Functional differences between aggregated and dispersed insulin-producing cells.

AIMS/HYPOTHESIS: Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. METHODS: MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. RESULTS: Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca(2+)]c response. CONCLUSIONS/INTERPRETATION: We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca(2+)]c.

cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells.

Activation of hormone receptors was recently found to evoke oscillations of the cAMP concentration ([cAMP]) beneath the plasma membrane of insulin-secreting cells. Here we investigate how different time courses of cAMP signals influence the generation of cytoplasmic Ca(2+) signals and nuclear translocation of the PKA (protein kinase A) catalytic subunit in individual INS-1 beta-cells. [cAMP] was measured with a fluorescent translocation biosensor and ratiometric evanescent wave microscopy. Analysis of PKA nuclear translocation was performed with epifluorescence microscopy and FlAsH (fluorescein arsenical helix binder) labelling of tetracysteine-tagged PKA-Calpha subunit. Both oscillatory and stable elevations of [cAMP] induced by intermittent or constant inhibition of phosphodiesterases with isobutylmethylxanthine evoked Ca(2+) spiking. During [cAMP] oscillations, the Ca(2+) spiking was restricted to the periods of elevated [cAMP]. In contrast, only stable [cAMP] elevation induced nuclear entry of FlAsH-labelled PKA-Calpha. These results indicate that oscillations of [cAMP] lead to selective target activation by restricting the spatial redistribution of PKA.

Inward-rectifier K+ current in guinea-pig ventricular myocytes exposed to hyperosmotic solutions.

Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K+ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K+ current (I(K1)), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrode's solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3-2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba(2+)-sensitive I(K1), an increase in the amplitude of outward I(K1), and a steepening of the slope of the inward I(K1)-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (G(K1)) and rectification, G(K1)-V data were fitted with Boltzmann functions to determine maximal G(K1) (G(K1)max) and voltage at one-half G(K1)max (V(0.5)). Superfusion with hyperosmotic sucrose solutions led to significant increases in G(K1)max (e.g., 28 +/- 2% with 1.8T), and significant negative shifts in V(0.5) (e.g., -6.7 +/- 0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that G(K1)max and V(0.5) were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K+. We conclude that the effects of hyperosmotic sucrose solutions on I(K1) are related to shrinkage-induced concentrating of intracellular K+.

Store-operated influx of Ca(2+) in pancreatic beta-cells exhibits graded dependence on the filling of the endoplasmic reticulum.

The store-operated pathway for Ca(2+) entry was studied in individual mouse pancreatic beta-cells by measuring the cytoplasmic concentrations of Ca(2+) ([Ca(2+)](i)) and Mn(2+) ([Mn(2+)](i)) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca(2+) sequestration. To avoid interference with voltage-dependent Ca(2+) entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca(2+) depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca(2+)](i) or from the rate of increase in [Mn(2+)](i) due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn(2+) depended on store filling in a graded manner. The store-operated influx of Ca(2+)/Mn(2+) was inhibited by Gd(3+) and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the beta-cell has direct implications for the control of membrane potential and insulin secretion.