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.     

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.     

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.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

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.     

In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes

Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO.     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

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.     

n-alkane oxidation by apseudomonas formation and β-oxidation of intermediate fatty acids

Summary From a culture broth ofPseudomonas aeruginosa (KSLA strain 473) grown on heptane as the sole source of carbon, fatty acids could be isolated after a period of decreased oxygen supply. The corresponding methyl esters—obtained by treatment with diazomethane—were separated by gas-liquid chromatography and identified by mass spectrometry. Heptylic, valeric and propionic acids were shown to be present in the original culture broth. Using the same techniques the formation of caproic acid from hexane was shown to occur, whereas the amount of butyric acid formed was extremely small and inconsistent. These results show conclusively that this microbiological oxidation of heptane and hexane proceeds by way of the corresponding fatty acids, which are further degraded by β-oxidation. The absence of caproic and valeric acids in heptane and hexane oxidation, respectively, shows that decarboxylation of fatty acids does not occur.     

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.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.     

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.     

Innermembrane association of three mitochondrial β-oxidation enzymes revealed by immunoelectron microscopic technique

Summary Ultrastructural localization of three mitochondrial β-oxidation enzymes, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase in rat liver was studied by a post-embedding immunocytochemical technique. Rat liver was fixed by perfusion. Vibratome sections (100 μm thick) were embedded in Lowicryl K4M. Ultrathin sections were separately incubated with antibody to each enzyme, followed by protein A-gold complex. Gold particles representing the antigenic sites for all enzymes examined were confined exclusively to mitochondria of hepatocytes and other sinus-lining cells. Peroxisomes were consistently negative for the immunolabelling. In the mitochondria the gold particles were localized in the matrical side of inner membrane. The control experiments confirmed the specificity of the immunolabelling. The results firstly indicate that the mitochondrial β-oxidation enzymes are present in the matrix of mitochondria and associated with the inner membrane.     

Impairment of mitochondrial β-oxidation in rats under cold-hypoxic environment

Mitochondrial ß-oxidation of fatty acid provides a major source of energy in mammals. High altitude (HA), characterized by hypobaric hypoxia and low ambient temperatures, causes alteration in metabolic homeostasis. Several studies have depicted that hypoxic exposure in small mammals causes hypothermia due to hypometabolic state. Moreover, cold exposure along with hypoxia reduces hypoxia tolerance in animals. The present study investigated the rate of β-oxidation and key enzymes, carnitine palmitoyl transferase-I (CPT-I) and hydroxyacyl CoA dehydrogenase (HAD), in rats exposed to cold-hypobaric hypoxic environment. Male Sprague Dawley rats (190–220 g) were randomly divided into eight groups (n?=?6 rats in each group): 1 day hypoxia (H1); 7  days hypoxia (H7); 1 day cold (C1); 7 days cold (C7); 1 day cold-hypoxia (CH1); 7 days cold-hypoxia (CH7) exposed; and unexposed control for 1 and 7 days (UC1 and UC7). After exposure, animals were anaesthetized with ketamine (50 mg/kg body weight) and xylazine (10 mg/kg body weight) intraperitonialy and sacrificed. Mitochondrial CPT-I, HAD, ¹⁴C-palmitate oxidation in gastrocnemius muscle and liver, and plasma leptin were measured. Mitochondrial CPT-I was significantly reduced in muscle and liver in CH1 and CH7 as compared to respective controls. HAD activity was significantly reduced in H1 and CH7, and in H1, H7, CH1, and CH7 as compared to unexposed controls in muscle and liver, respectively. A concomitant decrease in ¹⁴C-palmitate oxidation was found. Significant reduction in plasma leptin in hypoxia and cold-hypoxia suggested hypometabolic state. It can be concluded that ß-oxidation of fatty acids is reduced in rats exposed to cold-hypoxic environment due to the persisting hypometabolic state in cold-hypoxia exposure.     

Glyoxysomal β-oxidation of long-chain fatty acids: completeness of degradation

The ability of glyoxysomes from sunflower (Helianthusannuus L.) cotyledons to completely degrade long-chain fatty acids into their constituent acetyl units and the time courses of the appearance of acyl-CoA intermediates during β-oxidation have been studied using ¹⁴C-labelled substrates at non-saturating concentrations (1.3 to 1.8 μmol · l⁻¹). [¹⁴C]Acetyl-CoA was formed from [18-¹⁴C]oleate metabolized at a yield of up to 80%, and from [U-¹⁴C]palmitate and [U-¹⁴C]linoleate to an extent indicating that a maximum of 80% and 30%, respectively, of the substrate β-oxidized had been degraded beyond the C₄-CoA intermediate level. To obtain the latter values, an acetyl-CoA-removing system was required during β-oxidation. Constant re-oxidation of the NADH formed during the β-oxidation did not replace the effect of acetyl-CoA removal. Neither the completeness of the linoleate β-oxidation nor the rate of reaction were influenced by NADPH. Medium- and short-chain acyl-CoA intermediates were predominantly detected during β-oxidation of the long-chain substrates employed. The degradation of these intermediates appeared to be stimulated mainly in the presence of an acetyl-CoA-removing system. The time courses of the appearance of intermediates corresponded to a precursor-product relationship between intermediates of decreasing chain lengths. Received: 12 December 1997 / Accepted: 26 January 1998     

Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid β-oxidation cycle in Escherichia coli

Polyhydroxyalkanoates that contain the medium-chain-length monomers (mcl-PHAs) have a wide range of applications owing to their superior physical and mechanical properties. A challenge to synthesize such mcl-PHAs from unrelated and renewable sources is exploiting the efficient metabolic pathways that lead to the formation of precursor (R)-3-hydroxyacyl-CoA. Here, by engineering the reversed fatty acid β-oxidation cycle, we were able to synthesize mcl-PHAs in Escherichia coli directly from glucose. After deletion of the major thioesterases, the engineered E. coli produced 6.62 wt% of cell dry weight mcl-PHA heteropolymers. Furthermore, when a low-substrate-specificity PHA synthase from Pseudomonas stutzeri 1317 was employed, recombinant E. coli synthesized 12.10 wt% of cell dry weight scl–mcl PHA copolymers, of which 21.18 mol% was 3-hydroxybutyrate and 78.82 mol% was medium-chain-length monomers. The reversed fatty acid β-oxidation cycle offered an efficient metabolic pathway for mcl-PHA biosynthesis in E. coli and can be further optimized.     

Does membrane fatty acid composition modulate mitochondrial functions and their thermal sensitivities?

We investigated the effect of modifying fatty acid modification of heart mitochondrial membranes by dietary intervention on the functions and thermal sensitivity of electron transport system complexes embedded in the inner mitochondrial membrane. Four groups of rats were fed diets differing in their fat (coconut, olive or fish oil) and antioxidant (fish oil with or without probucol) contents. After 16 weeks of feeding, the coconut and olive oil groups had lower long-chain n-3 polyunsaturated fatty acids contents and a lower unsaturation index compared to both fish oil groups. These differences in fatty acid composition were not related to any differences in the mitochondrial respiration rate induced at Complexes I, II or IV, or to differences in their thermal sensitivity. The coconut oil group showed a lower mitochondrial affinity for pyruvate at 5 degrees C (k(mapp)=6.4+/-1.8) compared to any other groups (k(mapp)=3.8+/-0.5; 4.7+/-0.8; 3.6+/-1.1, for olive, fish oil and fish oil and probucol groups, respectively). At least in rat heart, our results do not support a major impact of the fatty acid composition of the mitochondrial membrane on the function of mitochondrial enzymatic complexes or on their temperature sensitivity.     

Lipocalin 13 protein protects against hepatic steatosis by both inhibiting lipogenesis and stimulating fatty acid β-oxidation

Obesity is associated with hepatic steatosis, partially due to increased lipogenesis and decreased fatty acid β-oxidation in the liver; however, the underlying mechanism of abnormal lipid metabolism is not fully understood. We reported previously that obesity is associated with LCN13 (lipocalin 13) deficiency. LCN13 is a lipocalin family member involved in glucose metabolism, and LCN13 deficiency appears to contribute to hyperglycemia in obese mice. Here, we show that LCN13 is also an important regulator of lipogenesis and β-oxidation in the liver. In primary hepatocytes, recombinant LCN13 directly suppressed lipogenesis and increased fatty acid β-oxidation, whereas neutralization of endogenous LCN13 had an opposite effect. Transgenic overexpression of LCN13 protected against hepatic steatosis in mice with either dietary or genetic (ob/ob) obesity. LCN13 transgenic overexpression also improved hyperglycemia, glucose intolerance, and insulin resistance in ob/ob mice. Short-term LCN13 overexpression via an adenovirus-mediated gene transfer similarly attenuated hepatic steatosis in db/db mice. LCN13 inhibited the expression of important lipogenic genes and stimulated the genes that promote β-oxidation. These results suggest that LCN13 decreases liver lipid levels by both inhibiting hepatic lipogenesis and stimulating β-oxidation. LCN13 deficiency is likely to contribute to fatty liver disease in obese mice.