Evaluation of Full-Length Versus V4-Region 16S rRNA Sequencing for Phylogenetic Analysis of Mouse Intestinal Microbiota After a Dietary Intervention.

The composition of microbial communities is commonly determined by sequence analyses of one of the variable (V) regions in the bacterial 16S rRNA gene. We aimed to assess whether sequencing the full-length versus the V4 region of the 16S rRNA gene affected the results and interpretation of an experiment. To test this, mice were fed a diet without and with the prebiotic inulin and from cecum samples, two primary data sets were generated: (1) a 16S rRNA full-length data set generated by the PacBio platform; (2) a 16S rRNA V4 region data set generated by the Illumina MiSeq platform. A third derived data set was generated by in silico extracting the 16S rRNA V4 region data from the 16S rRNA full-length PacBio data set. Analyses of the primary and derived 16S rRNA V4 region data indicated similar bacterial abundances, and α- and ß-diversity. However, comparison of the 16S rRNA full-length data with the primary and derived 16S rRNA V4 region data revealed differences in relative bacterial abundances, and α- and ß-diversity. We conclude that the sequence length of 16S rRNA gene and not the sequence analysis platform affected the results and may lead to different interpretations of the effect of an intervention that affects the microbiota.

Bone marrow transplantation induces changes in the gut microbiota that chronically increase the cytokine response pattern of splenocytes.

Bone marrow transplantation (BMT) involves conditioning regimens which acutely induce side effects, including systemic inflammation, intestinal damage and shifts in the gut microbial composition, some of which may persist chronically. As the gut microbiota affect systemic immune responses, we aimed to investigate whether, post-BMT, the peripheral immune system is modulated as a direct consequence of alterations in the gut microbiota. We show that 24 weeks post-BMT, splenocytes but not peritoneal macrophages display increased cytokine response patterns upon ex-vivo stimulation with various pathogens as compared to untreated controls. The pattern of BMT-induced cytokine responses was transferred to splenocytes, and not to peritoneal macrophages, of healthy controls via co-housing and transferred to germfree mice via transplantation of cecum content. Thus, BMT induces changes in gut microbiota that in their turn increase cytokine responsiveness of splenocytes. Thus, BMT establishes a dominant microbiota that attenuates normalization of the immune-response.

Akkermansia muciniphila Exerts Lipid-Lowering and Immunomodulatory Effects without Affecting Neointima Formation in Hyperlipidemic APOE*3-Leiden.CETP Mice.

SCOPE: Akkermansia muciniphila (A. muciniphila) is an intestinal commensal with anti-inflammatory properties both in the intestine and other organs. The aim is to investigate the effects of oral administration of A. muciniphila on lipid metabolism, immunity, and cuff-induced neointima formation in hyperlipidemic APOE*3-Leiden (E3L).CETP mice. METHODS AND RESULTS: Hyperlipidemic male E3L.CETP mice are daily treated with 2 × 108 CFU A. muciniphila by oral gavage for 4 weeks and the effects are determined on plasma lipid levels, immune parameters, and cuff-induced neointima formation and composition. A. muciniphila administration lowers body weight and plasma total cholesterol and triglycerides levels. A. muciniphila influences the immune cell composition in mesenteric lymph nodes, as evident from an increased total B cell population, while reducing the total T cell and neutrophil populations. Importantly, A. muciniphila reduces the expression of the activation markers MHCII on dendritic cells and CD86 on B cells. A. muciniphila also increases whole blood ex vivo lipopolysaccharide-stimulated IL-10 release. Finally, although treatment with A. muciniphila improves lipid metabolism and immunity, it does not affect neointima formation or composition. CONCLUSIONS: Four weeks of treatment with A. muciniphila exerts lipid-lowering and immunomodulatory effects, which are insufficient to inhibit neointima formation in hyperlipidemic E3L.CETP mice.

The prebiotic inulin modulates gut microbiota but does not ameliorate atherosclerosis in hypercholesterolemic APOE*3-Leiden.CETP mice.

Gut microbiota have been implicated in the development of atherosclerosis and cardiovascular disease. Since the prebiotic inulin is thought to beneficially affect gut microbiota, we aimed to determine the effect of inulin supplementation on atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice. Female E3L.CETP mice were fed a western-type diet containing 0.1% or 0.5% cholesterol with or without 10% inulin. The effects of inulin were determined on: microbiota composition, cecal short-chain fatty acid (SCFA) levels, plasma lipid levels, atherosclerosis development, hepatic morphology and hepatic inflammation. Inulin with 0.5% dietary cholesterol increased specific bacterial genera and elevated levels of cecal SCFAs, but did not affect plasma cholesterol levels or atherosclerosis development. Surprisingly, inulin resulted in mild hepatic inflammation as shown by increased expression of inflammation markers. However, these effects were not accompanied by increased hepatic macrophage number. Analogously, inulin induced mild steatosis and increased hepatocyte size, but did not affect hepatic triglyceride content. Inulin with 0.1% dietary cholesterol did not affect hepatic morphology, nor hepatic expression of inflammation markers. Overall, inulin did not reduce hypercholesterolemia or atherosclerosis development in E3L.CETP mice despite showing clear prebiotic activity, but resulted in manifestations of hepatic inflammation when combined with a high percentage of dietary cholesterol.

Dietary Mannan Oligosaccharides Modulate Gut Microbiota, Increase Fecal Bile Acid Excretion, and Decrease Plasma Cholesterol and Atherosclerosis Development.

SCOPE: Mannan oligosaccharides (MOS) have proven effective at improving growth performance, while also reducing hyperlipidemia and inflammation. As atherosclerosis is accelerated both by hyperlipidemia and inflammation, we aim to determine the effect of dietary MOS on atherosclerosis development in hyperlipidemic ApoE*3-Leiden.CETP (E3L.CETP) mice, a well-established model for human-like lipoprotein metabolism. METHODS AND RESULTS: Female E3L.CETP mice were fed a high-cholesterol diet, with or without 1% MOS for 14 weeks. MOS substantially decreased atherosclerotic lesions up to 54%, as assessed in the valve area of the aortic root. In blood, IL-1RA, monocyte subtypes, lipids, and bile acids (BAs) were not affected by MOS. Gut microbiota composition was determined using 16S rRNA gene sequencing and MOS increased the abundance of cecal Bacteroides ovatus. MOS did not affect fecal excretion of cholesterol, but increased fecal BAs as well as butyrate in cecum as determined by gas chromatography mass spectrometry. CONCLUSION: MOS decreased the onset of atherosclerosis development via lowering of plasma cholesterol levels. These effects were accompanied by increased cecal butyrate and fecal excretion of BAs, presumably mediated via interactions of MOS with the gut microbiota.

Loss of angiopoietin-like 4 (ANGPTL4) in mice with diet-induced obesity uncouples visceral obesity from glucose intolerance partly via the gut microbiota.

AIMS/HYPOTHESIS: Angiopoietin-like 4 (ANGPTL4) is an important regulator of triacylglycerol metabolism, carrying out this role by inhibiting the enzymes lipoprotein lipase and pancreatic lipase. ANGPTL4 is a potential target for ameliorating cardiometabolic diseases. Although ANGPTL4 has been implicated in obesity, the study of the direct role of ANGPTL4 in diet-induced obesity and related metabolic dysfunction is hampered by the massive acute-phase response and development of lethal chylous ascites and peritonitis in Angptl4-/- mice fed a standard high-fat diet. The aim of this study was to better characterise the role of ANGPTL4 in glucose homeostasis and metabolic dysfunction during obesity. METHODS: We chronically fed wild-type (WT) and Angptl4-/- mice a diet rich in unsaturated fatty acids and cholesterol, combined with fructose in drinking water, and studied metabolic function. The role of the gut microbiota was investigated by orally administering a mixture of antibiotics (ampicillin, neomycin, metronidazole). Glucose homeostasis was assessed via i.p. glucose and insulin tolerance tests. RESULTS: Mice lacking ANGPTL4 displayed an increase in body weight gain, visceral adipose tissue mass, visceral adipose tissue lipoprotein lipase activity and visceral adipose tissue inflammation compared with WT mice. However, they also unexpectedly had markedly improved glucose tolerance, which was accompanied by elevated insulin levels. Loss of ANGPTL4 did not affect glucose-stimulated insulin secretion in isolated pancreatic islets. Since the gut microbiota have been suggested to influence insulin secretion, and because ANGPTL4 has been proposed to link the gut microbiota to host metabolism, we hypothesised a potential role of the gut microbiota. Gut microbiota composition was significantly different between Angptl4-/- mice and WT mice. Interestingly, suppression of the gut microbiota using antibiotics largely abolished the differences in glucose tolerance and insulin levels between WT and Angptl4-/- mice. CONCLUSIONS/INTERPRETATION: Despite increasing visceral fat mass, inactivation of ANGPTL4 improves glucose tolerance, at least partly via a gut microbiota-dependent mechanism.

The Prebiotic Inulin Aggravates Accelerated Atherosclerosis in Hypercholesterolemic APOE*3-Leiden Mice.

The prebiotic inulin has proven effective at lowering inflammation and plasma lipid levels. As atherosclerosis is provoked by both inflammation and hyperlipidemia, we aimed to determine the effect of inulin supplementation on atherosclerosis development in hypercholesterolemic APOE*3-Leiden (E3L) mice. Male E3L mice were fed a high-cholesterol (1%) diet, supplemented with or without 10% inulin for 5 weeks. At week 3, a non-constrictive cuff was placed around the right femoral artery to induce accelerated atherosclerosis. At week 5, vascular pathology was determined by lesion thickness, vascular remodeling, and lesion composition. Throughout the study, plasma lipids were measured and in week 5, blood monocyte subtypes were determined using flow cytometry analysis. In contrast to our hypothesis, inulin exacerbated atherosclerosis development, characterized by increased lesion formation and outward vascular remodeling. The lesions showed increased number of macrophages, smooth muscle cells, and collagen content. No effects on blood monocyte composition were found. Inulin significantly increased plasma total cholesterol levels and total cholesterol exposure. In conclusion, inulin aggravated accelerated atherosclerosis development in hypercholesterolemic E3L mice, accompanied by adverse lesion composition and outward remodeling. This process was not accompanied by differences in blood monocyte composition, suggesting that the aggravated atherosclerosis development was driven by increased plasma cholesterol.

Butyrate reduces appetite and activates brown adipose tissue via the gut-brain neural circuit.

OBJECTIVE: Butyrate exerts metabolic benefits in mice and humans, the underlying mechanisms being still unclear. We aimed to investigate the effect of butyrate on appetite and energy expenditure, and to what extent these two components contribute to the beneficial metabolic effects of butyrate. DESIGN: Acute effects of butyrate on appetite and its method of action were investigated in mice following an intragastric gavage or intravenous injection of butyrate. To study the contribution of satiety to the metabolic benefits of butyrate, mice were fed a high-fat diet with butyrate, and an additional pair-fed group was included. Mechanistic involvement of the gut-brain neural circuit was investigated in vagotomised mice. RESULTS: Acute oral, but not intravenous, butyrate administration decreased food intake, suppressed the activity of orexigenic neurons that express neuropeptide Y in the hypothalamus, and decreased neuronal activity within the nucleus tractus solitarius and dorsal vagal complex in the brainstem. Chronic butyrate supplementation prevented diet-induced obesity, hyperinsulinaemia, hypertriglyceridaemia and hepatic steatosis, largely attributed to a reduction in food intake. Butyrate also modestly promoted fat oxidation and activated brown adipose tissue (BAT), evident from increased utilisation of plasma triglyceride-derived fatty acids. This effect was not due to the reduced food intake, but explained by an increased sympathetic outflow to BAT. Subdiaphragmatic vagotomy abolished the effects of butyrate on food intake as well as the stimulation of metabolic activity in BAT. CONCLUSION: Butyrate acts on the gut-brain neural circuit to improve energy metabolism via reducing energy intake and enhancing fat oxidation by activating BAT.

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.

BMT decreases HFD-induced weight gain associated with decreased preadipocyte number and insulin secretion.

Experimental bone marrow transplantation (BMT) in mice is commonly used to assess the role of immune cell-specific genes in various pathophysiological settings. The application of BMT in obesity research is hampered by the significant reduction in high-fat diet (HFD)-induced obesity. We set out to characterize metabolic tissues that may be affected by the BMT procedure and impair the HFD-induced response. Male C57BL/6 mice underwent syngeneic BMT using lethal irradiation. After a recovery period of 8 weeks they were fed a low-fat diet (LFD) or HFD for 16 weeks. HFD-induced obesity was reduced in mice after BMT as compared to HFD-fed control mice, characterized by both a reduced fat (-33%; p<0.01) and lean (-11%; p<0.01) mass, while food intake and energy expenditure were unaffected. As compared to control mice, BMT-treated mice had a reduced mature adipocyte volume (approx. -45%; p<0.05) and reduced numbers of preadipocytes (-38%; p<0.05) and macrophages (-62%; p<0.05) in subcutaneous, gonadal and visceral white adipose tissue. In BMT-treated mice, pancreas weight (-46%; p<0.01) was disproportionally decreased. This was associated with reduced plasma insulin (-68%; p<0.05) and C-peptide (-37%; p<0.01) levels and a delayed glucose clearance in BMT-treated mice on HFD as compared to control mice. In conclusion, the reduction in HFD-induced obesity after BMT in mice is at least partly due to alterations in the adipose tissue cell pool composition as well as to a decreased pancreatic secretion of the anabolic hormone insulin. These effects should be considered when interpreting results of experimental BMT in metabolic studies.

FcRγ-chain deficiency reduces the development of diet-induced obesity.

OBJECTIVE: Pathogenic immunoglobulins are produced during the development of obesity and contribute to the development of insulin resistance (IR). However, the mechanisms by which these antibodies affect IR are largely unknown. This study investigated whether Fc-receptors contribute to the development of diet-induced obesity and IR by studying FcRγ(-/-) mice that lack the γ-subunit necessary for signaling and cell surface expression of FcγR and FcεRI. METHODS: FcRγ(-/-) and wild-type (WT) mice were fed a high-fat diet (HFD) to induce obesity. At 4 and 11 weeks, body weight and insulin sensitivity were measured, and adipose tissue (AT) inflammation was determined. Furthermore, intestinal triglyceride (TG) uptake and plasma TG clearance were determined, and gut microbiota composition was analyzed. RESULTS: FcRγ(-/-) mice gained less weight after 11 weeks of HFD. They had reduced adiposity, adipose tissue inflammation, and IR. Interestingly, FcRγ(-/-) mice had higher lean mass compared to WT mice, which was associated with increased energy expenditure. Intestinal TG absorption was increased whereas plasma TG clearance was not affected in FcRγ(-/-) mice. Gut microbial composition differed significantly and might therefore have added to the observed phenotype. CONCLUSIONS: FcRγ-chain deficiency reduces the development of diet-induced obesity, as well as associated AT inflammation and IR at 11 weeks of HFD.