Intraintestinal fermentation of fructo- and galacto-oligosaccharides and the fate of short-chain fatty acids in humans.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

Milk fat globule membrane modulates inflammatory pathways in human monocytes: A crossover human intervention study.

BACKGROUND: Intake of high-fat foods raises postprandial plasma triglycerides and inflammatory markers, which may depend on the type of fat ingested. Dairy products are commonly consumed, but not much is known about the impact of milk fat and the milk fat globule membrane on postprandial inflammation. Here, we aimed to study the effect of milk fat with and without milk fat globule membrane and a vegetable fat blend on post-prandial inflammation, with a focus on blood monocyte gene expression. METHODS: We performed a randomized, double-blind cross-over trial in 37 middle-aged healthy male and female volunteers (BMI 22-27 kg/m2). The participants consumed a meal shake containing 95.5 g of fat consisting of either a vegetable fat blend (VEGE), anhydrous milk fat (AMF, without milk fat globule membrane), or cream (CREAM, containing milk fat globule membrane). Blood monocytes were collected at 0 h and 6 h postprandially and used for bulk RNA sequencing and ex vivo stimulation with LPS. RESULTS: Consumption of all three shakes significantly decreased the percentage of classical monocytes and increased the percentages of intermediate monocytes and non-classical monocytes. No differences in these measures were observed between shakes. Using a threshold of p < 0.01, 787 genes were differentially regulated postprandially between the three shakes. 89 genes were differentially regulated postprandially between AMF and VEGE, 373 genes between AMF and CREAM, and 667 genes between VEGE and CREAM, indicating that the effect of CREAM on monocyte gene expression was distinct from AMF and VEGE. Pathway analyses showed that VEGE significantly increased the expression of genes involved in inflammatory pathways, whereas this was less pronounced after AMF and not observed after CREAM. In addition, CREAM significantly down-regulated the expression of genes involved in energy metabolism-related pathways, such as glycolysis, TCA cycle, and oxidative phosphorylation, as well as HIF-1 signaling. CONCLUSION: Compared to the consumption of an anhydrous milk fat without milk fat globule membrane and a vegetable fat blend, the consumption of cream with milk fat globule membrane downregulated inflammatory pathways in blood monocytes, thus suggesting a potential inflammation inhibitory effect of milk fat globule membrane.

Systematic comparison of transcriptomes of Caco-2 cells cultured under different cellular and physiological conditions.

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host-microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data.

Exposure to low-dose perfluorooctanoic acid promotes hepatic steatosis and disrupts the hepatic transcriptome in mice.

OBJECTIVE: Perfluoroalkyl substances (PFAS) are man-made chemicals with demonstrated endocrine-disrupting properties. Exposure to perfluorooctanoic acid (PFOA) has been linked to disturbed metabolism via the liver, although the exact mechanism is not clear. Moreover, information on the metabolic effects of the new PFAS alternative GenX is limited. We examined whether exposure to low-dose PFOA and GenX induces metabolic disturbances in mice, including NAFLD, dyslipidemia, and glucose tolerance, and studied the involvement of PPARα. METHODS: Male C57BL/6J wildtype and PPARα-/- mice were given 0.05 or 0.3 mg/kg body weight/day PFOA, or 0.3 mg/kg body weight/day GenX while being fed a high-fat diet for 20 weeks. Glucose and insulin tolerance tests were performed after 18 and 19 weeks. Plasma metabolite levels were measured next to a detailed assessment of the liver phenotype, including lipid content and RNA sequencing. RESULTS: Exposure to high-dose PFOA decreased body weight and increased liver weight in wildtype and PPARα-/- mice. High-dose but not low-dose PFOA reduced plasma triglycerides and cholesterol, which for triglycerides was dependent on PPARα. PFOA and GenX increased hepatic triglycerides in a PPARα-dependent manner. RNA sequencing showed that the effects of GenX on hepatic gene expression were entirely dependent on PPARα, while the effects of PFOA were mostly dependent on PPARα. In the absence of PPARα, the involvement of PXR and CAR became more prominent. CONCLUSION: Overall, we show that long-term and low-dose exposure to PFOA and GenX disrupts hepatic lipid metabolism in mice. Whereas the effects of PFOA are mediated by multiple nuclear receptors, the effects of GenX are entirely mediated by PPARα. Our data underscore the potential of PFAS to disrupt metabolism by altering signaling pathways in the liver.

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate.

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 h. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy homeostasis, this is likely associated with the use of SCFAs as an energy source by the induced pluripotent stem cell derived intestinal epithelial cells similar to in vivo intestinal tissues where butyrate is an important energy source.

Macrophages take up VLDL-sized emulsion particles through caveolae-mediated endocytosis and excrete part of the internalized triglycerides as fatty acids.

Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDLs) and chylomicrons, which represent the triglyceride-rich lipoproteins. Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a proinflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of VLDL-sized emulsion particles is dependent on lipoprotein lipase (LPL) and requires the lipoprotein-binding C-terminal domain but not the catalytic N-terminal domain of LPL. Subsequent internalization of VLDL-sized emulsion particles by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. It is shown that STARD3 is required for the transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids.

Human gut microbiota composition and its predicted functional properties in people with western and healthy dietary patterns.

PURPOSE: Some dietary habits cluster together, and for this reason it is advised to study the impact of entire dietary patterns on human health, rather than that of individual dietary habits. The main objective of this study was to evaluate differences in gut microbiota composition and their predicted functional properties between people with a healthy (HDP) and western (WDP) dietary pattern. METHODS: A cross-sectional, observational study was carried out on 200 participants enrolled 2017-2018 in Poznan, Poland, equally distributed into HDP and WDP groups. Diet was estimated using 3-day food records and information on stool transit times was collected. Fecal microbiota composition was assessed by 16S rRNA gene sequencing and its functional properties were predicted by the PICRUSt2 workflow. RESULTS: The α-diversity did not differ between people with WDP and HDP, but ß-diversity was associated with dietary pattern. People with HDP had higher relative abundances (RA) of Firmicutes and Faecalibacterium and lower RA of Bacteroidota and Escherichia-Shigella than participants with WDP. Only a small proportion of the variance in microbiota composition (1.8%) and its functional properties (2.9%) could be explained by dietary intake (legumes, simple sugars and their sources, like fruit, soft drinks) and stool transit characteristics. CONCLUSION: Gut microbiota composition and predicted metabolic potential is shaped by overall diet quality as well as the frequency of defecation; however, the cumulative effect of these explain only a relatively low proportion of variance.

Exploring the associations between transcript levels and fluxes in constraint-based models of metabolism.

BACKGROUND: Several computational methods have been developed that integrate transcriptomics data with genome-scale metabolic reconstructions to increase accuracy of inferences of intracellular metabolic flux distributions. Even though existing methods use transcript abundances as a proxy for enzyme activity, each method uses a different hypothesis and assumptions. Most methods implicitly assume a proportionality between transcript levels and flux through the corresponding function, although these proportionality constant(s) are often not explicitly mentioned nor discussed in any of the published methods. E-Flux is one such method and, in this algorithm, flux bounds are related to expression data, so that reactions associated with highly expressed genes are allowed to carry higher flux values. RESULTS: Here, we extended E-Flux and systematically evaluated the impact of an assumed proportionality constant on model predictions. We used data from published experiments with Escherichia coli and Saccharomyces cerevisiae and we compared the predictions of the algorithm to measured extracellular and intracellular fluxes. CONCLUSION: We showed that detailed modelling using a proportionality constant can greatly impact the outcome of the analysis. This increases accuracy and allows for extraction of better physiological information.

A toolbox for the comprehensive analysis of small volume human intestinal samples that can be used with gastrointestinal sampling capsules.

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.

Immunomodulating effects of 13- and 16-hydroxylated docosahexaenoyl ethanolamide in LPS stimulated RAW264.7 macrophages.

Docosahexaenoyl ethanolamide (DHEA), the ethanolamine conjugate of the n-3 long chain polyunsaturated fatty acid docosahexaenoic acid, is endogenously present in the human circulation and in tissues. Its immunomodulating properties have been (partly) attributed to an interaction with the cyclooxygenase-2 (COX-2) enzyme. Recently, we discovered that COX-2 converts DHEA into two oxygenated metabolites, 13- and 16-hydroxylated-DHEA (13- and 16-HDHEA, respectively). It remained unclear whether these oxygenated metabolites also display immunomodulating properties like their parent DHEA. In the current study we investigated the immunomodulating properties of 13- and 16-HDHEA in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The compounds reduced production of tumor necrosis factor alpha (TNFα), interleukin (IL)-1ß and IL-1Ra, but did not affect nitric oxide (NO) and IL-6 release. Transcriptome analysis showed that the compounds inhibited the LPS-mediated induction of pro-inflammatory genes (InhbA, Ifit1) and suggested potential inhibition of regulators such as toll-like receptor 4 (TLR4), MyD88, and interferon regulatory factor 3 (IRF3), whereas anti-inflammatory genes (SerpinB2) and potential regulators IL-10, sirtuin 1 (Sirt-1), fluticasone propionate were induced. Additionally, transcriptome analysis of 13-HDHEA suggests a potential anti-angiogenic role. In contrast to the known oxylipin-lowering effects of DHEA, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses revealed that 13- and 16-HDHEA did not affect oxylipin formation. Overall, the anti-inflammatory effects of 13-HDHEA and 16-HDHEA are less pronounced compared to their parent molecule DHEA. Therefore, we propose that COX-2 metabolism of DHEA acts as a regulatory mechanism to limit the anti-inflammatory properties of DHEA.

Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia.

Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

B. burgdorferi sensu lato-induced inhibition of antigen presentation is mediated by RIP1 signaling resulting in impaired functional T cell responses towards Candida albicans.

Antigen presentation is a crucial innate immune cell function that instructs adaptive immune cells. Loss of this pathway severely impairs the development of adaptive immune responses. To investigate whether B. burgdorferi sensu lato. spirochetes modulate the induction of an effective immune response, primary human PBMCs were isolated from healthy volunteers and stimulated with B. burgdorferi s.l. Through cell entry, TNF receptor I, and RIP1 signaling cascades, B. burgdorferi s.l. strongly downregulated genes and proteins involved in antigen presentation, specifically HLA-DM, MHC class II and CD74. Antigen presentation proteins were distinctively inhibited in monocyte subsets, monocyte-derived macrophages, and dendritic cells. When compared to a range of other pathogens, B. burgdorferi s.l.-induced suppression of antigen presentation appears to be specific. Inhibition of antigen presentation interfered with T-cell recognition of B. burgdorferi s.l., and memory T-cell responses against Candidaalbicans. Re-stimulation of PBMCs with the commensal microbe C.albicans following B. burgdorferi s.l. exposure resulted in significantly reduced IFN-γ, IL-17 and IL-22 production. These findings may explain why patients with Lyme borreliosis develop delayed adaptive immune responses. Unravelling the mechanism of B. burgdorferi s.l.-induced inhibition of antigen presentation, via cell entry, TNF receptor I, and RIP1 signaling cascades, explains the difficulty to diagnose the disease based on serology and to obtain an effective vaccine against Lyme borreliosis.

Comparative study of the transcriptomes of Caco-2 cells cultured under dynamic vs. static conditions following exposure to titanium dioxide and zinc oxide nanomaterials.

Due to the widespread application of food-relevant inorganic nanomaterials, the gastrointestinal tract is potentially exposed to these materials. Gut-on-chip in vitro systems are proposed for the investigation of compound toxicity as they better recapitulate the in vivo human intestinal environment than static models, due to the added shear stresses associated with the flow of the medium. We aimed to compare cellular responses of intestinal epithelial Caco-2 cells at the gene expression level upon TiO2 (E171) and ZnO (NM110) nanomaterial exposure when cultured under dynamic and conventionally applied static conditions. Whole-genome transcriptome analyses upon exposure of the cells to TiO2 and ZnO nanomaterials revealed differentially expressed genes and related biological processes that were culture condition specific. The total number of differentially expressed genes (p < 0.01) and affected pathways (p < 0.05 and FDR < 0.25) after nanomaterial exposure was higher under dynamic culture conditions than under static conditions for both nanomaterials. The observed increase in nanomaterial-induced responses in the gut-on-chip model indicates that shear stress might be a major factor in cell susceptibility. This is the first report on the application of a gut-on-chip system in which gene expression responses upon TiO2 and ZnO nanomaterial exposure are evaluated and compared to a static system. It extends current knowledge on nanomaterial toxicity assessment and the influence of a dynamic environment on cellular responses. Application of the gut-on-chip system resulted in higher sensitivity of the cells and might thus be an attractive system for use in the toxicological hazard characterization of nanomaterials.

Minor Changes in the Composition and Function of the Gut Microbiota During a 12-Week Whole Grain Wheat or Refined Wheat Intervention Correlate with Liver Fat in Overweight and Obese Adults.

BACKGROUND: Whole grain wheat (WGW) products are advocated as a healthy choice when compared with refined wheat (RW). One proposed mechanism for these health benefits is via the microbiota, because WGW contains multiple fibers. WGW consumption has been proposed to ameliorate nonalcoholic fatty liver disease, in which microbiota might play a role. OBJECTIVES: We investigated the effect of WGW compared with RW intervention on the fecal microbiota composition and functionality, and correlated intervention-induced changes in bacteria with changes in liver health parameters in adults with overweight or obesity. METHODS: We used data of a 12-wk double-blind, randomized, controlled, parallel trial to examine the effects of a WGW (98 g/d) or RW (98 g/d) intervention on the secondary outcomes fecal microbiota composition, predicted microbiota functionality, and stool consistency in 37 women and men (aged 45-70 y, BMI 25-35 kg/m2). The changes in microbiota composition, measured using 16S ribosomal RNA gene sequencing, after a 12-wk intervention were analyzed with nonparametric tests, and correlated with changes in liver fat and circulating concentrations of liver enzymes including alanine transaminase, aspartate transaminase, γ-glutamyltransferase, and serum amyloid A. RESULTS: The WGW intervention increased the mean (± SD) relative abundances of Ruminococcaceae_UCG-014 (baseline: 2.2 ± 4.6%, differential change over time (Δ) 0.51 ± 4.2%), Ruminiclostridium_9 (baseline: 0.065 ± 0.11%, Δ 0.054 ± 0.14%), and Ruminococcaceae_NK4A214_group (baseline: 0.37 ± 0.56%, Δ 0.17 ± 0.83%), and also the predicted pathway acetyl-CoA fermentation to butyrate II (baseline: 0.23 ± 0.062%, Δ 0.035 ± 0.059%), compared with the RW intervention (P values <0.05). A change in Ruminococcaceae_NK4A214_group was positively correlated with the change in liver fat, in both the WGW (ρ = 0.54; P = 0.026) and RW (ρ = 0.67; P = 0.024) groups. CONCLUSIONS: In middle-aged overweight and obese adults, a 12-wk WGW intervention increased the relative abundance of a number of bacterial taxa from the family Ruminococcaceae and increased predicted fermentation pathways when compared with an RW intervention. Potential protective health effects of replacement of RW by WGW on metabolic organs, such as the liver, via modulation of the microbiota, deserve further investigation.This trial was registered at clinicaltrials.gov as NCT02385149.

Probing metabolic memory in the hepatic response to fasting.

Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here, we tested the hypothesis that a strong metabolic stimulus such as fasting may influence the hepatic response to a subsequent fast and thus elicit a memory effect. Overnight fasting in mice significantly increased plasma free fatty acids, glycerol, ß-hydroxybutyrate, and liver triglycerides, and decreased plasma glucose, plasma triglycerides, and liver glycogen levels. In addition, fasting dramatically changed the liver transcriptome, upregulating genes involved in gluconeogenesis and in uptake, oxidation, storage, and mobilization of fatty acids, and downregulating genes involved in fatty acid synthesis, fatty acid elongation/desaturation, and cholesterol synthesis. Fasting also markedly impacted the liver metabolome, causing a decrease in the levels of numerous amino acids, glycolytic-intermediates, TCA cycle intermediates, and nucleotides. However, these fasting-induced changes were unaffected by two previous overnight fasts. Also, no significant effect was observed of prior fasting on glucose tolerance. Finally, analysis of the effect of fasting on the transcriptome in hepatocyte humanized mouse livers indicated modest similarity in gene regulation in mouse and human liver cells. In general, genes involved in metabolic pathways were upregulated or downregulated to a lesser extent in human liver cells than in mouse liver cells. In conclusion, we found that previous exposure to fasting in mice did not influence the hepatic response to a subsequent fast, arguing against the concept of metabolic memory in the liver. Our data provide a useful resource for the study of liver metabolism during fasting.

Fermentation Kinetics of Selected Dietary Fibers by Human Small Intestinal Microbiota Depend on the Type of Fiber and Subject.

SCOPE: An underexplored topic is the investigation of health effects of dietary fibers via modulation of human small intestine (SI) microbiota. A few previous studies hint at fermentation of some dietary fibers in the distal SI of humans and pigs. Here the potential of human SI microbiota to degrade dietary fibers and produce metabolites in vitro is investigated. METHODS AND RESULTS: Fructans, galacto-oligosaccharides, lemon pectins, and isomalto/malto-polysaccharides are subjected to in vitro batch fermentations inoculated with ileostomy effluent from five subjects. Fiber degradation products, formation of bacterial metabolites, and microbiota composition are determined over time. Galacto- and fructo-oligosaccharides are rapidly utilized by the SI microbiota of all subjects. At 5h of fermentation, 31%-82% of galacto-oligosaccharides and 29%-89% fructo-oligosaccharides (degree of polymerization DP4-8) are utilized. Breakdown of fructo-oligosaccharides/inulin DP ≥ 10, lemon pectin, and iso-malto/maltopolysaccharides only started after 7h incubation. Degradation of different fibers result in production of mainly acetate, and changed microbiota composition over time. CONCLUSION: Human SI microbiota have hydrolytic potential for prebiotic galacto- and fructo-oligosaccharides. In contrast, the higher molecular weight fibers inulin, lemon pectin, and iso-malto/maltopolysaccharides show slow fermentation rate. Fiber degradation kinetics and microbiota responses are subject dependent, therefore personalized nutritional fiber based strategies are required.

Comparative transcriptome analysis of human skeletal muscle in response to cold acclimation and exercise training in human volunteers.

BACKGROUND: Cold acclimation and exercise training were previously shown to increase peripheral insulin sensitivity in human volunteers with type 2 diabetes. Although cold is a potent activator of brown adipose tissue, the increase in peripheral insulin sensitivity by cold is largely mediated by events occurring in skeletal muscle and at least partly involves GLUT4 translocation, as is also observed for exercise training. METHODS: To investigate if cold acclimation and exercise training overlap in the molecular adaptive response in skeletal muscle, we performed transcriptomics analysis on vastus lateralis muscle collected from human subjects before and after 10 days of cold acclimation, as well as before and after a 12-week exercise training intervention. RESULTS: Cold acclimation altered the expression of 756 genes (422 up, 334 down, P < 0.01), while exercise training altered the expression of 665 genes (444 up, 221 down, P < 0.01). Principal Component Analysis, Venn diagram, similarity analysis and Rank-rank Hypergeometric Overlap all indicated significant overlap between cold acclimation and exercise training in upregulated genes, but not in downregulated genes. Overlapping gene regulation was especially evident for genes and pathways associated with extracellular matrix remodeling. Interestingly, the genes most highly induced by cold acclimation were involved in contraction and in signal transduction between nerve and muscle cells, while no significant changes were observed in genes and pathways related to insulin signaling or glucose metabolism. CONCLUSIONS: Overall, our results indicate that cold acclimation and exercise training have overlapping effects on gene expression in human skeletal muscle, but strikingly these overlapping genes are designated to pathways related to tissue remodeling rather than metabolic pathways.

Effects of Casein, Chicken, and Pork Proteins on the Regulation of Body Fat and Blood Inflammatory Factors and Metabolite Patterns Are Largely Dependent on the Protein Level and Less Attributable to the Protein Source.

The impact of meat protein on metabolic regulation is still disputed and may be influenced by protein level. This study aimed to explore the effects of casein, pork, and chicken proteins at different protein levels (40% E vs 20% E) on body weight regulation, body fat accumulation, serum hormone levels, and inflammatory factors/metabolites in rats maintained on high-fat (45% E fat) diets for 84 d. Increased protein levels resulted in a significant reduction in body fat mass and an increase in the serum levels of the anti-inflammatory cytokine IL-10, independent of protein source. Analysis of blood via untargeted metabolomics analysis identified eight, four, and four metabolites significantly altered by protein level, protein source, and a protein level-source interaction, respectively. Together, the effects of casein, chicken, and pork protein on the regulation of body fat accumulation and blood metabolite profile are largely dependent on protein level and less attributable to the protein source.

Estrogen receptor alpha (ERα)-mediated coregulator binding and gene expression discriminates the toxic ERα agonist diethylstilbestrol (DES) from the endogenous ERα agonist 17ß-estradiol (E2).

Diethylstilbestrol (DES) is a synthetic estrogen and proven human teratogen and carcinogen reported to act via the estrogen receptor α (ERα). Since the endogenous ERα ligand 17ß-estradiol (E2) does not show these adverse effects to a similar extent, we hypothesized that DES' interaction with the ERα differs from that of E2. The current study aimed to investigate possible differences between DES and E2 using in vitro assays that detect ERα-mediated effects, including ERα-mediated reporter gene expression, ERα-mediated breast cancer cell (T47D) proliferation and ERα-coregulator interactions and gene expression in T47D cells. Results obtained indicate that DES and E2 activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, significant differences between DES- and E2-induced binding of the ERα to 15 coregulator motifs and in transcriptomic signatures obtained in the T47D cells were observed. It is concluded that differences observed in binding of the ERα with several co-repressor motifs, in downregulation of genes involved in histone deacetylation and DNA methylation and in upregulation of CYP26A1 and CYP26B1 contribute to the differential effects reported for DES and E2.

A20 Promotes Ripoptosome Formation and TNF-Induced Apoptosis via cIAPs Regulation and NIK Stabilization in Keratinocytes.

The ubiquitin-editing protein A20 (TNFAIP3) is a known key player in the regulation of immune responses in many organs. Genome-wide associated studies (GWASs) have linked A20 with a number of inflammatory and autoimmune disorders, including psoriasis. Here, we identified a previously unrecognized role of A20 as a pro-apoptotic factor in TNF-induced cell death in keratinocytes. This function of A20 is mediated via the NF-κB-dependent alteration of cIAP1/2 expression. The changes in cIAP1/2 protein levels promote NIK stabilization and subsequent activation of noncanonical NF-κB signaling. Upregulation of TRAF1 expression triggered by the noncanonical NF-κB signaling further enhances the NIK stabilization in an autocrine manner. Finally, stabilized NIK promotes the formation of the ripoptosome and the execution of cell death. Thus, our data demonstrate that A20 controls the execution of TNF-induced cell death on multiple levels in keratinocytes. This signaling mechanism might have important implications for the development of new therapeutic strategies for the treatment of A20-associated skin diseases.