Maruf Saddik

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)

The role of acetyl-coenzyme A carboxylase (ACC) in regulating fatty acid oxidation was investigated in isolated fatty acid perfused working rat hearts. Overall fatty acid oxidation rates were determined by addition of 1.2 mM [3H]palmitate to the perfusate of hearts in which the endogenous triglyceride pool was prelabeled with [14C]palmitate. Rates of both exogenous and endogenous fatty acid oxidation were measured by simultaneous measurement of 3H2O and 14CO2 production, respectively. A second series of hearts were perfused under similar conditions except that [U-14C]glucose was present in the perfusate for measurement of glucose oxidation rates. Addition of dichloroacetate (DCA, 1 mM) to the perfusate resulted in a dramatic stimulation of glucose oxidation (a 411% increase), with a parallel decrease in fatty acid oxidation (from 305 +/- 51 to 206 +/- 40 nmol/g dry weight.min.unit work). DCA treatment increased the contribution of glucose oxidation to ATP production from 7.1 to 30.6%, while decreasing the contribution of overall fatty acid oxidation from 92.9 to 69.4%. Tissue levels of malonyl-CoA in hearts treated with DCA were higher compared to controls (14.0 +/- 0.6 and 10.0 +/- 0.7 nmol/g dry weight, respectively) and were negatively correlated (r = -0.85) with overall fatty acid oxidation rates. Acetyl-CoA levels were also significantly higher in DCA-treated hearts, and a positive correlation (r = 0.88) was seen between myocardial acetyl-CoA and malonyl-CoA levels. This suggests that DCA treatment increased the supply of acetyl-CoA for ACC. Western blots revealed the presence of both the 280-kDa (ACC-280) and the 265-kDa (ACC-265) isoforms of ACC in cardiac tissue, with a predominance of ACC-280. The activity of ACC extracted from hearts was similar in both groups when assayed under optimal conditions of acetyl-CoA and citrate. However, using affinity purified ACC, it was demonstrated that heart ACC (predominantly ACC-280) had a higher Km for acetyl-CoA than ACC isolated from white adipose tissue (predominantly ACC-265). We conclude that ACC is an important regulator of fatty acid oxidation in the heart and that acetyl-CoA supply is a key determinant of heart ACC-280 activity. As acetyl-CoA levels increase, ACC-280 is activated resulting in an increase in malonyl-CoA inhibition of fatty acid oxidation.

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.

Although myocardial triacylglycerol may be a potentially important source of fatty acids for β-oxidation in diabetes, few studies have measured triacylglycerol turnover directly in hearts from diabetic animals. In this study, myocardial triacylglycerol turnover was directly measured in isolated working hearts from streptozotocin-induced acutely diabetic rats. Hearts were initially perfused in the presence of 1.2 mM [ 14 C]palmitate and 11 mM glucose for 1 h (pulse) to label the endogenous lipid pools, followed by a 10-min washout perfusion. Hearts were then perfused for another hour (chase) with buffer containing 11 mM glucose ± 1.2 mM [ 3 H]palmitate. During the chase, both 14 CO 2 and 3 H 2 O production (measures of endogenous and exogenous fatty acid oxidation, respectively) were determined. A second series of hearts were perfused using the same protocol, except that unlabeled palmitate was used during the pulse and 11 mM [ 14 C(U),5- 3 H]glucose ± unlabeled palmitate was present during the chase. Both glycolysis ( 3 H 2 O production) and glucose oxidation ( 14 CO 2 production) rates were measured in this series. Myocardial triacylglycerol levels were significantly higher in the diabetic rat hearts (77.5 ± 4.6 vs. 33.7 ± 4.1 μmol fatty acid/g dry mass in control hearts). In diabetic rat hearts chased with 1.2 mM palmitate, triacylglycerol lipolysis was increased, although endogenous [ 14 C]palmitate oxidation rates were similar to control hearts and contributed 10.1% of overall ATP production. The majority of fatty acids derived from triacylglycerol lipolysis were released into the perfusate. In the absence of palmitate, both triacylglycerol lipolysis and endogenous [ 14 C]palmitate oxidation rates were significantly increased in diabetic rat hearts, compared with control. Under these conditions, triacylglycerol fatty acid oxidation contributed 70% of steady-state ATP production in diabetic rat hearts, compared with 34% in control hearts. These results demonstrate that in diabetic rat hearts myocardial triacylglycerol lipolysis is significantly increased and can readily be used as a source of fatty acids for mitochondrial β-oxidation.Key words: heart, triacylglycerols, fatty acid oxidation, glucose oxidation, glycolysis.