Mitochondrial acyl-CoA Synthetase Research Articles

Sortilin-mediated translocation of mitochondrial ACSL1 impairs adipocyte thermogenesis and energy expenditure in male mice

Beige fat activation involves a fuel switch to fatty acid oxidation following chronic cold adaptation. Mitochondrial acyl-CoA synthetase long-chain family member 1 (ACSL1) localizes in the mitochondria and plays a key role in fatty acid oxidation; however, the regulatory mechanism of the subcellular localization remains poorly understood. Here, we identify an endosomal trafficking component sortilin (encoded by Sort1) in adipose tissues that shows dynamic expression during beige fat activation and facilitates the translocation of ACSL1 from the mitochondria to the endolysosomal pathway for degradation. Depletion of sortilin in adipocytes results in an increase of mitochondrial ACSL1 and the activation of AMPK/PGC1α signaling, thereby activating beige fat and preventing high-fat diet (HFD)-induced obesity and insulin resistance. Collectively, our findings indicate that sortilin controls adipose tissue fatty acid oxidation by substrate fuel selection during beige fat activation and provides a potential targeted approach for the treatment of metabolic diseases.

Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Abstract 13712: Imaging Short-Chain Fatty Acid Metabolism and Transport in the Failing Heart by PET

Introduction: Heart failure compromises the transport and oxidation of long chain fatty acids. Cardiomyocytes can compensate by using short-chain fatty acids (SCFA) which rely on other transport mechanisms. Based on this principle, we used 2-[ 18 F]fluoropropionic acid ([ 18 F]FPA), a radiolabeled SCFA analog, to assess these metabolic changes and image them by positron emission tomography (PET) in a doxorubicin-induced mouse model of cardiotoxicity. Hypothesis: We hypothesize that the increased demand for SCFA by the failing heart promotes cardiac accumulation of [ 18 F]FPA which can be imaged by PET. Methods: [ 18 F]FPA was injected intravenously into male C57BL/6J mice 10 weeks following intraperitoneal injections of 8 x 3 mg/kg doxorubicin (DOX). Dynamic PET acquisitions were performed from 60-120 min post injection of 9.25-11 MBq [ 18 F]FPA. These images were acquired in the presence or absence of 5 mg/kg AZD3965, an inhibitor of monocarboxylate transporters. Heart uptake of [ 18 F]FPA was determined by image-based quantitation and confirmed by a biodistribution study. Mechanistic [ 18 F]FPA uptake studies were also conducted in primary human cardiomyocyte (HCM) cultures. Results: Mice experiencing DOX-induced cardiotoxicity exhibited statistically significant increases in cardiac [ 18 F]FPA. AZD3965 did not significantly alter cardiac uptake but reduced uptake in all other tissues except the kidneys, the route of excretion. Additionally, [ 18 F]FPA uptake is lost in HCM for which mitochondrial acyl-CoA synthetase (ACSS) 1, but not cytosolic ACSS2, is inhibited. Conclusions: Our findings present both mechanistic and translational applications for [ 18 F]FPA. AZD3965 effectively reduced [ 18 F]FPA uptake in other tissues except for the heart and can therefore be co-administered to enhance the heart-to-background ratio. Altogether, our results indicate that [ 18 F]FPA may enter cardiac tissues by diffusion and become metabolically trapped in mitochondria.

Imaging Anaplerosis in the Failing Heart by PET

ObjectiveHeart failure is often preceded by distinct metabolic alterations. However, this shift cannot be sustained indefinitely and typically leads to cardiac dysfunction and arrest. These alterations are thought to be caused by mitochondrial dysfunction leading to the accumulation of acyl‐CoAs. To compensate, the injured heart shifts from oxidation of long chain fatty acids (C16) to metabolism of substrates that can replenish the TCA cycle. This is because these fatty acids are restricted to catabolism to acetyl‐CoA, for which the enzymes are saturated. By contrast, odd chain fatty acids are metabolized to propionate. The enzymes specific for propionate are freely available for metabolism to succinate leading to TCA cycle anaplerosis. Propionate conversion to propionyl‐CoA is the first and major step for its sequestration and metabolism. Moreover, the mitochondrial acyl‐CoA synthetase (ACSS) responsible for this conversion is highly expressed in the heart. Hence, we hypothesize that 2‐[18F]‐fluoropropionate ([18F]‐FPA) would significantly accumulate in the failing heart and can be imaged by positron emission tomography (PET). We addressed our hypothesis by studying the substrate behavior of FPA for ACSS, and by metabolic profiling of HepG2 cells incubated with FPA. We also successfully imaged heart uptake of [18F]‐FPA in healthy rats by PET.MethodsThe ability of acetate, propionate, and FPA to act as ACSS substrates was determined by a colorimetric pyrophosphate assay. Metabolites were determined by LC/MS in lysates of HepG2 cells incubated with basal media, 10 mM propionate or 10 mM FPA. Racemic 18F‐FPA was synthesized from ethyl‐2‐bromopropionate by standard nucleophilic substitution. For PET, Sprague‐Dawley rats were injected with 250‐500 µCi (9.25‐18.5 MBq) of [18F]‐FPA, and a 60‐minute dynamic PET scan was immediately acquired.ResultsThe kinetic characteristics for the reaction of ACSS acting on acetate (Km = 4.94 mM, Vmax = 0.25 nmols/sec), propionate (Km = 24.20 mM, Vmax = 0.19 nmols/sec), and 2‐fluoropropionoate (V = 0.05 nmols/sec) were determined. We also observed a reduction of free CoA in HepG2 cells incubated in 10 mM propionate (4.4 ± 1.2‐fold) or 10 mM FPA (3.09 ± 0.96‐fold). The kinetics of 18F‐FPA uptake into the healthy heart was assessed by PET and determined to be maximal at 10 minutes post‐injection.ConclusionsWe identified [18F]‐FPA, and potentially other odd chain fatty acids, as promising metabolic tracers for imaging anaplerosis in the injured heart. FPA effectively decreases free CoA in HepG2 cells, which indicates the formation of acyl‐CoAs. Moreover, FPA exhibits some substrate activity for ACSS, representing a major conduit by which it is metabolized in the heart. The poor substrate activity of FPA is ideal as it precludes metabolism by the highly active and specific ACSS enzymes in the liver. Albeit we expect FPA to accumulate in the heart due to its high ACSS expression which is induced in energy deficient states that are characteristic of heart failure. In support of this idea, we demonstrated that [18F]‐FPA is taken up by the healthy heart. We expect the metabolic alterations resulting from ensuing heart failure are sufficient to increase the uptake of [18F]‐FPA in these tissues. Our long‐term aim is to develop 18F‐labeled short chain fatty acids, such as [18F]‐FPA, that can be used to diagnose at‐risk patients. The early detection of heart failure by non‐invasive means is critical for the development of interventions to reduce the severity and aid recovery from cardiac disease.

Impaired mitochondrial medium-chain fatty acid oxidation drives periportal macrovesicular steatosis in sirtuin-5 knockout mice

Medium-chain triglycerides (MCT), containing C8–C12 fatty acids, are used to treat several pediatric disorders and are widely consumed as a nutritional supplement. Here, we investigated the role of the sirtuin deacylase Sirt5 in MCT metabolism by feeding Sirt5 knockout mice (Sirt5KO) high-fat diets containing either C8/C10 fatty acids or coconut oil, which is rich in C12, for five weeks. Coconut oil, but not C8/C10 feeding, induced periportal macrovesicular steatosis in Sirt5KO mice. 14C–C12 degradation was significantly reduced in Sirt5KO liver. This decrease was localized to the mitochondrial β-oxidation pathway, as Sirt5KO mice exhibited no change in peroxisomal C12 β-oxidation. Endoplasmic reticulum ω-oxidation, a minor fatty acid degradation pathway known to be stimulated by C12 accumulation, was increased in Sirt5KO liver. Mice lacking another mitochondrial C12 oxidation enzyme, long-chain acyl-CoA dehydrogenase (LCAD), also developed periportal macrovesicular steatosis when fed coconut oil, confirming that defective mitochondrial C12 oxidation is sufficient to induce the steatosis phenotype. Sirt5KO liver exhibited normal LCAD activity but reduced mitochondrial acyl-CoA synthetase activity with C12. These studies reveal a role for Sirt5 in regulating the hepatic response to MCT and may shed light into the pathogenesis of periportal steatosis, a hallmark of human pediatric non-alcoholic fatty liver disease.

Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4

Long-chain fatty acids are important metabolites for the generation of energy and the biosynthesis of lipids. The molecular mechanism of their cellular uptake has remained controversial. The fatty acid transport protein (FATP) family has been named according to its proposed function in mediating this process at the plasma membrane. Here, we show that FATP4 is in fact localized to the endoplasmic reticulum and not the plasma membrane as reported previously. Quantitative analysis confirms the positive correlation between expression of FATP4 and uptake of fatty acids. However, this is dependent on the enzymatic activity of FATP4, catalyzing the esterification of fatty acids with CoA. Monitoring fatty acid uptake at the single-cell level demonstrates that the ER localization of FATP4 is sufficient to drive transport of fatty acids. Expression of a mitochondrial acyl-CoA synthetase also enhances fatty acid uptake, suggesting a general relevance for this mechanism. Our results imply that cellular uptake of fatty acids can be regulated by intracellular acyl-CoA synthetases. We propose that the enzyme FATP4 drives fatty acid uptake indirectly by esterification. It is not a transporter protein involved in fatty acid translocation at the plasma membrane.

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation.

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.

Potent Peroxisome Proliferators Inhibit β-Oxidation in the Isolated Perfused Rat Liver

It is unknown whether peroxisome proliferators decrease hepatic fatty acid oxidation via uncoupling of respiration or if they inhibit extramitochondrial fatty acyl CoA synthesis. Therefore, the purpose of this study was to examine both processes simultaneously using the isolated perfused liver, a whole cell preparation where enzymes and biochemical processes can be monitored continuously under nearly physiological conditions. Accordingly, ketone body formation (β-hydroxybutyrate + acetoacetate) from lipid metabolism and oxygen uptake, which is increased by uncoupling agents, were monitored at the same time. 2-Bromooctanoate, a known inhibitor of acyl CoA synthetase, decreased ketone body formation in a dose-dependent manner without altering cellular respiration (half-maximal inhibition, ∼25 μM) and concomitantly increased protein kinase C nearly fourfold also in a dose-dependent fashion. Ketogenesis was also blocked maximally 50–66% with mono(ethylhexyl)phthalate, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643), and nafenopin, potent peroxisome proliferators and tumor promoters. These compounds also increased protein kinase C three- to fourfold without altering oxygen uptake significantly. Thus, lipid metabolism appears to be the prime target of potent peroxisome proliferators most likely on actions via acyl CoA synthetase rather than oxidative phosphorylation. In contrast, weak peroxisome proliferators and tumor promoters, di(ethylhexyl)phthalate and 2-ethylhexanol, did not affect ketogenesis, oxygen consumption, or protein kinase C at similar concentrations. Additionally, octanoate increased ketone body formation in the presence of nafenopin. Because octanoate is metabolized by mitochondrial acyl CoA synthetase independent of carnitine acyltransferase, these results indicate that nafenopin does not inhibit mitochondrial β-oxidation. Taken together, it is concluded that potent peroxisome proliferators preferentially block ketogenesis without altering cellular respiration in the liver. This phenomenona, which occurs due to inhibition of acyl CoA synthetase, leads to an elevation of free fatty acids that stimulates protein kinase C and promotes cell proliferation.

The function of acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor (DBI).

Acyl-CoA-binding protein has been isolated independently by five different groups based on its ability to (1) displace diazepam from the GABAA receptor, (2) affect cell growth, (3) induce medium-chain acyl-CoA-ester synthesis, (4) stimulate steroid hormone synthesis, and (5) affect glucose-induced insulin secretion. In this survey evidence is presented to show that ACBP is able to act as an intracellular acyl-CoA transporter and acyl-CoA pool former. The rat ACBP genomic gene consists of 4 exons and is actively expressed in all tissues tested with highest concentration being found in liver. ACBP consists of 86 amino acid residues and contains 4 alpha-helices which are folded into a boomerang type of structure with alpha-helices 1, 2 and 4 in the one arm and alpha-helix 3 and an open loop in the other arm of the boomerang. ACBP is able to stimulate mitochondrial acyl-CoA synthetase by removing acyl-CoA esters from the enzyme. ACBP is also able to desorb acyl-CoA esters from immobilized membranes and transport and deliver these for mitochondrial beta-oxidation. ACBP efficiently protects acetyl-CoA carboxylase and the mitochondrial ADP/ATP translocase against acyl-CoA inhibition. Finally, ACBP is shown to be able to act as an intracellular acyl-CoA pool former by overexpression in yeast. The possible role of ACBP in lipid metabolism is discussed.

Relations between fatty acid activation and adenine nucleotide translocase systems in mitochondria

Experiments with freshly isolated rat liver mitochondria and mitochondria aged by two different methods showed that the activity of the mitochondrial fatty acid activation system can lead to inhibition of adenine nucleotide (AN) transport through the inner mitochondrial membrane. Inhibition of AN translocase was abolished by preincubating the mitochondria with carnitine. A special feature distinguishing inhibition of AN translocase by aged mitochondria is that these mitochondrial membranes contain fatty acids in sufficient concentration to cause inhibition on the addition of CoA and ATP. The results suggest a role for acyl-CoA formed by mitochondrial acyl-CoA synthetase in the regulation of AN transport in the mitochondrion.

Regulation of liver acyl-coa synthetase activity

1. 1. Increased activity was observed upon addition of cell supernatant fraction during in vitro assay of rat liver microsomal acyl-CoA synthetase. However, when a constant fatty acid/protein ratio was maintained then the increased activity could be accounted for by the residual activity in the added supernatant alone. We therefore fail to confirm the existence of a “supernatant stimulating factor” 1–5. 2. 2. The acyl-CoA synthetase activities of whole homogenates prepared from livers of either fed or 48 h fasted rats were the same. We therefore fail to confirm the in vivo regulatory role attributed to the “supernatant stimulating factor” as determined by increased acyl-CoA synthetase activity of whole homogenates prepared from livers of 48 h fasted rats compared with the activity of homogenates prepared from livers of fed rats 4. 3. 3. Microsomal and mitochondrial acyl-CoA synthetase activities were not affected by 48 h fasting and only increased slightly (15–25%) after refeeding fasted rats a stock diet, a high fat diet (15% corn oil) or a fat free diet. We therefore conclude that acyl-CoA synthetases are constitutive enzymes of the endoplasmic reticulum and outer mitochondrial membrane whose activity does not vary with fasting or feeding various diets. 4. 4. The implications of these findings for fatty acid metabolism are discussed.