Effects of age, diet and obesity on insulin secretion from isolated perfused rat pancreas: Response to glucose, arginine and glucagon-like peptide 1 (7-37)

The insulin secretory responses to glucose, arginine and glucagon-like peptide (GLP)-1-(7- 37 have been evaluated from the isolated perfused pancreas of rats with either acquired or genetic obesity, ie, a) fed ad libitum 14-mo old Sprague-Dawley rats as compared to age-matched animals subjected to two types of dietary restriction (every-other-day feeding, EOD, and 40% restriction? 40% DR), and b) 2.5-mo old genetically obese fa/fa rats as compared to the lean counterpart, In mature fed ad libitum rats, the glucose-stimulated insulin release from the perfused pancreas was increased 5-fold by addition of 0.1 nM GLP-1 (7-37), a subsequent challenge with high glucose resulted in an improvement of the first phase of insulin release, In 40% DR rats, a similar pattern of secretion was observed, with the difference of a lower response to arginine than in fed ad libitum animals, In EOD rats, the overall secretory performance of the perfused pancreas was approximately 50% lower than in the fed ad libitum group but probably adequate to the reduced weight of the animals, In genetically obese young rats, both the response to GLP-1 (7-37) anti the total insulin secretion were higher than in the lean controls. Interestingly, the maximal insulin outputs from the perfused pancreases were observed in both the groups of overweight animals, In conclusion no impairment in the secretory responsiveness of beta-cells occurs in obese animals, Conversely, at least within the age limits of the present study, the endocrine pancreas develops a compensatory ability to match the augmented insulin demand due to the over-weight. In the light of the observed great sensitivity of the isolated perfused pancreas to GLP-1 (7-37), changes in the responsiveness of beta-cells to incretins might be involved in the modulation of the endocrine pancreatic function of obese rats

Control of Precursor Maturation and Disposal Is an Early Regulative Mechanism in the Normal Insulin Production of Pancreatic β-Cells

The essential folding and maturation process of proinsulin in β-cells is largely uncharacterized. To analyze this process, we improved approaches to immunoblotting, metabolic labeling, and data analysis used to determine the proportion of monomers and non-monomers and changes in composition of proinsulin in cells. We found the natural occurrence of a large proportion of proinsulin in various non-monomer states, i.e., aggregates, in normal mouse and human β-cells and a striking increase in the proportion of proinsulin non-monomers in Ins2+/Akita mice in response to a mutation (C96Y) in the insulin 2 (Ins2) gene. Proinsulin emerges in monomer and abundant dual-fate non-monomer states during nascent protein synthesis and shows heavy and preferential ATP/redox-sensitive disposal among secretory proteins during early post-translational processes. These findings support the preservation of proinsulin's aggregation-prone nature and low relative folding rate that permits the plentiful production of non-monomer forms with incomplete folding. Thus, in normal mouse/human β-cells, proinsulin's integrated maturation and degradation processes maintain a balance of natively and non-natively folded states, i.e., proinsulin homeostasis (PIHO). Further analysis discovered the high susceptibility of PIHO to cellular energy and calcium changes, endoplasmic reticulum (ER) and reductive/oxidative stress, and insults by thiol reagent and cytokine. These results expose a direct correlation between various extra-/intracellular influences and (a)typical integrations of proinsulin maturation and disposal processes. Overall, our findings demonstrated that the control of precursor maturation and disposal acts as an early regulative mechanism in normal insulin production, and its disorder is crucially linked to β-cell failure and diabetes pathogenesis

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway

Ablation of TSC2 Enhances Insulin Secretion by Increasing the Number of Mitochondria through Activation of mTORC1

) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells. mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes. mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1.Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells

Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+](i)) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+](i) followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+](i) mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+](i) control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.link_to_subscribed_fulltex

Caerulein causes translocation of protein kinase C in rat acini without increasing cytosolic free Ca2+

We investigated the relationships between changes in cytosolic free Ca2+ ([Ca2+](i)) and amylase secretion in dispersed rat pancreatic acini. Although 10 pM caerulein did not raise [Ca2+](i), higher concentrations (1 nM) of the peptide elicited a prompt, marked, but transient (2-3 min) elevation of [Ca2+](i). Both concentrations of caerulein caused an almost identical release of amylase over a 30-min period. To investigate the mechanism(s) underlying Ca2+-independent secretion, we measured the effect of the secretagogue on protein kinase C activity and found that both caerulein concentrations caused a significant translocation of protein kinase C from the cytosolic to the microsomal fraction. Because 1 nM caerulein induced a greater enzyme secretion than 10 pM caerulein during the first 2-5 min of stimulation, we explored further the role of [Ca2+](i) transients during the first minutes of secretion. Addition of ionomycin in the presence of 10 pM caerulein resulted in a rise in [Ca2+](i) and enhanced secretion as a result of caerulein in a near additive fashion during the first 2 min of stimulation. Second, we pretreated acini for 5 min with 1 μM 12-O-tetradecanoylphorbol-13-acetate. This maneuver inhibited both caerulein-induced inositol trisphosphate formation and [Ca2+](i) elevation. These findings were paralleled by a similar inhibition of caerulein-stimulated amylase release only during the first 5 min of secretion. These results indicate that 1) caerulein can stimulate amylase secretion independently of a concomitant [Ca2+](i) rise, possibly by activation of protein kinase C, and 2) an elevation of [Ca2+](i) serves as a trigger to enhance amylase release only during the initial phase of secretion.link_to_subscribed_fulltex

Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

BACKGROUND AND PURPOSE: Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action. EXPERIMENTAL APPROACH: We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER. KEY RESULTS: Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration. CONCLUSIONS AND IMPLICATIONS: Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives