Intestinal permeability and peripheral immune cell composition are altered by pregnancy and adiposity at mid- and late-gestation in the mouse.

It is clear that the gastrointestinal tract influences metabolism and immune function. Most studies to date have used male test subjects, with a focus on effects of obesity and dietary challenges. Despite significant physiological maternal adaptations that occur across gestation, relatively few studies have examined pregnancy-related gut function. Moreover, it remains unknown how pregnancy and diet can interact to alter intestinal barrier function. In this study, we investigated the impacts of pregnancy and adiposity on maternal intestinal epithelium morphology, in vivo intestinal permeability, and peripheral blood immunophenotype, using control (CTL) and high-fat (HF) fed non-pregnant female mice and pregnant mice at mid- (embryonic day (E)14.5) and late (E18.5) gestation. We found that small intestine length increased between non-pregnant mice and dams at late-gestation, but ileum villus length, and ileum and colon crypt depths and goblet cell numbers remained similar. Compared to CTL-fed mice, HF-fed mice had reduced small intestine length, ileum crypt depth and villus length. Goblet cell numbers were only consistently reduced in HF-fed non-pregnant mice. Pregnancy increased in vivo gut permeability, with a greater effect at mid- versus late-gestation. Non-pregnant HF-fed mice had greater gut permeability, and permeability was also increased in HF-fed pregnant dams at mid but not late-gestation. The impaired maternal gut barrier in HF-fed dams at mid-gestation coincided with changes in maternal blood and bone marrow immune cell composition, including an expansion of circulating inflammatory Ly6Chigh monocytes. In summary, pregnancy has temporal effects on maternal intestinal structure and barrier function, and on peripheral immunophenotype, which are further modified by HF diet-induced maternal adiposity. Maternal adaptations in pregnancy are thus vulnerable to excess maternal adiposity, which may both affect maternal and child health.

Gnotobiotic mice housing conditions critically influence the phenotype associated with transfer of faecal microbiota in a context of obesity.

OBJECTIVE: Faecal microbiota transplantation (FMT) in germ-free (GF) mice is a common approach to study the causal role of the gut microbiota in metabolic diseases. Lack of consideration of housing conditions post-FMT may contribute to study heterogeneity. We compared the impact of two housing strategies on the metabolic outcomes of GF mice colonised by gut microbiota from mice treated with a known gut modulator (cranberry proanthocyanidins (PAC)) or vehicle. DESIGN: High-fat high-sucrose diet-fed GF mice underwent FMT-PAC colonisation in sterile individual positive flow ventilated cages under rigorous housing conditions and then maintained for 8 weeks either in the gnotobiotic-axenic sector or in the specific pathogen free (SPF) sector of the same animal facility. RESULTS: Unexpectedly, 8 weeks after colonisation, we observed opposing liver phenotypes dependent on the housing environment of mice. Mice housed in the GF sector receiving the PAC gut microbiota showed a significant decrease in liver weight and hepatic triglyceride accumulation compared with control group. Conversely, exacerbated liver steatosis was observed in the FMT-PAC mice housed in the SPF sector. These phenotypic differences were associated with housing-specific profiles of colonising bacterial in the gut and of faecal metabolites. CONCLUSION: These results suggest that the housing environment in which gnotobiotic mice are maintained post-FMT strongly influences gut microbiota composition and function and can lead to distinctive phenotypes in recipient mice. Better standardisation of FMT experiments is needed to ensure reproducible and translatable results.

Human gut microbiota after bariatric surgery alters intestinal morphology and glucose absorption in mice independently of obesity.

OBJECTIVE: Bariatric surgery is an effective treatment for type 2 diabetes (T2D) that changes gut microbial composition. We determined whether the gut microbiota in humans after restrictive or malabsorptive bariatric surgery was sufficient to lower blood glucose. DESIGN: Women with obesity and T2D had biliopancreatic diversion with duodenal switch (BPD-DS) or laparoscopic sleeve gastrectomy (LSG). Faecal samples from the same patient before and after each surgery were used to colonise rodents, and determinants of blood glucose control were assessed. RESULTS: Glucose tolerance was improved in germ-free mice orally colonised for 7 weeks with human microbiota after either BPD-DS or LSG, whereas food intake, fat mass, insulin resistance, secretion and clearance were unchanged. Mice colonised with microbiota post-BPD-DS had lower villus height/width and crypt depth in the distal jejunum and lower intestinal glucose absorption. Inhibition of sodium-glucose cotransporter (Sglt)1 abrogated microbiota-transmissible improvements in blood glucose control in mice. In specific pathogen-free (SPF) rats, intrajejunal colonisation for 4 weeks with microbiota post-BPD-DS was sufficient to improve blood glucose control, which was negated after intrajejunal Sglt-1 inhibition. Higher Parabacteroides and lower Blautia coincided with improvements in blood glucose control after colonisation with human bacteria post-BPD-DS and LSG. CONCLUSION: Exposure of rodents to human gut microbiota after restrictive or malabsorptive bariatric surgery improves glycaemic control. The gut microbiota after bariatric surgery is a standalone factor that alters upper gut intestinal morphology and lowers Sglt1-mediated intestinal glucose absorption, which improves blood glucose control independently from changes in obesity, insulin or insulin resistance.

Dietary sugar lowers immunity and microbiota that protect against metabolic disease.

Diet influences intestinal microbiota, inflammation, and metabolism. Kawano et al. show that dietary sugar engaged upper gut innate lymphoid cells to replace segmented filamentous bacteria with a pathobiont. Added sugar worsened early metabolic disease by lowering protective Th17 immunity, thereby promoting intestinal lipid absorption and obesity in high-fat-diet-fed mice.

Life-long exercise training and inherited aerobic endurance capacity produce converging gut microbiome signatures in rodents.

High aerobic endurance capacity can be acquired by training and/or inherited. Aerobic exercise training (AET) and aging are linked to altered gut microbiome composition, but it is unknown if the environmental stress of exercise and host genetics that predispose for higher exercise capacity have similar effects on the gut microbiome during aging. We hypothesized that exercise training and host genetics would have conserved effects on the gut microbiome across different rodents. We studied young sedentary (Y-SED, 2-month-old) mice, old sedentary (O-SED, 26-month-old) mice, old mice with life-long AET (O-AET, 26-month-old), and aged rats selectively bred for high (HCR [High Capacity Runner], 21-month-old) and low (LCR [Low Capacity Runner], 21-month-old) aerobic capacity. Our results showed that O-SED mice had lower running capacity than Y-SED mice. The fecal microbiota of O-SED mice had a higher relative abundance of Lachnospiraceae, Ruminococcaceae, Turicibacteriaceae, and Allobaculum, but lower Bacteroidales, Alistipes, Akkermansia, and Anaeroplasma. O-AET mice had a higher running capacity than O-SED mice. O-AET mice had lower fecal levels of Lachnospiraceae, Turicibacteriaceae, and Allobaculum and higher Anaeroplasma than O-SED mice. Similar to O-AET mice, but despite no exercise training regime, aged HCR rats had lower Lachnospiraceae and Ruminococcaceae and expansion of certain Bacteroidales in the fecal microbiome compared to LCR rats. Our data show that environmental and genetic modifiers of high aerobic endurance capacity produce convergent gut microbiome signatures across different rodent species during aging. Therefore, we conclude that host genetics and life-long exercise influence the composition of the gut microbiome and can mitigate gut dysbiosis and functional decline during aging.

Determining the metabolic impact of postbiotics in mice.

Postbiotics cooperate to influence immune and metabolic outcomes in the host. Here we describe a protocol for in vivo assessment of blood glucose control following acute administration of lipopolysaccharide (LPS) and peptidoglycan (PGN) in mice. This protocol can be adapted for testing a broad range of microbial molecules and ligands for host immune receptors. Experience with mouse handling is required. For complete details on the use and execution of this protocol, please refer to Anhê et al. (2021) and Cavallari et al. (2017).

Consistent gut bacterial and short-chain fatty acid signatures in hypoabsorptive bariatric surgeries correlate with metabolic benefits in rats.

OBJECTIVE: The study aimed at comparing how changes in the gut microbiota are associated to the beneficial effects of the most clinically efficient hypoabsorptive bariatric procedures, namely Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion with duodenal switch (BPD-DS) and single anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S). METHODS: Diet-induced obese (DIO) male Wistar rats were divided into seven groups. In addition to the groups subjected to RYGB, BPD-DS and SADI-S, the following four control groups were included: SHAM-operated rats fed a high-fat diet (SHAM HF), SHAM fed a low-fat diet (SHAM LF), SHAM HF-pair-weighed to BPD-DS (SHAM HF-PW) and sleeve-gastrectomy (SG) rats. Body weight, food intake, glucose tolerance, insulin sensitivity/resistance, and L-cell secretion were assessed. The gut microbiota (16 S ribosomal RNA gene sequencing) as well as the fecal and cæcal contents of short-chain fatty acids (SCFAs) were also analyzed prior to, and after the surgeries. RESULTS: The present study demonstrates the beneficial effect of RYGB, BPD-DS and SADI-S on fat mass gain and glucose metabolism in DIO rats. These benefits were proportional to the effect of the surgeries on food digestibility (BPD-DS > SADI-S > RYGB). Notably, hypoabsorptive surgeries led to consonant microbial signatures characterized by decreased abundance of the Ruminococcaceae (Oscillospira and Ruminococcus), Oscillospiraceae (Oscillibacter) and Christensenellaceae, and increased abundance of the Clostridiaceae (Clostridium), Sutterellaceae (Sutterella) and Enterobacteriaceae. The gut bacteria following hypoabsorptive surgeries were associated with higher fecal levels of propionate, butyrate, isobutyrate and isovalerate. Increases in the fecal SCFAs were in turn positively and strongly correlated with the levels of peptide tyrosine-tyrosine (PYY) and with the beneficial effects of the surgery. CONCLUSION: The present study emphasizes the consistency with which the three major hypoabsorptive bariatric procedures RYGB, BPD-DS and SADI-S create a gut microbial environment capable of producing a SCFA profile favorable to the secretion of PYY and to beneficial metabolic effects.

Gut microbiota-based vaccination engages innate immunity to improve blood glucose control in obese mice.

OBJECTIVE: Obesity and diabetes increase circulating levels of microbial components derived from the gut microbiota. Individual bacterial factors (i.e., postbiotics) can have opposing effects on blood glucose. METHODS: We tested the net effect of gut bacterial extracts on blood glucose in mice using a microbiota-based vaccination strategy. RESULTS: Male and female mice had improved glucose and insulin tolerance five weeks after a single subcutaneous injection of a specific dose of a bacterial extract obtained from the luminal contents of the upper small intestine (SI), lower SI, or cecum. Injection of mice with intestinal extracts from germ-free mice revealed that bacteria were required for a microbiota-based vaccination to improve blood glucose control. Vaccination of Nod1-/-, Nod2-/-, and Ripk2-/- mice showed that each of these innate immune proteins was required for bacterial extract injection to improve blood glucose control. A microbiota-based vaccination promoted an immunoglobulin-G (IgG) response directed against bacterial extract antigens, where subcutaneous injection of mice with the luminal contents of the lower SI elicited a bacterial extract-specific IgG response that is compartmentalized to the lower SI of vaccinated mice. A microbiota-based vaccination was associated with an altered microbiota composition in the lower SI and colon of mice. Lean mice only required a single injection of small intestinal-derived bacterial extract, but high fat diet (HFD)-fed, obese mice required prime-boost bacterial extract injections for improvements in blood glucose control. CONCLUSIONS: Subversion of the gut barrier by vaccination with a microbiota-based extract engages innate immunity to promote long-lasting improvements in blood glucose control in a dose-dependent manner.

Lower brown adipose tissue activity is associated with non-alcoholic fatty liver disease but not changes in the gut microbiota.

In rodents, lower brown adipose tissue (BAT) activity is associated with greater liver steatosis and changes in the gut microbiome. However, little is known about these relationships in humans. In adults (n = 60), we assessed hepatic fat and cold-stimulated BAT activity using magnetic resonance imaging and the gut microbiota with 16S sequencing. We transplanted gnotobiotic mice with feces from humans to assess the transferability of BAT activity through the microbiota. Individuals with NAFLD (n = 29) have lower BAT activity than those without, and BAT activity is inversely related to hepatic fat content. BAT activity is not related to the characteristics of the fecal microbiota and is not transmissible through fecal transplantation to mice. Thus, low BAT activity is associated with higher hepatic fat accumulation in human adults, but this does not appear to have been mediated through the gut microbiota.

Metabolic endotoxemia is dictated by the type of lipopolysaccharide.

Lipopolysaccharides (LPSs) can promote metabolic endotoxemia, which is considered inflammatory and metabolically detrimental based on Toll-like receptor (TLR)4 agonists, such as Escherichia coli-derived LPS. LPSs from certain bacteria antagonize TLR4 yet contribute to endotoxemia measured by endotoxin units (EUs). We found that E. coli LPS impairs gut barrier function and worsens glycemic control in mice, but equal doses of LPSs from other bacteria do not. Matching the LPS dose from R. sphaeroides and E. coli by EUs reveals that only E. coli LPS promotes dysglycemia and adipose inflammation, delays intestinal glucose absorption, and augments insulin and glucagon-like peptide (GLP)-1 secretion. Metabolically beneficial endotoxemia promoted by R. sphaeroides LPS counteracts dysglycemia caused by an equal dose of E. coli LPS and improves glucose control in obese mice. The concept of metabolic endotoxemia should be expanded beyond LPS load to include LPS characteristics, such as lipid A acylation, which dictates the effect of metabolic endotoxemia.

Bacterial Postbiotics as Promising Tools to Mitigate Cardiometabolic Diseases.

Gut microbes dictate critical features of host immunometabolism. Certain bacterial components and metabolites (termed postbiotics) mitigate cardiometabolic diseases whereas others potentiate pathological processes. In this review, we discuss key aspects related to the usefulness of bacterial-related molecules strategically positioned as promising treatment strategies for cardiometabolic diseases.

Micronutrients impact the gut microbiota and blood glucose.

Micronutrients influence hormone action and host metabolism. Dietary minerals, trace elements, and vitamins can alter blood glucose and cellular glucose metabolism, and several micronutrients are associated with the risk and progression of type 2 diabetes. Dietary components, microbes, and host immune, endocrine, and metabolic responses all interact in the intestine. There has been a focus on macronutrients modifying the host-microbe relationship in metabolic disease. Micronutrients are positioned to alter host-microbe symbiosis that participates in host endocrine control of glucose metabolism. Minerals and trace elements can alter the composition of the intestinal microbiota, gut barrier function, compartmentalized metabolic inflammation, cellular glucose transport, and endocrine control of glucose metabolism, including insulin and thyroid hormones. Dietary vitamins also influence the composition of the intestinal microbiota and vitamins can be biotransformed by gut microbes. Host-microbe regulation of vitamins can alter immunity, lipid and glucose metabolism, and cell fate and function of pancreatic beta cells. Causal effects of micronutrients in host-microbe metabolism are still emerging, and the mechanisms linking dietary excess or deficiency of specific micronutrients to changes in gut microbes directly linked to metabolic disease risk are not yet clear. Dietary fiber, fat, protein, and carbohydrates are key dietary factors that impact how microbes participate in host glucose metabolism. It is possible that micronutrient and microbiota-derived factors also participate in host-microbe responses that tip the balance in the endocrine control of host glucose metabolism. Dietary micronutrients should be considered, tested, and controlled in pre-clinical and clinical studies investigating host-microbe factors in metabolic diseases.

Gut microbiota impairs insulin clearance in obese mice.

OBJECTIVE: Hyperinsulinemia can be both a cause and consequence of obesity and insulin resistance. Hyperinsulinemia can result from increased insulin secretion and/or reduced insulin clearance. While many studies have focused on mechanisms triggering insulin secretion during obesity, the triggers for changes in insulin clearance during obesity are less defined. In this study, we investigated the role of the microbiota in regulating insulin clearance during diet-induced obesity. METHODS: Blood glucose and insulin clearance were tested in conventional male mice treated with antibiotics and germ-free mice colonized with microbes from mice that were fed a control (chow) diet or an obesogenic high-fat diet (HFD). The composition of the fecal microbiota was analyzed using 16S rRNA sequencing. RESULTS: Short-term HFD feeding and aging did not alter insulin clearance in the mice. Oral antibiotics mitigated impaired blood insulin clearance in the mice fed an HFD for 12 weeks or longer. Germ-free mice colonized with microbes from HFD-fed donor mice had impaired insulin but not C-peptide clearance. Microbe-transmissible insulin clearance impairment was only observed in germ-free mice after more than 6 weeks post-colonization upon HFD feeding. Five bacterial taxa predicted >90% of the variance in insulin clearance. Mechanistically, impaired insulin clearance was associated with lower levels of hepatic Ceacam-1 but increased liver and skeletal muscle insulin-degrading enzyme (IDE) activity. CONCLUSIONS: Gut microbes regulate insulin clearance during diet-induced obesity. A small cluster of microbes or their metabolites may be targeted for mitigating defects in insulin clearance and hyperinsulinemia during the progression of obesity and type 2 diabetes.

Type 2 diabetes influences bacterial tissue compartmentalisation in human obesity.

Visceral obesity is a key risk factor for type 2 diabetes (T2D). Whereas gut dysbiosis appears to be instrumental for this relationship, whether gut-associated signatures translocate to extra-intestinal tissues and how this affects host metabolism remain elusive. Here we provide a comparative analysis of the microbial profile found in plasma, liver and in three distinct adipose tissues of individuals with morbid obesity. We explored how these tissue microbial signatures vary between individuals with normoglycaemia and those with T2D that were matched for body mass index. We identified tissue-specific signatures with higher bacterial load in the liver and omental adipose tissue. Gut commensals, but also environmental bacteria, showed tissue- and T2D-specific compartmentalisation. T2D signatures were most evident in mesenteric adipose tissue, in which individuals with diabetes displayed reduced bacterial diversity concomitant with fewer Gram-positive bacteria, such as Faecalibacterium, as opposed to enhanced levels of typically opportunistic Gram-negative Enterobacteriaceae. Plasma samples of individuals with diabetes were similarly enriched in Enterobacteriaceae, including the pathobiont Escherichia-Shigella. Our work provides evidence for the presence of selective plasma and tissue microbial signatures in individuals with severe obesity and identifies new potential microbial targets and biomarkers of T2D.

The NLRP3 inflammasome regulates adipose tissue metabolism.

Adipose tissue regulates metabolic homeostasis by participating in endocrine and immune responses in addition to storing and releasing lipids from adipocytes. Obesity skews adipose tissue adipokine responses and degrades the coordination of adipocyte lipogenesis and lipolysis. These defects in adipose tissue metabolism can promote ectopic lipid deposition and inflammation in insulin-sensitive tissues such as skeletal muscle and liver. Sustained caloric excess can expand white adipose tissue to a point of maladaptation exacerbating both local and systemic inflammation. Multiple sources, instigators and propagators of adipose tissue inflammation occur during obesity. Cross-talk between professional immune cells (i.e. macrophages) and metabolic cells (i.e. adipocytes) promote adipose tissue inflammation during metabolic stress (i.e. metaflammation). Metabolic stress and endogenous danger signals can engage pathogen recognition receptors (PRRs) of the innate immune system thereby activating pro-inflammatory and stress pathways in adipose tissue. The Nod-like receptor protein 3 (NLRP3) inflammasome can act as a metabolic danger sensor to a wide range of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). Activation of the NLRP3 inflammasome facilitates caspase-1 dependent production of the pro-inflammatory cytokines IL-1ß and IL-18. Activation of the NLRP3 inflammasome can promote inflammation and pyroptotic cell death, but caspase-1 is also involved in adipogenesis. This review discusses the role of the NLRP3 inflammasome in adipose tissue immunometabolism responses relevant to metabolic disease. Understanding the potential sources of NLRP3 activation and consequences of NLRP3 effectors may reveal therapeutic opportunities to break or fine-tune the connection between metabolism and inflammation in adipose tissue during obesity.

Glucose alters the symbiotic relationships between gut microbiota and host physiology.

Bacteria and mammals exhibit all aspects of symbiosis. Metabolic flux in bacteria and in specific host cells can influence host-microbe symbiotic relationships and tip the balance between mutualism, commensalism, and parasitism. The relationship between microbes and host metabolism is bidirectional: microbes can influence host blood glucose, but glucose levels can influence the microbiota and host response to specific bacteria. A key consideration determining symbiotic relationships is compartmentalization of bacterial niches by mucosal, chemical, and physical barriers of the gut. We propose that compartmentalization of glucose levels in the blood versus the intestinal lumen is another important factor dictating host-microbe symbiosis. Host glucose and specific bacteria can modify the intestinal barrier, immune function, and antimicrobial defenses, which can then break down compartmentalization of microbes, alter glucose levels and impact symbiosis. Determining how glucose metabolism promotes mutualistic, commensal, and parasitic relationships within the entire microbiota community is relevant to glucose control in diabetes and enteric infections, which occur more often and have worse outcomes in diabetics.

Treatment with camu camu (Myrciaria dubia) prevents obesity by altering the gut microbiota and increasing energy expenditure in diet-induced obese mice.

OBJECTIVE: The consumption of fruits is strongly associated with better health and higher bacterial diversity in the gut microbiota (GM). Camu camu (Myrciaria dubia) is an Amazonian fruit with a unique phytochemical profile, strong antioxidant potential and purported anti-inflammatory potential. DESIGN: By using metabolic tests coupled with 16S rRNA gene-based taxonomic profiling and faecal microbial transplantation (FMT), we have assessed the effect of a crude extract of camu camu (CC) on obesity and associated immunometabolic disorders in high fat/high sucrose (HFHS)-fed mice. RESULTS: Treatment of HFHS-fed mice with CC prevented weight gain, lowered fat accumulation and blunted metabolic inflammation and endotoxaemia. CC-treated mice displayed improved glucose tolerance and insulin sensitivity and were also fully protected against hepatic steatosis. These effects were linked to increased energy expenditure and upregulation of uncoupling protein 1 mRNA expression in the brown adipose tissue (BAT) of CC-treated mice, which strongly correlated with the mRNA expression of the membrane bile acid (BA) receptor TGR5. Moreover, CC-treated mice showed altered plasma BA pool size and composition and drastic changes in the GM (eg, bloom of Akkermansia muciniphila and a strong reduction of Lactobacillus). Germ-free (GF) mice reconstituted with the GM of CC-treated mice gained less weight and displayed higher energy expenditure than GF-mice colonised with the FM of HFHS controls. CONCLUSION: Our results show that CC prevents visceral and liver fat deposition through BAT activation and increased energy expenditure, a mechanism that is dependent on the GM and linked to major changes in the BA pool size and composition.

Targeting macrophage scavenger receptor 1 promotes insulin resistance in obese male mice.

Immune components can bridge inflammatory triggers to metabolic dysfunction. Scavenger receptors sense lipoproteins, but it is not clear how different scavenger receptors alter carbohydrate metabolism during obesity. Macrophage scavenger receptor 1 (MSR1) and macrophage receptor with collagenous structure (MARCO) are scavenger receptors that have been implicated in lipoprotein metabolism and cardiovascular disease. We assessed glucose control, tissue-specific insulin sensitivity, and inflammation in Msr1- and Marco-deficient mice fed with obesogenic diets. Compared to wild-type (WT) mice, Msr1-/- mice had worse blood glucose control that was only revealed after diet-induced obesity, not in lean mice. Obese Msr1-/- mice had worse insulin-stimulated glucose uptake in the adipose tissue, which occurred in the absence of overt differences in adipose inflammation compared to obese WT mice. Msr1 deletion worsened dysglycemia independently from bacterial cell wall insulin sensitizers, such as muramyl dipeptide. MARCO was dispensable for glycemic control in obese mice. Oral administration of the polysaccharide fucoidan worsened glucose control in obese WT mice, but fucoidan had no effect on glycemia in obese Msr1-/- mice. Therefore, MSR1 is a scavenger receptor responsible for changes in glucose control in response to the environmental ligand fucoidan. Given the interest in dietary supplements and natural products reducing inflammation or insulin resistance in metabolic disease during obesity, our results highlight the importance of understanding which ligand-receptor relationships promote versus those that protect against metabolic disease factors. Our results show that ligand or gene targeting of MSR1 exacerbates insulin resistance in obese mice.