Fatty Acid Oxidation Pathway Research Articles

Fatty acid oxidation in normotriglyceridemic men

Moderate hypertriglyceridemia is frequently associated with central obesity, insulin resistance, and atherogenic dyslipidemia. We showed previously that moderately obese men with hypertriglyceridemia have reduced fatty acid oxidation postabsorptively and postprandially. In the present study, we examined the oxidation of fatty acids in normotriglyceridemic men. The study objective was to determine the relation between plasma triglyceride levels and fatty acid oxidation in normotriglyceridemic men. Twenty-four healthy, nonobese White and African American men participated in a cross-sectional metabolic study for evaluation of fatty acid oxidation. Men were healthy, and none took hypolipidemic or hypoglycemic agents. They ingested 200 mg of fat/hour/kg of body weight over a 10-hour period. Plasma levels of triglyceride, nonesterified fatty acids, 3-β-hydroxybutyrate, insulin, and glucagon were measured postabsorptively and postprandially. Chylomicron-triglyceride halflife was also calculated. Nonobese White and African-American men had similar anthropometry, levels of plasma triglyceride, lipoprotein cholesterol, nonesterified fatty acids, 3-β-hydroxybutyrate, insulin, and glucagon postabsorptively and postprandially. For the group as a whole, there was a positive and significant correlation between plasma fatty acids and 3-β-hydroxybutyrate and an inverse association between plasma triglyceride levels and 3-β-hydroxybutyrate at baseline. All subjects had increased levels of metabolites of interest postprandially. However, there were no significant changes in plasma insulin, glucagon, or the ratio of insulin to glucagon. The postprandial levels of 3-β-hydroxybutyrate correlated positively with nonesterified fatty acids and inversely with the half-life of chylomicron triglyceride. Normotriglyceridemia is strongly associated with oxidation of fatty acids by the liver suggesting the possibility that the fatty acid oxidation pathway is a potential target of intervention to prevent hypertriglyceridemia and concomitant fatty liver.

Ribosomal Proteins RPS11 and RPS20, Two Stress-Response Markers of Glioblastoma Stem Cells, Are Novel Predictors of Poor Prognosis in Glioblastoma Patients.

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.

Chronic vitamin A-enriched diet feeding induces body weight gain and adiposity in lean and glucose-intolerant obese rats of WNIN/GR-Ob strain.

What is the central question of this study? Previously, we reported that chronic feeding of a vitaminA-enriched diet to euglycaemic obese rats (WNIN/Ob) ameliorated obesity. Does this diet exert similar effects even with a different genetic background, i.e. obese rats of the WNIN/GR-Ob strain with impaired glucose tolerance? What is the main finding and its importance? VitaminA-enriched diet aggravated weight gain and adiposity/obesity in both lean and glucose-intolerant obese rats of the WNIN/GR-Ob strain. Therefore, the role of genetic factors and their regulation by nutrients in determining health and disease conditions assumes greater significance in experimental and clinical research. Vitamin A and its metabolites are key regulators of the development of adipose tissue and its associated metabolic complications. Here, we tested, in a glucose-intolerant obese rat model (the WNIN/GR-Ob stain), whether feeding a vitaminA-enriched diet alters adiposity and its associated changes. For this purpose, 30-week-old male lean and obese rats were divided into two groups and received either stock diet or vitaminA-enriched diet [2.6 or 129mg vitaminA(kg diet)(-1) , respectively] for 14weeks. At the end, feeding of the vitaminA-enriched diet resulted in increased body weight gain/obesity and retroperitoneal white adipose tissue (RPWAT) in both lean and obese rats of the WNIN/GR-Ob strain, when compared with their respective control animals receiving stock diet, without affecting food intake. An improvement in hypertriglyceridaemia and circulatory non-esterified fatty acid levels and unaltered hepatic fatty acid oxidative and triglyceride secretory pathway proteins with vitamin A-enriched diet feeding are suggestive of enhanced hepatic clearance of circulatory lipids, resulting in increased hepatic triglyceride accumulation. Transcriptional analysis of RPWAT showed that feeding the vitaminA-enriched diet augmented the expression of adipogenic/adipose tissue-specific genes; peroxisome proliferator-activated receptor-γ, stearoyl CoA desaturase1, retinol saturase, leptin and lipoprotein lipase and vitaminA metabolic pathway genes; retinoic acid receptors, retinoid X receptors and cytochrome P450 26B1. Besides, RPWAT-lipoprotein lipase-mediated clearance of triglyceride could also have contributed to increased adiposity and improved hypertriglyceridaemia. In conclusion, chronic feeding of vitamin A-enriched diet induces weight gain and adiposity in both lean and obese rats of the WNIN/GR-Ob strain, possibly through transcriptional regulation of key adipogenic pathway genes of RPWAT, but improves dyslipidaemia.

Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias

AbstractCancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.

Exogenous administration of DLK1 ameliorates hepatic steatosis and regulates gluconeogenesis via activation of AMPK.

Activation of Notch signaling pathologically enhances lipogenesis and gluconeogenesis in the liver causing non-alcoholic fatty liver disease (NAFLD) and diabetes. Delta-like 1 homolog (DLK1), an imprinted gene that can modulate adipogenesis and muscle development in mice, was found as an inhibitory regulator of Notch signaling. Therefore, we investigated the metabolic effect of exogenous DLK1 in vitro and in vivo. A soluble DLK1 peptide was generated with fusion between a human Fc fragment and extracellular domain of DLK1. Male db/db mice were randomly assigned to two groups: vehicle treated and DLK1-treated group (25 mg kg(-1), intraperitoneal injection, twice a week for 4 weeks). Primary mice hepatocytes and HepG2 cells were used for in vitro experiments. After 4 weeks of DLK1 administration, hepatic triglyceride content and lipid droplets in liver tissues, as well as serum levels of liver enzymes, were markedly decreased in db/db mice. DLK1 treatment induced phosphorylation of AMPK and ACC and suppressed nuclear expression of SREBP-1c in the mouse liver or hepatocytes, indicating regulation of fatty acid oxidation and synthesis pathways. Furthermore, DLK1-treated mice showed significantly lower levels of fasting and random glucose, with improved glucose and insulin tolerance compared with the vehicle-treated group. Macrophage infiltration and proinflammatory cytokine levels in the epididymal fat were decreased in DLK1-treated db/db mice. Moreover, DLK1 suppressed glucose production from hepatocytes, which was blocked after co-administration of an AMPK inhibitor, compound C. DLK1-treated hepatocytes and mouse liver tissues showed lower PEPCK and G6Pase expression. DLK1 triggered AKT phosphorylation followed by cytosolic translocation of FOXO1 from the nucleus in hepatocytes. The present study demonstrated that exogenous administration of DLK1 reduced hepatic steatosis and hyperglycemia via AMPK activation in the liver. This result suggests that DLK1 may be a novel therapeutic approach for treating NAFLD and diabetes.

Abstract 84: Expression of the Transcription Factors PPAR Alpha and Gamma, and Their Target Genes in the Heart of Obese Mice

The Peroxisome-Proliferator Activated Receptors (PPARs) have been identified as key regulators of energy metabolism by sensing fatty acid availability and activating the transcription of enzymes involved in fatty acid and glucose metabolism. In the heart, PPARs have been proven to participate in the development of diseases, such as cardiac hypertrophy and diabetic cardiomyopathy, conditions that have been associated to obesity. Furthermore, the use of PPARs activator drugs is frequent for the treatment of obesity-dependent diseases, such as hypoglycemiant (fibrates) and anti-diabetic (thiazolidinediones) drugs. Therefore, the objective of this project was to characterize the expression of PPARs and some of their target genes involved in fatty acid and glucose metabolism in the heart of obese mice. Mice (C57BL/6) were fed with either a standard chow diet (control group) or a high fat diet (obese group) for three months. Hearts were weighed prior to quantifying protein expression of two PPARs (alpha and gamma), and both mRNA and protein expression of three enzymes (phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (GPD1) and glycerol-3-phosphate acyltransferase (GPAT)). Protein levels were measured by western blot, while mRNA levels were quantified by real-time PCR. The obese group presented 41% increase in body weight compared with control mice. However, no change was observed in obese hearts weight compared with control group, meaning that hypertrophy had not been developed yet. The expression of PPAR-alpha was increased 42% in obese mice compared with the control group, suggesting a possible activation of the fatty acid oxidation pathway. PPAR-gamma together with GPD1 and GPAT showed no differences at any level; however, PFK presented 90% increase in mRNA level and 54% increase in protein level in obese mice. Interestingly, decrease in PFK has been reported in any altered PPAR-alpha condition, contrary to our results. These results suggest that there is a dysregulation of the heart’s energetic pathways mediated by PPARs in earlier stages of obesity.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Diallyl trisulphide inhibits adipogenesis in 3T3-L1 adipocytes through lipogenesis, fatty acid transport, and fatty acid oxidation pathways

Garlic and its related products (such as black garlic) exhibit multiple biological functions and attributed to the presence of organo-sulphur compounds. The aim of this study was to investigate the inhibitory effects of garlic-derived organo-sulphur compounds [including diallyl sulphide (DAS), diallyl disulphide (DADS), diallyl trisulphide (DATS), S-allylcysteine (SAC), and S-allyl-L-cysteine sulphoxide (alliin)] on adipogenesis in 3T3-L1 adipocytes. The result indicated that DATS significantly decreased the intracellular triacylglycerol, oil red O-stained material (OROSM), and glycerol-3-phosphate dehydrogenase (GPDH) activity and significantly increased the adiponectin production in 3T3-L1 adipocytes. Real-time RT-PCR analysis showed that DATS decreased the gene levels of adipogenesis (C/EBPα, PPARγ, ACC, and FAS), fatty acid transport (aP2, CD36, and LPL), and adipocytokines (leptin, MCP-1, PAI-1, and resistin), and increased the gene levels of fatty acid oxidation (PGC-1β and UCP1) and adiponectin in 3T3-L1 adipocytes. DATS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through regulation of gene levels of lipogenesis, lipolysis, and adipokines.

A metabolomic study of the PPARδ agonist GW501516 for enhancing running endurance in Kunming mice.

Exercise can increase peroxisome proliferator-activated receptor-δ (PPARδ) expression in skeletal muscle. PPARδ regulates muscle metabolism and reprograms muscle fibre types to enhance running endurance. This study utilized metabolomic profiling to examine the effects of GW501516, a PPARδ agonist, on running endurance in mice. While training alone increased the exhaustive running performance, GW501516 treatment enhanced running endurance and the proportion of succinate dehydrogenase (SDH)-positive muscle fibres in both trained and untrained mice. Furthermore, increased levels of intermediate metabolites and key enzymes in fatty acid oxidation pathways were observed following training and/or treatment. Training alone increased serum inositol, glucogenic amino acids, and branch chain amino acids. However, GW501516 increased serum galactose and β-hydroxybutyrate, independent of training. Additionally, GW501516 alone raised serum unsaturated fatty acid levels, especially polyunsaturated fatty acids, but levels increased even more when combined with training. These findings suggest that mechanisms behind enhanced running capacity are not identical for GW501516 and training. Training increases energy availability by promoting catabolism of proteins, and gluconeogenesis, whereas GW501516 enhances specific consumption of fatty acids and reducing glucose utilization.

Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus

Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors.

Plasmacytoid dendritic cell activation is dependent upon type I IFN-driven fatty acid oxidation (IRM9P.456)

Abstract Plasmacytoid dendritic cells (pDCs) are specialized for the secretion of type I interferons (IFN-I) and thus are considered mediators of antiviral responses. pDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9 and the ligation of TLR7/9 leads to rapid cellular activation. Recently, we found that stimulation of pDCs with the TLR9 agonist CpG resulted in a marked increase in mitochondrial oxygen consumption rates (OCR) dependent on increased fatty acid oxidation (FAO). FAO is essential for full activation of CpG-stimulated pDCs because etomoxir or carnitine palmitoyltransferase 1A shRNA (short-hairpin RNA) severely attenuated IFN-alpha production by these cells. Using RNA-seq, we identified the expression of many interferon-stimulated genes (ISG) in CpG-stimulated pDCs, amongst which were genes encoding FAO pathway components. We found that IFNAR -/- pDCs failed to increase mitochondrial OCR in response to TLR9 agonists. Taken together, our data indicate that IFN-alpha-induces FAO which is essential for full pDC activation in response to CpG.

Copy number variations in high and low fertility breeding boars.

BackgroundIn this study we applied the extreme groups/selective genotyping approach for identifying copy number variations in high and low fertility breeding boars. The fertility indicator was the calculated Direct Boar Effect on litter size (DBE) that was obtained as a by-product of the national genetic evaluation for litter size (BLUP). The two groups of animals had DBE values at the upper (high fertility) and lower (low fertility) end of the distribution from a population of more than 38,000 boars. Animals from these two diverse phenotypes were genotyped with the Porcine SNP60K chip and compared by several approaches in order to prove the feasibility of our CNV analysis and to identify putative markers of fertility.ResultsWe have identified 35 CNVRs covering 36.5 Mb or ~1.3% of the porcine genome. Among these 35 CNVRs, 14 were specific to the high fertility group, while 19 CNVRs were specific to the low fertility group which overlap with 137 QTLs of various reproductive traits. The identified 35 CNVRs encompassed 50 genes, among them 40 were specific to the low fertility group, seven to the high fertility group, while three were found in regions that were present in both groups but with opposite gain/loss status. A functional analysis of several databases revealed that the genes found in CNVRs from the low fertility group have been significantly enriched in members of the innate immune system, Toll-like receptor and RIG-I-like receptor signaling and fatty acid oxidation pathways.ConclusionsWe have demonstrated that our analysis pipeline could identify putative CNV markers of fertility, especially in case of low fertility boars.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1473-9) contains supplementary material, which is available to authorized users.

Enhanced cardiac Akt/protein kinase B signaling contributes to pathological cardiac hypertrophy in part by impairing mitochondrial function via transcriptional repression of mitochondrion-targeted nuclear genes.

Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity.

Creatine supplementation prevents fatty liver in rats fed choline-deficient diet: a burden of one-carbon and fatty acid metabolism

AimTo examine the effects of creatine (Cr) supplementation on liver fat accumulation in rats fed a choline-deficient diet. MethodsTwenty-four rats were divided into 3 groups of 8 based on 4 weeks of feeding an AIN-93 control diet (C), a choline-deficient diet (CDD) or a CDD supplemented with 2% Cr. The CDD diet was AIN-93 without choline. ResultsThe CDD significantly increased plasma homocysteine and TNFα concentration, as well as ALT activity. In liver, the CDD enhanced concentrations of total fat (55%), cholesterol (25%), triglycerides (87%), MDA (30%), TNFα (241%) and decreased SAM concentrations (25%) and the SAM/SAH ratio (33%). Cr supplementation prevented all these metabolic changes, except for hepatic SAM and the SAM/SAH ratio. However, no changes in PEMT gene expression or liver phosphatidylcholine levels were observed among the three experimental groups, and there were no changes in hepatic triglyceride transfer protein (MTP) mRNA level. On the contrary, Cr supplementation normalized expression of the transcription factors PPARα and PPARγ that were altered by the CDD. Further, the downstream targets and fatty acids metabolism genes, UCP2, LCAD and CPT1a, were also normalized in the Cr group as compared to CDD-fed rats. ConclusionCr supplementation prevented fat liver accumulation and hepatic injures in rats fed with a CDD for 4 weeks. Our results demonstrated that one-carbon metabolism may have a small role in mitigating hepatic fat accumulation by Cr supplementation. The modulation of key genes related to fatty acid oxidation pathway suggests a new mechanism by which Cr prevents liver fat accumulation.

The circadian clock maintains cardiac function by regulating mitochondrial metabolism in mice.

Cardiac function is highly dependent on oxidative energy, which is produced by mitochondrial respiration. Defects in mitochondrial function are associated with both structural and functional abnormalities in the heart. Here, we show that heart-specific ablation of the circadian clock gene Bmal1 results in cardiac mitochondrial defects that include morphological changes and functional abnormalities, such as reduced enzymatic activities within the respiratory complex. Mice without cardiac Bmal1 function show a significant decrease in the expression of genes associated with the fatty acid oxidative pathway, the tricarboxylic acid cycle, and the mitochondrial respiratory chain in the heart and develop severe progressive heart failure with age. Importantly, similar changes in gene expression related to mitochondrial oxidative metabolism are also observed in C57BL/6J mice subjected to chronic reversal of the light-dark cycle; thus, they show disrupted circadian rhythmicity. These findings indicate that the circadian clock system plays an important role in regulating mitochondrial metabolism and thereby maintains cardiac function.

Multi-tissue computational modeling analyzes pathophysiology of type 2 diabetes in MKR mice.

Computational models using metabolic reconstructions for in silico simulation of metabolic disorders such as type 2 diabetes mellitus (T2DM) can provide a better understanding of disease pathophysiology and avoid high experimentation costs. There is a limited amount of computational work, using metabolic reconstructions, performed in this field for the better understanding of T2DM. In this study, a new algorithm for generating tissue-specific metabolic models is presented, along with the resulting multi-confidence level (MCL) multi-tissue model. The effect of T2DM on liver, muscle, and fat in MKR mice was first studied by microarray analysis and subsequently the changes in gene expression of frank T2DM MKR mice versus healthy mice were applied to the multi-tissue model to test the effect. Using the first multi-tissue genome-scale model of all metabolic pathways in T2DM, we found out that branched-chain amino acids' degradation and fatty acids oxidation pathway is downregulated in T2DM MKR mice. Microarray data showed low expression of genes in MKR mice versus healthy mice in the degradation of branched-chain amino acids and fatty-acid oxidation pathways. In addition, the flux balance analysis using the MCL multi-tissue model showed that the degradation pathways of branched-chain amino acid and fatty acid oxidation were significantly downregulated in MKR mice versus healthy mice. Validation of the model was performed using data derived from the literature regarding T2DM. Microarray data was used in conjunction with the model to predict fluxes of various other metabolic pathways in the T2DM mouse model and alterations in a number of pathways were detected. The Type 2 Diabetes MCL multi-tissue model may explain the high level of branched-chain amino acids and free fatty acids in plasma of Type 2 Diabetic subjects from a metabolic fluxes perspective.

Preclinical evaluation of 3-18F-fluoro-2,2-dimethylpropionic acid as an imaging agent for tumor detection.

Deregulated cellular metabolism is a hallmark of many cancers. In addition to increased glycolytic flux, exploited for cancer imaging with (18)F-FDG, tumor cells display aberrant lipid metabolism. Pivalic acid is a short-chain, branched carboxylic acid used to increase oral bioavailability of prodrugs. After prodrug hydrolysis, pivalic acid undergoes intracellular metabolism via the fatty acid oxidation pathway. We have designed a new probe, 3-(18)F-fluoro-2,2-dimethylpropionic acid, also called (18)F-fluoro-pivalic acid ((18)F-FPIA), for the imaging of aberrant lipid metabolism and cancer detection. Cell intrinsic uptake of (18)F-FPIA was measured in murine EMT6 breast adenocarcinoma cells. In vivo dynamic imaging, time course biodistribution, and radiotracer stability testing were performed. (18)F-FPIA tumor retention was further compared in vivo to (18)F-FDG uptake in several xenograft models and inflammatory tissue. (18)F-FPIA rapidly accumulated in EMT6 breast cancer cells, with retention of intracellular radioactivity predicted to occur via a putative (18)F-FPIA carnitine-ester. The radiotracer was metabolically stable to degradation in mice. In vivo imaging of implanted EMT6 murine and BT474 human breast adenocarcinoma cells by (18)F-FPIA PET showed rapid and extensive tumor localization, reaching 9.1% ± 0.5% and 7.6% ± 1.2% injected dose/g, respectively, at 60 min after injection. Substantial uptake in the cortex of the kidney was seen, with clearance primarily via urinary excretion. Regarding diagnostic utility, uptake of (18)F-FPIA was comparable to that of (18)F-FDG in EMT6 tumors but superior in the DU145 human prostate cancer model (54% higher uptake; P = 0.002). Furthermore, compared with (18)F-FDG, (18)F-FPIA had lower normal-brain uptake resulting in a superior tumor-to-brain ratio (2.5 vs. 1.3 in subcutaneously implanted U87 human glioma tumors; P = 0.001), predicting higher contrast for brain cancer imaging. Both radiotracers showed increased localization in inflammatory tissue. (18)F-FPIA shows promise as an imaging agent for cancer detection and warrants further investigation.

Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Abstract 422: The Lipid Droplet Associated Protein CIDEC/Fsp27 is Regulated by PPARα and Prevents Hepatic Triglyceride Utilization

The cell death-inducing DFFA-like effector c (CIDEC; known as FSP27 or fat-specific protein 27 in rodents) is a lipid droplet (LD)-associated protein that is mainly expressed in white adipose tissue, where it promotes triglyceride storage. Fsp27 levels are undetectable in the livers of fed mice, but rise during fasting or under hepatosteatosis in both obese mice and patients. The Peroxisome Proliferator Activated Receptor α (PPARα) is a nuclear receptor that controls the fatty acid oxidative pathway. Hepatic PPARα signaling is activated physiologically during fasting, and pharmacologically by fibrates, a class of hypotriglyceridemic drugs. Here we show that CIDEC/Fsp27 is induced in primary hepatocytes and in the liver by synthetic PPARα agonists, and promoter reporter and ChIP assays identified CIDEC/Fsp27 as a conserved, direct PPARα target. Importantly, adenoviral-mediated shRNA silencing of hepatic Fsp27 resulted in a strong reduction of fasting-induced hepatosteatosis. Likewise, in vivo shRNA against Fsp27 resulted in decreased hepatic triglyceride contents in mice fed a high-fat diet, and in mice dosed with a PPARα agonist. Further studies in mouse primary hepatocytes showed that loss of Fsp27 activity enhanced PPARα-stimulated fatty acid oxidation. Collectively, our results highlight the physiological role of CIDEC/Fsp27 in hepatic triglyceride homeostasis. Additionally, these data suggest that patients taking fibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient catabolism of hepatic triglycerides.

Human serum acylcarnitine profiles in different glucose tolerance states

AimsTo understand the relationship between serum acylcarnitine profiles and glucose tolerance status. MethodsWe analyzed 61 subjects who were divided into three groups based on their glucose tolerance status: normal glucose tolerance (NGT; n=20,M/F=9/11, mean age 48 years), pre-diabetes (Pre-DM; n=20,M/F=11/9, mean age 51 years), or newly diagnosed type 2 diabetes mellitus (T2DM; n=21,M/F=8/13, mean age 49 years). Fasting serum free carnitine and acylcarnitine concentrations were determined using isotope dilution electrospray ionization mass spectrometry coupled with high performance liquid chromatography. ResultsIn comparison with NGT subjects, Pre-DM and type 2 diabetes subjects showed serum metabonomic changes highlighted by dysregulation of mitochondrial fatty acid combustion. Of the long-chain carnitine esters, significantly higher palmitoylcarnitine (C16), 3-OH-hexadecanoylcarnitine (C16-OH), carnitine C20, carnitine C22, and carnitine C24 concentrations (all P<0.05) were noted in the newly diagnosed type 2 diabetes group, and even the pre-diabetes group. ConclusionsThis research provides further evidence of alterations in serum acylcarnitine profiles being associated with worse glucoseintolerance. The findings may suggest different degrees of involvement of dysregulated mitochondrial function and incomplete long-chain fatty acid oxidation pathways in the natural course of type 2 diabetes.