Counter-Regulatory Response to a Fall in Circulating Fatty Acid Levels in Rainbow Trout. Possible Involvement of the Hypothalamus-Pituitary-Interrenal Axis

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.     

Dietary carbohydrates induce changes in glucosensing capacity and food intake of rainbow trout

We hypothesize that variations in dietary carbohydrate levels produce changes in glucosensor parameters in previously characterized glucosensing areas (hypothalamus and hindbrain) related with the regulation of food intake of a carnivorous fish species like rainbow trout. Therefore, we fed trout with standard, carbohydrate-free (CF) or high-carbohydrate (HC) diets for 10 days to assess changes in glucosensing system and food intake. Fish fed CF diet displayed hypoglycemia and increased food intake. Fish fed a HC diet displayed hyperglycemia and decreased food intake. Changes in food intake due to dietary carbohydrates were accompanied in hypothalamus and hindbrain of fish fed with HC diet by changes in parameters involved in glucosensing, such as increased glucose, glucose 6-phosphate, and glycogen levels and increased glucokinase (GK), glycogen synthase, and pyruvate kinase activities as well as increased GK and GLUT2 expression. All those results address for the first time in fish, despite the relative intolerance to glucose of carnivorous species, that dietary carbohydrates are important regulators of the glucosensing system in carnivorous fish, suggesting that the information generated by this system can be associated with the changes observed in food intake.     

Evidence for the presence of a glucosensor in hypothalamus, hindbrain, and Brockmann bodies of rainbow trout

The aim of this study was to evaluate the existence of a glucosensor in different regions of the brain and in the Brockmann bodies (BB) of the rainbow trout, Oncorhynchus mykiss. Five groups (n = 12) of trout were injected intraperitoneally with saline alone (control) or saline-containing bovine glucagon (100 mug/kg), bovine insulin (4 mg/kg), 2-deoxy-d-glucose (100 mg/kg), or d-glucose (500 mg/kg) to promote hyperglycemia (glucagon, d-glucose, 2-deoxy-d-glucose) or hypoglycemia (insulin). Six hours after injection, samples from four brain regions (hypothalamus, telencephalon, hindbrain, and midbrain) and the entire BB were taken. Our results demonstrate within the BB and both the hypothalamus and hindbrain a metabolic response different to that observed in other tissues (midbrain, telencephalon) but similar to that described in tissues known to be glucosensors in mammals. The metabolic responses of these areas to changes in plasma glycemia were characterized by parallel changes in GLUT-2 expression, hexokinase-IV, or glucokinase activity and expression, glycolytic potential, and levels of glycogen and glucose. These changes are similar to those reported in mammalian pancreatic beta-cells and glucose-excited (GE) neurons, two cell types containing glucosensors. This study provides evidence for the presence of glucosensors responsive to hyper- and hypoglycemia in rainbow trout BB, hypothalamus, and hindbrain.     

Changes in food intake and glucosensing function of hypothalamus and hindbrain in rainbow trout subjected to hyperglycemic or hypoglycemic conditions

To evaluate the possible role of glucose in the control of food intake (FI) in fish and the involvement of glucosensing system in that role, we have subjected rainbow trout (via intraperitoneal injections) to control, hyperglycemic (500 mg kg(-1) glucose body mass) or hypoglycemic (4 mg kg(-1) bovine insulin) conditions for 10 days. The experimental design was appropriate since hypoglycemia and hyperglycemia were observed the first 5 days after treatment and changes observed in metabolic parameters in liver were similar to those of fish literature. Hyperglycemic conditions elicited small changes in FI accompanied by increased glucose and glycogen levels, glucokinase (GK) activity and glycolytic potential in hypothalamus and hindbrain. In contrast, hypoglycemic conditions elicited a marked increase in FI accompanied by decreased glucose and glycogen levels and GK activity in the same brain regions whereas both regions displayed different responses in glycolytic potential. These results allow us to hypothesize that, despite the relative intolerance to glucose of carnivorous fish, changes in plasma glucose levels in rainbow trout detected by glucosensing areas in brain regions (hypothalamus and hindbrain) are integrated in those or near areas eliciting a response in FI, which was more important under hypoglycemic than under hyperglycemic conditions.     

High dietary intake of medium-chain fatty acids during pregnancy in rats prevents later-life obesity in their offspring

We investigated the effects of dietary fatty acids of different chain lengths during pregnancy in the rat on the susceptibility of offspring to later-life obesity and the underlying mechanisms. Pregnant rats were fed three different diets: standard (STD), high medium-chain fatty acids (MCFA); and high long-chain fatty acids (LCFA). The male offspring were assigned to three groups: STD control, MCFA and LCFA according to the maternal diets and suckled by dams fed with STD during pregnancy and lactation. After weaning, the offspring were fed with STD from 3 to 8 weeks of age. At the age of 8 weeks, rats in three groups: high-fat diet (HFD) control, MCFA and LCFA were fed with HFD until 14 weeks of age in an attempt to induce obesity, and rats in the HFD control group were selected randomly from the STD control group. Body weight and body fat content were decreased in the MCFA group accompanied by down-regulated mRNA expression of fatty acid synthase and acetyl-coA carboxylase 1, and increased mRNA and protein expression of adenosine monophosphate (AMP)-activated protein kinase (AMPK), carnitine palmitoyltransferase 1 and uncoupling protein 3 compared with the corresponding controls at 3, 8 and 14 weeks of age. The results suggested that the MCFA diet during pregnancy prevented later-life obesity in the offspring when they were exposed to HFD in later life, which might be related to programming of the expression of genes involved in fatty acid metabolism.     

Brain serotonin and the control of food intake in rainbow trout (Oncorhynchus mykiss): effects of changes in plasma glucose levels

The levels of 5-hydroxytryptamine and its main metabolite 5-hydroxyindoleacetic acid were assessed in two brain regions, hypothalamus and telencephalon, of rainbow trout (Oncorhynchus mykiss) submitted to increases or decreases in plasma glucose levels through different experimental approaches. Thus, intraperitoneal glucose treatment (500 mg kg(-1)) increased 5-hydroxytryptamine telencephalic levels. Long-term food deprivation up to 3 weeks significantly increased hypothalamic (2 weeks and 3 weeks) and telencephalic (1 week, 2 weeks, and 3 weeks) levels of 5-hydroxyindoleacetic acid, whereas the ratio 5-hydroxyindoleacetic acid/5-hydroxytryptamine significantly increased throughout the food-deprivation period assessed. Intraperitoneal treatment with bovine insulin (4 mg kg(-1)) decreased the 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio in hypothalamus after 1 h. Intraperitoneal administration of fenfluramine (3 mg kg(-1)) caused a depression in food intake coincident with a significant decrease of the hypothalamic 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio. These data are discussed in the context of the involvement of serotonergic system in the control of food intake in rainbow trout.     

In vitro leptin treatment of rainbow trout hypothalamus and hindbrain affects glucosensing and gene expression of neuropeptides involved in food intake regulation

The aim of the present study was to evaluate in hypothalamus and hindbrain of rainbow trout in vitro the effect of leptin treatment on glucosensing capacity and the expression of orexigenic and anorexigenic peptides involved in the control of food intake. In a first experiment, the response of parameters involved in glucosensing (GK, PK and GSase activities; GK expression and glucose; glycogen and DHAP levels) and the expression of orexigenic (NPY) and anorexigenic (POMC, CART, CRF) peptides was assessed in hypothalami and hindbrain incubated for 1 h with 2, 4 or 8 mM d-glucose alone (controls) or with 10 nM leptin, or with 10 nM leptin plus inhibitors of leptin signaling pathways (50 nM wortmannin and 500 nM AG490). Leptin treatment increased levels in parameters involved in glucosensing. Leptin treatment decreased NPY mRNA levels in hypothalamus without affecting the expression of the other peptides assessed. Leptin effects were reverted in the presence of inhibitors for all parameters assessed suggesting the involvement of JAK/STAT and IRS-PI(3)K pathways. In a second experiment, we observed time-dependent (1-3 h) and dose (10, 20 and 50 nM)- effects of leptin treatment in decreasing NPY mRNA levels without affecting expression of the other peptides assessed. Considering the orexigenic action of NPY in fish, it seems that the anorexic effect of leptin can be mediated by reduced expression of NPY occurring in hypothalamus, and that change can be related to the activation of the glucosensing system occurring simultaneously.     

Plasma ghrelin levels in rainbow trout in response to fasting, feeding and food composition, and effects of ghrelin on voluntary food intake

Ghrelin, a peptide hormone which stimulates growth hormone (GH) release, appetite and adiposity in mammals, was recently identified in fish. In this study, the roles of ghrelin in regulating food intake and the growth hormone (GH)-insulin-like growth factor I (IGF-I) system of rainbow trout (Oncorhynchus mykiss) were investigated in three experiments: 1) Pre- and postprandial plasma levels of ghrelin were measured in relation to dietary composition and food intake through dietary inclusion of radio-dense lead-glass beads, 2) the effect of a single intraperitoneal (i.p.) injection with rainbow trout ghrelin on short-term voluntary food intake was examined and 3) the effect of one to three weeks fasting on circulating ghrelin levels and the correlation with plasma GH and IGF-I levels, growth and lipid content in the liver and muscle was studied. There was no postprandial change in plasma ghrelin levels. Fish fed a normal-protein/high-lipid (31.4%) diet tended to have higher plasma ghrelin levels than those fed a high-protein/low-lipid (14.1%) diet. Plasma ghrelin levels decreased during fasting and correlated positively with specific growth rates, condition factor, liver and muscle lipid content, and negatively with plasma GH and IGF-I levels. An i.p. ghrelin injection did not affect food intake during 12-hours post-injection. It is concluded that ghrelin release in rainbow trout may be influenced by long-term energy status, and possibly by diet composition. Further, in rainbow trout, ghrelin seems to be linked to growth and metabolism, but does not seem to stimulate short-term appetite through a peripheral action.     

Effects of resveratrol and genistein on growth,nutrient utilization and fatty acid composition of rainbow trout

The replacement of the finite and costly resource fish oil is an important task for aquaculture nutrition. A promising approach could be the use of plant bioactives that may have the potential to influence the metabolism and the synthesis of n-3 long chain polyunsaturated fatty acids, especially EPA (20:5n-3) and DHA (22:6n-3). In this study, the two phytochemicals resveratrol (RV) and genistein (G) were investigated for their effects on fish growth, nutrient utilization and body nutrient composition alongside their effects on whole body fatty acid (FA) composition. In a feeding trial lasting 8 weeks, rainbow trout (initial BW: 81.4±0.5 g) were held in a recirculating aquaculture system and fed six experimental diets with varying fish oil levels as plain variants or supplemented with 0.3% of dry matter (DM) of either RV or G. The six diets were as follows: diet F4 had 4% DM fish oil, diet F0 had 0% DM fish oil, diets F4+RV, F4+G, F0+RV and F0+G were equal to the diets F4 and F0, respectively, and supplemented with the phytochemicals RV and G. The feeding of the F0+RV diet resulted in reduced feed intake, growth rate and slightly reduced whole body lipid levels. At the same time, the amount of polyunsaturated FA and the n-3/n-6 ratio were significantly increased in whole body homogenates of rainbow trout fed diet F0+RV in comparison to the F0 control. The feeding of the F0+G diet led to reduced feed intake, slightly increased protein utilization but did not significantly affect the whole body FA composition. Overall, feeding the fish oil-free diet supplemented with the phytochemicals resulted in more pronounced effects on fish performance and FA composition than the single factors per se (dietary fish oil level or phytochemical). Present data indicate that G might not be of profitable use for trout nutrition. In terms of FA composition, RV could be a potentially useful complement for fish oil. However, the impairment of growth and performance parameters as observed in the present study discourages its use in trout diets.     

Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae

We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA beta-oxidation activity was markedly reduced in intact ypr128cDelta mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA beta-oxidation is impaired in ypr128cDelta cells. Since MCFA beta-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cDelta mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.     

In vitro evidences for glucosensing capacity and mechanisms in hypothalamus, hindbrain, and Brockmann bodies of rainbow trout

We aimed to support in vitro the glucosensing capacity observed in vivo in rainbow trout hypothalamus, hindbrain, and Brockmann bodies (BB) and to obtain preliminary evidence of the mechanisms involved. The response of parameters involved in the glucosensing capacity [hexokinase, hexokinase IV (glucokinase), and pyruvate kinase activities and glucose and glycogen levels] was assessed in these tissues incubated for 1 h with 2, 4, or 8 mM D-glucose alone (control) or with specific agonists/inhibitors of the steps involved in glucosensing capacity in mammals. These agents were a competitor for glucose phosphorylation (15 mM mannose), sulfonylurea receptor-1 effectors (500 microM tolbutamide or diazoxide), glycolytic intermediates (15 mM glycerol, lactate, or pyruvate), and inhibitors of glucose transport (10 microM cytochalasin B), glycolysis [20 mM 2-deoxy-D-glucose (2-DG)], and L-type calcium channel (1 microM nifedipine). Control incubations of the three tissues displayed increased glucose and glycogen levels and glucokinase activities in response to increased medium glucose, thus supporting our previous in vivo studies. Furthermore, critical components of the glucosensing mammalian machinery are apparently functioning in the three tissues. The responses in brain regions to all substances tested (except 2-DG and nifedipine) were similar to those observed in mammals, suggesting a similar glucosensing machinery. In contrast, in BB, only the effects of 2-DG, lactate, pyruvate, diazoxide, and nifedipine were similar to those of mammalian beta-cells, suggesting that some of the components of the piscine glucosensing model are different than those of mammals. Such differences may relate to the importance of amino acids rather than glucose signaling in the trout BB.     

Intestinal absorption, blood transport and hepatic and muscle metabolism of fatty acids in preruminant and ruminant animals

Current research on lipid metabolism in ruminants aims to improve the growth and health of the animals and the muscle characteristics associated with meat quality. This review, therefore, focuses on fatty acid (FA) metabolism from absorption to partitioning between tissues and metabolic pathways. In young calves, which were given high-fat milk diets, lipid absorption is delayed because the coagulation of milk caseins results in the retention of dietary fat as an insoluble clot in the abomasum. After weaning, the calves were fed forage- and cereal-based diets containing low levels of long-chain fatty acids (LCFA) but leading to high levels of volatile fatty acid (VFA) production by the rumen microflora. Such differences in dietary FA affect: i) the lipid transport system via the production of lipoproteins by the intestine and the liver, and (ii) the subsequent metabolism of lipids and FA by tissues. In preruminant calves, high-fat feed stimulates the secretion of triacylglycerols (TG)-rich lipoproteins (chylomicrons, very-low density lipoproteins (VLDL)). Diets rich in polyunsaturated FA (PUFA) stimulate the production of chylomicrons by the intestine (at peak lipid absorption) and of high density lipoproteins by the liver, leading to high blood concentrations of cholesterol. High levels of non-esterified FA (NEFA) uptake by the liver in high-yielding dairy cows in early lactation leads to TG infiltration of the hepatocytes (fatty liver). This is due to the low chronic capacity of the liver to synthesise and secrete VLDL particles. This abnormality in hepatic FA metabolism involves defects in apolipoprotein B synthesis and low availability of apolipoproteins and lipids for VLDL packaging. Fatty liver in calves is also caused by milk containing either soybean oil (rich in n-6 PUFA), or coconut oil (rich in C12:0 and C14:0). The ability of muscle tissue to use FA as an energy source depends on its mitochondrial content and, hence, on many physiological factors. The uptake and partitioning of LCFA between oxidation and storage in muscle is regulated by the activity of key intracellular enzymes and binding proteins. One such protein, carnitine palmitoyltransferase I (CPT I) controls the transport of LCFA into mitochondria. Metabolites derived from LCFA inhibit glucose oxidation, decrease the activity of CPT I and decrease the efficiency of ATP production by mitochondria. Most research on tissue lipid metabolism in ruminants is focused on: i) the partitioning of FA oxidation between intracellular peroxisomes and mitochondria in the liver and in muscles; (ii) the regulation of lipid metabolism by leptin, a recently discovered hormone secreted by mature adipocytes; and iii) the effects of activation of the nuclear receptors (PPARs and RXR) by LCFA or by phytol metabolites derived from chlorophyll.     

Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.     

A novel role for central ACBP/DBI as a regulator of long‐chain fatty acid metabolism in astrocytes

Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABA_A receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.

     

Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production

The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.     

Involvement of cortisol and sirtuin1 during the response to stress of hypothalamic circadian system and food intake-related peptides in rainbow trout,Oncorhynchus mykiss

Stress is conditioning animal welfare by negatively affecting a wide range of physiological and behavioral functions. This may be applied to circadian physiology and food intake. Cortisol, the stress-related hormone, may mediate such effect of stress, but other indirect mediators might be considered, such as sirtuin1. Then, either the independent modulatory effect or the existence of any interaction between mediators may be responsible. The circadian system is the main modulator of several integrative mechanisms at both central and peripheral levels that are rhythmically presented, thus influencing different processes such as food intake. In this way, food intake is controlled by the circadian system, as demonstrated by the persistence of such rhythms of food intake in the absence of environmental external cues. Our study aimed to evaluate the daily profile of hypothalamic mRNA abundance of circadian clock genes (clock1a, bmal1, per1 and rev-erbβ-like), and food intake regulators (crf, pomc-a1, cart, and npy) in rainbow trout (Oncorhynchus mykiss), the impact of stress on such rhythms, and the involvement of cortisol and sirtuin1 as mediators. Four cohorts of trout were subjected to 1) normal stocking density (control group), 2) high stocking density for 72 hours (stress group), 3) normal stocking density and implanted with mifepristone, a glucocorticoid receptors antagonist, and 4) mifepristone administered and stressed for 72 hours. Fish from each group were sampled every 4-h along the 24-h LD cycle, and cortisol, glucose and lactate plasma levels were evaluated. Hypothalamic mRNA abundance of clock genes, food intake regulators, glucocorticoid receptors and sirtuin1 were qPCR assayed. Our results reveal the impact of stress on most of the genes assayed, but different mechanisms appear to be involved. The rhythm of clock genes displayed decreased amplitude and averaged levels in stressed trout, with no changes of the acrophase being observed. This effect was not prevented by mifepristone. On the contrary, the effect of stress on the daily profile of crf, pomc-a1, and npy was totally prevented by mifepristone administration. Accordingly, cortisol appears to mainly mediate the effect of stress on food intake regulators through binding to specific glucocorticoid receptors within trout hypothalamus, whereas sirtuin1 is apparently mediating such effects on the circadian system in the same brain region. Further research must be performed to clarify those mechanisms through which stress influences food intake and the circadian oscillator within the same brain region, hypothalamus, in rainbow trout, and the interaction among them all.     

Engineering of FRT-lacZ fusion constructs: induction of the Pseudomonas aeruginosa fadAB1 operon by medium and long chain-length fatty acids

Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT-lacZ-KmR constructs, we constructed two novel FRT-lacZ-GmR plasmids. This allows easy engineering of Pseudomonas aeruginosa reporter-gene fusions, post-mutant construction, with the Flp-FRT system. We demonstrate the usefulness of one of these FRT-lacZ-GmR plasmids to study the regulation of the fadAB1 operon in P. aeruginosa at its native locus. The fadAB1 operon, involved in fatty acid (FA) degradation, was significantly induced in the presence of several medium chain-length fatty acids (MCFA) and, to a lesser degree, long chain-length fatty acids (LCFA). In addition to the previous work on the FRT-lacZ-KmR tools, these new constructs increase the repertoire of tools that can be applied to P. aeruginosa or other species and strains of bacteria where kanamycin resistance may not be appropriate.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.