Gut-derived bacterial flagellin induces beta-cell inflammation and dysfunction.

Hyperglycemia and type 2 diabetes (T2D) are caused by failure of pancreatic beta cells. The role of the gut microbiota in T2D has been studied, but causal links remain enigmatic. Obese individuals with or without T2D were included from two independent Dutch cohorts. Human data were translated in vitro and in vivo by using pancreatic islets from C57BL6/J mice and by injecting flagellin into obese mice. Flagellin is part of the bacterial locomotor appendage flagellum, present in gut bacteria including Enterobacteriaceae, which we show to be more abundant in the gut of individuals with T2D. Subsequently, flagellin induces a pro-inflammatory response in pancreatic islets mediated by the Toll-like receptor (TLR)-5 expressed on resident islet macrophages. This inflammatory response is associated with beta-cell dysfunction, characterized by reduced insulin gene expression, impaired proinsulin processing and stress-induced insulin hypersecretion in vitro and in vivo in mice. We postulate that increased systemically disseminated flagellin in T2D is a contributing factor to beta-cell failure in time and represents a novel therapeutic target.

Fecal microbiota transplantation does not alter bacterial translocation and visceral adipose tissue inflammation in individuals with obesity.

AIMS: Visceral adipose tissue inflammation is a fundamental mechanism of insulin resistance in obesity and type 2 diabetes. Translocation of intestinal bacteria has been suggested as a driving factor for the inflammation. However, although bacterial DNA was detected in visceral adipose tissue of humans with obesity, it is unclear to what extent this is contamination or whether the gut microbiota is causally involved. Effects of fecal microbiota transplantation (FMT) on bacterial translocation and visceral adipose tissue inflammation in individuals with obesity and insulin resistance were assessed. MATERIAL AND METHODS: Eight individuals with clinically severe obesity (body mass index [BMI] >35 kg/m2) and metabolic syndrome received lean donor FMT 4 weeks prior to elective bariatric surgery. The participants were age-, sex-, and BMI-matched to 16 controls that underwent no fecal transplantation. Visceral adipose tissue was collected during surgery. Bacterial translocation was assessed by 16S rRNA gene sequencing of adipose tissue and feces. Pro-inflammatory cytokine expression and histopathological analyses of visceral adipose tissue were performed to assess inflammation. RESULTS: Fecal microbiota transplantation significantly altered gut microbiota composition. Visceral adipose tissue contained a very low quantity of bacterial DNA in both groups. No difference in visceral bacterial DNA content between groups was observed. Also, visceral expression of pro-inflammatory cytokines and macrophage infiltration did not differ between groups. No correlation between inflammatory tone and bacterial translocation was observed. CONCLUSIONS: Visceral bacterial DNA content and level of inflammation were not altered upon FMT. Thus, bacterial translocation may not be the main driver of visceral adipose tissue inflammation in obesity.

Fecal microbiota transplantation as tool to study the interrelation between microbiota composition and miRNA expression.

The intestinal gut microbiota is important for human metabolism and immunity and can be influenced by many host factors. A recently emerged host factor is secreted microRNA (miRNA). Previously, it has been shown that secreted miRNAs can influence the growth of certain bacteria and conversely, that shifts in the microbiota can alter the composition of secreted miRNAs. Here, we sought to further investigate the interaction between the gut microbiota and secreted miRNAs by the use of fecal microbiota transplantation (FMT). Subjects with the metabolic syndrome received either an autologous (n = 4) or allogenic (n = 14) FMT. Fecal samples were collected at baseline and 6 weeks after FMT, from which the microbiome and miRNA composition were determined via 16S rRNA sequencing and miRNA sequencing, respectively. We observed a significant correlation between the fecal miRNA expression and microbiota composition, both before and after FMT. Our results suggest that the FMT-induced shift in microbiota altered the fecal miRNA profile, indicated by correlations between differentially abundant microbes and miRNAs. This idea of a shift in miRNA composition driven by changes in the microbiota was further strengthened by the absence of a direct effect of specific miRNAs on the growth of specific bacterial strains.

Compensatory intestinal immunoglobulin response after vancomycin treatment in humans.

Intestinal immunoglobulins (Ig) are abundantly secreted antibodies that bind bacteria and bacterial components in the gut. This binding is considered to accelerate bacterial transit time and prevent the interaction of potentially immunogenic compounds with intestinal immune cells. Ig secretion is regulated by alterations in gut microbiome composition, an event rarely mapped in an intervention setting in humans. Here, we determined the intestinal and systemic Ig response to a major intervention in gut microbiome composition. Healthy humans and humans with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Coinciding with a vancomycin-induced increase in Gram-negative bacteria, fecal levels of the immunogenic bacterial components lipopolysaccharide (LPS) and flagellin drastically increased. Intestinal antibodies (IgA and IgM) significantly increased, whereas peripheral antibodies (IgG, IgA, and IgM) were mostly unaffected by vancomycin treatment. Bacterial cell sorting followed by 16S rRNA sequencing revealed that the majority of Gram-negative bacteria, including opportunistic pathogens, were IgA-coated after the intervention. We suggest that the intestinal Ig response after vancomycin treatment prevents the intrusion of pathogens and bacterial components into systemic sites.

Faecal microbiota transplantation halts progression of human new-onset type 1 diabetes in a randomised controlled trial.

OBJECTIVE: Type 1 diabetes (T1D) is characterised by islet autoimmunity and beta cell destruction. A gut microbiota-immunological interplay is involved in the pathophysiology of T1D. We studied microbiota-mediated effects on disease progression in patients with type 1 diabetes using faecal microbiota transplantation (FMT). DESIGN: Patients with recent-onset (<6 weeks) T1D (18-30 years of age) were randomised into two groups to receive three autologous or allogenic (healthy donor) FMTs over a period of 4 months. Our primary endpoint was preservation of stimulated C peptide release assessed by mixed-meal tests during 12 months. Secondary outcome parameters were changes in glycaemic control, fasting plasma metabolites, T cell autoimmunity, small intestinal gene expression profile and intestinal microbiota composition. RESULTS: Stimulated C peptide levels were significantly preserved in the autologous FMT group (n=10 subjects) compared with healthy donor FMT group (n=10 subjects) at 12 months. Small intestinal Prevotella was inversely related to residual beta cell function (r=-0.55, p=0.02), whereas plasma metabolites 1-arachidonoyl-GPC and 1-myristoyl-2-arachidonoyl-GPC levels linearly correlated with residual beta cell preservation (rho=0.56, p=0.01 and rho=0.46, p=0.042, respectively). Finally, baseline CD4 +CXCR3+T cell counts, levels of small intestinal Desulfovibrio piger and CCL22 and CCL5 gene expression in duodenal biopsies predicted preserved beta cell function following FMT irrespective of donor characteristics. CONCLUSION: FMT halts decline in endogenous insulin production in recently diagnosed patients with T1D in 12 months after disease onset. Several microbiota-derived plasma metabolites and bacterial strains were linked to preserved residual beta cell function. This study provides insight into the role of the intestinal gut microbiome in T1D. TRIAL REGISTRATION NUMBER: NTR3697.

Parenteral nutrition impairs plasma bile acid and gut hormone responses to mixed meal testing in lean healthy men.

BACKGROUND & AIMS: To investigate the acute effects of intravenous vs enteral meal administration on circulating bile acid and gut hormone responses. METHODS: In a randomized crossover design, we compared the effects of duodenal (via a nasoduodenal tube) vs parenteral (intravenous) administration over 180 min of identical mixed meals on circulating bile acid and gut hormone concentrations in eight healthy lean men. We analysed the bile acid and gut hormone responses in two periods: the intraprandial period from time point (T) 0 until T180 during meal administration and the postprandial period from T180 until T360, after discontinuation of meal administration. RESULTS: Intravenous meal administration decreased the intraprandial (AUC (µmol/L∗min) duodenal 1469 ± 284 vs intravenous 240 ± 39, p < 0.01) and postprandial bile acid response (985 ± 240 vs 223 ± 5, p < 0.05) and was accompanied by decreased gut hormone responses including glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1, glucagon-like peptide 2 and fibroblast growth factor 19. Furthermore, intravenous meal administration elicited greater glucose concentrations, but similar insulin concentrations compared to enteral administration. CONCLUSIONS: Compared to enteral administration, parenteral nutrition results in lower postprandial bile acid and gut hormone responses in healthy lean men. This was accompanied by higher glucose concentrations in the face of similar insulin concentrations exposing a clear incretin effect of enteral mixed meal administration. The alterations in bile acid homeostasis were apparent after only one intravenous meal.

Donor metabolic characteristics drive effects of faecal microbiota transplantation on recipient insulin sensitivity, energy expenditure and intestinal transit time.

OBJECTIVE: Bariatric surgery improves glucose metabolism. Recent data suggest that faecal microbiota transplantation (FMT) using faeces from postbariatric surgery diet-induced obese mice in germ-free mice improves glucose metabolism and intestinal homeostasis. We here investigated whether allogenic FMT using faeces from post-Roux-en-Y gastric bypass donors (RYGB-D) compared with using faeces from metabolic syndrome donors (METS-D) has short-term effects on glucose metabolism, intestinal transit time and adipose tissue inflammation in treatment-naïve, obese, insulin-resistant male subjects. DESIGN: Subjects with metabolic syndrome (n=22) received allogenic FMT either from RYGB-D or METS-D. Hepatic and peripheral insulin sensitivity as well as lipolysis were measured at baseline and 2 weeks after FMT by hyperinsulinaemic euglycaemic stable isotope (2H2-glucose and 2H5-glycerol) clamp. Secondary outcome parameters were changes in resting energy expenditure, intestinal transit time, faecal short-chain fatty acids (SCFA) and bile acids, and inflammatory markers in subcutaneous adipose tissue related to intestinal microbiota composition. Faecal SCFA, bile acids, glycaemic control and inflammatory parameters were also evaluated at 8 weeks. RESULTS: We observed a significant decrease in insulin sensitivity 2 weeks after allogenic METS-D FMT (median rate of glucose disappearance: from 40.6 to 34.0 µmol/kg/min; p<0.01). Moreover, a trend (p=0.052) towards faster intestinal transit time following RYGB-D FMT was seen. Finally, we observed changes in faecal bile acids (increased lithocholic, deoxycholic and (iso)lithocholic acid after METS-D FMT), inflammatory markers (decreased adipose tissue chemokine ligand 2 (CCL2) gene expression and plasma CCL2 after RYGB-D FMT) and changes in several intestinal microbiota taxa. CONCLUSION: Allogenic FMT using METS-D decreases insulin sensitivity in metabolic syndrome recipients when compared with using post-RYGB-D. Further research is needed to delineate the role of donor characteristics in FMT efficacy in human insulin-resistant subjects. TRIAL REGISTRATION NUMBER: NTR4327.

Oral vancomycin treatment does not alter markers of postprandial inflammation in lean and obese subjects.

Intake of a high-fat meal induces a systemic inflammatory response in the postprandial which is augmented in obese subjects. However, the underlying mechanisms of this response have not been fully elucidated. We aimed to assess the effect of gut microbiota modulation on postprandial inflammatory response in lean and obese subjects. Ten lean and ten obese subjects with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Oral high-fat meal tests (50 g fat/m2 body surface area) were performed before and after vancomycin intervention. Gut microbiota composition, leukocyte counts, plasma lipopolysaccharides (LPS), LPS-binding protein (LBP), IL-6 and MCP-1 concentrations and monocyte CCR2 and cytokine expression were determined before and after the high-fat meal. Oral vancomycin treatment resulted in profound changes in gut microbiota composition and significantly decreased bacterial diversity in both groups (phylogenetic diversity pre- versus post-intervention: lean, 56.9 ± 7.8 vs. 21.4 ± 6.6, P < 0.001; obese, 53.9 ± 7.8 vs. 21.0 ± 5.9, P < 0.001). After intervention, fasting plasma LPS significantly increased (lean, median [IQR] 0.81 [0.63-1.45] EU/mL vs. 2.23 [1.33-3.83] EU/mL, P = 0.017; obese, median [IQR] 0.76 [0.45-1.03] EU/mL vs. 1.44 [1.11-4.24], P = 0.014). However, postprandial increases in leukocytes and plasma LPS were unaffected by vancomycin in both groups. Moreover, we found no changes in plasma LBP, IL-6 and MCP-1 or in monocyte CCR2 expression. Despite major vancomycin-induced disruption of the gut microbiota and increased fasting plasma LPS, the postprandial inflammatory phenotype in lean and obese subjects was unaffected in this study.

Pancreatic 18F-FDG uptake is increased in type 2 diabetes patients compared to non-diabetic controls.

INTRODUCTION: Increasing evidence indicates that the development of type 2 diabetes is driven by chronic low grade beta-cell inflammation. However, it is unclear whether pancreatic inflammation can be noninvasively visualized in type 2 diabetes patients. We aimed to assess pancreatic 18F-FDG uptake in type 2 diabetes patients and controls using 18F-fluorodeoxylglucose positron emission tomography/computed tomography (18F-FDG PET/CT). MATERIAL AND METHODS: In this retrospective cross-sectional study, we enrolled 20 type 2 diabetes patients and 65 controls who had undergone a diagnostic 18F-FDG PET/CT scan and obtained standardized uptake values (SUVs) of pancreas and muscle. Pancreatic SUV was adjusted for background uptake in muscle and for fasting blood glucose concentrations. RESULTS: The maximum pancreatic SUVs adjusted for background muscle uptake (SUVmax.m) and fasting blood glucose concentration (SUVglucose) were significantly higher in diabetes patients compared to controls (median 2.86 [IQR 2.24-4.36] compared to 2.15 [IQR 1.51-2.83], p = 0.006 and median 2.76 [IQR 1.18-4.34] compared to 1.91 [IQR 1.27-2.55], p<0.001, respectively). In linear regression adjusting for age and body mass index, diabetes remained the main predictor of SUVmax.m and SUVglucose. CONCLUSION: Pancreatic 18F-FDG uptake adjusted for background muscle uptake and fasting blood glucose concentration was significantly increased in type 2 diabetes patients.

Depicting the composition of gut microbiota in a population with varied ethnic origins but shared geography.

Trillions of microorganisms inhabit the human gut and are regarded as potential key factors for health1,2. Characteristics such as diet, lifestyle, or genetics can shape the composition of the gut microbiota2-6 and are usually shared by individuals from comparable ethnic origin. So far, most studies assessing how ethnicity relates to the intestinal microbiota compared small groups living at separate geographical locations7-10. Using fecal 16S ribosomal RNA gene sequencing in 2,084 participants of the Healthy Life in an Urban Setting (HELIUS) study11,12, we show that individuals living in the same city tend to share similar gut microbiota characteristics with others of their ethnic background. Ethnicity contributed to explain the interindividual dissimilarities in gut microbiota composition, with three main poles primarily characterized by operational taxonomic units (OTUs) classified as Prevotella (Moroccans, Turks, Ghanaians), Bacteroides (African Surinamese, South-Asian Surinamese), and Clostridiales (Dutch). The Dutch exhibited the greatest gut microbiota α-diversity and the South-Asian Surinamese the smallest, with corresponding enrichment or depletion in numerous OTUs. Ethnic differences in α-diversity and interindividual dissimilarities were independent of metabolic health and only partly explained by ethnic-related characteristics including sociodemographic, lifestyle, or diet factors. Hence, the ethnic origin of individuals may be an important factor to consider in microbiome research and its potential future applications in ethnic-diverse societies.

Intestinal Ralstonia pickettii augments glucose intolerance in obesity.

An altered intestinal microbiota composition has been implicated in the pathogenesis of metabolic disease including obesity and type 2 diabetes mellitus (T2DM). Low grade inflammation, potentially initiated by the intestinal microbiota, has been suggested to be a driving force in the development of insulin resistance in obesity. Here, we report that bacterial DNA is present in mesenteric adipose tissue of obese but otherwise healthy human subjects. Pyrosequencing of bacterial 16S rRNA genes revealed that DNA from the Gram-negative species Ralstonia was most prevalent. Interestingly, fecal abundance of Ralstonia pickettii was increased in obese subjects with pre-diabetes and T2DM. To assess if R. pickettii was causally involved in development of obesity and T2DM, we performed a proof-of-concept study in diet-induced obese (DIO) mice. Compared to vehicle-treated control mice, R. pickettii-treated DIO mice had reduced glucose tolerance. In addition, circulating levels of endotoxin were increased in R. pickettii-treated mice. In conclusion, this study suggests that intestinal Ralstonia is increased in obese human subjects with T2DM and reciprocally worsens glucose tolerance in DIO mice.

Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy.

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time- and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.

Fecal Microbiota Transplantation: Therapeutic Potential for a Multitude of Diseases beyond Clostridium difficile.

The human intestinal tract contains trillions of bacteria, collectively called the gut microbiota. Recent insights have linked the gut microbiota to a plethora of diseases, including Clostridium difficile infection (CDI), inflammatory bowel disease (IBD), and metabolic diseases such as obesity, type 2 diabetes (T2D), and nonalcoholic steatohepatitis (NASH). Fecal microbiota transplantation (FMT) is currently tested as a therapeutic option in various diseases and can also help to dissect association from causality with respect to gut microbiota and disease. In CDI, FMT has been shown to be superior to antibiotic treatment. For IBD, T2D, and NASH, several placebo-controlled randomized controlled trials are under way. Moreover, techniques and standardization are developing. With the extension of FMT as a treatment modality in diseases other than CDI, a whole new treatment option may be emerging. Moreover, correlating alterations in specific strains to disease outcome may prove pivotal in finding new bacterial targets. Thus, although causality of the gut microbiota in various diseases still needs to be proven, FMT may prove to be a powerful tool providing us with diagnostic and therapeutic leads.

Gut Microbiota and Energy Expenditure in Health and Obesity.

The contribution of intestinal bacterial strains (gut microbiota) to the development of obesity and obesity-related disorders is increasingly recognized as a potential diagnostic and pharmacologic target. Alterations in the intestinal bacterial composition have been associated with presence of chronic low-grade inflammation, a known feature of insulin resistance and type 2 diabetes mellitus. However, causality still needs to be proven. Fecal transplantation studies in germ-free mice have provided crucial insight into the causality of gut microbiota in development of obesity and obesity-related disorders. Moreover, fecal transplantation studies in conjunction with fecal sampling in prospectively followed cohorts will help identify causally involved intestinal bacterial strains in human obesity. Results from these studies will lead to characterization of novel diagnostic markers as well as therapeutic strategies that aim to treat obesity and obesity-related disorders.

Accuracy for optical diagnosis of small colorectal polyps in nonacademic settings.

BACKGROUND & AIMS: When small colorectal lesions are accurately characterized, adenomas can be removed and discarded without formal histopathology analysis. Previous studies in an academic setting showed that many lesions can be managed accurately on the basis of their endoscopic image (optical diagnosis). We performed a prospective study to assess the accuracy of optical diagnosis of small colorectal polyps in a nonacademic setting (the DISCOUNT trial) by using high-resolution endoscopy (HRE) and narrow-band imaging (NBI). METHODS: During colonoscopy, 1 of 3 nonacademic endoscopists characterized small lesions and declared whether this was done with low or high confidence. In cases of high confidence, the endoscopists decided whether lesions should be removed and discarded or whether they could be left in situ. A surveillance interval was then recommended on-site. RESULTS: Of 215 patients in the study, 108 were found to have 281 small lesions. Of these lesions, 231 were characterized with high confidence by using HRE or NBI; the level of corresponding sensitivity was 77.0% (95% confidence interval, 68.4-83.8), and specificity was 78.8% (95% confidence interval, 70.6-85.2). Of these lesions, 164 were assigned for removal, and 67 were assigned to remain in situ, including 9 adenomas. In 54 patients, a surveillance interval could be recommended on-site that was in line with Dutch guidelines for 44 patients. CONCLUSIONS: Even though many lesions were characterized by HRE or NBI with high confidence, optical diagnosis in a nonacademic setting proved to be disappointing, with a sensitivity of 77.0% and a specificity of 78.8%. Many lesions were accurately assigned to be removed or remain in situ, although few adenomas were assigned to remain in situ. Also, 19% of on-site recommendations for a surveillance interval proved to be inaccurate.

Toxins of cyanobacteria.

Blue-green algae are found in lakes, ponds, rivers and brackish waters throughout the world. In case of excessive growth such as bloom formation, these bacteria can produce inherent toxins in quantities causing toxicity in mammals, including humans. These cyanotoxins include cyclic peptides and alkaloids. Among the cyclic peptides are the microcystins and the nodularins. The alkaloids include anatoxin-a, anatoxin-a(S), cylindrospermopsin, saxitoxins (STXs), aplysiatoxins and lyngbyatoxin. Both biological and chemical methods are used to determine cyanotoxins. Bioassays and biochemical assays are nonspecific, so they can only be used as screening methods. HPLC has some good prospects. For the subsequent detection of these toxins different detectors may be used, ranging from simple UV-spectrometry via fluorescence detection to various types of MS. The main problem in the determination of cyanobacterial toxins is the lack of reference materials of all relevant toxins. In general, toxicity data on cyanotoxins are rather scarce. A majority of toxicity data are known to be of microcystin-LR. For nodularins, data from a few animal studies are available. For the alkaloids, limited toxicity data exist for anatoxin-a, cylindrospermopsin and STX. Risk assessment for acute exposure could be relevant for some types of exposure. Nevertheless, no acute reference doses have formally been derived thus far. For STX(s), many countries have established tolerance levels in bivalves, but these limits were set in view of STX(s) as biotoxins, accumulating in marine shellfish. Official regulations for other cyanotoxins have not been established, although some (provisional) guideline values have been derived for microcystins in drinking water by WHO and several countries.