The pancreatic ß-cell in ageing: Implications in age-related diabetes.

The prevalence of type 2 diabetes (T2D) and impaired glucose tolerance (IGT) increases with ageing. T2D generally results from progressive impairment of the pancreatic islets to adapt ß-cell mass and function in the setting of insulin resistance and increased insulin demand. Several studies have shown an age-related decline in peripheral insulin sensitivity. However, a precise understanding of the pancreatic ß-cell response in ageing is still lacking. In this review, we summarize the age-related alterations, adaptations and/or failures of ß-cells at the molecular, morphological and functional levels in mouse and human. Age-associated alterations include processes such as ß-cell proliferation, apoptosis and cell identity that can influence ß-cell mass. Age-related changes also affect ß-cell function at distinct steps including electrical activity, Ca2+ signaling and insulin secretion, among others. We will consider the potential impact of these alterations and those mediated by senescence pathways on ß-cells and their implications in age-related T2D. Finally, given the great diversity of results in the field of ß-cell ageing, we will discuss the sources of this heterogeneity. A better understanding of ß-cell biology during ageing, particularly at older ages, will improve our insight into the contribution of ß-cells to age-associated T2D and may boost new therapeutic strategies.

The Effects of Aging on Male Mouse Pancreatic ß-Cell Function Involve Multiple Events in the Regulation of Secretion: Influence of Insulin Sensitivity.

Aging is associated with a decline in peripheral insulin sensitivity and an increased risk of impaired glucose tolerance and type 2 diabetes. During conditions of reduced insulin sensitivity, pancreatic ß cells undergo adaptive responses to increase insulin secretion and maintain euglycemia. However, the existence and nature of ß-cell adaptations and/or alterations during aging are still a matter of debate. In this study, we investigated the effects of aging on ß-cell function from control (3-month-old) and aged (20-month-old) mice. Aged animals were further categorized into 2 groups: high insulin sensitive (aged-HIS) and low insulin sensitive (aged-LIS). Aged-LIS mice were hyperinsulinemic, glucose intolerant, and displayed impaired glucose-stimulated insulin and C-peptide secretion, whereas aged-HIS animals showed characteristics in glucose homeostasis similar to controls. In isolated ß cells, we observed that glucose-induced inhibition of KATP channel activity was reduced with aging, particularly in the aged-LIS group. Glucose-induced islet NAD(P)H production was decreased in aged mice, suggesting impaired mitochondrial function. In contrast, voltage-gated Ca2+ currents were higher in aged-LIS ß cells, and pancreatic islets of both aged groups displayed increased glucose-induced Ca2+ signaling and augmented insulin secretion compared with controls. Morphological analysis of pancreas sections also revealed augmented ß-cell mass with aging, especially in the aged-LIS group, as well as ultrastructural ß-cell changes. Altogether, these findings indicate that aged mouse ß cells compensate for the aging-induced alterations in the stimulus-secretion coupling, particularly by adjusting their Ca2+ influx to ensure insulin secretion. These results also suggest that decreased peripheral insulin sensitivity exacerbates the effects of aging on ß cells.

The second-generation antipsychotic drug aripiprazole modulates the serotonergic system in pancreatic islets and induces beta cell dysfunction in female mice.

AIMS/HYPOTHESIS: Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity. METHODS: We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5-6.0 mg kg-1 day-1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques. RESULTS: Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction. CONCLUSIONS/INTERPRETATION: Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.

AAV8 Ins1-Cre can produce efficient ß-cell recombination but requires consideration of off-target effects.

In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on ß-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired ß-cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient ß-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of ß-cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice (Ins1-/-;Ins2f/f). AAV-mediated expression of Cre in ß-cells provides an effective alternative to transgenic approaches for inducible knockout studies.

SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes.

SNAP23 is the ubiquitous SNAP25 isoform that mediates secretion in non-neuronal cells, similar to SNAP25 in neurons. However, some secretory cells like pancreatic islet ß cells contain an abundance of both SNAP25 and SNAP23, where SNAP23 is believed to play a redundant role to SNAP25. We show that SNAP23, when depleted in mouse ß cells in vivo and human ß cells (normal and type 2 diabetes [T2D] patients) in vitro, paradoxically increased biphasic glucose-stimulated insulin secretion corresponding to increased exocytosis of predocked and newcomer insulin granules. Such effects on T2D Goto-Kakizaki rats improved glucose homeostasis that was superior to conventional treatment with sulfonylurea glybenclamide. SNAP23, although fusion competent in slower secretory cells, in the context of ß cells acts as a weak partial fusion agonist or inhibitory SNARE. Here, SNAP23 depletion promotes SNAP25 to bind calcium channels more quickly and longer where granule fusion occurs to increase exocytosis efficiency. ß Cell SNAP23 antagonism is a strategy to treat diabetes.

Morphological and functional adaptations of pancreatic alpha-cells during late pregnancy in the mouse.

BACKGROUND: Pregnancy represents a major metabolic challenge for the mother, and involves a compensatory response of the pancreatic beta-cell to maintain normoglycemia. However, although pancreatic alpha-cells play a key role in glucose homeostasis and seem to be involved in gestational diabetes, there is no information about their potential adaptations or changes during pregnancy. MATERIAL AND METHODS: Non-pregnant (controls) and pregnant C57BL/6 mice at gestational day 18.5 (G18.5) and their isolated pancreatic islets were used for in vivo and ex vivo studies, respectively. The effect of pregnancy hormones was tested in glucagon-secreting α-TC1.9 cells. Immunohistochemical analysis was performed in pancreatic slices. Glucagon gene expression was monitored by RT-qPCR. Glucagon secretion and plasma hormones were measured by ELISA. RESULTS: Pregnant mice on G18.5 exhibited alpha-cell hypertrophy as well as augmented alpha-cell area and mass. This alpha-cell mass expansion was mainly due to increased proliferation. No changes in alpha-cell apoptosis, ductal neogenesis, or alpha-to-beta transdifferentiation were found compared with controls. Pregnant mice on G18.5 exhibited hypoglucagonemia. Additionally, in vitro glucagon secretion at low glucose levels was decreased in isolated islets from pregnant animals. Glucagon content was also reduced. Experiments in α-TC1.9 cells indicated that, unlike estradiol and progesterone, placental lactogens and prolactin stimulated alpha-cell proliferation. Placental lactogens, prolactin and estradiol also inhibited glucagon release from α-TC1.9 cells at low glucose levels. CONCLUSIONS: The pancreatic alpha-cell in mice undergoes several morphofunctional changes during late pregnancy, which may contribute to proper glucose homeostasis. Gestational hormones are likely involved in these processes.

AAV GCG-EGFP, a new tool to identify glucagon-secreting α-cells.

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

Early overnutrition reduces Pdx1 expression and induces ß cell failure in Swiss Webster mice.

Childhood obesity and early rapid growth increase the risk for type 2 diabetes. Such early overnutrition can be modeled in mice by reducing litter size. We investigated the effects of early overnutrition and increased dietary fat intake on ß cell function in Swiss Webster mice. On a moderate-fat diet, early overnutrition accelerated weight gain and induced hyperinsulinemia in pups. Early overnutrition males exhibited higher ß cell mass but reduced islet insulin content and Pdx1 expression. Males had a high diabetes incidence that was increased by early overnutrition, characterized by a progressive increase in insulin secretion as well as ß cell death, indicated by histological analysis and increased circulating miR-375 levels. Females maintained normoglycemia throughout life. High-fat diet (HFD) increased diabetes incidence in males, whereas low-fat diet was completely protective. This protective effect was abolished in early overnutrition males transiently exposed to HFD in early life. Although Swiss Webster mice are not known to be diabetes-prone, the high diabetes incidence suggests an underlying genetic susceptibility that can be induced by overnutrition and increased dietary fat intake in early life. Thus, the nutritional environment in early life may impact long-term ß cell function and increase diabetes risk, particularly in genetically susceptible individuals.

Timing of Exposure and Bisphenol-A: Implications for Diabetes Development.

Bisphenol-A (BPA) is one of the most widespread endocrine disrupting chemicals (EDCs). It is used as the base compound in the production of polycarbonate and other plastics present in many consumer products. It is also used as a building block in epoxy can coating and the thermal paper of cash register receipts. Humans are consistently exposed to BPA and, in consequence, this compound has been detected in the majority of individuals examined. Over the last decade, an enlarging body of evidence has provided a strong support for the role of BPA in the etiology of diabetes and other metabolic disorders. Timing of exposure to EDCs results crucial since it has important implications on the resulting adverse effects. It is now well established that the developing organisms are particularly sensitive to environmental influences. Exposure to EDCs during early life may result in permanent adverse consequences, which increases the risk of developing chronic diseases like diabetes in adult life. In addition to that, developmental abnormalities can be transmitted from one generation to the next, thus affecting future generations. More recently, it has been proposed that gestational environment may also program long-term susceptibility to metabolic disorders in the mother. In the present review, we will comment and discuss the contributing role of BPA in the etiology of diabetes. We will address the metabolic consequences of BPA exposure at different stages of life and comment on the final phenotype observed in different whole-animal models of study.

Mitochondria as target of endocrine-disrupting chemicals: implications for type 2 diabetes.

Type 2 diabetes is a chronic, heterogeneous syndrome characterized by insulin resistance and pancreatic ß-cell dysfunction or death. Among several environmental factors contributing to type 2 diabetes development, endocrine-disrupting chemicals (EDCs) have been receiving special attention. These chemicals include a wide variety of pollutants, from components of plastic to pesticides, with the ability to modulate endocrine system function. EDCs can affect multiple cellular processes, including some related to energy production and utilization, leading to alterations in energy homeostasis. Mitochondria are primarily implicated in cellular energy conversion, although they also participate in other processes, such as hormone secretion and apoptosis. In fact, mitochondrial dysfunction due to reduced oxidative capacity, impaired lipid oxidation and increased oxidative stress has been linked to insulin resistance and type 2 diabetes. Herein, we review the main mechanisms whereby metabolism-disrupting chemical (MDC), a subclass of EDCs that disturbs energy homeostasis, cause mitochondrial dysfunction, thus contributing to the establishment of insulin resistance and type 2 diabetes. We conclude that MDC-induced mitochondrial dysfunction, which is mainly characterized by perturbations in mitochondrial bioenergetics, biogenesis and dynamics, excessive reactive oxygen species production and activation of the mitochondrial pathway of apoptosis, seems to be a relevant mechanism linking MDCs to type 2 diabetes development.

A Calcium-Dependent Chloride Current Increases Repetitive Firing in Mouse Sympathetic Neurons.

Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9'-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.

Endocrine-disrupting chemicals and the regulation of energy balance.

Energy balance involves the adjustment of food intake, energy expenditure and body fat reserves through homeostatic pathways. These pathways include a multitude of biochemical reactions, as well as hormonal cues. Dysfunction of this homeostatic control system results in common metabolism-related pathologies, which include obesity and type 2 diabetes mellitus. Metabolism-disrupting chemicals (MDCs) are a particular class of endocrine-disrupting chemicals that affect energy homeostasis. MDCs affect multiple endocrine mechanisms and thus different cell types that are implicated in metabolic control. MDCs affect gene expression and the biosynthesis of key enzymes, hormones and adipokines that are essential for controlling energy homeostasis. This multifaceted spectrum of actions precludes compensatory responses and favours metabolic disorders. Herein, we review the main mechanisms used by MDCs to alter energy balance. This work should help to identify new MDCs, as well as novel targets of their action.

GPR55 and the regulation of glucose homeostasis.

Pathophysiological conditions such as obesity and type 2 diabetes (T2D) are reportedly associated to over-activation of the endocannabinoid system (ECS). Therefore, modulation of the ECS offers potential therapeutic benefits on those diseases. GPR55, the receptor for L-α-lysophosphatidylinositol (LPI) that has also affinity for various cannabinoid ligands, is distributed at the central and peripheral level and it is involved in several physiological processes. This review summarizes the localization and role of GPR55 in tissues that are crucial for the regulation of glucose metabolism, and provides an update on its contribution in obesity and insulin resistance. Finally, the therapeutic potential of targeting the GPR55 receptor is also discussed.

GPR55: a new promising target for metabolism?

GPR55 is a G-protein-coupled receptor (GPCR) that has been identified as a new cannabinoid receptor. Given the wide localization of GPR55 in brain and peripheral tissues, this receptor has emerged as a regulator of multiple biological actions. Lysophosphatidylinositol (LPI) is generally accepted as the endogenous ligand of GPR55. In this review, we will focus on the role of GPR55 in energy balance and glucose metabolism. We will summarize its actions on feeding, nutrient partitioning, gastrointestinal motility and insulin secretion in preclinical models and the scarce data available in humans. The potential of GPR55 to become a new pharmaceutical target to treat obesity and type 2 diabetes, as well as the foreseeing difficulties are also discussed.

The role of autonomic efferents and uncoupling protein 1 in the glucose-lowering effect of leptin therapy.

OBJECTIVE: Leptin reverses hyperglycemia in rodent models of type 1 diabetes (T1D). Direct application of leptin to the brain can lower blood glucose in diabetic rodents, and can activate autonomic efferents and non-shivering thermogenesis in brown adipose tissue (BAT). We investigated whether leptin reverses hyperglycemia through a mechanism that requires autonomic innervation, or uncoupling protein 1 (UCP1)-mediated thermogenesis. METHODS: To examine the role of parasympathetic and sympathetic efferents in the glucose-lowering action of leptin, mice with a subdiaphragmatic vagotomy or 6-hydroxydopamine induced chemical sympathectomy were injected with streptozotocin (STZ) to induce hyperglycemia, and subsequently leptin treated. To test whether the glucose-lowering action of leptin requires activation of UCP1-mediated thermogenesis in BAT, we administered leptin in STZ-diabetic Ucp1 knockout (Ucp1 (-/-)) mice and wildtype controls. RESULTS: Leptin ameliorated STZ-induced hyperglycemia in both intact and vagotomised mice. Similarly, mice with a partial chemical sympathectomy did not have an attenuated response to leptin-mediated glucose lowering relative to sham controls, and showed intact leptin-induced Ucp1 expression in BAT. Although leptin activated BAT thermogenesis in STZ-diabetic mice, the anti-diabetic effect of leptin was not blunted in Ucp1 (-/-) mice. CONCLUSIONS: These results suggest that leptin lowers blood glucose in insulin-deficient diabetes through a manner that does not require parasympathetic or sympathetic innervation, and thus imply that leptin lowers blood glucose through an alternative CNS-mediated mechanism or redundant target tissues. Furthermore, we conclude that the glucose lowering action of leptin is independent of UCP1-dependent thermogenesis.

Acute stimulation of brain mu opioid receptors inhibits glucose-stimulated insulin secretion via sympathetic innervation.

Pancreatic insulin-secreting ß-cells express opioid receptors, whose activation by opioid peptides modulates hormone secretion. Opioid receptors are also expressed in multiple brain regions including the hypothalamus, where they play a role in feeding behavior and energy homeostasis, but their potential role in central regulation of glucose metabolism is unknown. Here, we investigate whether central opioid receptors participate in the regulation of insulin secretion and glucose homeostasis in vivo. C57BL/6J mice were acutely treated by intracerebroventricular (i.c.v.) injection with specific agonists for the three main opioid receptors, kappa (KOR), delta (DOR) and mu (MOR) opioid receptors: activation of KOR and DOR did not alter glucose tolerance, whereas activation of brain MOR with the specific agonist DAMGO blunted glucose-stimulated insulin secretion (GSIS), reduced insulin sensitivity, increased the expression of gluconeogenic genes in the liver and, consequently, impaired glucose tolerance. Pharmacological blockade of α2A-adrenergic receptors prevented DAMGO-induced glucose intolerance and gluconeogenesis. Accordingly, DAMGO failed to inhibit GSIS and to impair glucose tolerance in α2A-adrenoceptor knockout mice, indicating that the effects of central MOR activation on ß-cells are mediated via sympathetic innervation. Our results show for the first time a new role of the central opioid system, specifically the MOR, in the regulation of insulin secretion and glucose metabolism.

Disrupted Leptin Signaling in the Lateral Hypothalamus and Ventral Premammillary Nucleus Alters Insulin and Glucagon Secretion and Protects Against Diet-Induced Obesity.

Leptin signaling in the central nervous system, and particularly the arcuate hypothalamic nucleus, is important for regulating energy and glucose homeostasis. However, the roles of extra-arcuate leptin responsive neurons are less defined. In the current study, we generated mice with widespread inactivation of the long leptin receptor isoform in the central nervous system via Synapsin promoter-driven Cre (Lepr(flox/flox) Syn-cre mice). Within the hypothalamus, leptin signaling was disrupted in the lateral hypothalamic area (LHA) and ventral premammillary nucleus (PMV) but remained intact in the arcuate hypothalamic nucleus and ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, and nucleus of the tractus solitarius. To investigate the role of LHA/PMV neuronal leptin signaling, we examined glucose and energy homeostasis in Lepr(flox/flox) Syn-cre mice and Lepr(flox/flox) littermates under basal and diet-induced obese conditions and tested the role of LHA/PMV neurons in leptin-mediated glucose lowering in streptozotocin-induced diabetes. Lepr(flox/flox) Syn-cre mice did not have altered body weight or blood glucose levels but were hyperinsulinemic and had enhanced glucagon secretion in response to experimental hypoglycemia. Surprisingly, when placed on a high-fat diet, Lepr(flox/flox) Syn-cre mice were protected from weight gain, glucose intolerance, and diet-induced hyperinsulinemia. Peripheral leptin administration lowered blood glucose in streptozotocin-induced diabetic Lepr(flox/flox) Syn-cre mice as effectively as in Lepr(flox/flox) littermate controls. Collectively these findings suggest that leptin signaling in LHA/PMV neurons is not critical for regulating glucose levels but has an indispensable role in the regulation of insulin and glucagon levels and, may promote the development of diet-induced hyperinsulinemia and weight gain.

Glucagon-Like Peptide 1 Analogs and their Effects on Pancreatic Islets.

Glucagon-like peptide 1 (GLP-1) exerts many actions that improve glycemic control. GLP-1 stimulates glucose-stimulated insulin secretion and protects ß cells, while its extrapancreatic effects include cardioprotection, reduction of hepatic glucose production, and regulation of satiety. Although an appealing antidiabetic drug candidate, the rapid degradation of GLP-1 by dipeptidyl peptidase 4 (DPP-4) means that its therapeutic use is unfeasible, and this prompted the development of two main GLP-1 therapies: long-acting GLP-1 analogs and DPP-4 inhibitors. In this review, we focus on the pancreatic effects exerted by current GLP-1 derivatives used to treat diabetes. Based on the results from in vitro and in vivo studies in humans and animal models, we describe the specific actions of GLP-1 analogs on the synthesis, processing, and secretion of insulin, islet morphology, and ß cell proliferation and apoptosis.