Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Glucagon-like peptide-1 stimulates GABA formation by pancreatic beta-cells at the level of glutamate decarboxylase

Pancreatic beta-cells are the major extraneural site of glutamate decarboxylase expression (GAD). During culture of isolated beta-cells, the GAD product gamma-aminobutyrate (GABA) is rapidly released in the medium, independently of insulin. It is considered as a possible mediator of beta-cell influences on alpha-cells, acinar cells, and/or infiltrating lymphocytes. In this perspective, we investigated the regulation of GABA release by rat beta-cells during a 24-h culture period. Glucose was previously reported to inhibit GABA release by diverting cellular GABA to mitochondrial breakdown through activation of GABA transferase (GABA-T). In the present study, glucagon-like peptide-1 (GLP-1) was shown to stimulate GABA formation at the level of GAD, its effect being suppressed by the GAD inhibitor allylglycine and remaining unaltered by the GABA-T inhibitor gamma-vinyl-GABA. The stimulatory action of GLP-1 is cAMP dependent, being reproduced by the adenylate cyclase activator forskolin and the cAMP analog N(6)-benzoyladenosine-3',5'-cAMP and inhibited by a PKA inhibitor. It is dependent on protein synthesis and associated with an increased expression of GAD67 but not GAD65. The GLP-1-induced stimulation of GAD activity in beta-cells can elevate medium GABA levels in conditions of glucose-driven intracellular GABA breakdown and thus maintain GABA-mediated beta-cell influences on neighboring cells.     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

TRPM4 controls insulin secretion in pancreatic beta-cells

TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.     

Oleanolic acid enhances insulin secretion in pancreatic beta-cells

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.     

High [Na+]i in cardiomyocytes from rainbow trout

Intracellular Na(+)-concentration, [Na(+)](i) modulates excitation-contraction coupling of cardiac myocytes via the Na(+)/Ca(2+) exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca(2+) influx via reverse-mode NCX contributes significantly to contraction when [Na(+)](i) is 16 mM but not 10 mM. However, physiological [Na(+)](i) has never been measured. We recorded [Na(+)](i) using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na(+)](i) at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21 degrees C). Surprisingly, we found that [Na(+)](i) was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na(+)](i) in resting cells. Thus, we report a resting in vivo [Na(+)](i) of 13.4 mM for rainbow trout cardiomyocytes. [Na(+)](i) increased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na(+)](i). Our results provide the first measurement of [Na(+)](i) in rainbow trout cardiomyocytes. This surprisingly high [Na(+)](i) is likely to result in physiologically significant Ca(2+) influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca(2+)-source will decrease with the action potential duration as temperature and contraction frequency increases.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells

Glucagon-like peptide 1 (GLP-1) is an intestine-derived insulinotropic hormone that stimulates glucose-dependent insulin production and secretion from pancreatic beta-cells. Other recognized actions of GLP-1 are to suppress glucagon secretion and hepatic glucose output, delay gastric emptying, reduce food intake, and promote glucose disposal in peripheral tissues. All of these actions are potentially beneficial for the treatment of type 2 diabetes mellitus. Several GLP-1 agonists are in clinical trials for the treatment of diabetes. More recently, GLP-1 agonists have been shown to stimulate the growth and differentiation of pancreatic beta-cells, as well as to exert cytoprotective, antiapoptotic effects on beta-cells. Recent evidence indicates that GLP-1 agonists act on receptors on pancreas-derived stem/progenitor cells to prompt their differentiation into beta-cells. These new findings suggest an approach to create beta-cells in vitro by expanding stem/progenitor cells and then to convert them into beta-cells by treatment with GLP-1. Thus GLP-1 may be a means by which to create beta-cells ex vivo for transplantation into patients with insulinopenic type 1 diabetes and severe forms of type 2 diabetes.     

Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells

Mitochondrial metabolism plays a central role in insulin secretion in pancreatic beta-cells. Generation of protonmotive force and ATP synthesis from glucose-originated pyruvate are critical steps in the canonical pathway of glucose-stimulated insulin secretion. Mitochondrial metabolism is intertwined with pathways that are thought to amplify insulin secretion with mechanisms distinct from the canonical pathway, and the relative importance of these two pathways is controversial. Here I show that glucose-induced mitochondrial membrane potential (MMP) hyperpolarization is necessary for, and predicts, the rate of insulin secretion in primary cultured human beta-cells. When glucose concentration is elevated, increased metabolism results in a substantial MMP hyperpolarization, as well as in increased rates of ATP synthesis and turnover marked by faster cell respiration. Using modular kinetic analysis I explored what properties of cellular energy metabolism enable a large glucose-induced change in MMP in human beta-cells. I found that an ATP-dependent pathway activates glucose or substrate oxidation, acting as a positive feedback in energy metabolism. This activation mechanism is essential for concomitant fast respiration and high MMP, and for a high magnitude glucose-induced MMP hyperpolarization and therefore for insulin secretion.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Galanin inhibition of cholecystokinin-8-induced increase in [Ca2+]i in individual rat pancreatic B-cells.

The intracellular free Ca2+ ion concentration [( Ca2+]i) was measured in individual rat pancreatic B-cells loaded with fura-2. The cells were prepared by enzymatic digestion and fluorescence-activated cell sorting. The resting concentration of [Ca2+]i in B-cells was 126.3 +/- 3.1 nM in the presence of 4.4 mM glucose. Addition of cholecystokinin-8 (CCK-8) resulted in rapid and transient rises in [Ca2+]i. Perifusion of B-cells with galanin attenuated the amplitude and duration of CCK-8-induced [Ca2+]i changes and this inhibitory effect was concentration-dependent and reversible. Perifusion of B-cells with nifedipine, a voltage-sensitive Ca2+ channel blocker, reduced the duration of the [Ca2+]i increase induced by CCK-8, indicating that the Ca2+ entry from the extracellular space was, at least in part, involved in CCK-8-induced increases in [Ca2+]i.     

Effect of glucagon-like peptide-1 on insulin secretion

The insulinotropic actions of two forms of glucagon-like peptide 1 (GLP-1) containing 31 and 37 amino acid residues on perfused rat pancreas were compared with that of gastric inhibitory polypeptide (GIP), hitherto the most potent intestinal insulinotropic polypeptide known. The smaller form, C-terminally amidated GLP-1-(7-36), strongly enhanced insulin secretion stimulated by 11.1 mM D-glucose at a concentration as low as 0.1 nM. Comparable effects of GIP and GLP-1-(1-37) on insulin secretion were observed at concentrations of 1.0 nM and 10.0 nM, respectively. At the doses tested, neither GLP-1s nor GIP had any effect on insulin secretion induced by 3.3 mM D-glucose. At a concentration of 1.0 nM, GLP-1-(7-36 amide) also enhanced insulin secretion induced by 5 mM L-arginine whereas at concentrations of up to 10.0 nM, GLP-1-(1-37) did not. The results show that the smaller form of GLP-1 is more strongly insulinotropic than GIP. These findings suggest that the smaller GLP-1 may have a physiologically more important role as a modulator of insulin release.     

Cytosolic ratios of free [NADPH]/[NADP+] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion.

When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed. Glucose had no effect on the cytosolic free [NADH]/[NAD+] ratio. Neither the cytosolic free [NADPH]/[NADP+] ratio nor the corresponding [NADH]/[NAD+] ratio was affected when the islets were incubated with 20 mM-fructose or with 3 mM-glucose + 20 mM-fructose, although the last-mentioned condition stimulated insulin release. The insulin secretagogue leucine (10 mM) stimulated insulin secretion, but lowered the cytosolic free [NADPH]/[NADP+] ratio; 10 mM-leucine + 10 mM-glutamine stimulated insulin release and significantly enhanced both the [NADPH]/[NADP+] ratio and the [NADH]/[NAD+] ratio. It is concluded that the cytosolic free [NADPH]/[NADP+] ratio may be involved in coupling beta-cell glucose metabolism to beta-cell depolarization and ensuing insulin secretion, but it may not be the sole or major coupling factor in nutrient-induced stimulation of insulin secretion.     

Glucose-induced increase of potassium in pancreatic beta-cells associated with reduced mobilization of the ion

The potassium contents of beta-cell-rich pancreatic islets from ob/ob-mice were measured with an integrating flame photometer. After exposure to 5 mM glucose islet potassium increased by 17 +/- 2%, no additional effect being seen with increase of the sugar to 20 mM. Glucose counteracted the loss of islet potassium obtained on removal of the ion from the incubation medium, halving the initial disappearance rate. Whereas the effect of glucose in suppressing the mobilisation of potassium was mimicked by tolbutamide and quinine, it was antagonized by diazoxide. It is concluded that the glucose interference with the outward transport of K+ is sufficient to raise the beta-cell content of the ion.     

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.