Measurements of fatty acid and carbohydrate metabolism in te isolated working rat heart

The isolated working rat heart is a useful experimental model which allows contractile function to be measured in hearts perfused at physiologically relevant workloads. To maintain these high workloads the heart is required to generate a tremendous amount of energy. In vivo this energy is derived primarily from the oxidation of fatty acids. In many experimental situations it is desirable to perfuse the isolated working heart in the presence of physiologically relevant concentrations of fatty acids. This is particularly important when studying energy metabolism in the heart, or in determining how fatty acids alter the outcome of myocardial ischemic injury [1, 2]. The other major source of energy for the heart is derived from the oxidation of carbohydrates (glucose and lactate), with a smaller amount of ATP also being derived from glycolysis. Two byproducts of both fatty acid and carbohydrate metabolism are H2O and CO2. By labeling the glucose, lactate, or fatty acids in the perfusate with 3H or 14C the experimenter can quantitatively collect either 3H2O or 14CO2 produced by the heart. By using radioisotopes that are labeled at specific hydrogen or carbon molecules on the various energy substrates, and by knowing the specific activity of the radiolabeled substrate used, it is possible to determine the actual rate of flux through these individual pathways. This paper will describe the experimental protocols for directly measuring fatty acid and carbohydrate metabolism in isolated working rat hearts.     

Effects of hypothermia on energy metabolism in rat and Richardson's ground squirrel hearts

Belke, Darrell D., Lawrence C. H. Wang, and Gary D. Lopaschuk. Effects of hypothermia on energy metabolism in rat and Richardson's ground squirrel hearts. J. Appl.Physiol. 82(4): 1210-1218, 1997.Glycolysis,glucose oxidation, palmitate oxidation, and cardiac function weremeasured in isolated working hearts from ground squirrels and ratssubjected to a hypothermia-rewarming protocol. Hearts wereperfused initially for 30 min at 37°C, followed by 2 h ofhypothermic perfusion at 15°C, after which hearts were rewarmed to37°C and further perfused for 30 min. Functional recovery in groundsquirrel hearts was greater than in rat hearts after rewarming.Hypothermia-rewarming had a similar general effect on the variousmetabolic pathways in both species. Despite these similarities, totalenergy substrate metabolic rates were greater in rat than groundsquirrel hearts during hypothermia despite a lower level of work beingperformed by the rat hearts, indicating that rat hearts are lessefficient than ground squirrel hearts during hypothermia.After rewarming, energy substrate metabolism recovered completely inboth species, although cardiac work remained depressed in rat hearts.The difference in functional recovery between rat and ground squirrelhearts after rewarming cannot be explained by general differences inenergy substrate metabolism during hypothermia or after rewarming.

     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Interaction between altered insulin and lipid metabolism in CEACAM1-inactive transgenic mice

Inactivation of CEACAM1 in L-SACC1 mice by a dominant-negative transgene in liver impairs insulin clearance and increases serum free fatty acid (FFA) levels, resulting in insulin resistance. The contribution of elevated FFAs in the pathogenesis of insulin resistance is herein investigated. Treatment of L-SACC1 female mice with carnitine restored plasma FFA content. Concomitantly, it normalized insulin levels without directly regulating receptor-mediated insulin internalization and prevented glucose tolerance in these mice. Similarly, treatment with nicotinic acid, a lipolysis inhibitor, restored insulin-stimulated receptor uptake in L-SACC1 mice. Taken together, these data suggest that chronic elevation in plasma FFAs levels contributes to the regulation of insulin metabolism and action in L-SACC1 mice.     

beta-Hydroxybutyrate inhibits myocardial fatty acid oxidation in vivo independent of changes in malonyl-CoA content

This study tested the hypothesis that an acute infusion of beta-hydroxybutyrate inhibits myocardial fatty acid uptake and oxidation in vivo. Anesthetized pigs were untreated (n = 6) or treated with an intravenous infusion of fat emulsion (n = 7) to elevate plasma free fatty acid levels. A third group received fat emulsion plus an intravenous infusion of beta-hydroxybutyrate (25 micromol.kg-1.min-1; n = 7) for 60 min. All animals received a continuous infusion of [3H]palmitate, and myocardial fatty acid oxidation was measured from the cardiac production of 3H2O. Plasma free fatty acid concentrations were elevated in the fat emulsion group (0.77 +/- 0.11 mM) compared with the untreated group (0.15 +/- 0.03 mM), which resulted in greater myocardial free fatty acid oxidation. In contrast, the group receiving beta-hydroxybutyrate in addition to fat emulsion had elevated beta-hydroxybutyrate concentration (0.87 +/- 0.11 vs. 0.04 +/- 0.01 mM), but suppressed fatty acid oxidation (0.053 +/- 0.013 micromol.g-1.min-1) (P < 0.05) compared with the fat emulsion group (0.116 +/- 0.029 micromol.g-1.min-1). There were no differences among the three groups in the tissue content for malonyl-CoA, acetyl-CoA, or free CoA or the activity of acetyl-CoA carboxylase; thus the inhibition of fatty acid oxidation by elevated beta-hydroxybutyrate did not appear to be due to malonyl-CoA inhibition of carnitine palmitoyl transferase-I or to an increase in the acetyl-CoA-to-free CoA ratio. In conclusion, fatty acid uptake and oxidation is blocked by an infusion of beta-hydroxybutyrate; this effect was not due to elevated myocardial malonyl-CoA content.     

AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart

Myocardial ischemia is the leading cause of all cardiovascular deaths in North America. Myocardial ischemia is accompanied by profound changes in metabolism including alterations in glucose and fatty acid metabolism, increased uncoupling of glucose oxidation from glycolysis and accumulation of protons within the myocardium. These changes can contribute to a poor functional recovery of the heart. One key player in the ischemia-induced alteration in fatty acid and glucose metabolism is 5'AMP-activated protein kinase (AMPK). Accumulating evidence suggest that activation of AMPK during myocardial ischemia both increases glucose uptake and glycolysis while also increasing fatty acid oxidation during reperfusion. Gain-of-function mutations of AMPK in cardiac muscle may also be causally related to the development of hypertrophic cardiomyopathies. Therefore, a better understanding of role of AMPK in cardiac metabolism is necessary to appropriately modulate its activity as a potential therapeutic target in treating ischemia reperfusion injuries. This review attempts to update some of the recent findings that delineate various pathways through which AMPK regulates glucose and fatty acid metabolism in the ischemic myocardium.     

Pyruvate prevents cardiac dysfunction and AMP-activated protein kinase activation by hydrogen peroxide in isolated rat hearts

Ischemia-reperfusion injury in the heart results in enhanced production of H2O2 and activation of AMP-activated protein kinase (AMPK). Since mutations in AMPK result in cardiovascular dysfunction, we investigated whether the activation of AMPK mediates the H2O2-induced reduction in cardiac mechanical function. Isolated working rat hearts were perfused at 37 degrees C with Krebs-Henseleit solution. Following a 20-minute equilibration period, a single bolus of H2O2 (300 micromol/L) was added and the hearts were perfused for an additional 5 min. H2O2 induced a dramatic and progressive reduction in cardiac function. This was accompanied by rapid and significant activation of AMPK, an increase in Thr-172 phosphorylation of AMPK, and an increase in the creatine to phosphocreatine (Cr/PCr) ratio. Addition of pyruvate (5 mmol/L) to the perfusate prevented the H2O2-mediated reduction in cardiac mechanical dysfunction, activation of myocardial AMPK activity, increase in AMPK phosphorylation and the increase in the Cr/PCr ratio. Hearts challenged with H2O2 (300 micromol/L) in presence of either AMPK inhibitor Compound C (10 micromol/L) or its vehicle (dimethyl sulfoxide (DMSO), 0.1%) showed reduced impairment in cardiac mechanical function. Compound C but not its vehicle significantly inhibited myocardial AMPK activity. Thus, H2O2 induces cardiac dysfunction via both AMPK-dependent and independent mechanisms.     

Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis

Leptin regulates fatty acid metabolism in liver, skeletal muscle, and pancreas by partitioning fatty acids into oxidation rather than triacylglycerol (TG) storage. Although leptin receptors are present in the heart, it is not known whether leptin also regulates cardiac fatty acid metabolism. To determine whether leptin directly regulates cardiac fatty acid metabolism, isolated working rat hearts were perfused with 0.8 mm [9,10-(3)H]palmitate and 5 mm [1-(14)C]glucose to measure palmitate and glucose oxidation rates. Leptin (60 ng/ml) significantly increased palmitate oxidation rates 60% above control hearts (p < 0.05) and decreased TG content by 33% (p < 0.05) over the 60-min perfusion period. In contrast, there was no difference in glucose oxidation rates between leptin-treated and control hearts. Although leptin did not affect cardiac work, oxygen consumption increased by 30% (p < 0.05) and cardiac efficiency was decreased by 42% (p < 0.05). AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels. Leptin has also been shown to increase fatty acid oxidation in skeletal muscle through the activation of AMPK. However, we demonstrate that leptin had no significant effect on AMPK activity, AMPK phosphorylation state, ACC activity, or malonyl-CoA levels. AMPK activity and its phosphorylation state were also unaffected after 5 and 10 min of perfusion in the presence of leptin. The addition of insulin (100 microunits/ml) to the perfusate reduced the ability of leptin to increase fatty acid oxidation and decrease cardiac TG content. These data demonstrate for the first time that leptin activates fatty acid oxidation and decreases TG content in the heart. We also show that the effects of leptin in the heart are independent of changes in the AMPK-ACC-malonyl-CoA axis.     

Olfactory contribution to Fos expression during mating in inexperienced male hamsters

Male hamsters are very dependent on chemosensory cues for normal mating behavior. We have previously reported that central, vomeronasal pathways are intensely and selectively activated during mating or pheromonal stimulation. The contribution of main olfactory sensory input to the patterns of c-fos activation was investigated in this study. Sexually inexperienced male hamsters were either made anosmic by intranasal infusion of zinc sulfate or remained intact. Fos protein immunoreactivity was analyzed in main olfactory and vomeronasal pathways of the zinc sulfate-treated, anosmic animals after mating with receptive females for 45 min, and compared with Fos patterns seen in intact mating animals, some of which have been described in a previous publication. The zinc sulfate-treated anosmic males described here all mated when given access to receptive females. Whether mated or unstimulated, anosmic males had little or no Fos expression in main olfactory pathways; significantly less even than in unstimulated intact animals. Mating did not increase Fos expression in main olfactory pathways of intact animals over that of unstimulated intact controls. However, Fos expression in central vomeronasal pathways was significantly higher in mating anosmic males, as in intact males, compared with appropriate non-mating controls. Fos expression was significantly different between intact and zinc sulfate-treated anosmic mating males in only one area studied. The rostral anterior medial amygdala, known to receive a small olfactory terminal field, had significantly lower Fos expression in zinc sulfate-treated anosmic males that mated when compared with intact-mating animals. Thus, functional main olfactory input to the rostral vomeronasal amygdala can be demonstrated but does not appear to be critical for mating behavior in previously inexperienced male hamsters with intact vomeronasal organs. Other main olfactory input appears to have a negligible contribution to Fos-patterns in such animals.      

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.