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.

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.

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.

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.

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.

The whole transcriptome effects of the PPARα agonist fenofibrate on livers of hepatocyte humanized mice.

BACKGROUND: The role of PPARα in gene regulation in mouse liver is well characterized. However, less is known about the role of PPARα in human liver. The aim of the present study was to better characterize the impact of PPARα activation on gene regulation in human liver. To that end, chimeric mice containing hepatocyte humanized livers were given an oral dose of 300 mg/kg fenofibrate daily for 4 days. Livers were collected and analyzed by hematoxilin and eosin staining, qPCR, and transcriptomics. Transcriptomics data were compared with existing datasets on PPARα activation in normal mouse liver, human primary hepatocytes, and human precision cut liver slices. RESULTS: Of the different human liver models, the gene expression profile of hepatocyte humanized livers most closely resembled actual human liver. In the hepatocyte humanized mouse livers, the human hepatocytes exhibited excessive lipid accumulation. Fenofibrate increased the size of the mouse but not human hepatocytes, and tended to reduce steatosis in the human hepatocytes. Quantitative PCR indicated that induction of PPARα targets by fenofibrate was less pronounced in the human hepatocytes than in the residual mouse hepatocytes. Transcriptomics analysis indicated that, after filtering, a total of 282 genes was significantly different between fenofibrate- and control-treated mice (P < 0.01). 123 genes were significantly lower and 159 genes significantly higher in the fenofibrate-treated mice, including many established PPARα targets such as FABP1, HADHB, HADHA, VNN1, PLIN2, ACADVL and HMGCS2. According to gene set enrichment analysis, fenofibrate upregulated interferon/cytokine signaling-related pathways in hepatocyte humanized liver, but downregulated these pathways in normal mouse liver. Also, fenofibrate downregulated pathways related to DNA synthesis in hepatocyte humanized liver but not in normal mouse liver. CONCLUSION: The results support the major role of PPARα in regulating hepatic lipid metabolism, and underscore the more modest effect of PPARα activation on gene regulation in human liver compared to mouse liver. The data suggest that PPARα may have a suppressive effect on DNA synthesis in human liver, and a stimulatory effect on interferon/cytokine signalling.

Modulation of the gut microbiota impacts nonalcoholic fatty liver disease: a potential role for bile acids.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids.

Characterization of stem cell-derived liver and intestinal organoids as a model system to study nuclear receptor biology.

Nuclear receptors (NRs) are ligand-activated transcription factors regulating a large variety of processes involved in reproduction, development, and metabolism. NRs are ideal drug targets because they are activated by lipophilic ligands that easily pass cell membranes. Immortalized cell lines recapitulate NR biology poorly and generating primary cultures is laborious and requires a constant need for donor material. There is a clear need for development of novel preclinical model systems that better resemble human physiology. Uncertainty due to technical limitations early in drug development is often the cause of preclinical drugs not reaching the clinic. Here, we studied whether organoids, mini-organs derived from the respective mouse tissue's stem cells, can serve as a novel model system to study NR biology and targetability. We characterized mRNA expression profiles of the NR superfamily in mouse liver, ileum, and colon organoids. Tissue-specific expression patterns were largely maintained in the organoids, indicating their suitability for NR research. Metabolic NRs Fxrα, Lxrα, Lxrß, Pparα, and Pparγ induced expression of and binding to endogenous target genes. Transcriptome analyses of wildtype colon organoids stimulated with Rosiglitazone showed that lipid metabolism was the highest significant changed function, greatly mimicking the PPARs and Rosiglitazone function in vivo. Finally, using organoids we identify Trpm6, Slc26a3, Ang1, and Rnase4, as novel Fxr target genes. Our results demonstrate that organoids represent a framework to study NR biology that can be further expanded to human organoids to improve preclinical testing of novel drugs that target this pharmacologically important class of ligand activated transcription factors.

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.

Nonlinear transcriptomic response to dietary fat intake in the small intestine of C57BL/6J mice.

BACKGROUND: A high caloric diet, in conjunction with low levels of physical activity, promotes obesity. Many studies are available regarding the relation between dietary saturated fats and the etiology of obesity, but most focus on liver, muscle and white adipose tissue. Furthermore, the majority of transcriptomic studies seek to identify linear effects of an external stimulus on gene expression, although such an assumption does not necessarily hold. Our work assesses the dose-dependent effects of dietary fat intake on differential gene expression in the proximal, middle and distal sections of the small intestine in C57BL/6J mice. Gene expression is analyzed in terms of either linear or nonlinear responses to fat intake. RESULTS: The highest number of differentially expressed genes was observed in the middle section. In all intestine sections, most of the identified processes exhibited a linear response to increasing fat intake. The relative importance of logarithmic and exponential responses was higher in the proximal and distal sections, respectively. Functional enrichment analysis highlighted a constantly linear regulation of acute-phase response along the whole small intestine, with up-regulation of Serpina1b. The study of gene expression showed that exponential down-regulation of cholesterol transport in the middle section is coupled with logarithmic up-regulation of cholesterol homeostasis. A shift from linear to exponential response was observed in genes involved in the negative regulation of caspase activity, from middle to distal section (e.g., Birc5, up-regulated). CONCLUSIONS: The transcriptomic signature associated with inflammatory processes preserved a linear response in the whole small intestine (e.g., up-regulation of Serpina1b). Processes related to cholesterol homeostasis were particularly active in the middle small intestine and only the highest fat intake down-regulated cholesterol transport and efflux (with a key role played by the down-regulation of ATP binding cassette transporters). Characterization of nonlinear patterns of gene expression triggered by different levels of dietary fat is an absolute novelty in intestinal studies. This approach helps identifying which processes are overloaded (i.e., positive, logarithmic regulation) or arrested (i.e., negative, exponential regulation) in response to excessive fat intake, and can shed light on the relationships linking lipid intake to obesity and its associated molecular disturbances.

Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2.

Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

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.

Resistant starch induces catabolic but suppresses immune and cell division pathways and changes the microbiome in the proximal colon of male pigs.

Consumption of resistant starch (RS) has been associated with various intestinal health benefits, but knowledge of its effects on global gene expression in the colon is limited. The main objective of the current study was to identify genes affected by RS in the proximal colon to infer which biologic pathways were modulated. Ten 17-wk-old male pigs, fitted with a cannula in the proximal colon for repeated collection of tissue biopsy samples and luminal content, were fed a digestible starch (DS) diet or a diet high in RS (34%) for 2 consecutive periods of 14 d in a crossover design. Analysis of the colonic transcriptome profiles revealed that, upon RS feeding, oxidative metabolic pathways, such as the tricarboxylic acid cycle and ß-oxidation, were induced, whereas many immune response pathways, including adaptive and innate immune system, as well as cell division were suppressed. The nuclear receptor peroxisome proliferator-activated receptor γ was identified as a potential key upstream regulator. RS significantly (P < 0.05) increased the relative abundance of several butyrate-producing microbial groups, including the butyrate producers Faecalibacterium prausnitzii and Megasphaera elsdenii, and reduced the abundance of potentially pathogenic members of the genus Leptospira and the phylum Proteobacteria. Concentrations in carotid plasma of the 3 main short-chain fatty acids acetate, propionate, and butyrate were significantly higher with RS consumption compared with DS consumption. Overall, this study provides novel insights on effects of RS in proximal colon and contributes to our understanding of a healthy diet.

A diet high in resistant starch modulates microbiota composition, SCFA concentrations, and gene expression in pig intestine.

Resistant starch (RS) is highly fermentable by microbiota in the colon, resulting in the production of SCFAs. RS is thought to mediate a large proportion of its health benefits, including increased satiety, through the actions of SCFAs. The aim of this study was to investigate the effects of a diet high in RS on luminal microbiota composition, luminal SCFA concentrations, and the expression of host genes involved in SCFA uptake, SCFA signaling, and satiety regulation in mucosal tissue obtained from small intestine, cecum, and colon. Twenty adult female pigs were either assigned to a digestible starch (DS) diet or a diet high in RS (34%) for a period of 2 wk. After the intervention, luminal content and mucosal scrapings were obtained for detailed molecular analysis. RS was completely degraded in the cecum. In both the cecum and colon, differences in microbiota composition were observed between DS- and RS-fed pigs. In the colon these included the stimulation of the healthy gut-associated butyrate-producing Faecalibacterium prausnitzii, whereas potentially pathogenic members of the Gammaproteobacteria, including Escherichia coli and Pseudomonas spp., were reduced in relative abundance. Cecal and colonic SCFA concentrations were significantly greater in RS-fed pigs, and cecal gene expression of monocarboxylate transporter 1 (SLC16A1) and glucagon (GCG) was induced by RS. In conclusion, our data show that RS modulates microbiota composition, SCFA concentrations, and host gene expression in pig intestine. Combined, our data provide an enhanced understanding of the interaction between diet, microbiota, and host.

Differential NF-kappaB pathways induction by Lactobacillus plantarum in the duodenum of healthy humans correlating with immune tolerance.

How do we acquire immune tolerance against food microorganisms and commensal bacteria that constitute the intestinal microbiota? We investigated this by stimulating the immune system of adults with commensal Lactobacillus plantarum bacteria. We studied the in vivo human responses to L. plantarum in a randomized double-blind placebo-controlled cross-over study. Healthy adults ingested preparations of living and heat-killed L. plantarum bacteria. Biopsies were taken from the intestinal duodenal mucosa and altered expression profiles were analyzed using whole-genome microarrays and by biological pathway reconstructions. Expression profiles of human mucosa displayed striking differences in modulation of NF-kappaB-dependent pathways, notably after consumption of living L. plantarum bacteria in different growth phases. Our in vivo study identified mucosal gene expression patterns and cellular pathways that correlated with the establishment of immune tolerance in healthy adults.

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.

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.

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.