Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimer's disease genes and by Aβ amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimer's disease targeting neuroprotection.

Secretion of the peptide hormone insulin from pancreatic β cells constitutes an important step in the regulation of body homeostasis. Insulin is stored in large dense core vesicles and released by exocytosis, a multistage process involving transport of vesicles to the plasma membrane, their docking, priming and finally their fusion with the plasma membrane. Some of the protein components necessary for this process have been identified in β cells. The export of potent and potentially harmful substances has to be tightly controlled. The secretory response in pancreatic β cells requires the concerted action of nutrients together with enteric hormones and neurotransmitters acting on G‐protein coupled receptors. It is well established that glucose and other metabolizable nutrients depolarize the β‐cell membrane and the ensuing Ca²⁺ influx through voltage‐dependent channels constitutes a main stimulus for insulin exocytosis. Theoretical considerations and recent observations suggest in addition an organizing role for the Ca²⁺ channel similar to neurotransmission. A second regulatory control on exocytosis is exerted by monomeric and heterotrimeric G‐proteins. The monomeric GTPase Rab3A controls insulin secretion through cycling between a guanosine triphosphate liganded vesicle‐bound form and a guanosine diphosphate liganded, cytosolic form. The effect of neurohormones is transduced by the heterotrimeric GTPases. Whereas pertussis‐toxin sensitive α‐subunits exert direct inhibition at the level of exocytosis, the Gβγ‐subunits are required for stimulation. It is possible that these GTPases exert immediate regulation, while protein kinases and phosphatases may modulate long‐term adaptation at the exocytotic machinery itself. The molecular nature of their activators and effectors still await identification. Insights into the progression of the exocytotic vesicle from docking to fusion and how these processes are precisely regulated by proteins and second messengers may provide the basis for new therapeutic principles.

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight 1 . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations 2 . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3-5. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulinsecreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in β-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunoflu-orescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decresed by the overexpression of wildtype Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human β-cells, a different Rab3 isoform but with homologous function may replace Rab3A.

The Ca2+‐ and phospholipid‐binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+‐affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 μM cytosolic free Ca2+. The well‐characterized synaptotagmin isoforms I and II are present in pancreatic β‐cell lines RINm5F, INS‐1 and HIT‐T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin‐containing secretory granules whereas only minor amounts were found on synaptic vesicle‐like microvesicles. Antibodies or Fab‐fragments directed against the Ca2+‐dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+‐stimulated, but not GTPγS‐induced exocytosis from streptolysin‐O‐permeabilized INS‐1 and HIT‐T15 cells. Transient expression of wild‐type synaptotagmin II did not alter exocytosis in HIT‐T15 cells. However, mutations in the Ca2+‐dependent phospholipid binding site of the first C2 domain (Δ180–183, D231S) again inhibited only Ca2+‐, but not GTPγS‐evoked exocytosis. In contrast, mutations in the IP4‐binding sites of the second C2 domain (Δ325–341; K327,328,332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Δ180–183/K327,328,332Q) induced greater inhibition than mutant Δ180–183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.