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.     

Does fatty acid-binding protein play a role in fatty acid transport?

Summary The possible property of fatty acid-binding proteins (FABPs) to transport fatty acid was investigated in various model systems with FABP preparations from liver and heart. An effect of FABP, however, was not detectable with a combination of oleic acid-loaded mitochondria and vesicles or liposomes due to the rapid spontaneous transfer. Therefore, the mitochondria were separated from the vesicles in an equilibrium dialysis cell. The spontaneous fatty acid transfer was much lower and addition of FABP resulted in an increase of fatty acid transport. Oleic acid was withdrawn from different types of monolayers by FABP with rates up to 10%/min. When two separate monolayers were used, FABP increased fatty acid transfer between these monolayers and an equilibrium was reached.Abbreviations FABP(s) fatty acid-binding protein(s) - PC phosphatidylcholine - PS phosphatidylserine - PE phosphatidylethanolamine     

Is fatty acid uptake in cardiomyocytes determined by physicochemical fatty acid partition between albumin and membranes?

Summary Palmitate uptake by isolated, calcium-resistant cardiomyocytes was measured by using a stimulation chamber in which cell contraction can be evoked electrically. Experiments were performed in a medium containing physiological interstitial concentration of albumin (2%) and palmitate/albumin (P/A) ratios ranging from 0.03 to 2.5, and were compared to experiments with fixed P/A ratio (– 1).Initial rate of uptake (V_i) was calculated from fitted uptake vs. time curves as measured by accumulation of radioactivity in the cells from ¹⁴C-labelled palmitate. V_i-vs.-concentration curves exhibited a saturable component, if albumin concentration was kept constant. Almost no change in V_i was observed in experiments performed at constant P/A. This is in contrast to the albumin receptor hypothesis.The ¹⁴C-palmitate content of the myocytes as estimated by thin-layer-chromatography did reach a plateau at 30 s and had the same value at 30 min after administration. The cellular content of labelled palmitate could be attributed to the membrane compartment as calculated from partition coefficient (Kc) of fatty acids (FA) between albumin and membranes. With electrical stimulation V_i-vs.-palmitate concentration kinetics showed a shift in apparent Km from 62 µM (P/A – 0.22) to 23 µM (P/A = 0.08), and presence of 2,4-dinitrophenol increases V_i.Our results suggest that FA-transfer across the sarcolemmal membranes is determined by a physicochemical equilibrium between the compartments of extracellular FA-albumin complex, the membrane lipid phase, intracellular FA binding proteins and the respective aqueous phases. Consequently in cell suspensions the rate of palmitate uptake is controlled by a step of fatty acid metabolism possibly the formation of Fa CoA by the enzyme FA acyl CoA synthetase which is localized in membranes of endoplasmatic reticulum and mitochondria. This step is influenced by the metabolic state of the cells and by FA concentration in membranes.     

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.     

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.     

Properties of Bacillus acidocaldarius mutants deficient in ω-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in -cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing -cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.     

Higher plasma n-3 fatty acid status in the moderately healthy elderly in southern Québec: Higher fish intake or aging-related change in n-3 fatty acid metabolism?

The elderly reportedly have a significantly higher % of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma and red cell lipids. However, these observations are from a few small studies and the health status of the elderly in these studies is for the most part unclear. Since the elderly are susceptible to cardiovascular and neurological illnesses that seem to be related in part to lower intake of n-3 fatty acids it seems paradoxical that their blood levels of EPA and DHA would be higher than in young adults. We report here plasma fatty acid profiles and their response to supplementation with two types of fish oils from several of our recent studies in the moderately healthy elderly. We define the moderately healthy elderly as those who were in good physical condition, had no cognitive decline and, if present, in whom hypothyroidism, hyperlipidemia and/or hypertension were well-controlled. As shown previously, we confirm the higher % EPA and % total n-3 fatty acids (but not DHA) in fasting plasma and extend these findings to include higher plasma concentrations (mg/L) of n-3 fatty acids as well. The EPA-predominant supplement raised DHA only in the young, whereas the DHA-predominant supplement raised EPA more in the young than in the elderly. The moderately healthy elderly clearly have higher plasma n-3 fatty acids but whether this reflects differences in intake versus aging-related changes in n-3 fatty acid metabolism remains to be elucidated.     

Physiological function and ecological aspects of fatty acid-amino acid conjugates in insects†

In tritrophic interactions, plants recognize herbivore-produced elicitors and release a blend of volatile compounds (VOCs), which work as chemical cues for parasitoids or predators to locate their hosts. From detection of elicitors to VOC emissions, plants utilize sophisticated systems that resemble the plant–microbe interaction system. Fatty acid–amino acid conjugates (FACs), a class of insect elicitors, resemble compounds synthesized by microbes in nature. Recent evidence suggests that the recognition of insect elicitors by an ancestral microbe-associated defense system may be the origin of tritrophic interactions mediated by FACs. Here we discuss our findings in light of how plants have customized this defense to be effective against insect herbivores, and how some insects have successfully adapted to these defenses.     

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.     

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.     

Adrenic acid Δ4 desaturation and fatty acid composition in the liver of marine-oil fed streptozotocin diabetic rats

The aim of the present study was to assess the effect of streptozotocin diabetes and insulin treatment on adrenic acid Δ4 desaturation and fatty acid composition of liver microsomes in Wistar rats fed a fat free semi-synthetic basal diet supplemented with 10% EPA-rich marine oil. Results showed that, in liver microsomes of hyperglycemic rats, the ratio in total lipids was elevated and desaturation of adrenic acid to n-6 docosapentaenoic acid was enhanced. Insulin treatment with 2.0 I.U./100 g body weight⁻¹ twice a day for 3 days resulted in hypoglycemia and suppressed both the increased Δ4 n-6 desaturation and ratio. It is concluded that the Δ4 desaturation enzyme system, which is activated by experimental diabetes, is regulated by mechanisms different from those regulating Δ6 and Δ5 desaturations.     

Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARδ displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARδ mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARδ is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARδ in muscle cells, we forced its expression and that of a dominant-negative PPARδ mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARδ agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARδ enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARδ in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.     

Is the fatty acid composition of Daphnia galeata determined by the fatty acid composition of the ingested diet?

1. The fatty acid (FA) composition of Daphnia galeata and their algal food was analysed and showed many similarities, however, some significant differences were also found in the relative abundance of the FA C16 : 4ω3 and docosahexaenoic acid (DHA). Their relative abundances were much lower in daphnids than in their algal diet.
2. When daphnids were fed three distinct emulsion particles with DHA : eicosapentaenoic acid (EPA) ratios of c. 0.7, 2 and 4, the final DHA : EPA ratio in the daphnids always favoured EPA. The increase of the food DHA : EPA ratio resulted in a minor increase of DHA (to c. 2%). Feeding the animals on emulsion particles with increasing ratios of DHA : EPA, caused a minor ( c. 2%) increase of DHA level but EPA levels remained high ( c. 10%).
3. When labelled with [¹⁴C]linoleic acid and [¹⁴C]linolenic acid daphnids showed low conversion of both essential FA into C20 polyunsaturated fatty acids (PUFAs). This low conversion activity might explain the importance of C20 PUFAs as dietary compounds in the food of Daphnia.
4. The results indicate the insignificance of DHA and C16 : 4ω3 for daphnids. As EPA can be derived from C18 : 3ω3 it is not strictly essential, although it might be a significant factor in food quality for Daphnia.     

Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.     

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.     

Regulation of fatty acid synthesis and Δ9-desaturation in senescence of human fibroblasts

AimsNormal human cells in culture progressively lose their capacity for replication, ending in an irreversible arrested state known as replicative senescence. Senescence has been functionally associated to the process of organismal ageing and is also considered a major tumor-suppressing mechanism. Although a great deal of knowledge has uncovered many of the molecular aspects of senescence, little is known about the regulation of lipid synthesis, particularly the biosynthesis and Δ9-desaturation of fatty acids, during the senescence process.Main methodsBy using immunoblotting and metabolic radiolabeling, we determined the senescence-associated changes in major lipogenic pathways.Key findingsThe levels of fatty acid synthase and stearoyl-CoA desaturase-1 and, consequently, the formation of monounsaturated fatty acids, were notably decreased in senescent cells when compared to proliferating (young) fibroblasts. Moreover, we detected a reduction in the de novo synthesis of phospholipids with a concomitant increase in the formation of cholesterol in senescent cells compared to young fibroblasts. Finally, it was found that exogenous fatty acids were preferentially incorporated into the triacylglycerol pool of senescent cells.SignificanceThis set of observations is the first demonstration of a profound modification in lipid metabolism, particularly fatty acid biosynthesis and desaturation, caused by the senescence process and contributes to the increasing body of evidence linking de novo lipogenesis with cellular proliferation.     

Detection of lactobacillic acid in low erucic rapeseed oil—A note of caution when quantifying cyclic fatty acid monomers in vegetable oils

The purpose of this work was to identify an unknown component which has been detected during the analysis of cyclic fatty acid monomers (CFAMs) in low erucic acid rapeseed oils (LEAR). A sample of crude LEAR was transformed into fatty acid methyl esters (FAMEs) and hydrogenated using PtO₂. The hydrogenated sample was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and the fraction containing the CFAMs transformed into picolinyl esters. Analysing these picolinyl derivatives by gas–liquid chromatography coupled to mass spectrometry (GC–MS) showed that the unknown product observed in LEAR is the 11,12-methylene-octadecanoic acid. This cyclic fatty acid was also found in crude LEAR and in the corresponding seeds but was not detected in crude soya and sunflower oils. As this acid is present in the same fraction as CFAMs, known to be formed during heat treatment, great care must therefore be taken for not including it when quantifying CFAMs. It is thus necessary to verify by mass spectrometry the structures of the CFAMs in the isolated cyclic fatty acid fraction prior to quantification.