Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.

Lactoferrin (Lf): Retinoid interactions in the mammary glands of transgenic mice overexpressing human Lf

Induction of protein expression in a tissue-specific manner by gene transfer over-expression techniques has been one means to define the function of a protein in a biological paradigm. Studies with retinoid reporter constructs transfected in mammary cell lines suggests that lactoferrin (Lf) affects retinoid signaling pathways and alters apoptosis. We tested the effects and interactions of over-expressed mammary-specific human lactoferrin (hLf) and dietary retinol palmitate on lactation and mammary gland development in mice. Increased retinol palmitate in the diet increased daily retinol equivalents (RE) to 2.6-fold over the normal mouse control diet. Transgene (Tg) expression in the dam fed control diet depressed pup weight gain. Severe depression of pup weight gain was observed when homozygote TgTg dams were fed the RE diet. Normal weight gain was restored when pups were placed with a wild type dam fed the RE diet; conversely, normal growing pups from the wild type dams showed declining weight gains when fostered to the TgTg RE-fed dams. Northern analysis of mammary tissue extracts showed a reduction in WAP and an increase in IGFBP-3 mRNA that was associated with the presence of the transgene. Histological evaluation of 3 days lactating mammary tissue showed mammary epithelial cells from TgTg animals contained excessive secretory products, suggesting a block in cellular secretion mechanisms. In addition, the mammary cells displayed a cellular apical membrane puckering that extended into the alveoli lumens. These studies demonstrate an in vivo interaction of Tg-hLf expression and dietary retinoids in mouse mammary glands. While normal mammary gland physiology may not be representative by these experiments because high Lf concentrations during early lactation are abnormal, the demonstrated biological interaction suggests that typical periods of high Lf concentrations may have impact upon developing and involuting mammary glands.

Intramyocellular lipid stores increase markedly in athletes after 1.5 days lipid supplementation and are utilized during exercise in proportion to their content

Intramyocellular lipids (IMCL) and muscle glycogen provide local energy during exercise (EX). The objective of this study was to clarify the role of high versus low IMCL levels at equal initial muscle glycogen on fuel selection during EX. After 3 h of depleting exercise, 11 endurance-trained males consumed in a crossover design a high-carbohydrate (7 g kg(-1) day(-1)) low-fat (0.5 g kg(-1) day(-1)) diet (HC) for 2.5 days or the same diet with 3 g kg(-1) day(-1) more fat provided during the last 1.5 days of diet (four meals; HCF). Respiratory exchange, thigh muscle substrate breakdown by magnetic resonance spectroscopy, and plasma FFA oxidation ([1-(13)C]palmitate) were measured during EX (3 h, 50% W (max)). Pre-EX IMCL concentrations were 55% higher after HCF. IMCL utilization during EX in HCF was threefold greater compared with HC (P < 0.001) and was correlated with aerobic power and highly correlated (P < 0.001) with initial content. Glycogen values and decrements during EX were similar. Whole-body fat oxidation (Fat(ox)) was similar overall and plasma FFA oxidation smaller (P < 0.05) during the first EX hour after HCF. Myocellular fuels contributed 8% more to whole-body energy demands after HCF (P < 0.05) due to IMCL breakdown (27% Fat(ox)). After EX, when both IMCL and glycogen concentrations were again similar across trials, a simulated 20-km time-trial showed no difference in performance between diets. In conclusion, IMCL concentrations can be increased during a glycogen loading diet by consuming additional fat for the last 1.5 days. During subsequent exercise, IMCL decrease in proportion to their initial content, partly in exchange for peripheral fatty acids.

Toxicity of valproic acid in mice with decreased plasma and tissue carnitine stores

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model†

OBJECTIVES The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability; however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC); brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury; however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. METHODS Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF); 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. RESULTS Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively; P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%; P < 0.01 for all) in LoR subgroups. CONCLUSIONS Effects of MPC depend on energy substrate availability; MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement.

OBJECTIVE Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency. METHODS Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay. RESULTS Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients' fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate - a potential derivative of acryloyl-CoA in the valine catabolic pathway - was significantly increased, indicating impaired valine oxidation. INTERPRETATION In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model

Gebiet: Chirurgie Abstract: OBJECTIVES: – The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability, however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC), brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury, however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. – – METHODS: – Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF), 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. – – RESULTS: – Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively, P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%, P < 0.01 for all) in LoR subgroups. – – CONCLUSIONS: – Effects of MPC depend on energy substrate availability, MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 μL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅ max) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample. Graphical Abstract Time courses for fatty acid ethyl ester (FAEE) concentrations in DBS and ethanol concentrations for subject 1 over a period of 7 h. Ethanol ingestion occured during the first hour of the time course.