Munc18-1 and Munc18-2 proteins modulate beta-cell Ca2+ sensitivity and kinetics of insulin exocytosis differently.

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in ß-cell stimulus-secretion coupling. We show that pancreatic ß-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in ß-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated ß-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).

Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

While serotonin (5-HT) co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+)-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

cAMP increases the sensitivity of exocytosis to Ca²+ primarily through protein kinase A in mouse pancreatic beta cells.

Cyclic AMP regulates the late step of Ca²+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca²+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca²+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C(m) change had typically two phases. We observed that the Ca²+-dependency of the rate of the first C(m) change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2 µM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca²+-dependency of the exocytotic burst to significantly lower [Ca²+](i). To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca²+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 µM 6-Phe-cAMP or inhibition with 500 µM Rp-cAMPs in beta cells significantly shifted the EC(50) in the opposite directions. Specific activation of Epac2 did not change Ca²+ sensitivity. Our findings suggest that cAMP modulates Ca²+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca²+](i); Epac2 may contribute to enhance the rates of secretory vesicle fusion.

Pancreatic beta cell lines and their applications in diabetes mellitus research.

During the past 30 years great effort has been put into establishing an insulin-secreting beta cell line that retains normal regulation of insulin secretion, but only few of these attempts have been successful. To overcome the limited availability of primary beta cells and to include the principles of the 3Rs into the field of diabetes mellitus research, numerous investigators used X-rays or viruses to induce insulinomas, in vitro transformation, derivation of cells from transgenic mice or even non-islet cells to produce immortalised beta cell lines. The most widely used insulin-secreting cell lines are RIN, HIT, MIN, INS-1 and TC cells. These cells produce insulin and small amounts of glucagon and somatostatin. Some of them are only poorly responsive to glucose, others respond to glucose well, but their concentration-dependence curve is markedly shifted to higher sensitivity. Despite problems associated with beta cell cultures, these cell lines have provided some valuable information about physiological processes. However, an urgent need to establish a "normal" beta cell line of human or pig origin remains.