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.     

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     

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.     

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.     

Peroxisomal β-oxidation enzymes

Peroxisomal fatty acid oxidation enzymes are summarized in comparison to their mitochondrial counterparts. The peroxisomal enzymes involved in the β-oxidation spiral are schematically classified into two groups. The first group consists of hitherto purified and characterized classical enzymes: palmitoyl-CoA oxidase, the L-bifunctional protein, and 3-ketoacyl-CoA. These enzymes are inducible and act on the straight chain substrates. The second group consists of recently identified enzymes, branched-chain oxidase, the d-bifunctional protein, and sterolcarrier protein x, which catalyze four reactions of β-oxidation cycle. These are noninducible and act on branched-chain substrates.     

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.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.     

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.     

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.     

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.     

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.     

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.     

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.     

Futile cycle of β-oxidation and de novo lipogenesis are associated with essential fatty acids depletion in lipoatrophy

Total absence of adipose tissue (lipoatrophy) is associated with the development of severe metabolic disorders including hepatomegaly and fatty liver. Here, we sought to investigate the impact of severe lipoatrophy induced by deletion of peroxisome proliferator-activated receptor gamma (PPARγ) exclusively in adipocytes on lipid metabolism in mice. Untargeted lipidomics of plasma, gastrocnemius and liver uncovered a systemic depletion of the essential linoleic (LA) and α-linolenic (ALA) fatty acids from several lipid classes (storage lipids, glycerophospholipids, free fatty acids) in lipoatrophic mice. Our data revealed that such essential fatty acid depletion was linked to increased: 1) capacity for liver mitochondrial fatty acid β-oxidation (FAO), 2) citrate synthase activity and coenzyme Q content in the liver, 3) whole-body oxygen consumption and reduced respiratory exchange rate in the dark period, and 4) de novo lipogenesis and carbon flux in the TCA cycle. The key role of de novo lipogenesis in hepatic steatosis was evidenced by an accumulation of stearic, oleic, sapienic and mead acids in liver. Our results thus indicate that the simultaneous activation of the antagonic processes FAO and de novo lipogenesis in liver may create a futile metabolic cycle leading to a preferential depletion of LA and ALA. Noteworthy, this previously unrecognized cycle may also explain the increased energy expenditure displayed by lipoatrophic mice, adding a new piece to the metabolic regulation puzzle in lipoatrophies.     

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.