Studies indicate that the pesticide malathion may have a role in diabetes. Herein, we determined the effects of different concentrations of malathion on survival, ultrastructure, and electrophysiologic islet cell parameters. Acutely, high concentrations of malathion (0.5 or 1 mM) increased cell death in rat islet cells, while low con-centrations (0.1 mM) caused signs of cell damage in pancreatic alpha and beta cells. Exposure of RINm5F cells to malathion for 24 or 48 h confirmed the reduction in beta-cell viability at lower concentrations (0.001-100 mu M). Chronic exposure of mouse pancreatic alpha and beta cells to 3 nM of malathion led to increased voltage-gated K+ (Kv) currents in alpha-cells. Our findings show a time and concentration dependency for the malathion effect on the reduction of islet cell viability and indicate that pancreatic alpha cells are more sensitive to malathion effects on Kv currents and cell death.

This data article contains supporting information regarding the research article entitled “High butter-fat diet and bisphenol A additively impair male rat spermatogenesis” (P. Tarapore, M. Hennessy, D. Song, J. Ying, B. Ouyang, V. Govindarajah, et al.,) [1]. Sprague–Dawley females were fed AIN, high fat butter, 17α-ethinyl estradiol, or high fat butter plus four bisphenol A doses (2500 µg/kg bw-d, 250 µg/kg bw-d, 25 µg/kg bw-d, and 2.5 µg/kg bw-d) before and during pregnancy. All diets were switched to AIN after the pups were born. Male offspring received testosterone (T)- and estradiol-17β (E2)-filled implants from postnatal day 70–210 for 20 weeks (T+E2 rat model). The testes were weighed, and examined for impairments in spermatogenesis.

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.

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 Reverb 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.

Leptin, 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.We 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.Leptin incubation (0.0625-18.75 nmol/l) for 24 h 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 5 days. 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.Our 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.

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.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme expressed in pancreatic islets responsible for the degradation of proteoglycans such as perlecan and dermatan sulfate. Previous findings of our group demonstrated the involvement of IDS in the normal pathway of lysosomal degradation of secretory peptides, suggesting a role of this enzyme in beta-cell secretory functionality. The present study was undertaken to characterize the effect of IDS overexpression on insulin release. INS1E cells were transiently transfected with a construct encoding human IDS (hIDS). hIDS overexpression was associated with a gain of function detected by a reduction in heparan sulfate content. hIDS potentiated the glucose-stimulated insulin secretory response compared with controls (61%) with no changes in insulin mRNA levels or insulin peptide content. Results on quantification of the exocytotic process showed a significant increase in hIDS-transfected cells compared with controls. Furthermore, ultramorphological analysis demonstrated an increase in the number of granules in the immediate vicinity of the plasma membrane in hIDS-transfected cells and a decrease in total vesicles per square micrometer. hIDS overexpression induced phosphorylation of protein kinase C (PKC) alpha and its newly myristoylated alanine-rich C kinase substrate, MARCKS. We conclude that IDS has a role in glucose-stimulated insulin secretion via a mechanism that involves the activation of exocytosis through phosphorylation of PKCalpha and MARCKS.

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca2+ signal. However, the source of Ca2+ in these cells has not been established. The present studies indicate that Phaeocystis’ secretory granules work as an intracellular Ca2+ oscillator. Optical tomography reveals that photo-stimulation induces InsP3-triggered periodic lumenal [Ca2+] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca2+] that trigger exocytosis. This Ca2+ dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca2+-sensitive ion channels located on the granule membrane: an InsP3-receptor-Ca2+ channel, and an apamin-sensitive K+ channel.

Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic alpha-, beta-, and delta-cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic beta-cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non-beta-cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified alpha-, beta-, and delta-cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in beta- and delta-cell populations in a dose-dependent manner. Conversely, no significant responses were observed in alpha-cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca2+ signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the alpha-, beta-, and delta-cell populations, which might be important in the signal transduction events that lead to hormone release.

The stimulus-secretion coupling for insulin secretion by pancreatic β cells in response to high glucose involves synchronic cytosolic calcium oscillations driven by bursting electrical activity. Calcium inside organelles can regulate additional functions, but analysis of subcellular calcium signals, specially at the single cell level, has been hampered for technical constrains. Here we have monitored nuclear calcium oscillations by bioluminescence imaging of targeted aequorin in individual cells within intact islets of Langerhans as well as in the whole islet. We find that glucose generates a pattern of nuclear calcium oscillations resembling those found in the cytosol. Some cells showed synchronous nuclear calcium oscillations suggesting that the islet of Langerhans may also regulate the activation of Ca2+-responsive nuclear processes, such as gene transcription, in a coordinated, synchronic manner. The nuclear Ca2+ oscillations are due to bursting electrical activity and activation of plasma membrane voltage-gated Ca2+ channels with little or no contribution of calcium release from the intracellular Ca2+ stores. Irregularities in consumption of aequorins suggests that depolarization may generate formation of steep Ca2+ gradients in both the cytosol and the nucleus, but further research is required to investigate the role of such high [Ca2+] microdomains.

The phenomenology of nuclear Ca(2+) dynamics has experienced important progress revealing the broad range of cellular processes that it regulates. Although several agonists can mobilize Ca(2+) from storage in the nuclear envelope (NE) to the intranuclear compartment (INC), the mechanisms of Ca(2+) signaling in the nucleus still remain uncertain. Here we report that the NE/INC complex can function as an inositol-1,4,5-trisphosphate (InsP(3))-controlled Ca(2+) oscillator. Thin optical sectioning combined with fluorescent labeling of Ca(2+) probes show in cultured airway epithelial ciliated cells that ATP can trigger periodic oscillations of Ca(2+) in the NE ([Ca(2+)](NE)) and corresponding pulses of Ca(2+) release to the INC. Identical results were obtained in InsP(3)-stimulated isolated nuclei of these cells. Our data show that [Ca(2+)](NE) oscillations and Ca(2+) release to the INC result from the interplay between the Ca(2+)/K(+) ion-exchange properties of the intralumenal polyanionic matrix of the NE and two Ca(2+)-sensitive ion channels-an InsP(3)-receptor-Ca(2+) channel and an apamin-sensitive K(+) channel. A similar Ca(2+) signaling system operating under the same functional protocol and molecular hardware controls Ca(2+) oscillations and release in/to the endoplasmic reticulum/cytosol and in/to the granule/cytosol complexes in airway and mast cells. These observations suggest that these intracellular organelles share a remarkably conserved mechanism of InsP(3)-controlled frequency-encoded Ca(2+) signaling.

Glucose and other nutrients regulate many aspects of pancreatic islet physiology. This includes not only insulin release, but also insulin synthesis and storage and other aspects of beta-cell biology, including cell proliferation, apoptosis, differentiation, and gene expression. This implies that in addition to the well-described signals for insulin release, other intracellular signaling mechanisms are needed. Here we describe the role of global and local Ca(2+) signals in insulin release, the regulation of these signals by new membrane receptors, and the generation of nuclear Ca(2+) signals involved in gene expression. An integrated view of these pathways should improve the present description of the beta-cell biology and provide new targets for novel drugs.

Diabetes Mellitus is by far one of the most propagated chronic diseases, affecting 150 million people worldwide. This affliction is caused by a malfunction of pancreatic endocrine cells, which provokes a failure in the insulin release and glucose homeostasis. Plasma membrane K-ATP channels have a key role in the stimulus-secretion coupling of pancreatic beta-cells. Consequently, many investigations have developed efficient drugs for the treatment of diabetes, such as sulphonylureas, which specifically close K-ATP channels leading to an enhanced insulin secretion. Recent studies show that, in addition to its well-known plasma membrane location, sulphonylurea receptors and sulphonylurea-sensitive K-ATP channels are also present in various intracellular sites including secretory granules, mitochondria, endoplasmic reticulum and more recently, the nucleus. What roles do they play in these organelles? Intracellular K-ATP channels and sulphonylurea receptors, which operate in conjunction with classical pathways, can provide specific signaling circuits to establish direct links between extracellular signals and different cell functions, such as secretion or gene expression. The study of these intracellular channels provides novel perspectives in the signal transduction of the pancreatic beta-cell, and may offer clues for the development of new strategies in diabetes therapy. In this review we will address this topic with special emphasis on the biophysical basis and functional implications in the pancreatic beta-cell.