Funktionelle Analyse der LC-FACS in Dictyostelium discoideum

"Funktionelle Analyse der LC-FACS in Dictyostelium discoideum" Das Dictyostelium discoideum Gen fcsA kodiert für ein 75 kDa großes Protein. Es kann durch Homologieanyalysen der Amino-säuresequenz zu den "long-chain fatty acyl-CoA"-Synthetasen ge-rechnet werden, die lang-kettige Fettsäuren durch die kovalente Bindung von Coenzym A akti-vie-ren und damit für diverse Reak-tionen in Stoffwechsel und Molekül-Synthese der Zelle verfügbar machen. Die hier untersuchte D. discoideum LC-FACS lokalisiert als peripher assoziiertes Protein an der cytosolischen Seite der Membran von Endo-somen und kleiner Vesikel. Bereits kurz nach der Bildung in der frühen sauren Phase kann die Lokalisation der LC-FACS auf Endosomen ge-zeigt werden. Sie dissoziiert im Laufe ihrer Neutra-li-sierung und kann auf späten Endosomen, die vor ihrer Exocytose stehen nicht mehr nach-gewiesen werden. Ein Teil der kleinen die in der gesamte Zelle verteilten kleinen Vesikel zeigt eine Kolokalisation mit lysosomalen Enzymen. Trotz des intrazellulären Verteilungs-mus-ters, das eine Beteiligung dieses Pro-teins an der Endocytose nahe-legt, konnte kein signifikanter Rückgang der Pino- und Phagocytose-Rate in LC-FACS Nullmutanten beobachtet werden. Der endo-cy-to-ti-sche Transit ist in diesen Zellen etwas verlängert, außerdem zeigen die Endosomen einen deutlich erhöhten pH-Wert, was zu einer weniger effektiven Prozessierung eines lysosomalen Enzyms führt (a-Mannosidase). Die Funktion der LC-FACS ist die Aufnahme von langkettigen Fettsäuren aus dem Lumen der Endosomen.

Biologische Charakterisierung von LC-FACS-Proteinen in Dictyostelium discoideum

Die Endozytose und die anschließende Verwertung der aufgenommenen Substanzen ist Gegenstand zahlreicher Untersuchungen. Dabei wird ein besonderes Augenmerk auf die Proteine gelegt, die an diesen Vorgängen beteiligt sind. In der hier vorliegenden Arbeit wird der Lipid-Status der Zelle und Enzyme des Lipid-Stoffwechsels berücksichtigt. Das Ausschalten einer Long Chain-Fatty Acyl CoA Synthetase 1 (LC-FACS), fcsB, in Dictyostelium discoideum hat eine Veränderung der Menge an neutralen Lipiden zur Folge. In diesen LC-FACS2 „Knock-Out“-Zellen wird ein Zusammenhang zwischen neutralen Lipiden und der Phagozytose von Hefen und Bakterien detektiert. Ein Einfluss auf den endozytotischen Transit kann in diesen Zellen nur induziert werden, wenn man zusätzlich den Triglycerid-Hydrolyse-Inhibitor LSD1 in den Zellen exprimiert. Mit Hilfe der Daten wird ein Modell erstellt, indem die Reduktion der Menge an neutralen Lipiden nicht direkt für diesen Phänotyp verantwortlich ist. Es ist vielmehr das Energie-Niveau der Zellen, das die Phagozytoserate beeinflusst. Möglich macht dies ein Pool aus Fettsäuren im Zytoplasma. Dieser besteht aus unaktivierten Fettsäuren und Acyl-CoAs. Auf ihn greifen Kompartimente wie Lipidtropfen, Mitochondrien und Peroxisomen zu, wenn Fettsäuren verstoffwechselt werden sollen. In LC-FACS2 „Knock-Out“-Zellen, wird das Gleichgewicht im Pool in Richtung der unaktivierten Fettsäuren verschoben. Anhand der Größe dieses Pools kann die Zelle ihren Energiestatus messen. Ein höherer Energie-Status führt dann zu einer Reduktion der Phagozytoserate. Vacuolin B Null Zellen (vacB-) zeigen eine extreme Verzögerung im endozytotischen Transit. Schaltet man in diesen Zellen die LC-FACS1 aus (vacB-/fcsA-), so reduziert man ebenfalls die Menge an Triglyceriden. Dies ist darauf zurückzuführen, dass der Acyl-CoA Anteil des Fettsäure-Pools reduziert ist. Diese Reduktion resultiert hier in einer Beschleunigung des endozytotischen Transits. Die Exozytose von vacB--Zellen und vacB-/ fcsA--Zellen unterscheidet sich nicht. Daher wird die Ursache für diese Beschleunigung in veränderten Fusions- bzw. Fissionseigenschaften der Endosomen vermutet. Somit führt das Ausschalten von LC-FACS-Proteinen in Dictyostelium zu einer veränderten Zusammensetzung des Fettsäure-Pools. Dies hat im Fall der LC-FACS1 Modifikationen der Membran-Dynamik und im Fall der LC-FACS2 Änderungen des Energie-Spiegels zur Folge.

Skeletal muscle fat metabolism during post-exercise recovery in humans

Recovery after prolonged or high-intensity exercise is characterised by a substantial increase in adipose tissue lipolysis, resulting in elevated rates of plasma-derived fat oxidation. Despite the large increase in circulating fatty acids (FAs) after exercise, only a small fraction of this is taken up by exercised muscle in the lower extremities. Indeed, the predominant fate of non-oxidised FAs derived from post-exercise lipolysis is reesteriflcation hi the liver. During recovery from endurance exercise, a number of changes also occur hi skeletal muscle that allow for a high metabolic priority towards glycogen resynthesis. Reducing muscle glycogen during exercise potentiates these effects, however the cellular and molecular mechanisms regulating substrate oxidation following exercise remain poorly defined. The broad arm of this thesis was to examine the regulation of fat metabolism during recovery from glycogen-lowering exercise hi the presence of altered fat and glucose availability. In study I, eight endurance-trained males completed a bout of exhaustive exercise followed by ingestion of carbohydrate (CHO)-rich meals (64-70% of energy from CHO) at 1, 4, and 7 h of recovery. Duplicate muscle biopsies were obtained at exhaustion and 3, 6 and 18 h of recovery. Despite the large intake of CHO during recovery (491 ± 28 g or 6.8 + 0.3 g • kg-1), respiratory exchange ratio values of 0.77 to 0.84 indicated a greater reliance on fat as an oxidative fuel. Intramuscular triacylglycerol (IMTG) content remained unchanged in the presence of elevated glucose and insulin levels during recovery , suggesting IMTG has a negligible role in contributing to the enhanced fat oxidation after exhaustive exercise. It appears that the partitioning of exogenous glucose towards glycogen resynthesis is of high metabolic priority during immediate post-exercise recovery, supported by the trend towards reduced pyruvate dehydrogenase (PDH) activity and increased fat oxidation. The effect of altering plasma FA availability during post-exercise recovery was examined in study II. Eight endurance-trained males performed three trials consisting of glycogen-lowering exercise, followed by infusion of either saline (CON), saline + nicotinic acid (NA) (LFA) or Intralipid and heparin (HFA). Muscle biopsies were obtained at the end of exercise (0 h) and at 3 and 6 h in recovery. Altering the availability of plasma FAs during recovery induced changes in whole-body fat oxidation that were unrelated to differences in skeletal muscle malonyl-CoA. Furthermore, fat oxidation and acetyl-CoA carboxylase (ACC) phosphorylation appear to be dissociated after exercise, suggesting mechanisms other than phosphorylation-mediated changes in ACC activity have an important role in regulating malonyl-CoA and fat metabolism in human skeletal muscle after exercise. Alternative mechanisms include citrate and long-chain fatty acyl-CoA mediated changes in ACC activity, or differences in malonyl-CoA decarboxylase (MCD) activity. Reducing plasma FA concentrations with NA attenuated the post-exercise increase in MCD and pyruvate dehydrogenase kinase 4 (PDK4) gene expression, suggesting that FAs and/or other factors induced by NA are involved hi the regulation of these genes. Despite marked changes hi plasma FA availability, no significant changes in IMTG concentration were detected, providing further evidence that plasma-derived FAs are the preferential fuel source contributing to the enhanced fat oxidation post-exercise during recovery. To further examine the effect of substrate availability after exercise, Study III investigated the regulation of fat metabolism during a 6 h recovery period with or without glucose infusion. Enhanced glucose availability significantly increased CHO oxidation compared with the fasted state, although no differences in whole-body fat oxidation were apparent. Consistent with the similar rates of fat metabolism, no difference hi AMPK or ACCβ phosphorylation were observed between trials. In addition, no significant treatment or time effects for IMTG concentration were detected during recovery. The large exercise-induced PDK4 gene expression was attenuated when plasma FAs were reduced during glucose infusion, supporting the hypothesis that PDK4 is responsive to sustained changes in lipid availability and/or changes in plasma insulin. Furthermore, the possibility exists that the suppression of PDK4 mRNA also reduced PDK activity and thus maintained PDH activity to account for the higher rates of CHO oxidation observed during glucose infusion compared with the control trial.

Improvements in insulin resistance with exercise training: a lipocentric approach

Traditional views on the metabolic derangements underlying insulin resistance and Type 2 diabetes have been largely “glucocentric” in nature, focusing on the hyperglycemic and/or hyperinsulinemic states that result from impaired glucose tolerance. But in addition to glucose intolerance, there is a coordinated breakdown in lipid dynamics in individuals with insulin resistance, manifested by elevated levels of circulating free fatty acids, diminished rates of lipid oxidation, and excess lipid accumulation in skeletal muscle and/or liver. This review examines the premise that an oversupply and/or accumulation of lipid directly inhibits insulin action on glucose metabolism via changes at the level of substrate competition, enzyme regulation, intracellular signaling, and/or gene transcription. If a breakdown in lipid dynamics is causal in the development of insulin resistance (rather than a coincidental feature resulting from it), it should be possible to demonstrate that interventions that improve lipid homeostasis cause reciprocal changes in insulin sensitivity. Accordingly, the efficacy of aerobic endurance training in human subjects in mediating the association between deranged lipid metabolism and insulin resistance will be examined. It will be demonstrated that aerobic exercise training is a potent and effective primary intervention strategy in the prevention and treatment of individuals with insulin resistance.

Disassociation of muscle triglyceride content and insulin sensitivity after exercise training in patients with Type 2 diabetes

Aim/hypothesis. We determined the effect of exercise training on insulin sensitivity and muscle lipids (triglyceride [TGm] and long-chain fatty acyl CoA [LCACoA] concentration) in patients with Type 2 diabetes. Methods. Seven patients with Type 2 diabetes and six healthy control subjects who were matched for age, BMI, % body fat and VO2peak participated in a 3 days per week training program for 8 weeks. Insulin sensitivity was determined pre- and post-training during a 120 min euglycaemic- hyperinsulinaemic clamp and muscle biopsies were obtained before and after each clamp. Oxidative enzyme activities [citrate synthase (CS), β-hydroxy-acyl- CoA (β-HAD)] and TGm were determined from basal muscle samples pre- and post training, while total LCACoA content was measured in samples obtained before and after insulin-stimulation, pre- and post training. Results. The training-induced increase in VO2peak (∼20%, p<0.01) was similar in both groups. Compared with control subjects, insulin sensitivity was lower in the diabetic patients before and after training (∼60%; p<0.05), but was increased to the same extent in both groups with training (∼30%; p<0.01). TGm was increased in patients with Type 2 diabetes (170%; p<0.05) before, but was normalized to levels observed in control subjects after training. Basal LCACoA content was similar between groups and was unaltered by training. Insulin-stimulation had no detectable effect on LCACoA content. CS and β-HAD activity were increased to the same extent in both groups in response to training (p<0.001). Conclusion/interpretation. We conclude that the enhanced insulin sensitivity observed after short-term exercise training was associated with a marked decrease in TGm content in patients with Type 2 diabetes. However, despite the normalization of TGm to levels observed in healthy individuals, insulin resistance was not completely reversed in the diabetic patients.

Muscle oxidative capacity is a better predictor of insulin sensitivity than lipid status

We determined whole-body insulin sensitivity, long-chain fatty acyl coenzyme A (LCACoA) content, skeletal muscle triglyceride (TGm) concentration, fatty acid transporter protein content, and oxidative enzyme activity in eight patients with type 2 diabetes (TYPE 2); six healthy control subjects matched for age (OLD), body mass index, percentage of body fat, and maximum pulmonary O2 uptake; nine well-trained athletes (TRAINED); and four age-matched controls (YOUNG). Muscle biopsies from the vastus lateralis were taken before and after a 2-h euglycemic-hyperinsulinemic clamp. Oxidative enzyme activities, fatty acid transporters (FAT/CD36 and FABPpm), and TGm were measured from basal muscle samples, and total LCACoA content was determined before and after insulin stimulation. Whole-body insulin-stimulated glucose uptake was lower in TYPE 2 (P < 0.05) than in OLD, YOUNG, and TRAINED. TGm was elevated in TYPE 2 compared with all other groups (P < 0.05). However, both basal and insulin-stimulated skeletal muscle LCACoA content were similar. Basal citrate synthase activity was higher in TRAINED (P < 0.01), whereas β-hydroxyacyl CoA dehydrogenase activity was higher in TRAINED compared with TYPE 2 and OLD. There was a significant relationship between the oxidative capacity of skeletal muscle and insulin sensitivity (citrate synthase, r = 0.71, P < 0.001; β-hydroxyacyl CoA dehydrogenase, r = 0.61, P = 0.001). No differences were found in FAT/CD36 protein content between groups. In contrast, FABPpm protein was lower in OLD compared with TYPE 2 and YOUNG (P < 0.05). In conclusion, despite markedly elevated skeletal muscle TGm in type 2 diabetic patients and strikingly different levels of whole-body glucose disposal, both basal and insulin-stimulated LCACoA content were similar across groups. Furthermore, skeletal muscle oxidative capacity was a better predictor of insulin sensitivity than either TGm concentration or long-chain fatty acyl CoA content.

A mouse model for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired β-oxidation of very long chain fatty acids (VLCFAs) and reduced function of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) that leads to severe and progressive neurological disability. The X-ALD gene, identified by positional cloning, encodes a peroxisomal membrane protein (adrenoleukodystrophy protein; ALDP) that belongs to the ATP binding cassette transporter protein superfamily. Mutational analyses and functional studies of the X-ALD gene confirm that it and not VLCS is the gene responsible for X-ALD. Its role in the β-oxidation of VLCFAs and its effect on the function of VLCS are unclear. The complex pathology of X-ALD and the extreme variability of its clinical phenotypes are also unexplained. To facilitate understanding of X-ALD pathophysiology, we developed an X-ALD mouse model by gene targeting. The X-ALD mouse exhibits reduced β-oxidation of VLCFAs, resulting in significantly elevated levels of saturated VLCFAs in total lipids from all tissues measured and in cholesterol esters from adrenal glands. Lipid cleft inclusions were observed in adrenocortical cells of X-ALD mice under the electron microscope. No neurological involvement has been detected in X-ALD mice up to 6 months. We conclude that X-ALD mice exhibit biochemical defects equivalent to those found in human X-ALD and thus provide an experimental system for testing therapeutic intervention.

Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23–28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

Study of the expression of different genes of long-chain polyunsaturated fatty acids (LC-PUFA) metabolism during the early paralarval development of the common octopus (Octopus vulgaris)

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2015

Nutritional regulation of long-chain PUFA biosynthetic genes in rainbow trout (Oncorhynchus mykiss)

Most studies on dietary vegetable oil in rainbow trout (Oncorhynchus mykiss) have been conducted on a background of dietary EPA (20 : 5n-3) and DHA (22 : 6n-3) contained in the fishmeal used as a protein source in aquaculture feed. If dietary EPA and DHA repress their endogenous synthesis from α-linolenic acid (ALA, 18 : 3n-3), then the potential of ALA-containing vegetable oils to maintain tissue EPA and DHA has been underestimated. We examined the effect of individual dietary n-3 PUFA on the expression of the biosynthetic genes required for metabolism of ALA to DHA in rainbow trout. A total of 720 juvenile rainbow trout were allocated to twenty-four experimental tanks and assigned one of eight diets. The effect of dietary ALA, EPA or DHA, in isolation or in combination, on hepatic expression of fatty acyl desaturase (FADS)2a(Δ6), FADS2b(Δ5), elongation of very long-chain fatty acid (ELOVL)5 and ELOVL2 was examined after 3 weeks of dietary intervention. The effect of these diets on liver and muscle phospholipid PUFA composition was also examined. The expression levels of FADS2a(Δ6), ELOVL5 and ELOVL2 were highest when diets were high in ALA, with no added EPA or DHA. Under these conditions ALA was readily converted to tissue DHA. Dietary DHA had the largest and most consistent effect in down-regulating the gene expression of all four genes. The ELOVL5 expression was the least responsive of the four genes to dietary n-3 PUFA changes. These findings should be considered when optimising aquaculture feeds containing vegetable oils and/or fish oil or fishmeal to achieve maximum DHA synthesis.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.