Yves Guiot

Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability

Background and aims: Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. Methods: Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. Results: Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. Conclusion: We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes.

Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity

Obesity and type 2 diabetes are characterized by altered gut microbiota, inflammation, and gut barrier disruption. Microbial composition and the mechanisms of interaction with the host that affect gut barrier function during obesity and type 2 diabetes have not been elucidated. We recently isolated Akkermansia muciniphila, which is a mucin-degrading bacterium that resides in the mucus layer. The presence of this bacterium inversely correlates with body weight in rodents and humans. However, the precise physiological roles played by this bacterium during obesity and metabolic disorders are unknown. This study demonstrated that the abundance of A. muciniphila decreased in obese and type 2 diabetic mice. We also observed that prebiotic feeding normalized A. muciniphila abundance, which correlated with an improved metabolic profile. In addition, we demonstrated that A. muciniphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier, and gut peptide secretion. Finally, we demonstrated that all these effects required viable A. muciniphila because treatment with heat-killed cells did not improve the metabolic profile or the mucus layer thickness. In summary, this study provides substantial insight into the intricate mechanisms of bacterial (i.e., A. muciniphila) regulation of the cross-talk between the host and gut microbiota. These results also provide a rationale for the development of a treatment that uses this human mucus colonizer for the prevention or treatment of obesity and its associated metabolic disorders.

Pancreatic β‐cell mass in European subjects with type 2 diabetes

Decreases in both β‐cell function and number can contribute to insulin deficiency in type 2 diabetes. Here, we quantified the β‐cell mass in pancreas obtained at autopsy of 57 type 2 diabetic (T2D) and 52 non‐diabetic subjects of European origin. Sections from the body and tail were immunostained for insulin. The β‐cell mass was calculated from the volume density of β‐cells (measured by point‐counting methods) and the weight of the pancreas. The pancreatic insulin concentration was measured in some of the subjects. β‐cell mass increased only slightly with body mass index (BMI). After matching for BMI, the β‐cell mass was 41% (BMI < 25) and 38% (BMI 26–40) lower in T2D compared with non‐diabetic subjects, and neither gender nor type of treatment influenced these differences. β‐cell mass did not correlate with age at diagnosis but decreased with duration of clinical diabetes (24 and 54% lower than controls in subjects with <5 and >15 years of overt diabetes respectively). Pancreatic insulin concentration was 30% lower in patients. In conclusion, the average β‐cell mass is about 39% lower in T2D subjects compared with matched controls. Its decrease with duration of the disease could be a consequence of diabetes that, with further impairment of insulin secretion, contributes to the progressive deterioration of glucose homeostasis. We do not believe that the small difference in β‐cell mass observed within 5 years of onset could cause diabetes in the absence of β‐cell dysfunction.

Dietary non-digestible carbohydrates promote L-cell differentiation in the proximal colon of rats

One of the challenges in type 2 diabetes treatment is to ensure pancreas functionality with gut peptides such as glucagon-like peptide-1 (GLP-1). We have recently shown that the endogenous GLP-1 production is promoted by dietary non-digestible carbohydrates (oligofructose), the higher GLP-1 secretion could participate in the control of obesity and associated disorders. This experimental study was designed to highlight the mechanisms of endogenous increase of GLP-1 following non-digestible carbohydrate feeding. Male Wistar rats were fed a standard diet (70.4 g/100 g total carbohydrates; controls) or the same diet supplemented with oligofructose (10 g/100 g diet) for 4 weeks. GLP-1-producing L-cells of the colon were quantified by immunohistochemistry. GLP-1 was quantified by ELISA, and proglucagon, neurogenin 3 and NeuroD mRNA were measured in the colon by quantitative RT-PCR. The number of GLP-1-expressing cells was doubled in the proximal colon of oligofructose-treated rats, a phenomenon correlated with the increase in proglucagon mRNA and peptide content in the tissue. Moreover, oligofructose increased the number of enteroendocrine L-cells in the proximal colon by a mechanism involving up-regulation of two differentiation factors: neurogenin 3 and NeuroD. It is the first demonstration that nutrients fermented in the gut may promote L-cell differentiation in the proximal colon, a phenomenon contributing to a higher endogenous GLP-1 production. These results suggest a new mechanism by which dietary fibres may lower food intake and fat mass development.

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept.

Background. Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates Methods. Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation Results. Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response Conclusions. Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

Persistent hyperinsulinaemic hypoglycaemia of infancy: a heterogeneous syndrome unrelated to nesidioblastosis

The syndrome of persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) was described more than 40 years ago by Mc Quarrie.1 Despite the inordinate amount of interest in this syndrome, the pathogenesis of the disease has not yet been completely elucidated. For decades, the disease has been ascribed to nesidioblastosis. This term, first coined by Laidlaw, describes the persistence of a diffuse and disseminated proliferation of islet cells budding off from ducts.2 The concept of nesidioblastosis as the underlying condition of hyperinsulinism is still deeply rooted in the mind of many clinicians, although it has been questioned by several authors.3-5 Indeed, observations based on quantitative immunohistochemical investigations have shown that nesidioblastosis is a common feature of the pancreas in normoglycaemic neonates and infants (fig 1).3 Figure 1 Nesidioblastosis in the pancreas of a normoglycaemic infant. B cells budding off from ducts stained by an anti-insulin antibody (immunoperoxidase; original magnification, ×140). Progress in genetics and molecular biology has increased our understanding of the syndrome. By means of linkage analysis, hyperinsulinism in familial cases has been mapped to chromosome 11.6 Genetic molecular analyses have demonstrated mutations of the genes encoding both the sulphonylurea receptor7-10 and the Kir 6.2 subunit,11 as well as maternal loss of the imprinted gene at the 11p15 region, which might explain the insulin hypersecretion characteristic of this syndrome.12-14 In addition, physiological analysis has demonstrated the absence of functional ATP dependent potassium channels in some of these infants with hypoglycaemia.10 15 Several studies have clearly demonstrated the existence of two forms of PHHI. One corresponds to a focal pancreatic adenomatous hyperplasia (focal PHHI) and the other is characterised by a diffuse β cell abnormality (diffuse PHHI).16-18 Up until now, these two forms could not be differentiated by clinical or biochemical data, although …

Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction.

Background—In the heart, nitric oxide synthases (NOS) modulate cardiac contraction in an isoform-specific manner, which is critically dependent on their cellular and subcellular localization. Defective NO production by NOS3 (endothelial NOS [eNOS]) in the failing heart may precipitate cardiac failure, which could be reversed by overexpression of NOS3 in the myocardium. Methods and Results—We studied the influence of NOS3 in relation to its subcellular localization on the function of cardiomyocytes isolated from transgenic mice overexpressing NOS3 under the &agr;-myosin heavy chain promoter (NOS3-TG). Immunoblot analysis demonstrated moderate (5-fold) NOS3 overexpression in cardiomyocytes from NOS3-TG heterozygotes. Caveolar localization of transgenic eNOS was demonstrated by immunofluorescence, coimmunoprecipitation with caveolin-3, sucrose gradient fractionation, and immunogold staining revealed by electron microscopy. Compared with wild-type littermate, contractility of NOS3-TG cardiomyocytes analyzed by videomicroscopy revealed a lower incidence of spontaneous arrhythmic contractions (n=32, P<0.001); an attenuation of the &bgr;-adrenergic positive inotropic response (isoproterenol, 10−7 mol/L: 62.1±7.8% versus 90.8±8.0% of maximum Ca2+ response; n=10 to 17; P<0.05); a potentiation of the muscarinic negative chronotropic response (carbamylcholine, 3.10−8 mol/L: −63.9±14% versus −27.7±5.6% of basal rate; n=8 to 10; P<0.05), confirmed by telemetry in vivo; and an attenuation of the accentuated antagonism of &bgr;-adrenergically stimulated contraction (−14.6±1.5% versus −3.5±1.5; n=7 to 11; P<0.05). Cardiomyocyte NOS inhibition reversed all 4 effects (P<0.05). Conclusions—Moderate overexpression of NOS3, targeted to caveolae in murine cardiomyocytes, potentiates the postsynaptic muscarinic response and attenuates the effect of high concentrations of catecholamines. Cardiomyocyte NOS3 may represent a promising therapeutic target to restore the sympathovagal balance and protect the heart against arrhythmia.

Glucose regulation of islet stress responses and β-cell failure in type 2 diabetes

Pancreatic β‐cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional β‐cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine‐induced apoptosis and hypoxia. The glucose regulation of oxidative stress‐response and integrated stress‐response genes in cultured rat islets follows an asymmetric V‐shaped profile parallel to that of β‐cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional β‐cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, β‐cell demise under glucotoxic conditions may also result from β‐cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non‐endocrine islet cells. A better understanding of the relative contribution of each type of stress to β‐cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional β‐cell mass in glucose intolerant and type 2 diabetic patients.

The GluCre-ROSA26EYFP mouse: A new model for easy identification of living pancreatic α-cells

The control of glucagon secretion by pancreatic α‐cells is poorly understood, largely because of the difficulty to recognize living α‐cells. We describe a new mouse model, referred to as GluCre‐ROSA26EYFP (or GYY), allowing easy α‐cell identification because of specific expression of EYFP. GYY mice displayed normal glycemic control during a fasting/refeeding test or intraperitoneal insulin injection. Glucagon secretion by isolated islets was normally inhibited by glucose and stimulated by adrenaline. [Ca2+]c responses to arginine, adrenaline, diazoxide and tolbutamide, were similar in GYY and control mice. Hence, this new mouse model is a reliable and powerful tool to specifically study α‐cells.

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

Background. To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. Methods. Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. Results. In rats and primates, expressing a high level of GLUT2 expression on &bgr; cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig &bgr;-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. Conclusion. These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature &bgr; cells and compensatory &bgr;-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.