Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by < 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP-I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7-37) or the related GLP-I(7-36)amide. These hormones have specific beta-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep beta-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the beta-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

IB1 reduces cytokine-induced apoptosis of insulin-secreting cells.

IB1/JIP-1 is a scaffold protein that interacts with upstream components of the c-Jun N-terminal kinase (JNK) signaling pathway. IB1 is expressed at high levels in pancreatic beta cells and may therefore exert a tight control on signaling events mediated by JNK in these cells. Activation of JNK by interleukin 1 (IL-1beta) or by the upstream JNK constitutive activator DeltaMEKK1 promoted apoptosis in two pancreatic beta cell lines and decreased IB1 content by 50-60%. To study the functional consequences of the reduced IB1 content in beta cell lines, we used an insulin-secreting cell line expressing an inducible IB1 antisense RNA that lead to a 38% IB1 decrease. Reducing IB1 levels in these cells increased phosphorylation of c-Jun and increased the apoptotic rate in presence of IL-1beta. Nitric oxide production was not stimulated by expression of the IB1 antisense RNA. Complementary experiments indicated that overexpression of IB1 in insulin-producing cells prevented JNK-mediated activation of the transcription factors c-Jun, ATF2, and Elk1 and decreased IL-1beta- and DeltaMEKK1-induced apoptosis. These data indicate that IB1 plays an anti-apoptotic function in insulin-producing cells probably by controlling the activity of the JNK signaling pathway.

Survival impact of lung transplantation for COPD

La bronchopneumopathie chronique obstructive (BPCO) est l'indication la plus fréquente de la transplantation pulmonaire. Néanmoins, le bénéfice de survie dans cette indication est toujours débattu. Le but de cette étude était d'analyser l'impact de la transplantation pulmonaire sur la survie de patients BPCO à l'aide d'une nouvelle méthode utilisant l'index de BODE, un indice validé dans la prédiction de la survie de patients BPCO. L'index de BODE est composé de 4 variables (indice de masse corporelle, obstruction bronchique, dyspnée, capacité d'effort) et son score s'échelonne de 0 à 10, une valeur élevée signifiant une maladie plus sévère et donc une probabilité de survie moindre.Cette étude rétrospective a porté sur 54 patients BPCO ayant consécutivement bénéficié d'une transplantation pulmonaire (unilatérale ou bilatérale) au Centre Hospitalier Universitaire Vaudois et aux Hôpitaux Universitaires de Genève entre 1994 et 2007, avec un suivi jusqu'au 30 juin 2009. Le score de BODE avant transplantation a été calculé pour chaque patient, à partir duquel une survie prédite a été dérivée. Cette survie prédite a été comparée à la survie réelle des patients transplantés.Une majorité de patient (67%) a présenté un bénéfice individuel de survie suite à la transplantation pulmonaire. Ceci s'est vérifié aussi bien dans le sous-groupe de patients avec un score de BODE > 7 que dans celui avec un score de BODE < 7. La survie médiane était significativement améliorée par la transplantation pulmonaire dans la cohorte totale et dans le sous-groupe avec un score de BODE > 7, mais pas dans celui avec un score de BODE < 7. De plus, 4 ans après la transplantation, un bénéfice de survie ne peut être escompté que chez les patients présentant un score de BODE > 7.Dans notre cohorte, la transplantation pulmonaire a donc conduit à un bénéfice individuel de survie chez la majorité des patients, quel que soit leur score de BODE avant l'intervention. Toutefois, un bénéfice global de survie n'a pu être démontré que dans le groupe de patients ayant la maladie la plus sévère. Chez les patients moins sévèrement atteints, les risques liés à l'intervention sont plus importants que le bénéfice de survie escompté à long terme. Ces résultats confortent l'utilisation de l'index de BODE comme critère de sélection pour la transplantation pulmonaire chez les patients BPCO.

Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles.

In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Involvement of long non-coding RNAs in beta cell failure at the onset of type 1 diabetes in NOD mice.

AIMS/HYPOTHESIS: Exposure of pancreatic beta cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. As little is known about their role in insulin-secreting cells, this study aimed to evaluate their contribution to beta cell dysfunction. METHODS: The expression of lncRNAs was determined by microarray in the MIN6 beta cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells. RESULTS: MIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase in these lncRNAs was also seen in prediabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favoured beta cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB). CONCLUSIONS/INTERPRETATION: Our study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitises beta cells to apoptosis, probably contributing to their failure during the initial phases of the disease.

Normal skin of HIV-infected individuals contains increased numbers of dermal CD8 T cells and normal numbers of Langerhans cells

Dysregulation of the skin immune system (SIS) could explain the high prevalence of skin disorders in HIV+ individuals. The present study was carried out to determine whether alterations in the cell population of SIS and epidermal immunoactivation occur in the normal skin of HIV+ individuals. Forty-five biopsies were taken from the normal upper arm skin of 45 HIV+ patients and of 15 healthy controls. HIV+ individuals were divided into three categories according to their CD4 cell blood count (<200, 200-499 and ³500/µl). Hematoxylin-eosin was used to stain tissue sections for morphological analysis and immunohistochemistry was used for the evaluation of the frequency of macrophages, Langerhans cells, and CD lymphocyte subsets. In addition, semiquantitative analysis of LFA-1, ICAM-1 and HLA-DR was determined in epidermal cells. Macrophages, Langerhans cells, and CD lymphocyte subsets did not differ significantly between any of the patient categories and the control group. When all HIV+ individuals were compared as a group to the control group, a significant increase in dermal CD8+ T lymphocytes (P < 0.01) and lower CD4-CD8 ratios (P < 0.01) were observed in the HIV+ individuals. Epidermal ICAM-1 and HLA-DR expression was negative in both HIV+ and normal skin biopsies. No evidence of a depletion of the SIS population or of epidermal immunoactivation in normal skin from HIV+ individuals was demonstrable, suggesting that alterations in the central immune system are not necessarily reflected in the SIS of HIV-infected patients.

Function of the immunoregulatory CD4-CD8- T cells in the context of autoimmune diabetes

La tolérance immunitaire dépend de la distinction entre le soi et le non soi par le système immunitaire. Un bris dans la tolérance immunitaire mène à l'auto-immunité, qui peut provoquer la destruction des organes, des glandes, des articulations ou du système nerveux central. Le diabète auto-immun, également connu sous le nom diabète juvénile et diabète de type 1, résulte d'une attaque auto-immune sur les cellules β pancréatiques sécrétrices d’insuline, localisées au niveau des îlots de Langerhans du pancréas. Bien que le diabète auto-immun soit traitable par une combinaison d’injections quotidiennes d’insuline d’origine exogène, de régime et d'exercices, beaucoup de complications chroniques peuvent se manifester chez les patients, y compris, mais non limitées à, la cécité, les maladies cardiovasculaires, l’insuffisance rénale et l'amputation. En raison des nombreuses complications liées au diabète auto-immun à long terme, la recherche continue afin de mieux comprendre tous les facteurs impliqués dans la progression de la maladie dans le but de développer de nouvelles thérapies qui empêcheront, renverseront et/ou traiteront cette maladie. Un rôle primordial dans la génération et l'entretien de la tolérance immunitaire a été attribué au nombre et à la fonction des sous-populations de cellules régulatrices. Une de ces populations est constituée de cellules T CD4-CD8- (double négatives, DN), qui ont été étudiées chez la souris et l'humain pour leur contribution à la tolérance périphérique, à la prévention des maladies et pour leur potentiel associé à la thérapie cellulaire. En effet, les cellules de T DN sont d'intérêt thérapeutique parce qu'elles montrent un potentiel immunorégulateur antigène-spécifique dans divers cadres expérimentaux, y compris la prévention du diabète auto-immun. D’ailleurs, en utilisant un système transgénique, nous avons démontré que les souris prédisposées au diabète auto-immun présentent peu de cellules T DN, et que ce phénotype contribue à la susceptibilité au diabète auto-immun. En outre, un transfert des cellules T DN est suffisant pour empêcher la progression vers le diabète chez les souris prédisposées au diabète auto-immun. Ces résultats suggèrent que les cellules T DN puissent présenter un intérêt thérapeutique pour les patients diabétiques. Cependant, nous devons d'abord valider ces résultats en utilisant un modèle non-transgénique, qui est plus physiologiquement comparable à l'humain. L'objectif principal de cette thèse est de définir la fonction immunorégulatrice des cellules T DN, ainsi que le potentiel thérapeutique de celles-ci dans la prévention du diabète auto-immun chez un modèle non-transgénique. Dans cette thèse, on démontre que les souris résistantes au diabète auto-immun présentent une proportion et nombre absolu plus élevés de cellules T DN non-transgéniques, lorsque comparées aux souris susceptibles. Cela confirme une association entre le faible nombre de cellules T DN et la susceptibilité à la maladie. On observe que les cellules T DN éliminent les cellules B activées in vitro par une voie dépendante de la voie perforine et granzyme, où la fonction des cellules T DN est équivalente entre les souris résistantes et prédisposées au diabète auto-immun. Ces résultats confirment que l'association au diabète auto-immun est due à une insuffisance en terme du nombre de cellules T DN, plutôt qu’à une déficience fonctionnelle. On démontre que les cellules T DN non-transgéniques éliminent des cellules B chargées avec des antigènes d'îlots, mais pas des cellules B chargées avec un antigène non reconnu, in vitro. Par ailleurs, on établit que le transfert des cellules T DN activées peut empêcher le développement du diabète auto-immun dans un modèle de souris non-transgénique. De plus, nous observons que les cellules T DN migrent aux îlots pancréatiques, et subissent une activation et une prolifération préférentielles au niveau des ganglions pancréatiques. D'ailleurs, le transfert des cellules T DN entraîne une diminution d'auto-anticorps spécifiques de l'insuline et de cellules B de centres germinatifs directement dans les îlots, ce qui corrèle avec les résultats décrits ci-dessus. Les résultats présentés dans cette thèse permettent de démontrer la fonction des cellules T DN in vitro et in vivo, ainsi que leur potentiel lié à la thérapie cellulaire pour le diabète auto-immun.

Decreased [ 3 H] YM-09151-2 binding to dopamine D2 receptors in the hypothalamus, brainstem and pancreatic islets of streptozotocin -induced diabetic rats

In the present study dopamine was measured in the hypothalamus, brainstem, pancreatic islets and plasma, using HPLC. Dopamine D2 receptor changes in the hypothalamus, brainstem and pancreatic islets were studied using [3H] YM-09151-2 in streptozotocin-induced diabetic and insulintreated diabetic rats. There was a significant decrease in dopatnine content in the hypothalamus (P<0.001), brainstem (P<0.001), pancreatic islets (P<0.001) and plasma (P<0.00I) in diabetic rats when compared to control. Scatchard analysis of [3H] YM-09151-2 in the hypothalamus of diabetic rats showed a significant decrease in Bax (P<0.001) and Kd, showing an increased affinity of D2 receptors when compared to control. Insulin treatment did not completely reverse the changes that occurred during diabetes. There was a significant decrease in B,nax (P<0.01) with decreased affinity in the brainstem of diabetic rats. The islet membrane preparation of diabetic rats showed a significant decrease (P<0.001) in the binding of [3H] YM-09 151-2 with decreased Kd (P<0.001) compared to control. The increase in affinity of D2 receptors in hypothalamus and pancreatic islets and the decreased affinity in brainstem were confirmed by competition analysis. Thus our results suggest that the decreased dopamine D, receptor function in the hypothalamus, brainstem and pancreas affects insulin secretion in diabetic rats, which has immense clinical relevance to the management of diabetes.

“Adrenergic receptors and monoamines in the brain and pancreatic islets of streptozotoein diabetic rats: Role in insulin secretion as a function of age”

I) To study the changes in the content of brain rrrorroamirres in streptozotocirr-irrduced tliabetes as a lirnction of age and to lirrd the role oliadrenal lrornroncs in diabetic state. 2) To assess the adrenergic receptor function in the brain stem ofstreptozotocin-induced diabetic rats ofdillerent ages. 3) To study the changes in the basal levels of second messenger cAMP in the brain stenr ofstreptozotocin-induced diabetic rats as a function of age. 4) To study the changes occurring in the content ofmorroamines and their metabolites in whole pancreas and isolated pancreatic islets of streptozotocin-diabetic rats as a function ofage and the effect of adrenal hormones. 5) To study the adrenergic receptors and basal levels of cAMP in isolated pancreatic islets in young and old streptozotoein-diabetic rats. 6) The in virro study of CAMP content in pancreatic islets of young and old rats and its ellect on glucose induced insulin secretion. 7) 'lhe in vitro study on the involvement of dopamine and corticosteroids in glucose induced insulin secretion in pancreatic islets as a function of age.