Henrik Kindmark

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

SummaryPlasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca2+]i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca2+]i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca2+]i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca2+]i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca2+]i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca2+]i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.

Protein kinase C activity affects glucose‐induced oscillations in cytoplasmic free Ca2+ in the pancreatic B‐cell

Acute stimulation of protein kinase C (PKC) inhibited glucose‐induced slow oscillations in cytoplasmic free Ca2+‐concentration, [Ca2+]i, in mouse pancreatic B‐cells. In PKC‐depleted cells glucose induced rapid transients in [Ca2+]i, lasting for approximately 10 s, superimposed on the slow oscillations in [Ca2+]i. It was demonstrated that the transients did not occur in the absence of extracellular Ca2+. Each transient typically was preceded by a slow increase in [Ca2+]i, representing the rising phase of an ordinary glucose‐induced slow oscillation, and the [Ca2+]i, immediately after a transient was lower than just before the spike. These data further emphasize the interplay between voltage‐dependent Ca2+‐channels and the phospholipase C system in the regulation of B‐cell [Ca2+]i‐oscillations.

Regulation of cytoplasmic free Ca2+ in insulin-secreting cells

The cytoplasmic free Ca2+ concentration([Ca2+]i) has a fundamental role in the β-cell stimulus-secretion coupling and is regulated by a sophisticated interplay between nutrients, hormones and neurotransmitters. Metabolism of glucose and other nutrients leads to ATP generation, closure of ATP-regulated K+-channels, depolarization, opening of voltage-activated L-type Ca2+-channels, increase in [Ca2+]i and insulin release (1,2). Hormones and neurotransmitters affect the β-cell through the activation of receptors coupled to various effector systems, such as the adenylate cyclase (AC) or phospholipase C (PLC) system (2). Upon activation of these systems, cAMP is formed or phosphatidyl inositol 4,5-bisphosphate is hydrolysed, resulting in the formation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). Whereas InsP3 mobilizes intracellularly bound Ca2+, most probably from the endoplasmic reticulum, DAG activates protein kinase C (PKC) (1–3). Although InsP3 increases [Ca2+]i, there is little effect on insulin release, suggesting that the trisphosphate is not primarily involved as a signal for exocytosis in the β-cell (3). With regard to PKC, the physiological role is more clear and this enzyme is involved as a modulator of multiple steps in the β-cell signal-transduction pathway (1–3).

Dissociation between exocytosis and Ca2+-channel activity in mouse pancreatic β-cells stimulated with calmidazolium (compound R24571)

Calmidazolium, a calmodulin inhibitor, suppressed influx of Ca2+ through voltage‐gated Ca2+ channels in mouse pancreatic β‐cells. Despite this fact, calmidazolium stimulated insulin release from β‐cells at basal glucose concentration. This effect was not mediated by protein kinase C (PKC), since it persisted in PKC‐depleted cells. RpcAMPS significantly attenuated tha calmidazolium‐stimulated insulin secretion, indicating that calmidazolium acts, at least partly, through PKA. The compound also stimulated insulin secretion from electropermeabilized β‐cells, indicating effects on distal steps in the stimulus‐secretion coupling. The use of calmidazolium offers possibilities to investigate the mechanisms activating exocytosis under conditions where the cytoplasmic‐free Ca2+ concentration does not increase.

The Imidazoline Derivative Calmidazolium Inhibits Voltage-Gated Ca2+-Channels and Insulin Release But Has No Effect on the Phospholipase C System in Insulin Producing RINm5F-cells.

The present study shows that the calmodulin antagonist calmidazolium inhibited influx of Ca2+ through voltage-gated Ca2+-channels in clonal insulin producing RINm5F-cells. The mechanism of inhibition may involve both Ca2+-calmodulin-dependent protein kinases and direct binding of calmidazolium to the Ca2+-channel. Calmidazolium did not affect uptake of Ca2+ into intracellular Ca2+-pools, inositol 1,4,5-trisphosphate (InsP3) formation or action on intracellular Ca2+-pools. The calmodulin inhibitor also did not affect glucose utilization or oxidation in RINm5F-cells, speaking against an unspecific toxic effect of the compound. KCl-and ATP-stimulated insulin release from RINm5F-cells was attenuated by calmidazolium, whereas basal hormone secretion was unaffected.

Short- and long-term effects of β-cellulin and transforming growth factor-α on β-cell function in cultured fetal rat pancreatic islets

The polypeptide β-cellulin, identified in conditioned media from insulinoma cell cultures and produced by pancreatic islet cells, was recently identified as a possible autocrine growth factor for the pancreatic islet β-cell. In this study, we investigated the short- and long-term actions of β-cellulin, and the structurally related transforming growth factor-α (TGF-α), on β-cell function in fetal rat pancreatic islets in vitro. We found that neither β-cellulin nor TGF-α (10 nMeach), in contrast to glucose (20 mM), acutely influenced β-cell levels of cytosolic-free Ca2+. Additionally, whereas glucose markedly increased short-term (60-min) insulin release, neither β-cellulin nor TGF-α (10nM each) influenced the rate of hormone secretion at basal (3 mM) or stimulatory (20 mM) concentrations of glucose. Likewise, long-term (24-h) exposure of islets to a high glucose concentration significantly augmented the secretion of insulin. This effect was slightly potentiated by TGF-α (10 nM), but not β-celluin (10 nM), at high (but not low) glucose concentrations. Conversely, the islet insulin content was not significantly affected by β-cellulin or TGF-α at any glucose concentration tested. We conclude that, although β-cellulin is produced by islet cells, the peptide does not seem to be of importance for the regulation of insulin production by isolated pancreatic β-cells.