Fat to the fire: the regulation of lipid oxidation with exercise and environmental stress

Lipids are an important fuel for submaximal aerobic exercise. The ways in which lipid oxidation is regulated during locomotion is an area of active investigation. Indeed, the integration between cellular regulation of lipid metabolism and whole-body exercise performance is a fascinating but often overlooked research area. Additionally, the interaction between environmental stress, exercise, and lipid oxidation has not been sufficiently examined. There are many functional and structural steps as fatty acids are mobilized, transported, and oxidized in working muscle, which may serve either as regulatory points for responding to acute or chronic stimuli or as raw material for natural selection. At the whole-animal level, the partitioning of lipids and carbohydrates across exercise intensities is remarkably similar among mammals, which suggests that there is conservation in regulatory mechanisms. Conversely, the proportions of circulatory and intramuscular fuels differ between species and across exercise intensities. Responses to acute and chronic environmental stress likely involve the interaction of genetic and nongenetic changes in the fatty acid pathway. Determining which of these factors help regulate the fatty acid pathway and what impact they have on whole-animal lipid oxidation and performance is an important area of future research. Using an integrative approach to complete the information loop from gene to physiological function provides the most powerful mode of analysis.     

The metabolic effects of thia fatty acids in rat liver depend on the position of the sulfur atom

The effects on oxidation and composition of fatty acids in rat liver were compared after administration of fatty acids with sulfur substituted in different positions. It has been hypothesized that drugs with hydrophobic backbone have lipid-lowering effects because they are not easily catabolized by mitochondrial beta-oxidation. Thia fatty acids cannot be beta-oxidized when sulfur is in 3-position, but beta-oxidation is possible when sulfur is positioned further from the carboxyl group. To investigate whether catabolism of thia fatty acids would affect their ability to influence lipid metabolism, a series of thia fatty acids were synthesized and administered by oral gavage to male Wistar rats (300 mg/kg bodyweight/day for 7 days). Depending on the position of the sulfur atom and the chain length, the thia fatty acids were beta-oxidized, desaturated and/or elongated, and the accumulated amounts were lower as the sulfur atom were positioned further from the carboxyl group. All thia fatty acids led to high peroxisomal beta-oxidation of endogenous fatty acids, whereas the mitochondrial beta-oxidation was high when sulfur was in 3-position, low when sulfur was in 4-position and similar to controls when sulfur was in 5- or 7-position. The changes in hepatic fatty acid composition were more pronounced when sulfur was positioned close to the carboxyl group. In conclusion, both the position of the sulfur atom and the chain length appear to determine the catabolic fate of thia fatty acids, and the non-beta-oxidizable thia fatty acids were most potent in regulating oxidation and composition of endogenous fatty acids in rat liver.     

Changes in short‐chain acyl‐coA dehydrogenase during rat cardiac development and stress

This study was designed to investigate the expression of short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time‐dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim‐trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down‐regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator‐activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down‐regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for ‘the recapitulation of foetal energy metabolism’. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.     

Lipid metabolism and toxicity in the heart

The heart has both the greatest caloric needs and the most robust oxidation of fatty acids (FAs). Under pathological conditions such as obesity and type 2 diabetes, cardiac uptake and oxidation are not balanced and hearts accumulate lipid potentially leading to cardiac lipotoxicity. We will first review the pathways utilized by the heart to acquire FAs from the circulation and to store triglyceride intracellularly. Then we will describe mouse models in which excess lipid accumulation causes heart dysfunction and experiments performed to alleviate this toxicity. Finally, the known relationships between heart lipid metabolism and dysfunction in humans will be summarized.     

Lipid metabolism in skeletal muscle: generation of adaptive and maladaptive intracellular signals for cellular function

Fatty acids derived from adipose tissue lipolysis, intramyocellular triacylglycerol lipolysis, or de novo lipogenesis serve a variety of functions in skeletal muscle. The two major fates of fatty acids are mitochondrial oxidation to provide energy for the myocyte and storage within a variety of lipids, where they are stored primarily in discrete lipid droplets or serve as important structural components of membranes. In this review, we provide a brief overview of skeletal muscle fatty acid metabolism and highlight recent notable advances in the field. We then 1) discuss how lipids are stored in and mobilized from various subcellular locations to provide adaptive or maladaptive signals in the myocyte and 2) outline how lipid metabolites or metabolic byproducts derived from the actions of triacylglycerol metabolism or β-oxidation act as positive and negative regulators of insulin action. We have placed an emphasis on recent developments in the lipid biology field with respect to understanding skeletal muscle physiology and discuss unanswered questions and technical limitations for assessing lipid signaling in skeletal muscle.     

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.     

Metabolic Partitioning of Endogenous Fatty Acid in Adipocytes

Objective: To develop an accurate new method to measure the partitioning of adipocyte endogenous fatty acids among different metabolic pathways, a critical step toward understanding the regulatory mechanism by which fat disposition is modulated. Research Methods and Procedures: Isolated primary rat adipocytes were pre‐incubated with isotope‐labeled fatty acids. This allows determination of the specific activity of labeled fatty acids in the endogenous lipid pool. After the removal of exogenous fatty acids, the disposition of endogenous fatty acids into the three major metabolic pathways, namely, oxidation, re‐esterification, and release into the medium, was measured independently. This was compared with the total lipolytic release of endogenous fatty acids, as measured by glycerol release. Adipocytes from normal fed and fasted animals were used to determine the effects of physiological variations on the metabolic fate of endogenous fatty acids. Results: In normal fed animals, 0.2% of endogenous fatty acids were oxidized, 50.1% were released, and 49.7% were re‐esterified. Fasting doubled the partitioning of fatty acids toward oxidation (p < 0.05) in association with increased lipolysis (1.4‐fold increase) (p < 0.05). This effect was completely abolished by the addition of insulin to the cells (61% reduction) (p < 0.05). Discussion: The endogenous fatty acids in adipocytes are actively oxidized. This process can be regulated by altered physiological conditions or by insulin. Over time, it is possible that a small shift of fatty acids toward oxidation could have a significant impact on body fuel economy. This hypothesis needs to be tested.     

The role of triacylglycerol in cardiac energy provision

Triacylglycerols (TAGs) constitute the main energy storage resource in mammals, by virtue of their high energy density. This in turn is a function of their highly reduced state and hydrophobicity. Limited water solubility, however, imposes specific requirements for delivery and uptake mechanisms on TAG-utilising tissues, including the heart, as well as intracellular disposition. TAGs constitute potentially the major energy supply for working myocardium, both through blood-borne provision and as intracellular TAG within lipid droplets, but also provide the heart with fatty acids (FAs) which the myocardium cannot itself synthesise but are required for glycerolipid derivatives with (non-energetic) functions, including membrane phospholipids and lipid signalling molecules. Furthermore they serve to buffer potentially toxic amphipathic fatty acid derivatives. Intracellular handling and disposition of TAGs and their FA and glycerolipid derivatives similarly requires dedicated mechanisms in view of their hydrophobic character. Dysregulation of utilisation can result in inadequate energy provision, accumulation of TAG and/or esterified species, and these may be responsible for significant cardiac dysfunction in a variety of disease states. This review will focus on the role of TAG in myocardial energy provision, by providing FAs from exogenous and endogenous TAG sources for mitochondrial oxidation and ATP production, and how this can change in disease and impact on cardiac function. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Malonyl CoA control of fatty acid oxidation in the newborn heart in response to increased fatty acid supply

The concentration of fatty acids in the blood or perfusate is a major determinant of the extent of myocardial fatty acid oxidation. Increasing fatty acid supply in adult rat increases myocardial fatty acid oxidation. Plasma levels of fatty acids increase post-surgery in infants undergoing cardiac bypass operation to correct congenital heart defects. How a newborn heart responds to increased fatty acid supply remains to be determined. In this study, we examined whether the tissue levels of malonyl CoA decrease to relieve the inhibition on carnitine palmitoyltransferase (CPT) I when the myocardium is exposed to higher concentrations of long-chain fatty acids in newborn rabbit heart. We then tested the contribution of the enzymes that regulate tissue levels of malonyl CoA, acetyl CoA carboxylase (ACC), and malonyl CoA decarboxylase (MCD). Our results showed that increasing fatty acid supply from 0.4 mmol/L (physiological) to 1.2 mmol/L (pathological) resulted in an increase in cardiac fatty acid oxidation rates and this was accompanied by a decrease in tissue malonyl CoA levels. The decrease in malonyl CoA was not related to any alterations in total and phosphorylated acetyl CoA carboxylase protein or the activities of acetyl CoA carboxylase and malonyl CoA decarboxylase. Our results suggest that the regulatory role of malonyl CoA remained when the hearts were exposed to high levels of fatty acids.     

Cytoplasmic unsaturated free fatty acids inhibit ATP-dependent gating of the G protein-gated K(+) channel

This study reports the identification of an endogenous inhibitor of the G protein-gated (K(ACh)) channel and its effect on the K(ACh) channel kinetics. In the presence of acetylcholine in the pipette, K(ACh) channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated K(ACh) channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the K(ACh) channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of approximately 40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.2-2 microM) reversibly inhibited the ATP-dependent gating of native K(ACh) channels in atrial cells and hippocampal neurons, and of recombinant K(ACh) channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4, 5-bisphosphate (PIP(2))-induced changes in K(ACh) channel kinetics but were ineffective against ATP-activated background K(1) channels and PIP(2)-activated K(ATP) channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal K(ACh) channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G protein-gated K(+) channel.     

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.     

Lipids in the heart: a source of fuel and a source of toxins

PURPOSE OF REVIEW: How do lipids arrive in the heart and other tissues? This review focuses on new information on pathways of lipid uptake into the heart. RECENT FINDINGS: Fatty acids, the major cardiac fuel, are obtained from either lipoproteins or free fatty acids associated with albumin. The heart is the tissue with the most robust expression of lipoprotein lipase, and recent data attest to the importance of this enzyme in supplying optimal amounts of fatty acids for the heart. Genetic deletion of CD36 also shows that this transporter is important for cardiac uptake of lipids. Retinoid acquisition by the heart involves pathways parallel to those used for fatty acid uptake: a pathway for acquisition of core lipoprotein retinyl ester and another for nonlipoprotein retinol. Dilated lipotoxic cardiomyopathy is the consequence of excess lipid uptake. SUMMARY: Genetic modifications that affect lipid uptake, oxidation, and storage are being exploited to elucidate the pathophysiology of cardiomyopathies and to discover how lipids relate to heart failure in humans with obesity and diabetes mellitus. This information is likely to lead to new diagnostic categories of cardiomyopathy and more pathophysiologically appropriate treatments.     

Role of fatty acid in the control of protein synthesis in liver cells

Concentrations of oleate (0.2-1 mM) within the physiological range of plasma free fatty acids induced a dose dependent statistically significant inhibition of protein labelling in isolated liver cells. The inhibitory effect was as high as 50% and it was not impeded when long chain fatty acid oxidation was prevented. Experiments carried out with hepatocytes from 48 h fasted rats, incubated in the absence of any exogenous energy source, show that the inhibition of endogenous long chain fatty acid oxidation induced a decreased rate of protein synthesis apparently related to changes in the cellular energy state. It is concluded that fatty acids play a dual role in the regulation of protein synthesis in liver cells: 1. endogenous fatty acids appear to be the main energy fuel for protein synthesis when no other exogenous substrate is present and the carbohydrate stores are low; 2. exogenous fatty acids seem to control protein synthesis by interacting with some key regulatory step.     

Perfused heart studies to investigate lipid metabolism

The isolated perfused heart preparation is an invaluable model for investigating metabolism in a variety of physiological and pathological states. It avoids confounding systemic factors (e.g. endocrine, metabolic and work load changes) and permits simultaneous measurement of mechanical function. The ability to measure arteriovenous concentration differences across the myocardium and the coronary flow rate, together with the use of radiolabelled substrates, permits assessment of substrate assimilation and disposition of most potential energetic substrates. In the case of lipids, metabolism of non-esterified fatty acids has been extensively investigated in the perfused rat heart, but fatty acids may also be derived from circulating triacylglycerols (TAG) in lipoproteins [chylomicrons, very-low-density-lipoprotein (VLDL)]. TAG requires initial hydrolysis by the endothelial enzyme lipoprotein lipase and hence an intact heart preparation is vital to maintain tissue structural integrity. Chylomicron-TAG utilization and fate (oxidation, tissue-lipid deposition) in isolated working hearts has been studied using chylomicrons obtained from thoracic-duct catheters. However, lack of availability of sufficient quantities of VLDL has hindered examination of their cardiac utilization; the recent development of a technique to produce large quantities of radio-labelled rat VLDL has facilitated these studies and established that VLDL-TAG is an important metabolic substrate for working heart. Results relating to myocardial utilization of VLDL-TAG under varying physiological (lactation) and pathological (endotoxinaemia) conditions will be presented. The putative role of VLDL as a regulator of cardiac lipid metabolism will also be discussed.     

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.     

The dynamic of lipid oxidation in human myotubes

Both endogenous and exogenous lipid levels may be regulators of total lipid oxidation in skeletal muscles. We studied the dynamics of lipid oxidation in human myotubes established from healthy, lean subjects exposed to acutely and chronically increased palmitate concentrations. The intramyocellular triacylglycerol content increased with chronic palmitate exposure. Both, ectopically increased intracellular and extracellular lipid levels were simultaneously oxidized and could partly suppress each other's oxidation. Overall, the highest acute palmitate treatments stimulated fatty acid oxidation whilst the highest chronic treatments decreased total lipid oxidation. Intracellular lipids showed a more complete oxidation than exogenous lipids. Endogenous lipids reduced insulin-mediated glucose oxidation. Thus, both endogenous and exogenous lipid concentrations regulated each other's oxidation and total lipid oxidation in human myotubes. A reduced exogenous lipid oxidation, secondary to increased triacylglycerol levels, may redirect free fatty acids into esterification and oxidation from intracellular stores, thereby protecting myotubes from FFA lipotoxic effects.