No effect of 25-hydroxyvitamin D supplementation on the skeletal muscle transcriptome in vitamin D deficient frail older adults.

OBJECTIVE: Vitamin D deficiency is common among older adults and has been linked to muscle weakness. Vitamin D supplementation has been proposed as a strategy to improve muscle function in older adults. The aim of this study was to investigate the effect of calcifediol (25-hydroxycholecalciferol) on whole genome gene expression in skeletal muscle of vitamin D deficient frail older adults. METHODS: A double-blind placebo-controlled trial was conducted in vitamin D deficient frail older adults (aged above 65), characterized by blood 25-hydroxycholecalciferol concentrations between 20 and 50 nmol/L. Subjects were randomized across the placebo group and the calcifediol group (10 µg per day). Muscle biopsies were obtained before and after 6 months of calcifediol (n = 10) or placebo (n = 12) supplementation and subjected to whole genome gene expression profiling using Affymetrix HuGene 2.1ST arrays. RESULTS: Expression of the vitamin D receptor gene was virtually undetectable in human skeletal muscle biopsies, with Ct values exceeding 30. Blood 25-hydroxycholecalciferol levels were significantly higher after calcifediol supplementation (87.3 ± 20.6 nmol/L) than after placebo (43.8 ± 14.1 nmol/L). No significant difference between treatment groups was observed on strength outcomes. The whole transcriptome effects of calcifediol and placebo were very weak, as indicated by the fact that correcting for multiple testing using false discovery rate did not yield any differentially expressed genes using any reasonable cut-offs (all q-values ~ 1). P-values were uniformly distributed across all genes, suggesting that low p-values are likely to be false positives. Partial least squares-discriminant analysis and principle component analysis was unable to separate treatment groups. CONCLUSION: Calcifediol supplementation did not significantly affect the skeletal muscle transcriptome in frail older adults. Our findings indicate that vitamin D supplementation has no effects on skeletal muscle gene expression, suggesting that skeletal muscle may not be a direct target of vitamin D in older adults. TRIAL REGISTRATION: This study was registered at clinicaltrials.gov as NCT02349282 on January 28, 2015.

Global testing of shifts in metabolic phenotype.

INTRODUCTION: Current metabolomics approaches to unravel impact of diet- or lifestyle induced phenotype variation and shifts predominantly deploy univariate or multivariate approaches, with a posteriori interpretation at pathway level. This however often provides only a fragmented view on the involved metabolic pathways. OBJECTIVES: To demonstrate the feasibility of using Goeman's global test (GGT) for assessment of variation and shifts in metabolic phenotype at the level of a priori defined pathways. METHODS: Two intervention studies with identified phenotype variations and shifts were examined. In a weight loss (WL) intervention study obese subjects received a mixed meal challenge before and after WL. In a polyphenol (PP) intervention study obese subjects received a high fat mixed meal challenge (61E% fat) before and after a PP intervention. Plasma samples were obtained at fasting and during the postprandial response. Besides WL- and PP-induced phenotype shifts, also correlation of plasma metabolome with phenotype descriptors was assessed at pathway level. The plasma metabolome covered organic acids, amino acids, biogenic amines, acylcarnitines and oxylipins. RESULTS: For the population of the WL study, GGT revealed that HOMA correlated with the fasting levels of the TCA cycle, BCAA catabolism, the lactate, arginine-proline and phenylalanine-tyrosine pathways. For the population of the PP study, HOMA correlated with fasting metabolite levels of TCA cycle, fatty acid oxidation and phenylalanine-tyrosine pathways. These correlations were more pronounced for metabolic pathways in the fasting state, than during the postprandial response. The effect of the WL and PP intervention on a priori defined metabolic pathways, and correlation of pathways with insulin sensitivity as described by HOMA was in line with previous studies. CONCLUSION: GGT confirmed earlier biological findings in a hypothesis led approach. A main advantage of GGT is that it provides a direct view on involvement of a priori defined pathways in phenotype shifts.

ANGPTL4 promotes bile acid absorption during taurocholic acid supplementation via a mechanism dependent on the gut microbiota.

Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4-/- mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4-/- mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4-/- mice than in TCA-supplemented wild-type mice. These changes in the Angptl4-/- mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4-/- mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4-/- mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4-/- and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4-/- and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota.

Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion.

Caspase-1 is known to activate the proinflammatory cytokines IL-1ß and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we investigated whether caspase-1 plays a role in triglyceride-(TG)-rich lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1 deficiency reduced the postprandial TG response to an oral lipid load, whereas TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [(3)H]TG-labeled very low-density lipoprotein (VLDL)-like emulsion particles. An oral gavage of [(3)H]TG-containing olive oil revealed that caspase-1 deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1 deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1 deficiency did not affect FA oxidation or FA uptake but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.

Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans.

Resveratrol is a natural compound that affects energy metabolism and mitochondrial function and serves as a calorie restriction mimetic, at least in animal models of obesity. Here, we treated 11 healthy, obese men with placebo and 150 mg/day resveratrol (resVida) in a randomized double-blind crossover study for 30 days. Resveratrol significantly reduced sleeping and resting metabolic rate. In muscle, resveratrol activated AMPK, increased SIRT1 and PGC-1α protein levels, increased citrate synthase activity without change in mitochondrial content, and improved muscle mitochondrial respiration on a fatty acid-derived substrate. Furthermore, resveratrol elevated intramyocellular lipid levels and decreased intrahepatic lipid content, circulating glucose, triglycerides, alanine-aminotransferase, and inflammation markers. Systolic blood pressure dropped and HOMA index improved after resveratrol. In the postprandial state, adipose tissue lipolysis and plasma fatty acid and glycerol decreased. In conclusion, we demonstrate that 30 days of resveratrol supplementation induces metabolic changes in obese humans, mimicking the effects of calorie restriction.

Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress.

RATIONALE: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart. OBJECTIVE: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences. METHODS AND RESULTS: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding. CONCLUSIONS: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

Effect of synthetic dietary triglycerides: a novel research paradigm for nutrigenomics.

BACKGROUND: The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-kappaB, RXRs, LXRs, FXR, HNF4alpha, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPARalpha. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPARalpha agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip(R) Arrays, fatty acid- and WY14643-induced interactions between PPARalpha and coregulators were found to be highly similar, although several PPARalpha-coactivator interactions specific for WY14643 were identified. CONCLUSIONS/SIGNIFICANCE: We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPARalpha and mimic those of synthetic PPARalpha agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.

Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: a combined virtual screening and biological assay approach.

Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPARgamma) improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides additionally bind to PPARgamma and exhibit PPARgamma agonistic activity. This activity was predicted in silico by virtual screening and confirmed in vitro in a binding assay, a transactivation assay, and by measuring the expression of PPARgamma target genes. Among the measured compounds, gliquidone and glipizide (two sulfonylureas), as well as nateglinide (a glinide), exhibit PPARgamma agonistic activity at concentrations comparable with those reached under pharmacological treatment. The most active of these compounds, gliquidone, is shown to be as potent as pioglitazone at inducing PPARgamma target gene expression. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, because it provides evidence that drugs can be designed that target both the sulfonylurea receptor and PPARgamma. Targeting both receptors could increase pancreatic insulin secretion and improve insulin resistance. Glinides, sulfonylureas, and other acidified sulfonamides may be promising leads in the development of new PPARgamma agonists. In addition, we provide a unified concept of the PPARgamma binding ability of seemingly disparate compound classes.

Peroxisome proliferator-activated receptor-alpha-null mice have increased white adipose tissue glucose utilization, GLUT4, and fat mass: Role in liver and brain.

Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.