Roles of plasma membrane ryanodine receptors in pancreatic beta cells.

Type 2 diabetes, as its alternative name (maturity-onset diabetes) suggests, was once a disease of older people. But obesity, the major pre-disposing factor for type 2 diabetes, is increasing alarmingly among all age-groups and so too is the prevalence of type 2 diabetes, which increased by at least 25% in the west and by 50% or more elsewhere since 2000. Diabetes requires lifelong therapy and its complications reduce quality or life and life-expectancy. The disease presents when beta-cells in the pancreas are no longer able to release sufficient insulin to control effectively the blood glucose concentration. Most drugs used to treat diabetes work by promoting insulin release, but they cannot faithfully replicate the oscillatory changes in insulin secretion that provide the most effective regulation of glucose uptake by target tissues. Our work has identified a channel (the type 2 ryanodine receptor, RyR2) in the membrane that surrounds the beta-cell. We suggest that RyR2 may be inserted into this membrane when the vesicles that release insulin fuse with it, and that the RyR2 may then control the flux of calcium ions that regulates insulin release. This unexpected contribution from RyR2 may thereby form part of a regulatory loop contributing to oscillatory control of insulin release. The accessibility of RyR2 (on the surface of the cell) may allow it to become a target for novel drugs to promote insulin release. Our work is concerned with unravelling the roles of RyR2 in control of insulin release.

Type 2 diabetes mellitus is an increasingly prevalent health burden. It arises when insulin release from pancreatic beta-cells is insufficient to allow effective control of plasma glucose in the presence of increasing insulin resistance. Glucose evokes the pulsatile insulin release that is optimal for effective regulation of target tissues principally via its metabolism to ATP in beta-cells, which closes KATP channels, causing depolarization, activation of voltage-gated Ca2+ channels and exocytosis of insulin-containing vesicles. Incretins, via cAMP, potentiate glucose-evoked insulin release. Other pathways, including those involving intracellular Ca2+ channels (ryanodine and IP3 receptors, RyR, IP3R), also contribute to the oscillatory changes in cytosolic [Ca2+] and cAMP that evoke insulin release. But their roles are less clearly defined. IP3R3 (the only IP3R isoform in beta-cells) are expressed only in the ER, whereas RyR are also expressed in secretory vesicles and/or endosomes. Hitherto, the substantial evidence supporting roles for RyR in insulin secretion assumed that RyR are conventionally located in intracellular stores. But our recent work using patch-clamp recording, measurements of Ca2+ and Mn2+ entry, and RNAi has unexpectedly demonstrated that small numbers of functional RyR2 are expressed in the plasma membrane (PM) of RINm5F insulinoma cells and freshly isolated rat beta-cells. We propose that RyR2 may, in association with insulin secretion, be dynamically trafficked to the PM and that within the PM signals evoked by glucose and incretins (ATP, cAMP, etc) may acutely regulate the gating of PM RyR2. This may allow PM RyR2 to regulate membrane potential and directly mediate Ca2+ entry. This novel sequence may account for some of the KATP-independent regulation of insulin release. Using INS-1E insulinoma cells and rat beta-cells, and combining confocal microscopy, patch-clamp recording, subcellular fractionation and measurements of Ca2+ signals and insulin release, we will address three questions: 1. What determines the differential distribution of RyR2 and IP3R3 in beta-cells? 2. Do physiological regulators of insulin secretion control trafficking of RyR2 to and from the PM? 3. Do RyR within the PM contribute to the Ca2+ signals that regulate insulin secretion?