Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.     

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.     

Combined defects in oxidative phosphorylation and fatty acid β-oxidation in mitochondrial disease

Mitochondria provide the main source of energy to eukaryotic cells, oxidizing fats and sugars to generate ATP. Mitochondrial fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two metabolic pathways which are central to this process. Defects in these pathways can result in diseases of the brain, skeletal muscle, heart and liver, affecting approximately 1 in 5000 live births. There are no effective therapies for these disorders, with quality of life severely reduced for most patients. The pathology underlying many aspects of these diseases is not well understood; for example, it is not clear why some patients with primary FAO deficiencies exhibit secondary OXPHOS defects. However, recent findings suggest that physical interactions exist between FAO and OXPHOS proteins, and that these interactions are critical for both FAO and OXPHOS function. Here, we review our current understanding of the interactions between FAO and OXPHOS proteins and how defects in these two metabolic pathways contribute to mitochondrial disease pathogenesis.     

Heat stress deteriorates mitochondrial β-oxidation of long-chain fatty acids in cultured fibroblasts with fatty acid β-oxidation disorders

Mitochondrial fatty acids β-oxidation disorder (FAOD) has become popular with development of tandem mass spectrometry (MS/MS) and enzymatic evaluation techniques. FAOD occasionally causes acute encephalopathy or even sudden death in children. On the other hand, hyperpyrexia may also trigger severe seizures or encephalopathy, which might be caused by the defects of fatty acid β-oxidation (FAO). We investigated the effect of heat stress on FAO to determine the relationship between serious febrile episodes and defect in β-oxidation of fatty acid in children. Fibroblasts from healthy control and children with various FAODs, were cultured in the medium loaded with unlabelled palmitic acid for 96 h at 37 °C or 41 °C. Acylcarnitine (AC) profiles in the medium were determined by MS/MS, and specific ratios of ACs were calculated. Under heat stress (at 41 °C), long-chain ACs (C12, C14, or C16) were increased, while medium-chain ACs (C6, C8, or C10) were decreased in cells with carnitine palmitoyl transferase II deficiency, very-long-chain acyl-CoA dehydrogenase deficiency and mitochondrial trifunctional protein deficiency, whereas AC species from short-chain (C4) to long-chain (C16) were barely affected in medium-chain acyl-CoA dehydrogenase and control. While long-chain ACs (C12–C16) were significantly elevated, short to medium-chain ACs (C4–C10) were reduced in multiple acyl-CoA dehydrogenase deficiency. These data suggest that patients with long-chain FAODs may be more susceptible to heat stress compared to medium-chain FAOD or healthy control and that serious febrile episodes may deteriorate long-chain FAO in patients with long-chain FAODs.     

Possible involvement of fatty acid binding protein in peroxisomal β-oxidation of fatty acids

The localization of β-oxidation of fatty acids in isolated peroxisomes from rat liver was investigated. The enzyme system is soluble in the luminal compartment and carnitine does not appear to be involved in the transfer of the CoA derivatives through the peroxisomal membrane. Experiments involving proteolysis, inhibitors and competitive inhibition suggest that a fatty acid binding protein is responsible for the carrier process. This carrier protein seems to be present in increased amounts both in the supernatant and in the peroxisomes after clofibrate induction.     

Sulfides impair short chain fatty acid β-oxidation at acyl-CoA dehydrogenase level in colonocytes: Implications for ulcerative colitis

The disease process of ulcerative colitis (UC) is associated with a block in -oxidation of short chain fatty acid in colonic epithelial cells which can be reproduced by exposure of cells to sulfides. The aim of the current work was to assess the level in the -oxidation pathway at which sulfides might be inhibitory in human colonocytes. Isolated human colonocytes from cases without colitis (n = 12) were exposed to sulfide (1.5 mM) in the presence or absence of exogenous CoA and ATP. Short chain acyl-CoA esters were measured by a high performance liquid chromatographic assay. ¹⁴CO₂ generation was measured from [1-¹⁴C]butyrate and [6-¹⁴C]glucose. ¹⁴CO₂ from butyrate was significantly reduced (p < 0.001) by sulfide. When colonocytes were incubated with hydrogen sulfide in the presence of CoA and ATP, butyryl-CoA concentration was increased (p < 0.01), while crotonyl-CoA (p < 0.01) and acetyl-CoA (p < 0.01) concentrations were decreased. These results show that sulfides inhibit short chain acyl-CoA dehydrogenase. As oxidation of n-butyrate governs the epithelial barrier function of colonocytes the functional activity of short chain acyl-CoA dehydrogenase may be critical in maintaining colonic mucosal integrity. Maintaining the functional activity of dehydrogenases could be an important determinant in the expression of ulcerative colitis.     

Cilostazol induces mitochondrial fatty acid β-oxidation in C2C12 myotubes

Cilostazol is a drug licensed for the treatment of intermittent claudication. Its main action is to elevate intracellular levels of cyclic monophosphate (cAMP) by inhibiting the activity of type III phosphodiesterase, a cAMP-degrading enzyme. The effects of cilostazol on fatty acid oxidation (FAO) are as yet unknown. In this study, we report that cilostazol can elevate complete FAO and decrease both triacylglycerol (TAG) accumulation and TAG secretion. This use of cilostazol treatment increases expression of PGC-1α and, subsequently, its target genes, such as ERRα, NOR1, CD36, CPT1, MCAD, and ACO. Expression of these factors is linked to fatty acid β-oxidation but this effect is inhibited by H-89, a specific inhibitor of the PKA/CREB pathway. Importantly, knockdown of PGC-1α using siRNA abolished the effects of cilostazol in fatty acid oxidation (FAO) and TAG metabolism. These findings suggested that the PKA/CREB/PGC-1α pathway plays a critical role in cilostazol-induced fatty acid oxidation and TAG metabolism.     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Rapid β-oxidation of eicosapentaenoic acid in mouse brain: An in situ study

Analyses of brain phospholipid fatty acid profiles reveal a selective deficiency and enrichment in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. In order to account for this difference in brain fatty acid levels, we hypothesized that EPA is more rapidly β-oxidized upon its entry into the brain. Wild-type C57BL/6 mice were perfused with either ¹⁴C-EPA or ¹⁴C-DHA via in situ cerebral perfusion for 40 s, followed by a bicarbonate buffer to wash out the residual radiolabeled polyunsaturated fatty acid (PUFA) in the capillaries. ¹⁴C-PUFA-perfused brains were extracted for chemical analyses of neutral lipid and phospholipid fatty acids. Based on the radioactivity in aqueous, total lipid, neutral lipid and phospholipid fractions, volume of distribution (V_D, μl/g) was calculated. The V_D between ¹⁴C-EPA- and ¹⁴C-DHA-perfused samples was not statistically different for total lipid, neutral lipids or total phospholipids. However, the V_D of ¹⁴C-EPA in the aqueous fraction was 2.5 times higher than that of ¹⁴C-DHA (p=0.025), suggesting a more extensive β-oxidation than DHA. Furthermore, radiolabeled palmitoleic acid, a fatty acid that can be synthesized de novo, was detected in brain phospholipids from ¹⁴C-EPA but not from ¹⁴C-DHA-perfused mice suggesting that β-oxidation products of EPA were recycled into endogenous fatty acid biosynthetic pathways. These findings suggest that low levels of EPA in brain phospholipids compared to DHA may be the result of its rapid β-oxidation upon uptake by the brain.     

Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid β-oxidation

The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.     

Peroxisomal β-oxidation of fatty acids in bovine and rat liver

Hepatic peroxisomal β-oxidation rates were compared in liver homogenates from cows and rats during different nutritional and physiological states. Peroxisomal oxidation in liver homogenates from cows represented 50% and 77% of the total capacity for the initial cycle of β-oxidation of palmitate and octanoate, respectively, but only 26% and 65% for rats. Lactation or food deprivation did not alter rates of hepatic peroxisomal β-oxidation of palmitate or octanoate in cows. Fasting and clofibrate treatment increased rates of total and peroxisomal β-oxidation of palmitate and octanoate in rat liver.     

Benzafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal β-oxidation in rat white adipose tissue

Rats treated with bezafibrate, a PPAR activator, gain less body weight and increase daily food intake. Previously, we have related these changes to a shift of thermogenesis from brown adipose tissue to white adipose tissue attributable to bezafibrate, which induces uncoupling proteins (UCP), UCP-1 and UCP-3, in rat white adipocytes. Nevertheless, UCP induction was weak, implying additional mechanisms in the change of energy homeostasis produced by bezafibrate. Here we show that bezafibrate, in addition to inducing UCPs, modifies energy homeostasis by directly inducing aco gene expression and peroxisomal fatty acid -oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPAR or ob gene in white adipose tissue. These results indicate that bezafibrate reduces the amount of fatty acids available for triglyceride synthesis in white adipose tissue.     

Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Increased peroxisomal fatty acid β-oxidation and enhanced expression of peroxisome proliferator-activated receptor-α in diabetic rat liver

To determine whether the increased fatty acid -oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR- and its target genes responsible for the metabolism of fatty acids.     

LSDP5 enhances triglyceride storage in hepatocytes by influencing lipolysis and fatty acid β-oxidation of lipid droplets

Lipid storage droplet protein 5 (LSDP5) is a lipid droplet-associated protein of the PAT (perilipin, adipophilin, and TIP47) family that is expressed in the liver in a peroxisome proliferator-activated receptor alpha (PPARα)-dependent manner; however, its exact function has not been elucidated. We noticed that LSDP5 was localized to the surface of lipid droplets in hepatocytes. Overexpression of LSDP5 enhanced lipid accumulation in the hepatic cell line AML12 and in primary hepatocytes. Knock-down of LSDP5 significantly decreased the triglyceride content of lipid droplets, stimulated lipolysis, and modestly increased the mitochondrial content and level of fatty-acid β-oxidation in the mitochondria. The expression of PPARα was increased in LSDP5-deficient cells and required for the increase in the level of fatty acid β-oxidation in LSDP5-deficient cells. Using serial deletions of LSDP5, we determined that the lipid droplet-targeting domain and the domain directing lipid droplet clustering overlapped and were localized to the 188 amino acid residues at the N-terminus of LSDP5. Our findings suggest that LSDP5, a novel lipid droplet protein, may contribute to triglyceride accumulation by negatively regulating lipolysis and fatty acid oxidation in hepatocytes.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed β-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of Δ9- and Δ6-desaturases in liver microsomes of rats fed diets supplemented with β-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg β-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for Δ9-desaturase and Δ6-desaturase activities. The activity of Δ9-desaturase was lower in liver microsomes of rats fed β-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal Δ6-desaturase activity was, however, higher in liver of rats fed 13-cis-retinoic acid; there was no effect of β-carotene on Δ6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding β-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Mitochondrial,but not peroxisomal, β-oxidation of fatty acids is conserved in coenzyme A-Deficient rat liver

Growth hormone (GH) exerts acute insulin-like effects, such as increased lipogenesis and inhibition of catecholamine-induced lipolysis, in rat adipocytes that have not been exposed to GH during the preceding three hours. We found that OPC3911, a highly specific inhibitor of the cGMP-inhibited cAMP phosphodiesterase, completely blocked the antilipolytic but not the lipogenic effect of GH. This indicates that the antilipolytic effect of GH is mediated through activation of this phosphodiesterase leading to reduction of cAMP levels in the same manner as has been shown for insulin.     

Cimicifuga racemosa impairs fatty acid β-oxidation and induces oxidative stress in livers of ovariectomized rats with renovascular hypertension

The aim of this work was to evaluate the effects of therapeutic doses of Cimicifuga racemosa on cardiovascular parameters and on liver lipid metabolism and redox status in an animal model of estrogen deficiency associated with hypertension, a condition that could make the liver more vulnerable to drug-induced injuries. Female Wistar rats were subjected to the surgical procedures of bilateral ovariectomy (OVX) and induction of renovascular hypertension (two-kidneys, one-clip; 2K1C). These animals (OVX + 2K1C) were treated with daily doses of a C. racemosa extract, using a dose that is similar to that recommended to postmenopausal women (0.6mg/kg), over a period of 15 days. The results were compared to those of untreated OVX + 2K1C, OVX, and control rats. The treatment with C. racemosa caused a significant reduction in blood pressure. In the liver, treatment did not prevent the development of steatosis, and it reduced the mitochondrial and peroxisomal capacity to oxidize octanoyl-CoA compared to the untreated animals. In addition, C. racemosa caused numerous undesirable effects on the liver redox status: it increased the mitochondrial reactive oxygen species generation, an event that was not accompanied by an increase in the activity of superoxide dismutase, and it induced a decrease in peroxisomal catalase activity. Although the reduced glutathione content had not been affected, a phenomenon that probably reflected the restoration of glucose-6-phosphate dehydrogenase activity by C. racemosa, oxidative damage was evidenced by the elevated level of thiobarbituric acid-reactive substances found in the liver of treated animals.