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Ryanodine receptors in human pancreatic ß cells: localization and effects on insulin secretion ¹

Division of Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

²Correspondence: Division of Metabolism, Washington University School of Medicine, Box 8126, 8831 Wohl Clinic, 660 S. Euclid, St. Louis, MO 63110, USA. E-mail: polonsky{at}im.wustl.edu or jim{at}jimjohnson.ca

SPECIFIC AIMS

Pancreatic ß cell secretory dysfunction is a key determinant of disease progression in type 2 diabetes. In diabetes, the expression and function of ryanodine receptor Ca²⁺ release channels (RyR) are reduced. The present studies were undertaken to define the subcellular location and the role of RyR in the control of stimulated and basal insulin release from human pancreatic ß cells.

PRINCIPAL FINDINGS

1. Localization of functional RyR in human pancreatic ß cells
Using a monoclonal antibody that recognizes both RyR isoforms present in ß cells (RyR1, RyR2), we found RyR in 84% of insulin positive human islet cells. Confocal microscopy demonstrated RyR staining in a punctate vesicular pattern in human (Fig. 1 A, B) and mouse ß cells (not shown). Although RyR immunofluorescence was found in close proximity to the insulin secretory granules, there was no colocalization between RyR and insulin (Fig. 1A ), challenging the conclusions of previous work on the MIN6 ß cell line. Next we tested whether RyR are found in endosomes, using the early endosomal antigen 1 (EEA1) as a marker. RyR was found on a subpopulation of EEA1-positive endosomes (Fig. 1B ; colocalization in 42±6% of pixels, n=3 cells). Endosomes likely represent a small subpopulation of ryanodine-sensitive Ca²⁺ stores, as only 20 ± 4% of RyR immunofluorescence colocalized with EEA1.

View larger version (44K): [in this window] [in a new window]   Figure 1. Localization and function of RyR in human ß cells. A) Dispersed human islet cells were labeled with antiserum to RyR (green) and insulin (red). B) The vesicles that stained for RyR also showed significant staining for the early endosomal antigen 1 (EEA1). C) 1 nM ryanodine (white bar) increased [Ca2+]c in 40% of human islet cells. D) Blocking RyR with 10 µM ryanodine did not increase cytosolic Ca2+ in ß cells.

We tested the hypothesis that human ß cells contain functional RyR using the stimulatory properties of nanomolar concentrations of ryanodine. Activating RyR with 1 nM ryanodine evoked transient Ca²⁺ signals (peak 128±18 nM above baseline) in dispersed human ß cells (Fig. 1C ). The elevations in global cytosolic Ca²⁺ evoked by 1 nM ryanodine in 3 mM glucose were much smaller than the subsequent Ca²⁺ responses to glucose. Micromolar concentrations of ryanodine known to block the RyR did not alter cytosolic Ca²⁺ in unstimulated cells (Fig. 1D ).

2. Ryanodine stimulates insulin release independently of endoplasmic reticulum Ca²⁺ stores
The effects of directly stimulating RyR on insulin release are unknown. Thus, we examined insulin release in the presence of a wide range of ryanodine concentrations. Low doses of ryanodine (0.1–10 nM) stimulated significant insulin release from dispersed human ß cells and intact islets in the presence of 3 mM glucose. In 15 mM glucose, no significant effect of ryanodine could be detected at any dose. Unexpectedly, concentrations of ryanodine known to inhibit RyR (10–100 µM) also evoked significant transient insulin release. The time courses of insulin release stimulated by 1 nM and 10 µM ryanodine were different at the first and last time points, suggesting partially distinct mechanisms.

Previous studies, including an investigation of Ca²⁺ uptake into the ryanodine-sensitive Ca²⁺ stores of MIN6 cells, have suggested that ryanodine-sensitive Ca²⁺ stores include those independent of thapsigargin-sensitive sarco/endoplasmic reticulum ATPase (SERCA) pumps in the insulin granule or endosomal compartments. In human ß cells, thapsigargin (2 µM) potentiated, rather than blocked, ryanodine-stimulated insulin release (Fig. 2 A), suggesting that ryanodine-induced insulin release was mediated by a non-ER compartment. We have shown that endosomes, a putative thapsigargin-independent Ca²⁺ store, contain RyR, making this organelle a good candidate for mediating the effects of ryanodine on insulin release.

View larger version (26K): [in this window] [in a new window]   Figure 2. Mechanisms of ryanodine-induced insulin release. A) Insulin release evoked by 1 nM ryanodine or 10 µM ryanodine are not blocked by the addition of 2 µM thapsigargin (n=6). Stars denote significant differences from control. Double stars denote significant differences compared with no thapsigargin. B) Effects of 1 nM ryanodine, 10 µM ryanodine and 15 mM glucose are differentially affected by 30 min preincubation with the cytosolic Ca2+ chelator BAPTA-AM (n=12). C) 100 µM ryanodine stimulated insulin release under basal conditions, but not from perifused human islets depolarized with 30 mM KCl (n=4). D) Cytosolic Ca2+ was measured during a similar experimental protocol, where 50 µM ryanodine was applied after stimulation with 30 mM KCl.

3. Activation or inhibition of RyR promotes insulin release through cytosolic Ca²⁺-dependent or cytosolic Ca²⁺-independent mechanisms, respectively
We tested whether insulin release induced by high and low concentrations of ryanodine depended on a rise in cytosolic Ca²⁺, rather than changes in luminal Ca²⁺. Insulin release evoked by 1 nM ryanodine was abolished by preincubation with BAPTA-AM (Fig. 2B ), demonstrating a requirement for the elevations in cytosolic Ca²⁺ described above. On the other hand, insulin release due to 10 µM ryanodine was not blocked by BAPTA treatment, consistent with observation that 10 µM ryanodine did not generate cytosolic Ca²⁺ signals. These findings are consistent with the idea that Ca²⁺ in the lumen of ryanodine-sensitive Ca²⁺ stores may play a role in insulin release.

The effects of elevating cytosolic Ca²⁺ on the temporal dynamics of insulin release induced by blocking RyR were examined using an islet perifusion system. Blocking RyR with 100 µM ryanodine evoked a rapid increase in insulin secretion from unstimulated islets. In contrast, insulin release may have been slightly reduced by ryanodine when islets were depolarized with 30 mM KCl (Fig. 2C ). In the same experiments, glucagon release was not affected by ryanodine. Thus, the secretagogue effects of inhibitory concentrations of ryanodine are unrelated to classical Ca²⁺-induced Ca²⁺ release phenomena. In fact, 10 µM ryanodine induced an increase in cytosolic Ca²⁺ in the presence of KCl (Fig. 2D ), an effect that is opposite to what would be expected if these channels were involved in Ca²⁺-induced Ca²⁺ release under these conditions. This was not due to direct effects on voltage-gated Ca²⁺ currents or mimicked when RyR were opened with 1 nM ryanodine or when IP₃R were blocked with xestospongin C. These experiments suggest that RyR play a role in Ca²⁺ buffering (i.e., Ca²⁺-induced Ca²⁺ uptake).

4. RyR do not mediate glucose signaling in human ß cells
Having determined that direct modulation of secretory granule ryanodine receptors can increase exocytosis, we asked whether glucose signaling or glucose-stimulated insulin release require ryanodine-sensitive Ca²⁺ stores, as has been inferred from studies in MIN6 cells and CD38^–/– mice. In primary human and mouse ß cells, 10–100 µM ryanodine did not inhibit glucose-evoked Ca²⁺ signals (not shown). Similarly, neither ryanodine nor dantrolene inhibited glucose-stimulated insulin release from dispersed primary islet cells or perifused islets (not shown), strongly suggesting that RyR does not play a role in glucose signaling under normal conditions. Together these results suggest that the effects of RyR on insulin exocytosis are glucose independent and that the CD38/RyR pathway is not involved in acute glucose signaling.

CONCLUSIONS AND SIGNIFICANCE

The presence and function of ryanodine receptors in pancreatic ß cells has been the subject of much debate. The present study, which used ryanodine rather than caffeine or other less specific RyR agonists, has demonstrated that RyR are involved in the control of insulin secretion in human ß cells. Our results lead us to propose new models for RyR function in nonmuscle cells (Fig. 3 ) and call into question two major assumptions regarding the role of RyR in ß cells, namely, that RyR activation is absolutely dependent on glucose or cAMP and that RyR exert their effects in ß cells wholly via Ca²⁺-induced Ca²⁺ release. The observation of ‘Ca²⁺-induced Ca²⁺ uptake’ suggests the involvement of an acidic organelle with low luminal Ca²⁺, strategically located near the plasma membrane and capable of the rapid uptake of Ca²⁺ from the cytosol. The only organelles known to fit this description are endosomes. Together, the localization of RyR to endosomes and implication of non-ER Ca²⁺ stores in ryanodine-induced insulin release suggests that ryanodine does not act by altering a Ca²⁺-induced Ca²⁺ release process. The second assumption is challenged by the finding that ryanodine stimulates insulin release under basal conditions and not when ß cells are stimulated with glucose or KCl. The observation that nanomolar concentrations of ryanodine led to an increase in cytosolic Ca²⁺ sufficient to release insulin from human ß cells supports the controversial hypothesis that strategically located intracellular Ca²⁺ stores are capable of stimulating secretion from endocrine cells. This novel pathway appears to be completely separate from the well-known mechanism involving influx of extracellular Ca²⁺ through voltage-gated Ca²⁺ channels that is activated during glucose-stimulated insulin release. Although RyR may play an important role in glucose signaling in both the MIN6 and HIT ß cell lines, our examination of primary human ß cells provides strong evidence that the cADPr/RyR arm of CD38 signaling is not the primary glucose signaling pathway.

View larger version (52K): [in this window] [in a new window]   Figure 3. Working model of the role of RyR on human ß cell physiology. A) Putative mechanism for the effects of ryanodine cytosolic Ca2+ homeostasis during depolarizing stimuli. Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) flows into closely situated acidic organelles, such as endosomes, which act as an initial temporary buffer. Thereafter, Ca2+ is transferred through the RyR to a secondary buffer, possibly the mitochondria. B) When the flow of Ca2+ is blocked at the level of the ryanodine receptor (RyR) Ca2+ would accumulate within the endosomes and in the neighboring cytosol (red). However, since blocking RyR does not induce insulin secretion during depolarization, this Ca2+ signal may be spatially or temporally inadequate. C) Effects of ryanodine on insulin release during unstimulated conditions. Directly activating non-ER RyR with nanomolar ryanodine elicits a highly localized Ca2+ signal sufficient to induce the recruitment and/or fusion of insulin containing secretory granules. Blocking non-ER RyR transmits an unknown signal (light blue arrow) that also stimulates insulin granule exocytosis. D) The signal transduction cascade mediating glucose-stimulated insulin release that requires active metabolism, the KATP channel and VDCC, is independent of RyR and CD38.

Surprisingly, when RyR were blocked with micromolar concentrations of ryanodine, basal but not stimulated insulin secretion was increased through a distinct cytosolic Ca²⁺-independent mechanism. Since blocking concentrations of ryanodine do not increase cytosolic Ca²⁺, we propose that the regulation of luminal Ca²⁺ by RyR plays an important role in insulin exocytosis. Our results agree with a proposed model for the interaction between luminal Ca²⁺ and exocytosis in the INS-1 ß cell line. Previous data has shown that depletion of the IP₃/thapsigargin-insensitive acidic Ca²⁺ stores, which would include endosomes and secretory granules, led to a reduction in insulin exocytosis. Therefore, blocking Ca²⁺ efflux through RyR using micromolar ryanodine or dantrolene would stimulate insulin exocytosis, as we observed in the present study. However, it seems unlikely given our colocalization studies that the ryanodine-sensitive Ca²⁺ stores in human ß cells are present in the secretory granules themselves. This contrasts with conclusions from work on the MIN6 cell line where ryanodine-sensitive Ca²⁺ stores were found in VAMP2 containing structures, thought to be primarily the insulin secretory granules. However, VAMP2 may also be expressed in endosomes and we observed a limited degree of RyR colocalization with EEA1.

Although the exact mechanism by which luminal Ca²⁺ within the endosomes influences the exocytosis of insulin granules is unclear, these results point a previously unexpected link between Ca²⁺ signaling and insulin release. Loss of RyR activity in diabetes, may therefore contribute to the basal hyperinsulinemia associated with the disease. Basal hyperinsulinemia may have several deleterious ramifications, including the desensitization of insulin signaling (i.e., insulin resistance) and ß cell exhaustion. These defects would impair the ability of the ß cell to correctly respond to glucose and may contribute to the pathophysiology of diabetes. Thus, future studies should be directed at understanding the role of RyR in vivo and exploring intracellular Ca²⁺ release channels as novel therapeutic targets.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1280fje;

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