Postbiotic impact on host metabolism and immunity provides therapeutic potential in metabolic disease.

The gut microbiota influences aspects of metabolic disease, including tissue inflammation, adiposity, blood glucose, insulin, and endocrine control of metabolism. Prebiotics or probiotics are often sought to combat metabolic disease. However, prebiotics lack specificity and can have deleterious bacterial community effects. Probiotics require live bacteria to find a colonization niche sufficient to influence host immunity or metabolism. Postbiotics encompass bacterial-derived components and molecules, which are well-positioned to alter host immunometabolism without relying on colonization efficiency or causing widespread effects on the existing microbiota. Here, we summarize the potential for beneficial and detrimental effects of specific postbiotics related to metabolic disease and the underlying mechanisms of action. Bacterial cell wall components such as lipopolysaccharides, muropeptides, lipoteichoic acids and flagellin have context-dependent effects on host metabolism by engaging specific immune responses. Specific types of postbiotics within broad classes of compounds such as lipopolysaccharides, muropeptides can have opposing effects on endocrine control of host metabolism where certain postbiotics are insulin sensitizers and others promote insulin resistance. Bacterial metabolites such as short chain fatty acids, bile acids, lactate, glycerol, succinate, ethanolamine, and ethanol can be substrates for host metabolism. Postbiotics can fuel host metabolic pathways directly or influence endocrine control of metabolism through immunomodulation or mimicking host-derived hormones. The interaction of postbiotics in the host-microbe relationship should be considered during metabolic inflammation and metabolic disease.

Intermittent fasting increases fat oxidation and promotes metabolic flexibility in lean mice but not obese type 2 diabetic mice.

Obesity and type 2 diabetes (T2D) are associated with metabolic inflexibility, characterized by an impaired ability to switch between substrate storage and utilization pathways. Metabolic inflexibility during obesity is typified by lower engagement of fatty acid metabolism despite an ample supply of stored lipids. Intermittent fasting (IF) can promote metabolic flexibility. However, it is not clear how obesity and T2D alter metabolic flexibility after repeated IF. Male obese db/db and control db/+ mice were fasted for 24 h twice a week for 10 wk. This 5:2 IF regimen did not alter body mass, body composition, food intake, or physical activity in db/db or db/+ mice. After IF, db/db mice had lower fatty acid oxidation and higher carbohydrate oxidation in the fed state, indicating metabolic inflexibility to metabolize lipids. After IF, control db/+ mice had higher fatty acid oxidation and lower carbohydrate oxidation in the fed state, characteristic of metabolic flexibility, and increased engagement of lipid metabolism. In the fasted state, IF lowered carbohydrate oxidation and increased fatty acid oxidation in control db/+ mice but not in obese db/db mice. After IF, db/db mice also had lower serum ß-hydroxybutyrate than control db/+ mice. Ten weeks of IF decreased adipocyte size in visceral adipose tissue of control db/+ mice, but this IF regimen did not change adipocyte size in obese db/db mice. Therefore, IF increases fatty acid oxidation and metabolic flexibility in lean mice, but this adaptation is absent in a mouse model of obesity and type 2 diabetes.NEW & NOTEWORTHY We show that a 5:2 intermittent fasting regimen can increase lipid oxidation without altering body mass in lean mice. Therefore, repeated intermittent fasting can increase metabolic flexibility without the need for (or prior to) weight loss. Intermittent fasting did not increase lipid oxidation in mice with obesity and type 2 diabetes, highlighting that obesity and/or type 2 diabetes limit changes in metabolic flexibility and mitigate increased fatty acid oxidation without weight loss during intermittent fasting.

Bruton's tyrosine kinase (BTK) inhibitors alter blood glucose and insulin in obese mice but reduce inflammation independent of BTK.

Obesity is associated with metabolic inflammation, which can contribute to insulin resistance, higher blood glucose, and higher insulin indicative of prediabetes progression. The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a metabolic danger sensor implicated in metabolic inflammation. Many features of metabolic disease can activate the NLRP3 inflammasome; however, it is not yet clear which upstream triggers to target, and there are no clinically approved NLRP3 inflammasome inhibitors for metabolic disease. Bruton's tyrosine kinase (BTK) mediates activation of the NLRP3 inflammasome. Ibrutinib is the most-studied pharmacological inhibitor of BTK, and it can improve blood glucose control in obese mice. However, inhibitors of tyrosine kinases are permissive, and it is unknown if BTK inhibitors require BTK to alter endocrine control of metabolism or metabolic inflammation. We tested whether ibrutinib and acalabrutinib, a new generation BTK inhibitor with higher selectivity, require BTK to inhibit the NLRP3 inflammasome, metabolic inflammation, and blood glucose in obese mice. Chronic ibrutinib administration lowered fasting blood glucose and improved glycemia, whereas acalabrutinib increased fasting insulin levels and increased markers of insulin resistance in high-fat diet-fed CBA/J mice with intact Btk. These metabolic effects of BTK inhibitors were absent in CBA/CaHN-Btkxid/J mice with mutant Btk. However, ibrutinib and acalabrutinib reduced NF-κB activity, proinflammatory gene expression, and NLRP3 inflammasome activation in macrophages with and without functional BTK. These data highlight that the BTK inhibitors can have divergent effects on metabolism and separate effects on metabolic inflammation that can occur independently of actions on BTK.NEW & NOTEWORTHY Bruton's tyrosine kinase (BTK) is involved in immune function. It was thought that BTK inhibitors improve characteristics of obesity-related metabolic disease by lowering metabolic inflammation. However, tyrosine kinase inhibitors are permissive, and it was not known if different BTK inhibitors alter host metabolism or immunity through actions on BTK. We found that two BTK inhibitors had divergent effects on blood glucose and insulin via BTK, but inhibition of metabolic inflammation occurred independently of BTK in obese mice.

Intermittent fasting influences immunity and metabolism.

Intermittent fasting (IF) modifies cell- and tissue-specific immunometabolic responses that dictate metabolic flexibility and inflammation during obesity and type 2 diabetes (T2D). Fasting forces periods of metabolic flexibility and necessitates increased use of different substrates. IF can lower metabolic inflammation and improve glucose metabolism without lowering obesity and can influence time-dependent, compartmentalized changes in immunity. Liver, adipose tissue, skeletal muscle, and immune cells communicate to relay metabolic and immune signals during fasting. Here we review the connections between metabolic and immune cells to explain the divergent effects of IF compared with classic caloric restriction (CR) strategies. We also explore how the immunometabolism of metabolic diseases dictates certain IF outcomes, where the gut microbiota triggers changes in immunity and metabolism during fasting.

Wearable Aptalyzer Integrates Microneedle and Electrochemical Sensing for In Vivo Monitoring of Glucose and Lactate in Live Animals.

Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel MN arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of type 1 diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and the need for reliable and multiplexed bioanalytical monitoring in vivo.

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.

Microbiota and Nod-like receptors balance inflammation and metabolism during obesity and diabetes.

Gut microbiota influence host immunity and metabolism during obesity. Bacterial sensors of the innate immune system relay signals from specific bacterial components (i.e., postbiotics) that can have opposing outcomes on host metabolic inflammation. NOD-like receptors (NLRs) such as Nod1 and Nod2 both recruit receptor-interacting protein kinase 2 (RIPK2) but have opposite effects on blood glucose control. Nod1 connects bacterial cell wall-derived signals to metabolic inflammation and insulin resistance, whereas Nod2 can promote immune tolerance, insulin sensitivity, and better blood glucose control during obesity. NLR family pyrin domain containing (NLRP) inflammasomes can also generate divergent metabolic outcomes. NLRP1 protects against obesity and metabolic inflammation potentially because of a bias toward IL-18 regulation, whereas NLRP3 appears to have a bias toward IL-1ß-mediated metabolic inflammation and insulin resistance. Targeting specific postbiotics that improve immunometabolism is a key goal. The Nod2 ligand, muramyl dipeptide (MDP) is a short-acting insulin sensitizer during obesity or during inflammatory lipopolysaccharide (LPS) stress. LPS with underacylated lipid-A antagonizes TLR4 and counteracts the metabolic effects of inflammatory LPS. Providing underacylated LPS derived from Rhodobacter sphaeroides improved insulin sensitivity in obese mice. Therefore, certain types of LPS can generate metabolically beneficial metabolic endotoxemia. Engaging protective adaptive immunoglobulin immune responses can also improve blood glucose during obesity. A bacterial vaccine approach using an extract of the entire bacterial community in the upper gut promotes protective adaptive immune response and long-lasting improvements in blood glucose control. A key future goal is to identify and combine postbiotics that cooperate to improve blood glucose control.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

Kinase inhibitors for cancer alter metabolism, blood glucose, and insulin.

Small molecule kinase inhibitors (SMKIs) are a class of therapeutic drugs that target protein kinases in diseases such as cancer. SMKIs are often designed to inhibit kinases involved in cell proliferation, but these drugs alter cell metabolism and the endocrine control of organismal metabolism. SMKI treatment in diabetic cancer patients reveals that certain SMKIs improve blood glucose levels and can mitigate insulin dependence or diabetic medication requirements in both type 1 diabetes (T1D) and type 2 diabetes (T2D). Certain SMKIs can preserve functional ß-cell mass and increase insulin secretion or insulin sensitivity. It is not yet clear why different SMKIs can have opposing effects on insulin and blood glucose. Understanding the therapeutic effects of these drugs in T1D and T2D is complicated by overlapping off-target effects of SMKIs. The potency of inhibition of the intended protein kinase and inhibition of multiple off-target kinases may underpin conflicting reports of how certain SMKIs alter blood glucose and insulin. We summarize the effects of SMKIs on the intended and off-target kinases that can alter blood glucose and insulin, including c-Abl, c-Kit, EGFR, and VEGF. Inhibition of PDGFRß consistently lowers blood glucose in T1D and T2D. The effects of SMKIs on the kinases that regulate immune pathways, such as BTK and RIPKs, mediate many of the diverse effects of these drugs on metabolism. We highlight that inhibition of RIPK2 by SMKIs is a central node in metabolism that influences key metabolic pathways including lipolysis, blood glucose control, insulin secretion, and insulin resistance.

Chronic exposure to synthetic food colorant Allura Red AC promotes susceptibility to experimental colitis via intestinal serotonin in mice.

Chemicals in food are widely used leading to significant human exposure. Allura Red AC (AR) is a highly common synthetic colorant; however, little is known about its impact on colitis. Here, we show chronic exposure of AR at a dose found in commonly consumed dietary products exacerbates experimental models of colitis in mice. While intermittent exposure is more akin to a typical human exposure, intermittent exposure to AR in mice for 12 weeks, does not influence susceptibility to colitis. However, exposure to AR during early life primes mice to heightened susceptibility to colitis. In addition, chronic exposure to AR induces mild colitis, which is associated with elevated colonic serotonin (5-hydroxytryptamine; 5-HT) levels and impairment of the epithelial barrier function via myosin light chain kinase (MLCK). Importantly, chronic exposure to AR does not influence colitis susceptibility in mice lacking tryptophan hydroxylase 1 (TPH1), the rate limiting enzyme for 5-HT biosynthesis. Cecal transfer of the perturbed gut microbiota by AR exposure worsens colitis severity in the recipient germ-free (GF) mice. Furthermore, chronic AR exposure elevates colonic 5-HT levels in naïve GF mice. Though it remains unknown whether AR has similar effects in humans, our study reveals that chronic long-term exposure to a common synthetic colorant promotes experimental colitis via colonic 5-HT in gut microbiota-dependent and -independent pathway in mice.

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.

High-fructose feeding suppresses cold-stimulated brown adipose tissue glucose uptake independently of changes in thermogenesis and the gut microbiome.

Diets rich in added sugars are associated with metabolic diseases, and studies have shown a link between these pathologies and changes in the microbiome. Given the reported associations in animal models between the microbiome and brown adipose tissue (BAT) function, and the alterations in the microbiome induced by high-glucose or high-fructose diets, we investigated the potential causal link between high-glucose or -fructose diets and BAT dysfunction in humans. Primary outcomes are changes in BAT cold-induced thermogenesis and the fecal microbiome (clinicaltrials.gov, NCT03188835). We show that BAT glucose uptake, but not thermogenesis, is impaired by a high-fructose but not high-glucose diet, in the absence of changes in the gastrointestinal microbiome. We conclude that decreased BAT glucose metabolism occurs earlier than other pathophysiological abnormalities during fructose overconsumption in humans. This is a potential confounding factor for studies relying on 18F-FDG to assess BAT thermogenesis.

Postbiotics engage IRF4 in adipocytes to promote sex-dependent changes in blood glucose during obesity.

Postbiotics are microbial-derived components or metabolites that can influence host immunity and metabolism. Some postbiotics can improve blood glucose control and lower inflammation during bacterial or nutritional stress. Bacterial cell wall-derived muramyl dipeptide (MDP) is a potent insulin-sensitizing postbiotic that engages NOD2, RIPK2, and requires interferon regulatory factor 4 (IRF4) to lower inflammation and improve blood glucose. However, the sex-dependent effects of this postbiotic and the cell type required for IRF4 to cause inflammatory versus glycemic responses to MDP were unknown. Here, we measured how MDP injection altered glucose tolerance and adipose tissue inflammation during low-level endotoxemia and high fat diet (HFD)-induced obesity in male and female adipocyte-specific IRF4 knockout mice (AdipoIRF4fl/fl ) compared to WTfl/fl mice. Adipocyte IRF4 was required for the blood glucose-lowering effects of MDP during endotoxemia and HFD-induced obesity in male mice. However, MDP did not alter blood glucose in female WTfl/fl and AdipoIRF4fl/f mice during endotoxemia. Unexpectedly, female HFD-fed AdipoIRF4fl/f mice had lower blood glucose after MDP treatment compared to WTfl/fl mice. MDP lowered inflammatory gene expression in adipose tissue of HFD-fed WTfl/fl and AdipoIRF4fl/fl mice of both sexes. Therefore, MDP-mediated lowering of adipose inflammation does not require adipocyte IRF4 and was independent of sex. Together, these data show that injection of MDP, an insulin-sensitizing postbiotic, lowers adipose tissue inflammation in male and female mice, but lower adipose inflammation is not always associated with improved blood glucose. The blood glucose-lowering effect of the postbiotic MDP and dependence on adipocyte IRF4 is sex-dependent.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

Metformin-induced reductions in tumor growth involves modulation of the gut microbiome.

BACKGROUND/PURPOSE: Type 2 diabetes and obesity increase the risk of developing colorectal cancer. Metformin may reduce colorectal cancer but the mechanisms mediating this effect remain unclear. In mice and humans, a high-fat diet (HFD), obesity and metformin are known to alter the gut microbiome but whether this is important for influencing tumor growth is not known. METHODS: Mice with syngeneic MC38 colon adenocarcinomas were treated with metformin or feces obtained from control or metformin treated mice. RESULTS: We find that compared to chow-fed controls, tumor growth is increased when mice are fed a HFD and that this acceleration of tumor growth can be partially recapitulated through transfer of the fecal microbiome or in vitro treatment of cells with fecal filtrates from HFD-fed animals. Treatment of HFD-fed mice with orally ingested, but not intraperitoneally injected, metformin suppresses tumor growth and increases the expression of short-chain fatty acid (SCFA)-producing microbes Alistipes, Lachnospiraceae and Ruminococcaceae. The transfer of the gut microbiome from mice treated orally with metformin to drug naïve, conventionalized HFD-fed mice increases circulating propionate and butyrate, reduces tumor proliferation, and suppresses the expression of sterol response element binding protein (SREBP) gene targets in the tumor. CONCLUSION: These data indicate that in obese mice fed a HFD, metformin reduces tumor burden through changes in the gut microbiome.

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.

Insulin resistance corresponds with a progressive increase in NOD1 in high fat diet-fed mice.

PURPOSE: Innate immune components participate in obesity-induced inflammation, which can contribute to endocrine dysfunction during metabolic diseases. However, the chronological activation of specific immune proteins such as Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and relevance to cellular crosstalk during the progression of obesity-associated insulin resistance (IR) is not known. METHODS: The NOD1 signaling in various insulin-sensitive metabolic tissues during the progression of diet-insulin resistance was assessed in C57BL/6J mice fed with 60% high-fat diet (HFD) for 4, 8, 12, and 16 weeks. Intestinal permeability was measured using FITC-dextran. NOD1 activating potential was analyzed using HEK-Blue mNOD1 cells. RESULTS: HFD-fed mice showed progressive induction of glucose intolerance and impairment of insulin signaling in key metabolic tissues. We found a time-dependent increase in intestinal permeability coupled with transport and accumulation of NOD1 activating ligand in the serum of HFD-fed mice. We also observed a progressive accumulation of γ-D-glutamyl-meso-diaminopimelic acid (DAP), a microbial peptidoglycan ligand known to activate NOD1, in serum samples of the HFD-fed mice. There was also a progressive increase in transcripts levels of NOD1 in bone marrow-derived macrophages during HFD-feeding. In addition, skeletal muscle, adipose and liver, the key insulin sensitive metabolic tissues also had a time-dependent increase in transcripts of NOD1 and Rip2 and a corresponding activation of pro-inflammatory responses in these tissues. CONCLUSION: These data highlight the correlation of inflammation and insulin resistance to NOD1 activation in the bone marrow derived macrophages and insulin responsive metabolic tissues during high fat diet feeding in mice.

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).