Induction Of Fatty Acid Oxidation Research Articles

Lipotoxicity: what is the fate of fatty acids?

Accumulation of lipid in nonadipose tissues plays a critical role in the pathogenesis of a diverse array of chronic diseases, including diabetes and heart failure. Normal cellular homeostasis relies on a critical balance of fuel uptake and utilization that is controlled by elaborate transcriptional networks, which ensure that cellular energy needs are consistently met and toxic intermediates do not accumulate. This process is particularly important for the heart, which has a continuous high energy demand. The heart meets this demand by dynamically shifting its preference for glucose and fatty acids in various physiologic and pathologic circumstances. This change is driven by alterations in signaling networks and regulated expression of key metabolic genes. While the heart predominantly relies on fatty acids as a fuel source, under normal conditions the myocardium is not a site of significant lipid storage. Moreover, conditions that promote fatty acid excess, such as insulin resistance and diabetes, are associated with detrimental effects on cardiac function. Evidence has emerged that fatty acid overload may damage the myocardium through excessive fatty acid oxidation and accumulation of toxic lipid species within the myocardium. These toxic effects have been termed “lipotoxicity” (1). A number of investigators have implicated specific lipid species, such as long-chain saturated fatty acids (palmitate), in the pathogenesis of cardiomyocyte dysfunction (2, 3), while unsaturated fatty acids (oleate) are considered cardioprotective (4). The mechanism whereby some fatty acid species exert more toxic effects than others is poorly understood. In this issue of The Journal of Lipid Research, Lockridge et al. (5) report microarray data of the differential effects of several fatty acid species in adult cardiac myocytes. Previous investigations have primarily evaluated direct effects of individual fatty acid species in neonatal rat cardiac myocytes, where the expression of key metabolic regulators is still changing. Thus, the work by Lockridge et al. provides new insight into effects in a mature cardiac myocyte and does this in a systematic and unbiased manner. The most striking finding in this study was the dramatic effect of the long-chain saturated fatty acid palmitate on gene expression for enzymes involved in the endoplasmic reticulum (ER) stress response and apoptosis pathways as well as markers of DNA damage. These findings are consistent with other recent data in CHO cells, in which palmitate was found to be trafficked to the ER and to have dramatic effects on ER structure and integrity as well as to disrupt mitochondrial function (6). Furthermore, ER stress has been implicated in apoptosis of pancreatic b cells from type 2 diabetic models (7). Taken together, this recent evidence strongly implicates ER stress in the cellular dysfunction associated with palmitate exposure. Further studies specifically evaluating the ER stress response in cardiac myocytes will be important in understanding the mechanisms of cardiac dysfunction associated with lipid overload and in targeting potential therapies to alleviate cardiomyopathy associated with diabetes. Interestingly, Borradaile et al. (6) also found that induction of fatty acid oxidation minimized the effects on ER integrity upon exposure to palmitate. Lockridge et al. (5) demonstrated differential effects of oleate versus palmitate on gene expression for a number of enzymes that play a role in upregulating mitochondrial fatty acid oxidation pathways (e.g., Cpt1a, Fatp1, and Ucp3). While palmitate also upregulated many components of mitochondrial oxidative pathways, the more robust effect of oleate on some of these genes is intriguing. One of the genes the authors highlight that is specifically induced by oleate is Ucp3. They suggest that upregulation of Ucp3 may result in increased recycling of free CoA and thus promote the oxidation of oleate, whereas palmitate may be more likely to accumulate in the mitochondria, leading to cytotoxic effects. Further studies to evaluate this hypothesis will be important for understanding the fate of fatty acid species within the cell and potentially to assist in identifying targets for cellular rescues. One such study might investigate the potential for unsaturated fatty acids to serve as preferential ligands for the peroxisome proliferator-activated receptor family, which is known to modulate fatty acid uptake and oxidation.

Transplacental induction of fatty acid oxidation in newborn pigs by clofibrate

This study examined the effect of maternal feeding of clofibrate on fatty acid oxidation in newborn pigs. Pregnant sows were fed a commercial diet with or without 0.8% clofibrate for 7 days. Blood samples were collected from the utero‐ovarian artery of the sows and the umbilical vein of the intact term fetuses as they were removed from the sows by c‐section on day 113 of gestation. HPLC analysis clearly identified clofibric acid was present in the plasma of the clofibrate fed sow and the fetuses. The maternal‐fed clofibric acid had no impact on the liver weight of the newborn pigs, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate significantly increased 14C‐accumulation in CO2 and ASP by 2.9‐fold from [1‐14C]‐oleic acid and 1.6‐fold from [1‐14C]‐lignoceric acid. Correspondingly, hepatic carnitine palmitoyltransferase (CPT) and fatty acid oxidase (FAO) activities were increased also by 36% and 42% compared to the newborn pigs from the control sow. These data demonstrate that clofibrate crosses the placental membrane of the sow and enters fetal circulation to induce hepatic fatty acid oxidation by increasing CPT and FAO activities of the newborn. Supported by CSREES, USDA NRI program award 2007‐35206‐17897.

The effects of clofibrate on malonyl‐CoA inhibition of carnitine pamitoyltransferase I (CPT I) and fatty acid oxidation in liver of neonatal pigs

Previous studies demonstrated that clofibrate increased CPT I activity and fatty acid oxidation in piglets, but the role of malonyl‐CoA, a physiological inhibitor of CPT I, has not been clearly elucidated. Thirty colostrum‐deprived newborn pigs were fed milk replacer and orally gavaged with either vehicle (2 % Tween 80) or clofibrate (75 mg /kg body weight) +/− etomoxir (5 mg/ kg body weight) once daily for up to 7 days, and the sensitivity of liver CPT I to malonyl‐CoA inhibition and fatty acid oxidation were determined using 1‐14C‐octanoic, oleic and erucic acids. Administration of clofibrate significantly increased liver CPT I activity (60%) and malonyl‐CoA IC50 values (56%). Addition of etomoxir potently inhibited CPT I activity, but malonyl‐CoA IC50 remained unchanged. Consistent with CPT I activity, clofibrate increased mitochondrial fatty acid oxidation by 53%, but did not affect the rate of peroxisomal beta‐oxidation. Etomoxir did not alter the rate of fatty acid oxidation despite a 30% reduction in CPT I activity. These results indicate that the increase in fatty acid oxidation induced by clofibrate is due to both an increase in CPT I activity and a decrease in sensitivity of CPT I to malonyl‐CoA inhibition, suggesting that malonyl‐CoA plays an important role in the induction of fatty acid oxidation by clofibrate. Supported by CSREES, USDA NRI program award 2007‐35206‐1 7897.

Estrogen-related Receptors Stimulate Pyruvate Dehydrogenase Kinase Isoform 4 Gene Expression

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression.

Short-term activation of peroxysome proliferator-activated receptor β/δ increases fatty acid oxidation but does not restore insulin action in muscle cells from type 2 diabetic patients

Defective fatty acid oxidation in skeletal muscle is one of the possible causes of insulin resistance. Peroxisome proliferator-activated receptor beta activators are strong inducers of fatty acid oxidation. The aim of this study was to verify whether activation of fatty acid oxidation by PPARbeta agonists in human skeletal muscle cells prepared from type 2 diabetic patients could improve the reduced responses to insulin that characterized this cell model. GW0742 (10 nM) significantly increased fatty acid oxidation and oxidative gene expression in myotubes prepared from both healthy subjects and type 2 diabetic patients. In cells from control subjects, incubation with the agonist for 48 h affected neither insulin-induced rate of glycogen synthesis nor the phosphorylation state of protein kinase B (PKB serine 473). Myotubes from type 2 diabetic patients displayed marked reduction in the effects of insulin on glycogen synthesis and on PKB phosphorylation. However, treatment with PPARbeta agonists did not restore these defects. Therefore, these results indicate that induction of fatty acid oxidation with PPARbeta activators during short-term exposition is not sufficient to correct for insulin resistance in muscle cells from type 2 diabetic patients. This suggests that additional studies are needed to better characterize the link between fatty acid oxidation and insulin sensitivity in humans.

Delayed induction of Δ-6 and Δ-5 desaturases by a peroxisome proliferator

Δ-6 Desaturase (D6D) is the key enzyme for the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic acid (AA) and docosahexaenoic acid (DHA) in mammals. Transcription of D6D gene is activated by both sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferators (PP). This response of D6D is paradoxical because SREBP-1c transactivates genes for fatty acid synthesis in liver, while PP induce enzymes for fatty acid oxidation. We hypothesized that the induction of D6D gene by PP is a compensatory response to the increased HUFA demand caused by peroxisome proliferation and induction of fatty acid oxidation. We investigated the time-course effects of a PP, Wy14643, on the induction of HUFA metabolizing genes and HUFA profile in rat liver. The mRNA of fatty acid oxidation enzymes in the Wy14643 fed group became significantly higher than controls at 4 h and reached maximum within 28 h. In contrast, the mRNA of Δ-6 and Δ-5 desaturases in the Wy14643 group was not significantly higher than control at 4 h and took >28 h to reach the maximum. Despite the induction of HUFA synthetic pathway, the concentration of end products (AA and DHA) remained unchanged throughout the 4-day period in liver phospholipids and non-esterified fatty acids. Taken together, this study supports our hypothesis and suggests that peroxisome proliferation and induction of fatty acid oxidation enzymes are the major mechanisms of the induction of HUFA synthesis by PP.

Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

The regulation of ketogenesis.

Ketone bodies accumulate in the plasma in conditions of fasting and uncontrolled diabetes. The initiating event is a change in the molar ratio of glucagon:insulin. Insulin deficiency triggers the lipolytic process in adipose tissue with the result that free fatty acids pass into the plasma for uptake by liver and other tissues. Glucagon appears to be the primary hormone involved in the induction of fatty acid oxidation and ketogenesis in the liver. It acts by acutely dropping hepatic malonyl-CoA concentrations as a consequence of inhibitory effects exerted in the glycolytic pathway and on acetyl-CoA carboxylase (EC 6.4.1.2). The fall in malonyl-CoA concentration activates carnitine acyltransferase I (EC 2.3.1.21) such that long-chain fatty acids can be transported through the inner mitochondrial membrane to the enzymes of fatty acid oxidation and ketogenesis. The latter are high-capacity systems assuring that fatty acids entering the mitochondria are rapidly oxidized to ketone bodies. Thus, the rate-controlling step for ketogenesis is carnitine acyltransferase I. Administration of food after a fast, or of insulin to the diabetic subject, reduces plasma free fatty acid concentrations, increases the liver concentration of malonyl-CoA, inhibits carnitine acyltransferase I and reverses the ketogenic process.