Maternal high fat diet induces early cardiac hypertrophy and alters cardiac metabolism in Sprague Dawley rat offspring.

BACKGROUND AND AIM: Maternal high fat diets (mHFD) have been associated with an increased offspring cardiovascular risk. Recently we found that the class IIa HDAC-MEF2 pathway regulates gene programs controlling fatty acid oxidation in striated muscle. This same pathway controls hypertrophic responses in the heart. We hypothesized that mHFD is associated with activation of signal controlling class II a HDAC activity and activation of genes involved in fatty acid oxidation and cardiac hypertrophy in offspring. METHODS AND RESULTS: Female Sprague Dawley rats were fed either normal fat diet (12%) or high fat diet (43%) three weeks prior to mating, remaining on diets until study completion. Hearts of postnatal day 1 (PN1) and PN10 pups were collected. Bioenergetics and respiration analyses were performed in neonatal ventricular cardiomyocytes (NVCM). In offspring exposed to mHFD, body weight was increased at PN10 accompanied by increased body fat percentage and blood glucose. Heart weight and heart weight to body weight ratio were increased at PN1 and PN10, and were associated with elevated signalling through the AMPK-class IIa HDAC-MEF2 axis. The expression of the MEF2-regulated hypertrophic markers ANP and BNP were increased as were expression of genes involved in fatty acid oxidation. However this was only accompanied by an increased protein expression of fatty acid oxidation enzymes at PN10. NVCM isolated from these pups exhibited increased glycolysis and an impaired substrate flexibility. CONCLUSION: Combined, these results suggest that mHFD induces signalling and transcriptional events indicative of reprogrammed cardiac metabolism and of cardiac hypertrophy in Sprague Dawley rat offspring.

Mitochondrial pharmacology: energy, injury and beyond.

While the mitochondrion has long fascinated biologists and the sheer diversity of druggable targets has made it attractive for potential drug development, there has been little success translatable to the clinic. Given the diversity of inborn errors of metabolism and mitochondrial diseases, mitochondrially mediated oxidative stress (myopathies, reperfusion injury, Parkinson's disease, ageing) and the consequences of disturbed energetics (circulatory shock, diabetes, cancer), the potential for meaningful gain with novel drugs targeting mitochondrial mechanisms is huge both in terms of patient quality of life and health care costs. In this themed issue of the British Journal of Pharmacology, we highlight the key directions of the contemporary advances in the field of mitochondrial biology, emerging drug targets and new molecules which are close to clinical application. Authors' contributions are diverse both in terms of species and organs in which the mitochondrially related studies are performed, and from the perspectives of mechanisms under study. Defined roles of mitochondria in disease are updated and previously unknown contributions to disease are described in terms of the interface between basic science and pathological relevance.

Mitochondrial fatty acid oxidation alterations in heart failure, ischaemic heart disease and diabetic cardiomyopathy.

Heart disease is a leading cause of death worldwide. In many forms of heart disease, including heart failure, ischaemic heart disease and diabetic cardiomyopathies, changes in cardiac mitochondrial energy metabolism contribute to contractile dysfunction and to a decrease in cardiac efficiency. Specific metabolic changes include a relative increase in cardiac fatty acid oxidation rates and an uncoupling of glycolysis from glucose oxidation. In heart failure, overall mitochondrial oxidative metabolism can be impaired while, in ischaemic heart disease, energy production is impaired due to a limitation of oxygen supply. In both of these conditions, residual mitochondrial fatty acid oxidation dominates over mitochondrial glucose oxidation. In diabetes, the ratio of cardiac fatty acid oxidation to glucose oxidation also increases, although primarily due to an increase in fatty acid oxidation and an inhibition of glucose oxidation. Recent evidence suggests that therapeutically regulating cardiac energy metabolism by reducing fatty acid oxidation and/or increasing glucose oxidation can improve cardiac function of the ischaemic heart, the failing heart and in diabetic cardiomyopathies. In this article, we review the cardiac mitochondrial energy metabolic changes that occur in these forms of heart disease, what role alterations in mitochondrial fatty acid oxidation have in contributing to cardiac dysfunction and the potential for targeting fatty acid oxidation to treat these forms of heart disease.

Glucagon and a glucagon-GLP-1 dual-agonist increases cardiac performance with different metabolic effects in insulin-resistant hearts.

BACKGROUND AND PURPOSE: The prevalence of heart disease continues to rise, particularly in subjects with insulin resistance (IR), and improved therapies for these patients is an important challenge. In this study we evaluated cardiac function and energy metabolism in IR JCR:LA-cp rat hearts before and after treatment with an inotropic compound (glucagon), a glucagon-like peptide-1 (GLP-1) receptor agonist (ZP131) or a glucagon-GLP-1 dual-agonist (ZP2495). EXPERIMENTAL APPROACH: Hearts from IR and lean JCR:LA rats were isolated and perfused in the working heart mode for measurement of cardiac function and metabolism before and after addition of vehicle, glucagon, ZP131 or ZP2495. Subsequently, cardiac levels of nucleotides and short-chain CoA esters were measured by HPLC. KEY RESULTS: Hearts from IR rats showed decreased rates of glycolysis and glucose oxidation, plus increased palmitate oxidation rates, although cardiac function and energy state (measured by ATP/AMP ratios) was normal compared with control rats. Glucagon increased glucose oxidation and glycolytic rates in control and IR hearts, but the increase was not enough to avoid AMP and ADP accumulation in IR hearts. ZP131 had no significant metabolic or functional effects in either IR or control hearts. In contrast, ZP2495 increased glucose oxidation and glycolytic rates in IR hearts to a similar extent to that of glucagon but with no concomitant accumulation of AMP or ADP. CONCLUSION AND IMPLICATIONS: Whereas glucagon compromised the energetic state of IR hearts, glucagon-GLP-1 dual-agonist ZP2495 appeared to preserve it. Therefore, a glucagon-GLP-1 dual-agonist may be beneficial compared with glucagon alone in the treatment of severe heart failure or cardiogenic shock in subjects with IR.

Enhanced glucose uptake via GLUT4 fuels recovery from calcium overload after ischaemia-reperfusion injury in sevoflurane- but not propofol-treated hearts.

BACKGROUND: So far, no study has explored the effects of sevoflurane, propofol, and Intralipid on metabolic flux rates of fatty acid oxidation (FOX) and glucose oxidation (GOX) in hearts exposed to ischaemia-reperfusion. METHODS: Isolated paced working rat hearts were exposed to 20 min of ischaemia and 30 min of reperfusion. Peri-ischaemic sevoflurane (2 vol%) and propofol (100 µM) in the formulation of 1% Diprivan(®) were assessed for their effects on oxidative energy metabolism and intracellular diastolic and systolic Ca(2+) concentrations. Substrate flux was measured using [(3)H]palmitate and [(14)C]glucose and [Ca(2+)] using indo-1AM. Western blotting was used to determine the expression of the sarcolemmal glucose transporter GLUT4 in lipid rafts. Biochemical analyses of nucleotides, ceramides, and 32 acylcarnitines were also performed. RESULTS: Sevoflurane, but not propofol, improved the recovery of left ventricular work (P=0.008) and myocardial efficiency (P=0.008) compared with untreated ischaemic hearts. This functional improvement was accompanied by reduced increases in post-ischaemic diastolic and systolic intracellular Ca(2+) concentrations (P=0.008). Sevoflurane, but not propofol, increased GOX (P=0.009) and decreased FOX (P=0.019) in hearts exposed to ischaemia-reperfusion. GLUT4 expression was markedly increased in lipid rafts of sevoflurane-treated hearts (P=0.016). Increased GOX closely correlated with reduced Ca(2+) overload. Intralipid alone decreased energy charge and increased long-chain and hydroxyacylcarnitine tissue levels, whereas sevoflurane decreased toxic ceramide formation. CONCLUSIONS: Enhanced glucose uptake via GLUT4 fuels recovery from Ca(2+) overload after ischaemia-reperfusion in sevoflurane- but not propofol-treated hearts. The use of a high propofol concentration (100 µM) did not result in similar protection.

AMP-activated protein kinase control of energy metabolism in the ischemic heart.

Myocardial ischemia produces an energy-deficient state in heart muscle, which if not corrected can lead to cardiomyocyte death. AMP-activated protein kinase (AMPK) is a key kinase that can increase energy production in the ischemic heart. During ischemia a rapid activation of AMPK occurs, resulting in an activation of both myocardial glucose uptake and glycolysis, as well as an increase in fatty acid oxidation. This activation of AMPK has the potential to increase energy production, thereby protecting the heart during ischemic stress. However, at clinically relevant high levels of fatty acids, ischemia-induced activation of AMPK also stimulates fatty acid oxidation during and following ischemia. This can contribute to ischemic injury secondary to an inhibition of glucose oxidation, which results in a decrease in cardiac efficiency. As a result, AMPK activation has the potential to be either beneficial or harmful in the ischemic heart.

Mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice.

The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.

Short- and long-term beneficial effects of trimetazidine in patients with diabetes and ischemic cardiomyopathy.

BACKGROUND: Trimetazidine (TMZ) has been shown to partially inhibit free fatty acid oxidation by shifting substrate utilization from fatty acid to glucose. The aim of this study was to assess the effects of TMZ in patients with diabetes and ischemic cardiomyopathy. METHODS: Sixteen patients with diabetes and ischemic hypokinetic cardiomyopathy (all males) on conventional therapy were randomized to receive either placebo or TMZ (20 mg 3 times per day), each arm lasting 15 days, and then again to receive either placebo or TMZ for 2 additional 6-month periods, according to a double-blind, crossover design. At the end of each period, all patients underwent exercise testing, 2-dimensional echocardiography, and hyperinsulinemic/euglycemic clamp. Among the others, New York Heart Association class, ejection fraction, exercise time, fasting blood glucose, end-clamp M value (index of total body glucose disposal) and endothelin-1 levels were evaluated. RESULTS: Both in the short and long term (completed by 13 patients), on TMZ compared to placebo, ejection fraction (47 +/- 7 vs 41 +/- 9 and 45 +/- 8 vs 36 +/- 8%, P <.001 for both) and M value (4.0 +/- 1.8 vs 3.3 +/- 1.6, P =.003, and 3.5 +/- 1.5 vs 2.7 +/- 1.6 mg/kg body weight/min, P <.01) increased, while fasting blood glucose (121 +/- 30 vs 136 +/- 40, P =.02 and 125 +/- 36 vs 140 +/- 43, P =.19) and endothelin-1 (8.8 +/- 3.8 vs 10.9 +/- 3.8, P <.001 and 6.2 +/- 2.4 vs 9.2 +/- 4.3 pg/mL, P =.03) decreased. In the short term, 10 patients decreased 1 class on the NYHA scale during treatment with TMZ (P =.019 vs placebo). Eight patients decreased 1 NYHA class while on long-term TMZ treatment, while on placebo 1 patient increased 1 NYHA class and none improved (P =.018 vs placebo). CONCLUSIONS: In a short series of patients with diabetes and ischemic cardiomyopathy, TMZ improved left ventricular function, symptoms, glucose metabolism, and endothelial function. Shifting energy substrate preference away from fatty acid metabolism and toward glucose metabolism by TMZ appears an effective adjunctive treatment in patients with diabetes with postischemic cardiomyopathy.

AMP-activated protein kinase regulation of fatty acid oxidation in the ischaemic heart.

The heart relies predominantly on a balance between fatty acids and glucose to generate its energy supply. There is an important interaction between the metabolic pathways of these two substrates in the heart. When circulating levels of fatty acids are high, fatty acid oxidation can dominate over glucose oxidation as a source of energy through feedback inhibition of the glucose oxidation pathway. Following an ischaemic episode, fatty acid oxidation rates increase further, resulting in an uncoupling between glycolysis and glucose oxidation. This uncoupling results in an increased proton production, which worsens ischaemic damage. Since high rates of fatty acid oxidation can contribute to ischaemic damage by inhibiting glucose oxidation, it is important to maintain proper control of fatty acid oxidation both during and following ischaemia. An important molecule that controls myocardial fatty acid oxidation is malonyl-CoA, which inhibits uptake of fatty acids into the mitochondria. The levels of malonyl-CoA in the heart are controlled both by its synthesis and degradation. Three enzymes, namely AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD), appear to be extremely important in this process. AMPK causes phosphorylation and inhibition of ACC, which reduces the production of malonyl-CoA. In addition, it is suggested that AMPK also phosphorylates and activates MCD, promoting degradation of malonyl-CoA levels. As a result malonyl-CoA levels can be dramatically altered by activation of AMPK. In ischaemia, AMPK is rapidly activated and inhibits ACC, subsequently decreasing malonyl-CoA levels and increasing fatty acid oxidation rates. The consequence of this is a decrease in glucose oxidation rates. In addition to altering malonyl-CoA levels, AMPK can also increase glycolytic rates, resulting in an increased uncoupling of glycolysis from glucose oxidation and an enhanced production of protons and lactate. This decreases cardiac efficiency and contributes to the severity of ischaemic damage. Decreasing the ischaemic-induced activation of AMPK or preventing the downstream decrease in malonyl-CoA levels may be a therapeutic approach to treating ischaemic heart disease.

Regulation of myocardial fatty acid oxidation by substrate supply.

We tested the hypothesis that myocardial substrate supply regulates fatty acid oxidation independent of changes in acetyl-CoA carboxylase (ACC) and 5'-AMP-activated protein kinase (AMPK) activities. Fatty acid oxidation was measured in isolated working rat hearts exposed to different concentrations of exogenous long-chain (0.4 or 1.2 mM palmitate) or medium-chain (0.6 or 2.4 mM octanoate) fatty acids. Fatty acid oxidation was increased with increasing exogenous substrate concentration in both palmitate and octanoate groups. Malonyl-CoA content only rose as acetyl-CoA supply from octanoate oxidation increased. The increases in octanoate oxidation and malonyl-CoA content were independent of changes in ACC and AMPK activity, except that ACC activity increased with very high acetyl-CoA supply levels. Our data suggest that myocardial substrate supply is the primary mechanism responsible for alterations in fatty acid oxidation rates under nonstressful conditions and when substrates are present at physiological concentrations. More extreme variations in substrate supply lead to changes in fatty acid oxidation by the additional involvement of intracellular regulatory pathways.

Optimizing cardiac energy metabolism: how can fatty acid and carbohydrate metabolism be manipulated?

Optimizing energy metabolism in the heart is a novel approach for the management of ischaemic heart disease, especially in conjunction with optimizing or restoring coronary flow. In particular, promoting myocardial glucose metabolism can enhance heart function, lessen injury to tissue, or both. Several pharmacological agents that directly stimulate myocardial glucose oxidation or indirectly stimulate glucose oxidation secondary to inhibition of oxidation of fatty acids are now available. Trimetazidine is the first compound in the class of 3-ketoacyl-coenzyme A thiolase inhibitors to see wide-spread clinical use. This agent increases glucose metabolism in the heart secondary to a direct inhibition of fatty acid metabolism. Considering results of experimental and clinical studies on other agents, it is clear that metabolic agents may provide a new approach to treating cardiovascular disease that should complement and improve existing therapies.

Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak.

Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 +/- 2.2 mV) versus aerobic (185.8 +/- 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 +/- 2.9 mV). At the maximal attainable membrane potential, O(2) consumption (nmol O(2) x mg(-1) x min(-1)) did not differ between untreated or DCA-treated hearts (128.3 +/- 7.5 and 120.6 +/- 7.6, respectively) but was significantly greater than aerobic hearts (76.6 +/- 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H(+) production from glucose metabolism to 53% of untreated hearts. Because H(+) production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.

Role of fatty acids in the recovery of cardiac function during resuscitation from hemorrhagic shock.

This study tested the hypothesis that removal of fatty acids as a fuel source would improve cardiac efficiency at the expense of reduced cardiac contractile function in the isolated working heart after hemorrhage-retransfusion. Non-heparinized male Sprague-Dawley rats were anesthetized with ketamine-xylazine and were hemorrhaged to a mean arterial blood pressure of 40 mmHg for 1 h. Two-thirds volume of shed blood was reinfused together with 0.9% NaCl in a volume equal to 2.3 times the shed blood volume, followed by continuous infusion of 0.9% NaCl at 10 mL/kg per h for 3 h. Hearts were removed and perfused in closed, recirculating working mode for 60 min to measure hydraulic work and cardiac efficiency. Rates of glycolysis and glucose oxidation were assessed with [5-3H/U-14C] glucose (11 mM) in the absence or presence of 0.4 mM palmitate. Compared to baseline measurements, hemorrhage-retransfusion significantly reduced arterial blood glucose (228+/-7 versus 118+/-12 mg/dL) and non-esterified fatty acid concentrations (0.36+/-0.01 versus 0.30+/-0.02 mM), while elevating blood lactate (0.8+/-0.1 versus 2.5+/-0.4 mM). Perfusion of sham hearts with glucose-only did not alter cardiac work compared to shams perfused with glucose plus palmitate. However, shocked hearts perfused with glucose-only demonstrated a significant reduction in cardiac work compared to shocked hearts perfused with glucose plus palmitate and compared to sham hearts perfused with glucose only (P < 0.05, repeated measures ANOVA). Shocked hearts perfused with glucose plus palmitate showed no reduction in cardiac work compared to shams. Shocked hearts perfused with glucose-only had increased glucose oxidation rates compared to shams perfused with glucose plus palmitate. In sham hearts perfused with glucose-only, myocardial glycogen and triacylglycerol contents were significantly reduced compared to hearts freeze-clamped in situ. These endogenous fuels were not decreased in shocked hearts. These data indicate that hemorrhagic shock renders the heart unable to mobilize endogenous fuels, and suggest that withdrawal of fatty acid oxidation will impair myocardial energy metabolism during resuscitation.

Malonyl CoA control of fatty acid oxidation in the diabetic rat heart.

Increased fatty acid metabolism can decrease cardiac function and efficiency, and may therefore contribute to the outcome of ischemic injury in the diabetic. Alterations in the control of myocardial malonyl CoA levels is an important contributing factor to these high fatty acid oxidation rates. This includes alterations in AMPK, ACC, and MCD activity in the diabetic rat heart. A further understanding of how malonyl CoA controls fatty acid oxidation in the diabetic heart should help identify new targets for pharmacological intervention which decreases the reliance of the heart on fatty acid oxidation, and ultimately improves heart function.

Influence of beta-adrenoceptor tone on the cardioprotective efficacy of adenosine A(1) receptor activation in isolated working rat hearts.

This study investigated the role of beta-adrenoceptors in the cardioprotective and metabolic actions of adenosine A(1) receptor stimulation. Isolated paced (300 beats min(-1)) working rat hearts were perfused with Krebs-Henseleit solution containing 1.2 mM palmitate. Left ventricular minute work (LV work), O(2) consumption and rates of glycolysis and glucose oxidation were measured during reperfusion (30 min) following global ischaemia (30 min) as well as during aerobic conditions. Relative to untreated hearts, N(6)-cyclohexyladenosine (CHA, 0.5 microM) improved post-ischaemic LV work (158%) and reduced glycolysis and proton production (53 and 42%, respectively). CHA+propranolol (1 microM) had similar beneficial effects, while propranolol alone did not affect post-ischaemic LV work or glucose metabolism. Isoprenaline (10 nM) impaired post-ischaemic function and after 25 min ischaemia recovery was comparable with 30 min ischaemia in untreated hearts (41 and 53%, respectively). Relative to isoprenaline alone, CHA+isoprenaline improved recovery of LV work (181%) and reduced glycolysis and proton production (64 and 60%, respectively). In aerobic hearts, CHA, propranolol or CHA+propranolol had no effect on LV work or glucose oxidation. Glycolysis was inhibited by CHA, propranolol and CHA+propranolol (50, 53 and 52%, respectively). Isoprenaline-induced increases in heart rate, glycolysis and proton production were attenuated by CHA (85, 57 and 53%, respectively). The cardioprotective efficacy of CHA was unaffected by antagonism or activation of beta-adrenoceptors. Thus, the mechanism of protection by adenosine A(1) receptor activation does not involve functional antagonism of beta-adrenoceptors.

Dichloroacetate improves postischemic function of hypertrophied rat hearts.

OBJECTIVES: We sought to determine whether improving coupling between glucose oxidation and glycolysis by stimulating glucose oxidation during reperfusion enhances postischemic recovery of hypertrophied hearts. BACKGROUND: Low rates of glucose oxidation and high glycolytic rates are associated with greater postischemic dysfunction of hypertrophied as compared with nonhypertrophied hearts. METHODS: Heart function, glycolysis and glucose oxidation were measured in isolated working control and hypertrophied rat hearts for 30 min before 20 min of global, no-flow ischemia and during 60 min of reperfusion. Selected control and hypertrophied hearts received 1.0 mmol/liter dichloroacetate (DCA), an activator of pyruvate dehydrogenase, at the time of reperfusion to stimulate glucose oxidation. RESULTS: In the absence of DCA, glycolysis was higher and glucose oxidation and recovery of function were lower in hypertrophied hearts than in control hearts during reperfusion. Dichloroacetate stimulated glucose oxidation during reperfusion approximately twofold in both groups, while significantly reducing glycolysis in hypertrophied hearts. It also improved function of both hypertrophied and control hearts. In the presence of DCA, recovery of function of hypertrophied hearts was comparable to or better than that of untreated control hearts. CONCLUSIONS: Dichloroacetate, given at the time of reperfusion, normalizes postischemic function of hypertrophied rat hearts and improves coupling between glucose oxidation and glycolysis by increasing glucose oxidation and decreasing glycolysis. These findings support the hypothesis that low glucose oxidation rates and high glycolytic rates contribute to the exaggerated postischemic dysfunction of hypertrophied hearts.

Lactate improves cardiac efficiency after hemorrhagic shock.

This study was undertaken to examine the role of lactate on cardiac function and metabolism after severe acute hemorrhagic shock. Anesthetized, nonheparinized rats were bled to a mean arterial pressure of 25-30 mm Hg for 1 h; controls were not bled. Their hearts were removed, and cardiac work and efficiency (work/oxygen consumption) were measured in the isolated working heart mode for 60 min. The hearts were perfused with one of five substrate combinations: 1) glucose (11 mM), 2) glucose + 0.4 mM palmitate, 3) glucose + 0.4 mM palmitate + 8.0 mM lactate, 4) glucose + 1.2 mM palmitate, or 5) glucose + 1.2 mM palmitate + 8.0 mM lactate. After perfusion, hearts were freeze-clamped, and tissue contents of free coenzyme-A (CoA), acetyl CoA, and succinyl CoA were measured, as was myocardial pyruvate dehydrogenase (PDH) activity. The addition of 8.0 mM lactate significantly improved cardiac work in shocked hearts perfused with 0.4 mM palmitate and increased cardiac efficiency in the presence of either 0.4 mM or 1.2 mM palmitate. Compared to control hearts, shocked hearts exhibited a 20-30% decrease in PDH activity. Shocked hearts perfused with lactate demonstrated no increase in acetyl CoA content but did have a significant increase in tissue succinyl CoA compared to control hearts perfused with lactate or shocked hearts perfused without lactate. In the heart recovering from severe hemorrhagic shock, lactate improves cardiac efficiency in the presence of free fatty acids, possibly by a anaplerosis of the tricarboxylic acid cycle.

Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism.

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.

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.