1) Release and degradation of the two incretin hormones show dissociated changes in obesity: GLP-1 but not GIP secretion is lower after meal ingestion and oral glucose, whereas GIP but not GLP-1 metabolism is increased after meal ingestion. 2) Increased plasma DPP-4 activity in obesity is not associated with a generalized augmented incretin hormone metabolism.

The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic ␤-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.diabetes ͉ mutagenesis ͉ soluble N-ethylmaleimide-sensitive factor attachment protein receptor ͉ schizophrenia ͉ exocytosis I ntracellular membrane fusion events within eukaryotic cells are mediated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. The prototypic SNARE-mediated fusion event is synaptic vesicle exocytosis, in which the membrane-associated SNAP-25 and syntaxin-1A interact with the vesicle-associated membrane protein (VAMP) to create a stable ternary core complex with 1:1:1 stoichiometry (1). This interaction has been extensively studied in various systems including neurons, neuroendocrine chromaffin cells, and pancreatic ␤-islet cells, all of which use the neuronal core complex proteins to regulate secretion (2, 3). In vivo knockout and mutagenesis studies in invertebrate systems have allowed SNARE function to be analyzed at the level of the synapse (4), but their scope has been limited by the failure of these organisms to model higher neurological functions in which SNARE function is strongly implicated. A major goal, therefore, has been the generation of a mammalian model of SNARE dysfunction in which the effects on complex neurological and behavioral phenotypes can be assessed.Both Vamp2 and Snap25 knockouts are perinatally lethal in homozygous mice, and heterozygotes show no apparent phenotype, so detailed analysis of viable affected individuals has not been possible (5, 6). The spontaneous mouse mutant coloboma carries a heterozygous deletion spanning four genes including Snap25 (7), but its locomotor hyperactivity phenotype is likely to depend on the additional loss of one or more of the other genes within the deleted region. Most recently, a viable syntaxin-1a knockout mouse has been described with subtle defects in long-term potentiation and fear-memory behavior, although normal synaptic transmission is unaffected likely because of functional compensation by syntaxin-1b (8). Existing mammalian SNARE mutations have therefore offered only very limited insights into the consequences of SNARE dysfunction in an adult system and have yielded no informat...

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.

The early release of GLP-1 and GIP are more pronounced in the morning than in the afternoon. This may contribute to the more rapid early insulin response, more pronounced potentiation of beta-cell function, and lower glucose after the morning meal.

Cholesterol-rich clusters of SNARE (soluble NSF attachment protein receptor) proteins have been implicated as being important for exocytosis. Here we demonstrate the significance of cholesterol for normal biphasic insulin secretion in mouse beta cells by removal of cholesterol from the plasma membrane using methyl-beta-cyclodextrin (MBCD). Maximal inhibition of insulin secretion in static incubations was achieved using 0.1 mM MBCD. In in situ pancreatic perfusion measurements, both first and second phase insulin secretions were reduced by approximately 50% (P<0.05). This was accompanied by a reduced number of docked large dense core vesicles (LDCVs) (approximately 40%; P<0.01) and a reduced exocytotic response (>50%; P<0.01). Further, subcellular fractionations demonstrated movement of the synaptosomal protein of 25 kDa (SNAP-25) from the plasma membrane to the cytosol after MBCD treatment. The inhibitory actions of MBCD were counteracted by subsequent addition of cholesterol. We hypothesize that desorption of cholesterol leads to the disturbance of a basic exocytotic mechanism partly due to migration of SNAP-25, and we conclude that insulin secretion is highly sensitive to changes in plasma membrane cholesterol.