Ivan Quesada

Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes

Glucocorticoids (GCs) are broadly prescribed for numerous pathological conditions because of their anti-inflammatory, antiallergic and immunosuppressive effects, among other actions. Nevertheless, GCs can produce undesired diabetogenic side effects through interactions with the regulation of glucose homeostasis. Under conditions of excess and/or long-term treatment, GCs can induce peripheral insulin resistance (IR) by impairing insulin signalling, which results in reduced glucose disposal and augmented endogenous glucose production. In addition, GCs can promote abdominal obesity, elevate plasma fatty acids and triglycerides, and suppress osteocalcin synthesis in bone tissue. In response to GC-induced peripheral IR and in an attempt to maintain normoglycaemia, pancreatic β-cells undergo several morphofunctional adaptations that result in hyperinsulinaemia. Failure of β-cells to compensate for this situation favours glucose homeostasis disruption, which can result in hyperglycaemia, particularly in susceptible individuals. GC treatment does not only alter pancreatic β-cell function but also affect them by their actions that can lead to hyperglucagonaemia, further contributing to glucose homeostasis imbalance and hyperglycaemia. In addition, the release of other islet hormones, such as somatostatin, amylin and ghrelin, is also affected by GC administration. These undesired GC actions merit further consideration for the design of improved GC therapies without diabetogenic effects. In summary, in this review, we consider the implication of GC treatment on peripheral IR, islet function and glucose homeostasis.

Bisphenol-A: a new diabetogenic factor?

The aim of this review was to analyze the potential effects of environmental chemicals on homeostatic control related to glycemia and energy balance. Many of the environmental chemicals can mimic or interfere with the action of hormones and are generally referred to as “endocrine disruptors”. Among these compounds, polychlorinated biphenyls, dioxins, phthalates and bisphenol-A have been correlated with alterations in blood glucose homeostasis in humans. In rodents it has been demonstrated that small doses of bisphenol-A have profound effects on glucose metabolism. Therefore, this altered blood glucose homeostasis may enhance the development of type 2 diabetes.

Involvement of the clock gene Rev-erb alpha in the regulation of glucagon secretion in pancreatic alpha-cells.

Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60–70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.

Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

Lysophosphatidic acid induces Ca2+ mobilization and c-Myc expression in mouse embryonic stem cells via the phospholipase C pathway.

Embryonic stem cells (ESC) are pluripotent and could be maintained in vitro in a self-renewing state indefinitely, at the same time preserving their potential to differentiate towards more specific lineages. Despite the progress in the field, the complex network of signalling cascades involved in the maintenance of the self-renewing and pluripotent state remains not fully understood. In the present study, we have investigated the role of lysophosphatidic acid (LPA), a potent mitogen present in serum, in Ca(2+) signalling and early gene activation in mouse ESC (mESC). In these cells, we detected the expression of the G-protein coupled LPA receptor subtypes LPA(1), LPA(2) and LPA(3). Using fluorescence Ca(2+) imaging techniques, we showed that LPA induced an increase in intracellular Ca(2+) concentration. This increase was also observed in the absence of extracellular Ca(2+), suggesting the involvement of internal stores. Pre-treatment with BAPTA-AM, thapsigargin or U-73122 efficiently blocked this Ca(2+) release, indicating that LPA was evoking Ca(2+) mobilization from the endoplasmic reticulum via the phospholipase C (PLC) pathway. Interestingly, this signalling cascade initiated by LPA was involved in inducing the expression of the Ca(2+)-dependent early response gene c-myc, a key gene implicated in ESC self-renewal and pluripotency. Additionally, LPA increased the proliferation rate of mESC. Our findings therefore outline the physiological role of LPA in mESC.

Reduced Insulin Secretion in Protein Malnourished Mice Is Associated with Multiple Changes in the β-Cell Stimulus-Secretion Coupling

The mechanism by which protein malnutrition impairs glucose-stimulated insulin secretion in the pancreatic beta-cell is not completely known but may be related to alterations in the signaling events involved in insulin release. Here, we aimed to study the stimulus-secretion coupling of beta-cells from mice fed with low-protein (LP) diet or normal-protein (NP) diet for 8 wk after weaning. Patch-clamp measurements in isolated cells showed that beta-cells from LP mice had a resting membrane potential that was more hyperpolarized than controls. Additionally, depolarization and generation of action potentials in response to stimulatory glucose concentrations were also impaired in beta-cells of LP mice. All these alterations in the LP group were most likely attributed to higher ATP-dependent K(+) (K(ATP)) channel activity in resting conditions and lower efficiency of glucose to induce the closure of these channels. Moreover, a Western blot analysis revealed higher protein levels of the sulphonylurea receptor of the K(ATP) channel in islets of LP mice. Because beta-cell Ca(2+) signals depend on electrical activity, intracellular Ca(2+) oscillations were measured by fluorescence microscopy in intact islets, indicating a lower response to glucose in the LP group. Finally, cell-to-cell synchrony of Ca(2+) signals was analyzed by confocal microscopy. Islets from LP mice exhibited a decreased level of coupling among beta-cells, which was probably due to the low expression levels of connexin 36. Therefore, low-protein diet leads to several alterations in the stimulus-secretion coupling of pancreatic beta-cells that might explain the diminished insulin secretion in response to glucose in this malnutrition state.

Leptin downregulates expression of the gene encoding glucagon in alphaTC1-9 cells and mouse islets

Aims/hypothesisLeptin, released by adipocytes, can modulate glucose homeostasis through direct actions on pancreatic alpha and beta cells. Although this hormone rapidly regulates alpha cell exocytosis, its long-term effects on glucagon gene expression are currently unknown.MethodsWe analysed glucagon mRNA levels and protein content in alphaTC1-9 cells and isolated mouse islets cultured with leptin, as well as in islets from mice treated in vivo with leptin. We also studied the involvement of the signal transducers and activators of transcription (STAT) pathway by western blot, immunofluorescence and interference RNA.ResultsLeptin incubation (0.0625–18.75xa0nmol/l) for 24xa0h inhibited glucagon gene expression in alphaTC1-9 cells. This inhibitory effect was also observed in isolated mouse islets cultured with leptin, as well as in islets from mice treated with leptin for 5xa0days. In contrast, no changes were detected in islets from db/db mice, which lack leptin receptors. Leptin treatment also reduced the glucagon protein content in alphaTC1-9 cells and mouse islets. Moreover, leptin induced phosphorylation of STAT3 and its translocation to the nucleus, which was confirmed by western blot analysis in alphaTC1-9 cells and by immunofluorescence in isolated alpha cells. Interestingly, the effect of leptin on glucagon mRNA levels was significantly reduced by Stat3 knockdown. In contrast, pharmacological inhibition of the phosphoinositide 3-kinase pathway did not affect leptin actions.Conclusions/interpretationOur results demonstrate that leptin can regulate glucagon gene expression in alpha cells via a STAT3 pathway, and are important for understanding the role of leptin in glucose homeostasis.

Clock genes, pancreatic function, and diabetes

Circadian physiology is responsible for the temporal regulation of metabolism to optimize energy homeostasis throughout the day. Disturbances in the light/dark cycle, sleep/wake schedule, or feeding/activity behavior can affect the circadian function of the clocks located in the brain and peripheral tissues. These alterations have been associated with impaired glucose tolerance and type 2 diabetes. Animal models with molecular manipulation of clock genes and genetic studies in humans also support these links. It has been demonstrated that the endocrine pancreas has an intrinsic self-sustained clock, and recent studies have revealed an important role of clock genes in pancreatic β cells, glucose homeostasis, and diabetes.

Glucagon Increases Beating Rate but Not Contractility in Rat Right Atrium. Comparison with Isoproterenol.

This study evaluated the chronotropic and inotropic responses to glucagon in spontaneously beating isolated right atria of rat heart. For comparison, we also investigated the effects resulting from stimulating β-adrenoceptors with isoproterenol in this tissue. Isoproterenol increased both atrial frequency and contractility but glucagon only enhanced atrial rate. The transcript levels of glucagon receptors were about three times higher in sinoatrial node than in the atrial myocardium. Chronotropic responses to glucagon and isoproterenol were blunted by the funny current (If) inhibitor ZD 7288. Inhibitors of protein kinase A, H-89 and KT-5720 reduced the chronotropic response to glucagon but not to isoproterenol. Inhibition of ryanodine receptors and calcium/calmodulin dependent protein kinase II (important regulators of sarcoplasmic reticulum Ca2+ release), with ruthenium red and KN-62 respectively, failed to alter chronotropic responses of either glucagon or isoproterenol. Non selective inhibition of phosphodiesterase (PDE) with 3-isobutylmethylxantine or selective inhibition of PDE3 or PDE4 with cilostamide or rolipram respectively did not affect chronotropic effects of glucagon or isoproterenol. Our results indicate that glucagon increases beating rate but not contractility in rat right atria which could be a consequence of lower levels of glucagon receptors in atrial myocardium than in sinoatrial node. Chronotropic responses to glucagon or isoproterenol are mediated by If current but not by sarcoplasmic reticulum Ca2+ release, neither are regulated by PDE activity.

Role of the clock gene Rev-erbα in metabolism and in the endocrine pancreas.

Several hormones are regulated by circadian rhythms to adjust the metabolism to the light/dark cycles and feeding/activity patterns throughout the day. Circadian rhythms are mainly governed by the central clock located in the suprachiasmatic nucleus but also by clocks present in peripheral organs, like the endocrine pancreas. Plasma glucose levels and the main pancreatic hormones insulin and glucagon also exhibit daily variations. Alterations in circadian rhythms are associated with metabolic disturbances and pathologies such as obesity and diabetes. The molecular components of central and peripheral clocks and their regulatory mechanisms are well established. Among the different clock genes, Rev‐erbα is considered one of the key links between circadian rhythms and metabolism. Rev‐erbα is a critical part of a negative feedback loop in the core circadian clock and modulates the clock oscillatory properties. In addition, Rev‐erbα plays an important role in the regulation of lipid and glucose metabolism, thermogenesis, adipocyte and muscle differentiation as well as mitochondrial function. In the endocrine pancreas, Rev‐erbα regulates insulin and glucagon secretion and pancreatic β‐cell proliferation. In the present review, we discuss all these subjects and, particularly, the role of the clock gene Rev‐erbα in the endocrine pancreas.