Studies On Insulin Exocytosis Regulation In Munc13-1-deficient Mice

Insulin release from pancreaticβcells in response to glucose is characterized by a biphasic time course. A full biphasic insulin response is the ultimate indication of a well-coupled healthyβcell. Interestingly, diabetes is associated with disturbance in the biphasic pattern of insulin secretion, resulting in a diminished first phase and a blunted second phase. Although first described almost 40 years ago, the cell biology of the two phase of insulin secretion remains debated. Thus it is of great importance to understand the mechanisms whereby the biphasic insulin response is generated and regulated. However, as the generation of biphasic insulin release probably involves concerted action of multi-factors, the significance of different functional LDCVs in the contribution of biphasic insulin release under glucose stimulation remains to be clarified. Munc13 proteins constitute a family of 4 mammalian homologues of C. elegans Unc-13 and Drosophila Dunc-13. Genetic deletion of Munc13-1/unc13 causes total arrest of synaptic transmission due to a complete loss of fusion-competent synaptic vesicles. Munc13 proteins contain C2 domain and a DAG binding C1 domain. The binding of DAG/phorbol-ester to C1 domain leads to enhanced priming activity of Munc13-1. The requirement of Munc13s for LDCVs, however, has not been established.In the current study, we have aimed at elucidating the role of Munc13-1 in the insulin secretion and dissecting its site of action using Munc13-1 knockout and Munc13-1H567K knockin mice. Secretion was analyzed by a combination of capacitance and dynamic insulin release measurements. Main results of the study are as fellows.1.Munc13-1-defecient mice die within a few hours of birth. Hence, we had to isolateβcells from newborn mice. we found isolatedβcells from newborn mice apparently developed typicalβcell characteristics including Na+ channel inactivation and response to glucose after 3 days in culture. The establishment of the experimental system will open possibility for elucidating the molecular mechanism of insulin secretion control using various knockout mice that are lethal at birth.2. Although Munc13-1 is absolutely necessary for neurotransmitter release from synaptic vesicles, the requirement of Munc13-1 for large dense core vesicles (LDCVs) has not been demonstrated. Cm measurements combined with flash photolysis was employed to assess the exocytosis from different pools of LDCVs. We found that the sustained component of exocytosis was almost completely abolished in Munc13-1-deficientβcells, and to a lesser extent in KI cells. The exocytotic burst, which represents the releasable vesicles, was not significantly influenced. We thus conclude that Munc13-1 is required for the second phase of insulin secretion in response to glucose stimulation. This is the first evidence that Munc13-1 is required for the supply of LDCVs during prolonged stimulus. Whereas knockout of Munc13-1 causes complete loss of readily releasable synaptic vesicles, we found that readily releasable LDCVs are normal in Munc13-1-deficient beta cells. These results suggest different requirement of Munc13-1 for synaptic vesicles and LDCVs.3. The mechanism underlying the generation of biphasic insulin response is elusive. The ultimate answer to this question would have to wait for the identification of molecular players that are specifically involved in the production of the biphasic insulin release. We now show that genetic ablation of Munc13-1 preferentially abolishes the second phase of insulin response to glucose, providing direct evidence that the priming of LDCVs from an unprimed pool constitutes the rate limiting step in the second phase of insulin release.4. It has been demonstrated that metabolic signals, other than the membrane-depolarizing signal (generated by closing KATP channels), are required for the production (or amplification) of the second phase. However the identities of these signals remain to be identified. Our results showing that the sustained insulin release is impaired in DAG-binding-deficient Munc13-1H567K KI mice suggest that glucose-activated DAG signaling is involved in the second phase insulin response to glucose and that Munc13-1 acts as a key target of DAG/phorbol-ester. It is interesting that the effect of PMA is completely abolished in the Munc13-1 KI mice, which seems to suggest that PKC activation by PMA plays no role in the control of exocytosis in mouseβcells. However, an alternative explanation could be that PKC acts upstream of Munc13-1. The conclusive role of PKC in insulin secretion will have to wait for further detailed studies through combined efforts from genetic manipulation to integrated physiological analysis.