₂O production) during reperfusion were similar in diabetic and control hearts (1623 ± 359 and 2071 ± 288 nmol/min/g dry wt, respectively). If DCA (1 mM) was added at reperfusion, hearts from control animals exhibited a significant improvement in function (HR × PSP × 10^? recovered to 20 ± 4 beats/mm Hg/min) that was accompanied by a 4-fold increase in glucose oxidation (from 208 ± 42 to 753 ± 111 nmol/min/g dry wt). DCA was without effect on functional recovery of diabetic rat hearts during reperfusion but did significantly increase glucose oxidation from 39 ± 6 to 179 ± 44 nmol/min/g dry wt). These data suggests that, unlike control hearts, low glucose oxidation rates are not an important factor in reperfusion recovery of previouskly ischemic diabetic rat hearts.     

The effects of chronic trimetazidine treatment on mechanical function and fatty acid oxidation in diabetic rat hearts

Clinical and experimental evidence suggest that increased rates of fatty acid oxidation in the myocardium result in impaired contractile function in both normal and diabetic hearts. Glucose utilization is decreased in type 1 diabetes, and fatty acid oxidation dominates for energy production at the expense of an increase in oxygen requirement. The objective of this study was to examine the effect of chronic treatment with trimetazidine (TMZ) on cardiac mechanical function and fatty acid oxidation in streptozocin (STZ)-diabetic rats. Spontaneously beating hearts from male Sprague-Dawley rats were subjected to a 60-minute aerobic perfusion period with a recirculating Krebs-Henseleit solution containing 11 mmol/L glucose, 100 muU/mL insulin, and 0.8 mmol/L palmitate prebound to 3% bovine serum albumin (BSA). Mechanical function of the hearts, as cardiac output x heart rate (in (mL/min).(beats/min).10-2), was deteriorated in diabetic (73 +/- 4) and TMZ-treated diabetic (61 +/- 7) groups compared with control (119 +/- 3) and TMZ-treated controls (131 +/- 6). TMZ treatment increased coronary flow in TMZ-treated control (23 +/- 1 mL/min) hearts compared with untreated controls (18 +/- 1 mL/min). The mRNA expression of 3-ketoacyl-CoA thiolase (3-KAT) was increased in diabetic hearts. The inhibitory effect of TMZ on fatty acid oxidation was not detected at 0.8 mmol/L palmitate in the perfusate. Addition of 1 mumol/L TMZ 30 min into the perfusion did not affect fatty acid oxidation rates, cardiac work, or coronary flow. Our results suggest that higher expression of 3-KAT in diabetic rats might require increased concentrations of TMZ for the inhibitory effect on fatty acid oxidation. A detailed kinetic analysis of 3-KAT using different concentrations of fatty acid will determine the fatty acid inhibitory concentration of TMZ in diabetic state where plasma fatty acid levels are increased.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

Accelerated triacylglycerol turnover kinetics in hearts of diabetic rats include evidence for compartmented lipid storage

Triacylglycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode (13)C- NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14,16-(13)C(8)]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for >or=2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. Consequently, TAG turnover rate was determined by fitting an exponential model to enrichment data rather than conventional two-point linear analysis. In the high-substrate group, both turnover rate (DR 820+/- 330, NR 190 +/-150 nmol.min(-1).g(-1) dry wt; P< 0.001) and [TAG] content (DR 78 +/-10, NR 32+/- 4 micromol/g dry wt; P< 0.001) were greater in the diabetic group. At lower substrate concentrations, turnover was greater in diabetics (DR 530+/-300, NR 160+/- 30; P<0.05). However, this could not be explained by simple mass action, because [TAG] content was similar between groups [DR 34+/- 7, NR 39+/- 9 micromol/g dry wt; not significant (NS)]. Consistent with exponential enrichment data, (13)C fractional enrichment of TAG was lower in diabetics (low- substrate groups: DR 4+/-1%, NR 10+/- 4%, P<0.05; high-substrate groups: DR 8+/- 3%, NR 14+/- 9%, NS), thereby supporting earlier speculation that TAG is compartmentalized in the diabetic heart.