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Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules.

ABSTRACT: Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long ( approximately 250 ms) stimuli was unaffected. Under normal conditions, Ca(2+) influx elicited by brief membrane depolarizations increases [Ca(2+)](i) to high levels within discrete microdomains and triggers the exocytosis of closely associated insulin granules. We found that these domains of localized Ca(2+) entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca(2+) increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the beta cell action potential was pharmacologically prolonged.

SUBMITTER: Hoppa MB

PROVIDER: S-EPMC2814048 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules.

Hoppa Michael B MB Collins Stephan S Ramracheya Reshma R Hodson Leanne L Amisten Stefan S Zhang Quan Q Johnson Paul P Ashcroft Frances M FM Rorsman Patrik P

Cell metabolism 20091201 6

Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long ( approximately 250 ms) stimuli was unaffected. Under normal conditions, Ca(2+) influx elicited by brief membrane depolarizations increases [Ca(2+)](i) to high levels ...[more]

PMID: 19945403

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Project description:Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

Project description:We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.

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Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules.

Publications

Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules.

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