Regulation of Insulin Exocytosis by Munc13-1

Abstract

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1 H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.

H3 Domain of Syntaxin 1A Inhibits KATP Channels by Its Actions on the Sulfonylurea Receptor 1 Nucleotide-Binding Folds-1 and -2

“…The likely reason is that the wild type Syn-1A when dialyzed exist in the free form, which Margittai et al (18) reported to be capable of adopting reversibly the closed or open conformation within a millisecond. Syn-1A could be "arrested" in one conformational state by regulatory proteins, such as Munc18a, which fixes Syn-1A into the closed conformation (15), and Munc13-1 (26,28), which would stabilize the open conformation. To more convincingly distinguish the distinct actions between the closed form and open form Syn-1A will require a constitutively closed conformation Syn-1A mutant, which, to our knowledge, has not been reported.…”

Munc13-1 Deficiency Reduces Insulin Secretion and Causes Abnormal Glucose Tolerance

“…Islet levels of syntaxin 1A and the nSec1/ Munc18-1 proteins are also reduced. Overexpression of Munc13-1 in insulinoma cells greatly potentiated insulin exocytosis evoked by glucose and phorbol-ester stimulation (20). This led us to postulate that reduced levels of Munc13-1 and SNARE proteins may directly contribute to the dysregulated insulin release from diabetic islets.…”

“…We recently reported that the levels of Munc13-1 are severely reduced in pancreatic islets of type 2 diabetic GK and fa/fa Zucker rats (20) as well as in diabetic human islets (21). Islet levels of syntaxin 1A and the nSec1/ Munc18-1 proteins are also reduced.…”

“…The heterozygous mice do not exhibit neurological deficits (13,19). The expression levels of Munc13-1 in Munc13-1 ϩ/ϩ , Munc13-1 ϩ/Ϫ , and Munc13-1 H567K/ϩ mouse brains were determined by Western blotting using a rabbit antibody generated against the NH 2 -terminal amino acids 1-305 of Munc13-1 (19,20). Consistent with previous reports (19), Munc13-1 levels were significantly reduced by 20% (n ϭ 2) in the brains of Munc13-1 ϩ/Ϫ mice compared with wild-type Munc13-1 ϩ/ϩ control mice (Fig.…”

“…Using Munc13-1-deficient mice (Munc13-1 Ϫ/Ϫ ) and knockin mice (Munc13-1 H567K/H567K ), the latter carrying a single-point mutation (H567K) within the Munc13-1 C 1 domain that abolishes DAG and phorbol ester binding, we showed previously that Munc13 proteins are the primary presynaptic phorbol ester receptors and mediate the priming of synaptic vesicles (19,22). Since mice homozygous for either one of these mutations die shortly after birth, we have used in the present study heterozygous mice (Munc13-1 ϩ/Ϫ and Munc13-1 H567K/ϩ ) to examine the islet Munc13-1 actions in insulin secretory granule priming, assuming that the reduced islet levels of Munc13-1 in heterozygous deletion mutant mice would mimic the Munc13-1 deficiency observed in type 2 diabetes (20,21). We show that heterozygous Munc13-1-deficient (Munc13-1 ϩ/Ϫ ) mice exhibit glucose intolerance that is caused by impaired insulin secretion.…”