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Aims Visceral adipose tissue inflammation is a fundamental mechanism of insulin resistance in obesity and type 2 diabetes. Translocation of intestinal bacteria has been suggested as a driving factor for the inflammation. However, although bacterial DNA was detected in visceral adipose tissue of humans with obesity, it is unclear to what extent this is contamination or whether the gut microbiota is causally involved. Effects of fecal microbiota transplantation (FMT) on bacterial translocation and visceral adipose tissue inflammation in individuals with obesity and insulin resistance were assessed. Material and Methods Eight individuals with clinically severe obesity (body mass index [BMI] >35 kg/m2) and metabolic syndrome received lean donor FMT 4 weeks prior to elective bariatric surgery. The participants were age‐, sex‐, and BMI‐matched to 16 controls that underwent no fecal transplantation. Visceral adipose tissue was collected during surgery. Bacterial translocation was assessed by 16S rRNA gene sequencing of adipose tissue and feces. Pro‐inflammatory cytokine expression and histopathological analyses of visceral adipose tissue were performed to assess inflammation. Results Fecal microbiota transplantation significantly altered gut microbiota composition. Visceral adipose tissue contained a very low quantity of bacterial DNA in both groups. No difference in visceral bacterial DNA content between groups was observed. Also, visceral expression of pro‐inflammatory cytokines and macrophage infiltration did not differ between groups. No correlation between inflammatory tone and bacterial translocation was observed. Conclusions Visceral bacterial DNA content and level of inflammation were not altered upon FMT. Thus, bacterial translocation may not be the main driver of visceral adipose tissue inflammation in obesity.

Objective: Insulin resistance develops prior to the onset of overt type 2 diabetes, making its early detection vital. Direct accurate evaluation is currently only possible with complex examinations like the stable isotope-based hyperinsulinemic euglycemic clamp (HIEC). Metabolomic profiling enables the detection of thousands of plasma metabolites, providing a tool to identify novel biomarkers in human obesity. Design: Liquid chromatography mass spectrometry–based untargeted plasma metabolomics was applied in 60 participants with obesity with a large range of peripheral insulin sensitivity as determined via a two-step HIEC with stable isotopes [6,6-2H2]glucose and [1,1,2,3,3-2H5]glycerol. This additionally enabled measuring insulin-regulated lipolysis, which combined with metabolomics, to the knowledge of this research group, has not been reported on before. Results: Several plasma metabolites were identified that significantly correlated with glucose and lipid fluxes, led by plasma (gamma-glutamyl)citrulline, followed by betaine, beta-cryptoxanthin, fructosyllysine, octanylcarnitine, sphingomyelin (d18:0/18:0, d19:0/17:0) and thyroxine. Subsequent machine learning analysis showed that a panel of these metabolites derived from a number of metabolic pathways may be used to predict insulin resistance, dominated by non-essential amino acid citrulline and its metabolite gamma-glutamylcitrulline. Conclusion: This approach revealed a number of plasma metabolites that correlated reasonably well with glycemic and lipolytic flux parameters, measured using gold standard techniques. These metabolites may be used to predict the rate of glucose disposal in humans with obesity to a similar extend as HOMA, thus providing potential novel biomarkers for insulin resistance.

Over the past decades maternal nutritional intake has been proven to affect the prenatal and postnatal development of the fetus as well as its long-term health (see (Jimenez-Chillaron et al. 2012) and (Lillycrop & Burdge. 2015) for recent overviews). In this journal, we have recently shown that maternal Western style diet gives rise to long lasting programming of the liver in adult mouse offspring (Pruis et al. 2014), affecting mostly the males. Moreover, we demonstrated that several aspects of sex-specific programming are already present in two-week old offspring (Mischke et al. 2013), i.e., well before puberty. The question arising now is if this sexual dimorphism was already present during prenatal development, and which factors contributed to its manifestation. This article is protected by copyright. All rights reserved.

Biliary lipid secretion plays an important role in gallstone disease and reverse cholesterol transport (RCT). Using Sr-bI/Abcg5 double knockout mice (dko), the present study investigated the differential contribution of two of the most relevant transporters: adenosine triphosphate (ATP)-binding cassette subfamily G member 5 and 8 (ABCG5/G8) and scavenger receptor class B type I (SR-BI) to sterol metabolism and RCT. Plasma cholesterol levels increased in the following order, mainly due to differences in high density lipoprotein (HDL): Abcg5 ko Sr-bI ko (-16%) > Abcg5 ko (-75%) > Sr-bI/Abcg5 dko (-94%), all at least P

Atherosclerosis is characterized by excessive cholesterol accumulation in the vessel wall. Current therapies mainly aim at decreasing influx through lowering plasma LDL-cholesterol levels. The challenge is to develop therapeutic interventions to increase efflux of excess cholesterol from the vessel wall. The pathway that mediates this efflux from vessel wall to final excretion in the feces is called reverse cholesterol transport. Recently, it has become apparent that the intestine plays an important regulatory role in this pathway. This article describes in detail a variety of experimental approaches to measure cholesterol fluxes in the hepatobiliary system as well as in the intestinal pathway. Curr. Protoc. Mouse Biol. 1:413-427 © 2011 by John Wiley & Sons, Inc. Keywords: mouse; fecal cholesterol excretion; biliary excretion; transintestinal cholesterol; excretion

Obesity and type 2 diabetes mellitus (T2DM) are attributed to a combination of genetic susceptibility and lifestyle factors. Their increasing prevalence necessitates further studies on modifiable causative factors and novel treatment options. The gut microbiota has emerged as an important contributor to the obesity--and T2DM--epidemic proposed to act by increasing energy harvest from the diet. Although obesity is associated with substantial changes in the composition and metabolic function of the gut microbiota, the pathophysiological processes remain only partly understood. In this review we will describe the development of the adult human microbiome and discuss how the composition of the gut microbiota changes in response to modulating factors. The influence of short-chain fatty acids, bile acids, prebiotics, probiotics, antibiotics and microbial transplantation is discussed from studies using animal and human models. Ultimately, we aim to translate these findings into therapeutic pathways for obesity and T2DM in humans.

Observations of predose spikes in transaminases from a phase I study prompted our review of other clinical trials, including 250 healthy participants from 4 studies at 3 sites. Six of these participants (2.4%) displayed elevated alanine aminotransferase/aspartate aminotransferase (ALT/AST) levels immediately predose despite normal values

Ezetimibe stimulates faecal neutral sterol (FNS) excretion in mice, which cannot be explained by cholesterol absorption inhibition alone. We investigated whether these effects are mediated via the sterol exporter ATP binding cassette transporter G8 (abcg8). Ezetimibe increased FNS excretion 2.7-fold in WT mice and 1.5-fold in abcg8(-/-) mice, without affecting biliary cholesterol secretion. Daily FNS excretion exceeded the sum of dietary cholesterol intake and biliary secretion by about 60%. Ezetimibe enhanced this 'extra' FNS excretion by 3.5-fold and 1.5-fold in wildtype (WT) and abcg8(-/-)mice, respectively. Ezetimibe stimulates fecal sterol excretion of non-biliary and non-dietary origin, probably through stimulation of trans-intestinal cholesterol excretion. We show that this effect depends on intact abcg8 function. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Objective.Studies in mice suggest that plasma apoM is lowered in hyperinsulinaemic diabetes and that apoM stimulates formation of pre-beta-HDL. Pre-beta-HDL is an acceptor of cellular cholesterol and may be critical for reverse cholesterol transport. Herein, we examined whether patients with type 2 diabetes have reduced plasma apoM and whether apoM is associated with pre-beta-HDL formation and cellular cholesterol efflux.Design.In 78 patients with type 2 diabetes and 89 control subjects, we measured plasma apoM with ELISA, pre-beta-HDL and pre-beta-HDL formation, phospholipid transfer protein (PLTP) activity and the ability of plasma to promote cholesterol efflux from cultured fibroblasts.Results.ApoM was similar to 9% lower in patients with type 2 diabetes compared to controls (0.025 +/- 0.006 vs. 0.027 +/- 0.007 g L(-1), P = 0.01). The difference in apoM was largely attributable to diabetes-associated obesity. ApoM was positively related to both HDL (r = 0.16; P = 0.04) and LDL cholesterol (r = 0.28; P = 0.0003). Pre-beta-HDL and pre-beta-HDL formation were not different between diabetic and control subjects. ApoM predicted pre-beta-HDL (r = 0.16; P = 0.04) and pre-beta-HDL formation (r = 0.19; P = 0.02), even independently of positive relationships with apoA-I, HDL-cholesterol and PLTP activity. Cellular cholesterol efflux to plasma was positively related to pre-beta-HDL and PLTP activity but not significantly to apoM.Conclusions.Plasma apoM is modestly reduced in type 2 diabetes. Pre-beta-HDL and pre-beta-HDL formation are positively associated with apoM, supporting the hypothesis that apoM plays a role in HDL remodelling in humans. Lower apoM may provide a mechanism to explain why pre-beta-HDL formation is not increased in type 2 diabetes despite elevated PLTP activity.

Summary Loss of mitochondrial respiratory flux is a hallmark of skeletal muscle aging, contributing to a progressive decline of muscle strength. Endurance exercise alleviates the decrease in respiratory flux, both in humans and in rodents. Here, we dissect the underlying mechanism of mitochondrial flux decline by integrated analysis of the molecular network. Mice were given a lifelong ad libitum low-fat or high-fat sucrose diet and were further divided into sedentary and running-wheel groups. At 6, 12, 18 and 24 months, muscle weight, triglyceride content and mitochondrial respiratory flux were analysed. Subsequently, transcriptome was measured by RNA-Seq and proteome by targeted LC-MS/MS analysis with 13C-labelled standards. In the sedentary groups, mitochondrial respiratory flux declined with age. Voluntary running protected the mitochondrial respiratory flux until 18 months of age. Beyond this time point, all groups converged. Regulation Analysis of flux, proteome and transcriptome showed that the decline of flux was equally regulated at the proteomic and at the metabolic level, while regulation at the transcriptional level was marginal. Proteomic regulation was most prominent at the beginning and at the end of the pathway, namely at the pyruvate dehydrogenase complex and at the synthesis and transport of ATP. Further proteomic regulation was scattered across the entire pathway, revealing an effective multisite regulation. Finally, reactions regulated at the protein level were highly overlapping between the four experimental groups, suggesting a common, post-transcriptional mechanism of muscle aging.

Cholesterol is of crucial importance during fetal development and relies on both maternal transport and fetal synthesis. During gestation, Lipoprotein related receptor protein type 2 (Megalin or LR...

Metabolic programming via the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model we aimed to characterise the role...

The small apical (canalicular) domains of hepatocytes form a luminal meshwork of tubules between adjacent hepatocytes and are the sites of primary bile formation. Organic compounds are transported across this membrane domain against high concentration gradients. It has been recognized in recent years that the hepatocyte is harnessed with a set of canalicular ATP-dependent transport proteins, specialized in this uphill transport. Bile salts, organic anions, cations, and neutral amphipaths are all pumped into the bile via such primary active transporters. Functionally, these transporters resemble ABC transporters overexpressed in cells with the multidrug resistance phenotype. Indeed, those transporters that have been characterized at the molecular level turn out to be new, or already recognized, members of this family. Phospholipid secretion across the canalicular membrane of the mouse is also mediated by a member of this family, mdr2 P-glycoprotein. This was demonstrated by the absence of phospholipid secretion into bile of mice with a disrupted mdr2 gene and by subsequent demonstration of phospholipid translocation in cells that overexpress this protein. The recognition of mdr2 P-glycoprotein as a phospholipid flippase sheds new light on the function of P-glycoproteins and is an important step in understanding the mechanism of biliary lipid secretion.

The bioavailability of penicillin and dihydrostreptomycin from three penicillin/ aminoglycoside fixed combination products for intramuscular injection was investigated in a four-way, randomized, crossover experiment in rabbits. Attention is focused on bioequivalence based on plasma concentration vs. time profiles to study whether the rabbit is a good model to detect differences in in vivo delivery of penicillin and/or dihydrostreptomycin after intramuscular administration of different products. In all products, penicillin was present as a suspension. Although the extent of absorption of penicillin did not differ between the three products, large differences in the rates of absorption were observed. With respect to dihydrostreptomycin, no significant differences were observed between the products. The results from this study demonstrate that the rabbit is a good model to detect differences in bioavailability of suspended penicillin from penicillin /dihydrostreptomycin fixed combination products for intramuscular injection. A study with the same products is presently being carried out in calves to investigate whether bioequivalence studies in rabbits could replace studies in the target animals.

Conscious male Wistar SPF Riv:TOX rats were dosed intravenously with 2.5, 5, or 10 mg/kg rac-propranolol.HCl, or with 5 mg/kg of either (-)-(S)- or (+)-(R)-propranolol.HCl. Disposition of (-)-(S)- and (+)-(R)-propranolol after dosing of rac-propranolol was linear in the dose range examined. Total plasma clearance was not changed in animals dosed with the individual enantiomers compared to the animals that were dosed with rac-propranolol. However, for (-)-(S)-propranolol both volume of distribution and elimination half-life decreased, whereas for (+)-(R)-propranolol increases were observed for these characteristics, in animals dosed with the individual enantiomers. Our observations suggest that the (+)-(R)-enantiomer competes with (-)-(S)-propranolol for plasma protein binding sites, resulting in lower plasma protein binding of the (-)-(S)-enantiomer when the racemate is administered. From recent toxicological experiments, it was concluded that rac-propranolol is more toxic than the individual enantiomers in the rat, when dosed iv at the same total mass. It is concluded that the observed potentiation of toxic effects of propranolol enantiomers when administered as a racemate can at least partly be explained by a pharmacokinetic interaction.

When isolated rat-liver cells were incubated for 1 min at 37 degrees C with filipin at a concentration of 50 microM, the plasma membrane became permeable to sucrose, inulin, glycerol 3-phosphate and other low-molecular-weight compounds. Upon removal of the filipin and subsequent incubation of the cells at 37 degrees C there was a gradual leakage of lactate dehydrogenase from the cells. However, the leakage of lactate dehydrogenase could be prevented for about 10 min by including glutathione and ATP in the incubation medium. The filipin-treated cells were able to metabolize phosphorylated sugars. The conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, glucose-6-phosphate and glucose was inhibited by AMP but not by high concentrations of fructose 1,6-bisphosphate. The results indicate that filipin-treated cells can be used to study the kinetic parameters of enzymes in their macromolecular environment in situ.

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.