Clinical Approaches to Preserve beta-Cell Function in Diabetes

In type 2 diabetes (DM2) there is progressive deterioration in beta-cell function and mass. It was found that islet function was about 50% of normal at the time of diagnosis and reduction in beta-cell mass of about 60% at necropsy (accelerated apoptosis). Among the interventions to preserve the beta-cells, those to lead to short-term improvement of beta-cell secretion are weight loss, metformin, sulfonylureas, and insulin. The long-term improvement was demonstrated with short-term intensive insulin therapy of newly diagnosed DM2, the use of antiapoptotic drugs such as glitazones, and the use of glucagon-like peptide-1 receptor agonists (GLP-1 mimetics), not inactivated by the enzyme dipeptidyl peptidase 4 and/or to inhibit that enzyme (GLP-1 enhancers). The incretin hormones are released from the gastrointestinal tract in response to nutrient ingestion to enhance glucose-dependent insulin secretion from the pancreas and overall maintenance of glucose homeostasis. From the two major incretins, GLP-1 and GIP (glucose-dependent insulinotropic polypeptide), only the first one or its mimetics or enhancers can be used for treatment. The GLP-1 mimetics exenatide and liraglutide as well as the DPP4 inhibitors (sitagliptin and vildagliptin) were approved for treatment of DM2.

Complexin I regulates glucose-induced secretion in pancreatic beta-cells.

The neuronal-specific protein complexin I (CPX I) plays an important role in controlling the Ca(2+)-dependent neurotransmitter release. Since insulin exocytosis and neurotransmitter release rely on similar molecular mechanisms and that pancreatic beta-cells and neuronal cells share the expression of many restricted genes, we investigated the potential role of CPX I in insulin-secreting cells. We found that pancreatic islets and several insulin-secreting cell lines express high levels of CPX I. The beta-cell expression of CPX I is mediated by the presence of a neuron restrictive silencer element located within the regulatory region of the gene. This element bound the transcriptional repressor REST, which is found in most cell types with the exception of mature neuronal cells and beta-cells. Overexpression of CPX I or silencing of the CPX I gene (Cplx1) by RNA interference led to strong impairment in beta-cell secretion in response to nutrients such as glucose, leucine and KCl. This effect was detected both in the early and the sustained secretory phases but was much more pronounced in the early phase. We conclude that CPX I plays a critical role in beta-cells in the control of the stimulated-exocytosis of insulin.

Low levels of sex hormone-binding globulin and hyperproinsulinemia as markers of increased pancreatic ß-cell demand in men

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic ß-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic ß-cell secretion in men with different body compositions. Eighteen young men (30.0 ± 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P<0.05), proinsulin (r = -0.47, P<0.05), C-peptide (r = -0.55, P<0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P<0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P<0.05). When subjects were divided into obese (BMI >28 kg/m2, N = 8) and nonobese (BMI £25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic ß-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic ß-cell demand.

Plasma free fatty acids do not provide the link between obesity and insulin resistance or β-cell dysfunction: results of the Reading, Imperial, Surrey, Cambridge, Kings (RISCK) study

Aims To investigate the relationship between adiposity and plasma free fatty acid levels and the influence of total plasma free fatty acid level on insulin sensitivity and β-cell function. Methods An insulin sensitivity index, acute insulin response to glucose and a disposition index, derived from i.v. glucose tolerance minimal model analysis and total fasting plasma free fatty acid levels were available for 533 participants in the Reading, Imperial, Surrey, Cambridge, Kings study. Bivariate correlations were made between insulin sensitivity index, acute insulin response to glucose and disposition index and both adiposity measures (BMI, waist circumference and body fat mass) and total plasma free fatty acid levels. Multivariate linear regression analysis was performed, controlling for age, sex, ethnicity and adiposity. Results After adjustment, all adiposity measures were inversely associated with insulin sensitivity index (BMI: β = −0.357; waist circumference: β = −0.380; body fat mass: β = −0.375) and disposition index (BMI: β = −0.215; waist circumference: β = −0.248; body fat mass: β = −0.221) and positively associated with acute insulin response to glucose [BMI: β = 0.200; waist circumference: β = 0.195; body fat mass β = 0.209 (P values <0.001)]. Adiposity explained 13, 4 and 5% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. After adjustment, no adiposity measure was associated with free fatty acid level, but total plasma free fatty acid level was inversely associated with insulin sensitivity index (β = −0.133), acute insulin response to glucose (β = −0.148) and disposition index [β = −0.218 (P values <0.01)]. Plasma free fatty acid concentration accounted for 1.5, 2 and 4% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. Conclusions Plasma free fatty acid levels have a modest negative association with insulin sensitivity, β-cell secretion and disposition index but no association with adiposity measures. It is unlikely that plasma free fatty acids are the primary mediators of obesity-related insulin resistance or β-cell dysfunction.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Vagotomy Ameliorates Islet Morphofunction And Body Metabolic Homeostasis In Msg-obese Rats.

The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Colleters in monocots: New record for Orchidaceae

Colleters are widely occurring in eudicots showing relevant taxonomic importance in several families. Nevertheless, there are few records in monocots, restricted to only one description of these glands in Orchidaceae. The genus Oncidium is polyphyletic, currently the subject of taxonomic studies. In this context, the secretory structures can be an important diagnostic character that may help in the delineation of this group. O. flexuosum Sims presents colleters in vegetative - leaf primordium of protocorms, apical and axillary buds in the mature rhizomes - and reproductive organs - at the base of bracts, bracteoles and sepals. All the colleters observed are finger-like trichomes, composed of two uniseriated cells, where the apical one is elongated and possesses dense cytoplasm. The exsudate accumulates in a subcuticular space. causing displacement of the cuticle. Histochemical tests indicate the presence of mucilage in association with lipophilic and proteinic compounds inside the secretory cell. Secretion is abundant, hyaline and slightly viscous. The localization of the trichomes and their exsudate indicate the involvement of these colleters with the protection of meristematic regions in vegetative and reproductive organs. These results can be useful in the taxonomy of the genus Oncidium and for future studies about colleters in monocots. (C) 2010 Elsevier GmbH. All rights reserved.

GLUT2, glucose sensing and glucose homeostasis.

The glucose transporter isoform GLUT2 is expressed in liver, intestine, kidney and pancreatic islet beta cells, as well as in the central nervous system, in neurons, astrocytes and tanycytes. Physiological studies of genetically modified mice have revealed a role for GLUT2 in several regulatory mechanisms. In pancreatic beta cells, GLUT2 is required for glucose-stimulated insulin secretion. In hepatocytes, suppression of GLUT2 expression revealed the existence of an unsuspected glucose output pathway that may depend on a membrane traffic-dependent mechanism. GLUT2 expression is nevertheless required for the physiological control of glucose-sensitive genes, and its inactivation in the liver leads to impaired glucose-stimulated insulin secretion, revealing a liver-beta cell axis, which is likely to be dependent on bile acids controlling beta cell secretion capacity. In the nervous system, GLUT2-dependent glucose sensing controls feeding, thermoregulation and pancreatic islet cell mass and function, as well as sympathetic and parasympathetic activities. Electrophysiological and optogenetic techniques established that Glut2 (also known as Slc2a2)-expressing neurons of the nucleus tractus solitarius can be activated by hypoglycaemia to stimulate glucagon secretion. In humans, inactivating mutations in GLUT2 cause Fanconi-Bickel syndrome, which is characterised by hepatomegaly and kidney disease; defects in insulin secretion are rare in adult patients, but GLUT2 mutations cause transient neonatal diabetes. Genome-wide association studies have reported that GLUT2 variants increase the risks of fasting hyperglycaemia, transition to type 2 diabetes, hypercholesterolaemia and cardiovascular diseases. Individuals with a missense mutation in GLUT2 show preference for sugar-containing foods. We will discuss how studies in mice help interpret the role of GLUT2 in human physiology.

Mécanismes moléculaires de la sécrétion d'insuline : implication de Slac2c/MyRIP, protéine effectrice de Rab27a, et rôle des phosphoinositides dans le processus d'exocytose

Résumé : La sécrétion de l'insuline en réponse au glucose circulant dans le sang est la fonction principale de la cellule β. La perte de cette fonction est une des caractéristiques du diabète de type 2. L'exocytose est une fonction cellulaire indispensable au renouvellement des composants lipidiques et protéiques de la membrane cellulaire, à la communication entre les cellules et au maintien d'un environnement adéquat. On peut distinguer deux types d'exocytose : l'exocytose constitutive et l'exocytose régulée. Cette dernière est déclenchée par des stimuli externes. L'exocytose régulée est contrôlée au niveau de la fusion des vésicules de sécrétion avec la membrane plasmique. Certains composants moléculaires impliqués dans ce processus font partie de la famille des GTPases Rab. Les deux membres de cette famille impliqués sont Rab3 et Rab27. Nous avons étudié le rôle de la GTPase Rab27 dans les cellules INS-1E, une lignée cellulaire pancréatique β qui sécrète de l'insuline de façon régulée. Nous avons trouvé que la diminution d'expression de la protéine en utilisant le technique de « RNA interference » diminue la sécrétion stimulée, mais que la distribution des granules n'est nullement affectées par ce changement d'activité intrinsèque. Un des effecteurs identifiés de cette GTPase est Slac2c/MyRIP. Cette protéine possède plusieurs domaines fonctionnels dont un qui lui permet de se lier à l'actine, constituant du cytosquelette cellulaire. L'ensemble de nos résultats suggèrent que Rab27 et MyRIP font partie d'un complexe permettant l'interaction de la granule de sécrétion avec le cytosquelette d'actine corticale et participent à la régulation des dernières étapes de l'exocytose d'insuline. Ensuite, nous avons étudié les phosphoinositides (PI). Les phosphoinositides sont d'importantes molécules impliquées dans le régulation du trafic vésiculaire. Nous avons trouvé que le phosphatidylinosito1-4-phosphate (PI4P) et le phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) augmentent la sécrétion sous l'action de 10µM de Ca2+ dans les cellules INS-1E perméabilisées avec la streptolysine-O. En plus, nous avons démontré que l'exocytose est diminuée dans les cellules intactes exprimant une protéine qui séquestre le PI(4,5)P2. Une diminution similaire est observée en diminuant l'expression de deux enzymes impliquées dans la production du PI(4,5)P2, la PI4Kinase β type III et la PIP5Kinase γ type I. Pour clarifier le mécanisme d'action des PI, nous avons investigué l'implication de trois cibles potentielles des PI, la PLD1, CAPS1 et Mint1. Pour ce faire, nous avons réduit le niveau d'expression endogène de ces protéines, ce qui inhibe la libération d'hormones provoquée par le glucose. Tout ceci indique donc que la production du PI(4,5)P2 est nécessaire pour le contrôle de la sécrétion et suggère qu'une partie de l'effet du PI sur la sécrétion pourrait être exercé par l'activation de la PLD1, CAPS1 et Mint1. Abstract Insulin release from pancreatic β-cells plays an essential role in the achievement of blood glucose homeostasis and defects in the regulation of this process lead to profound metabolic disorders and hyperglycaemia (eg. type 2 diabetes). Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment¬-to-moment requirements of the organism. Some of the molecular components involved in this process have been identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells. We investigated the involvement of Rab27 GTPase in the secretory process of the insulin-secreting cell line INS-1E. We found that selective reduction of Rab27 expression by RNA interference did not alter granule distribution but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors revealed that Slac2c/MyRIP is associated with granules and attenuation of Slac2c expression severely impaired hormone release. This protein contains several functional domains, including, a binding domain for the cellular cytoskeleton constituent actin. Taken together our data suggest the Rab27 and MyRIP are part of a complex mediating the interaction of secretory granules with cortical cytoskeleton and participate to the regulation of the final steps in insulin exoctytosis. In the second part of the thesis, we studied phosphoinositides (PI). Phosphoinositides are important molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinosito1-4-phosphate (PI4P) and phosphatidylinosito1-4,5-biphosphate (PI(4,5)P2) increase the secretory response triggered by 10µM Ca2+ in streptolysin-O permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P2. A similar decrease was observed after silencing of two enzymes involved in PI(4,5)P2 production, type III PI4Kinase β and type I PIP5Kinase γ, by RNA interference. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of exocytosis of three potential PI targets, PLD1, CAPS1 and Mint1. Transfection of cells with silencers capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose. Our data indicate that the production PI(4,5)P2 is necessary for proper control of p-cell secretion and suggest that at least part of the effects of PI on insulin exocytosis could be exerted through the activation of PLD1, CAPS1 and Mint1.

Central control of glucose homeostasis: the brain--endocrine pancreas axis.

A large body of data gathered over the last decades has delineated the neuronal pathways that link the central nervous system with the autonomic innervation of the endocrine pancreas, which controls alpha- and beta-cell secretion activity and mass. These are important regulatory functions that are certainly keys for preserving the capacity of the endocrine pancreas to control glucose homeostasis over a lifetime. Identifying the cells involved in controlling the autonomic innervation of the endocrine pancreas, in response to nutrient, hormonal and environmental cues and how these cues are detected to activate neuronal activity are important goals of current research. Elucidation of these questions may possibly lead to new means for preserving or restoring defects in insulin and glucagon secretion associated with type 2 diabetes.

Bradykinin enhances membrane electrical activity of pancreatic beta cells in the presence of low glucose concentrations

In most of cells bradykinin (BK) induces intracellular calcium mobilization. In pancreatic beta cells intracellular calcium is a major signal for insulin secretion. In these cells, glucose metabolism yields intracellular ATP which blocks membrane potassium channels. The membrane depolarizes, voltage-dependent Ca2+ channels are activated and the intracellular calcium load allows insulin secretion. Repolarization occurs due to activation of the Ca2+-dependent K+ channel. The insulin secretion depends on the integrity of this oscillatory process (bursts). Therefore, we decided to determine whether BK (100 nM) induces bursts in the presence of a non-stimulatory glucose concentration (5.6 mM). During continuous membrane voltage recording, our results showed that bursts were obtained with 11 mM glucose, blocked with 5.6 mM glucose and recovered with 5.6 mM glucose plus 100 nM BK. Thus, the stimulatory process obtained in the presence of BK and of a non-stimulatory concentration of glucose in the present study suggests that BK may facilitate the action of glucose on beta cell secretion.

Vagotomy ameliorates islet morphofunction and body metabolic homeostasis in MSG-obese rats

The parasympathetic nervous system is important for β-cell secretion and mass regulation. Here, we characterized involvement of the vagus nerve in pancreatic β-cell morphofunctional regulation and body nutrient homeostasis in 90-day-old monosodium glutamate (MSG)-obese rats. Male newborn Wistar rats received MSG (4 g/kg body weight) or saline [control (CTL) group] during the first 5 days of life. At 30 days of age, both groups of rats were submitted to sham-surgery (CTL and MSG groups) or subdiaphragmatic vagotomy (Cvag and Mvag groups). The 90-day-old MSG rats presented obesity, hyperinsulinemia, insulin resistance, and hypertriglyceridemia. Their pancreatic islets hypersecreted insulin in response to glucose but did not increase insulin release upon carbachol (Cch) stimulus, despite a higher intracellular Ca2+ mobilization. Furthermore, while the pancreas weight was 34% lower in MSG rats, no alteration in islet and β-cell mass was observed. However, in the MSG pancreas, increases of 51% and 55% were observed in the total islet and β-cell area/pancreas section, respectively. Also, the β-cell number per β-cell area was 19% higher in MSG rat pancreas than in CTL pancreas. Vagotomy prevented obesity, reducing 25% of body fat stores and ameliorated glucose homeostasis in Mvag rats. Mvag islets demonstrated partially reduced insulin secretion in response to 11.1 mM glucose and presented normalization of Cch-induced Ca2+ mobilization and insulin release. All morphometric parameters were similar among Mvag and CTL rat pancreases. Therefore, the higher insulin release in MSG rats was associated with greater β-cell/islet numbers and not due to hypertrophy. Vagotomy improved whole body nutrient homeostasis and endocrine pancreatic morphofunction in Mvag rats.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)). (C) 2008 Elsevier B.V. All rights reserved.

Salivary glands of Amblyomma cajennense (Acari: Ixodidae): a histological and an ultrastructural overview

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)