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Ryanodine receptors of pancreatic ß-cells mediate a distinct context-dependent signal for insulin secretion¹

JOSEPH D. BRUTON^*, RAF LEMMENS, CHUN-LIANG SHI, SOLVEIG PERSSON-SJÖGREN, HAKAN WESTERBLAD^*, MASROOR AHMED^¶, NIGEL J. PYNE^¶, MHAIRI FRAME^¶, BRIAN L. FURMAN^¶ and MD. SHAHIDUL ISLAM²

^* Department of Physiology, Karolinska Institutet, Stockholm, Sweden;
Department of Molecular Medicine, Karolinska Institutet, Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden;
Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, Umeå, Sweden; and
^¶ Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, UK

²Correspondence: Department of Molecular Medicine, Karolinska Institutet, Department of Endocrinology, Karolinska Hospital L6B:01, S-171 76 Stockholm, Sweden. E-mail: Shahidul.Islam{at}molmed.ki.se

SPECIFIC AIMS

Stimulation of insulin secretion from pancreatic ß cells by caffeine has been attributed solely to inhibition of cAMP-phosphodiesterases (cAMP-PDEs). An alternative hypothesis tested here is that caffeine stimulates insulin secretion primarily by sensitization of the RY receptors and that Ca²⁺-induced Ca²⁺ release through these channels mediates a context-dependent signal for insulin secretion.

PRINCIPAL FINDINGS

1. Caffeine stimulates insulin secretion in a glucose-dependent manner
Caffeine (2.5 mM) did not alter insulin secretion in the presence of 3 mM glucose but stimulated insulin secretion in the presence of 11.2 mM glucose. Stimulation of insulin secretion by caffeine was observed even when the effect of glucose on ATP-sensitive K⁺ channel (K_ATP) was bypassed by diazoxide (100 µM) and KCl (30 mM).

2. Stimulation of insulin secretion by caffeine is not due to inhibition of cAMP-phosphodiesterases
We used two caffeine analogs that have been reported not to inhibit cAMP-PDEs but sensitize the RY receptors. Isocaffeine, a 9-substituted isomer of caffeine (2.5 mM), did not inhibit cAMP-PDEs but still stimulated insulin secretion. Another 9-substituted methylxanthine, 3,9-dimethylxanthine (2.5 mM), was slightly less potent than caffeine in inhibiting cAMP-PDEs but was just as effective as caffeine in stimulating insulin secretion. There was a negative correlation between caffeine-induced cAMP-PDE-inhibition and stimulation of insulin secretion (Fig. 1 ). At a concentration as low as 0.25 mM, caffeine induced near-maximal stimulation of insulin secretion but inhibited cAMP-PDEs by only 18%. Caffeine did not increase cAMP content in these cells.

View larger version (30K): [in this window] [in a new window]   Figure 1. Concentration-response relationship showing the extent of inhibition of cAMP-PDEs and of stimulation of insulin secretion by caffeine. A) Extent of inhibition of cAMP-PDEs in the pellets (•) and supernatants () obtained from insulinoma cells. Values are mean ± SE (n=3). B) Effect of caffeine on glucose-dependent stimulation of insulin secretion. Values are mean ± SE (n=4). C) The extent of inhibition of cAMP-PDEs by caffeine was negatively correlated with stimulation of insulin secretion by the same concentrations of the xanthine drug (r=-0.93).

3. Stimulation of insulin secretion by caffeine is dependent on its ability to release Ca²⁺ from the intracellular stores
We imaged fluo-3-loaded clonal rat insulinoma cells (INS-1E) by confocal laser scanning microscopy. Caffeine (5 mM) induced a rapid and transient increase in [Ca²⁺]_i when applied in the presence of 5 mM glucose. Forskolin (5 µM) did not increase [Ca²⁺]_i under similar conditions. We examined the effects of 1.5 mM caffeine on Fura-2-loaded single mouse ß cells stimulated by 11.2 mM glucose. Caffeine initiated Ca²⁺-induced Ca²⁺ release (CICR) in the form of regenerative Ca²⁺ spikes that were superimposed on the glucose-induced increase in [Ca²⁺]_i. Isocaffeine (1.5 mM) induced similar Ca²⁺ spikes, but they were less frequent with isocaffeine than with caffeine. To examine whether release of Ca²⁺ from the ER was involved in stimulation of insulin secretion by caffeine, we tested the effect of caffeine on cells whose ER Ca²⁺ stores were depleted by prolonged inhibition of sarcoendoplasmic reticulum Ca²⁺ ATPase by thapsigargin. Basal insulin secretion in Ca²⁺-depleted cells was not different from that in controls cells. Glucose-dependent, caffeine-stimulated secretion was significantly reduced in thapsigargin-treated cells. Glucose-dependent stimulation of insulin secretion by caffeine was abolished in the presence of dantrolene (75 µM), a membrane-permeable inhibitor of RY receptors.

4. 9-Methyl 7-bromo eudistomin D (MBED) a caffeine-like sanitizer of RY receptors did not inhibit cAMP-PDEs but stimulated insulin secretion in a glucose-dependent manner
MBED, which sensitizes CICR by acting on the caffeine binding site of the RY receptors did not inhibit cAMP-PDEs in INS-1E cells. MBED (50 µM) increased [Ca²⁺]_i at localized sites that eventually resulted in a global increase which subsequently returned to the resting level despite the continued presence of the compound. MBED also stimulated insulin secretion in a glucose-dependent manner. Stimulation of insulin secretion by MBED was concentration dependent and, in this regard, more potent than caffeine (Fig. 2 ). Since insulinoma cells may differ from native ß cells, we tested the effects of caffeine and MBED on insulin secretion in primary ß cells of mouse islets. In the presence of 11.2 mM glucose, caffeine and MBED stimulated insulin secretion from islets. Caffeine and MBED increased both the first and the second phases of insulin secretion. MBED was clearly more potent than caffeine, the effect of 6 µM MBED being roughly comparable to that of 2.5 mM caffeine. MBED did not increase cAMP content in INS-1E cells.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of MBED, a caffeine-like sensitizer of RY receptor on insulin secretion from INS-1E cells. A) MBED (6 µM) stimulated insulin secretion when glucose was 11.2 mM. The magnitude of stimulation was similar to that obtained with caffeine (2.5 mM). B) Concentration-response relationship of MBED and glucose-dependent stimulation of insulin secretion.

CONCLUSIONS AND SIGNIFICANCE

The roles of RY receptors are best understood in the excitation-contraction coupling in muscles, but their roles in stimulus-secretion coupling have remained unclear. Since caffeine inhibits cAMP-PDEs and cAMP is known to stimulate insulin secretion, investigators attributed the effect of caffeine on insulin secretion solely to the inhibition of the cAMP-PDEs. We demonstrate that caffeine stimulates insulin secretion from ß cells and that such stimulation occurs only with a high concentration of glucose. Such stimulation of insulin secretion is due primarily to sensitization of the RY receptor by caffeine and not the inhibition of cAMP-PDEs. Thus, stimulation of insulin secretion by different concentrations of caffeine was not positively correlated with their inhibitory effects on cAMP-PDEs. Isocaffeine did not inhibit, but rather activated cAMP-PDEs. Nevertheless, it stimulated insulin secretion.

Our results indicate that caffeine stimulation of insulin secretion is due primarily to a Ca²⁺-dependent rather than a cAMP-dependent phenomenon. Thus, a high concentration of caffeine (5 mM) that can directly activate the RY receptors, increased [Ca²⁺]_i in these cells in the presence of 5 mM glucose. This increase in [Ca²⁺]_i evoked by caffeine was not due to cAMP since forskolin did not increase [Ca²⁺]_i under the similar conditions. Our finding that depletion of ER Ca²⁺ by thapsigargin inhibited the context-dependent stimulation of insulin secretion strongly suggests that caffeine stimulated insulin secretion by releasing Ca²⁺ from the ER. This is further supported by the finding that dantrolene, a membrane-permeable inhibitor of RY receptors, inhibited glucose-dependent stimulation of insulin secretion by caffeine.

The main experimental obstacle in unraveling the mechanism of action of caffeine on insulin secretion was the lack of availability of analogs that could sensitize the RY receptors without inhibiting cAMP-PDEs. We circumvented this by using MBED, which acts on the caffeine binding site of the RY receptors. MBED increased [Ca²⁺]_i but did not inhibit cAMP-PDEs and still stimulated insulin secretion in a context-dependent manner, mimicking the effect of caffeine. Finally, we demonstrated a glucose-dependent stimulation of insulin secretion by caffeine and MBED from primary ß cells of mouse islets.

Our results indicate that CICR mediated by the RY receptors stimulates insulin secretion in a characteristic context-dependent manner, thus providing the cells with a mechanism to trigger insulin secretion only when glucose concentration is high. Endogenous modulators of RY receptor (cAMP, cyclic ADP-ribose, palmitoyl CoA, and fructose 1,6,-diphosphate) may affect this mode of signaling and modulate insulin secretion in a context-dependent manner. Available insulin secretagogues used to treat type 2 diabetes act mainly on the K_ATP channel and stimulate insulin secretion irrespective of the prevailing blood glucose concentration, thereby increasing the risks for hypoglycemia. CICR mediated by the RY receptor may be a distinct target for insulin secretagogues currently under development that may allow insulin secretion to be triggered only in a glucose-dependent manner. Thus, CICR is a novel target for developing insulin secretagogues that will not cause hypoglycemia; in this respect, compounds like MBED will be important lead compounds for developing such drugs.

View larger version (25K): [in this window] [in a new window]   Figure 3. Scheme illustrating the role of RY receptors in stimulating insulin secretion. Insulin secretion can occur as a direct result of Ca2+ entry through the voltage-gated Ca2+ channels in the plasma membrane (A) or as a result of CICR through the RY receptors (B). The latter process is not readily triggered in ß cells when glucose concentration is low. CICR in ß cells is thus a context-dependent phenomenon, which requires sensitization by a variety of mechanisms and a higher filling of the ER Ca2+ pools. RY receptors thus mediate a signal for insulin secretion only when ambient glucose concentration is high. Some molecules generated from glucose metabolism and known to sensitize RY receptors are listed in the box. Factors that sensitize RY receptors thus are likely to stimulate insulin secretion only in the context of a high concentration of glucose. SC, secretory granules; FDP, fructose 2,6 bisphosphate.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0481fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0481fje

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