The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (≤2-fold stimulation by 15 m m o l / l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2-to 5-fold stimulation), and 16% were strongly responsive (5-to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (~7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 µmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/p a l m i t a t e (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the AT P -

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116 [1], which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), andCDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER)depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E.siRNA-mediated Ufm1 or Ufbp1knockdown enhances apoptosis upon ER stress.Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1participate in preventing ER stress-induced apoptosis in protein secretory cells.

The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet ␤-cell development, but its effects on adult ␤-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3 H] thymidine and 5-bromo-2-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-␤GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMVNkx6.1-treated islets are ␤ cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucosestimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [ 3 H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating ␤-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of ␤-cell mass in treatment of both major forms of diabetes.Type 1 diabetes results from autoimmune destruction of insulin-producing ␤ cells in the islets of Langerhans, whereas type 2 diabetes involves loss of glucose-stimulated insulin secretion (GSIS) and a gradual diminution of ␤-cell mass (45). Insulin injection therapy has been the standard treatment for type 1 diabetes since the discovery of the hormone more than 80 years ago. Islet transplantation has been investigated as an alternative to insulin injection, but a major obstacle to broad application of this approach has been an inadequate supply of human islets (21). Pharmacotherapy of type 2 diabetes includes administration of agents that enhance insulin secretion, but these drugs often lose efficacy over time and cause complications such as hypoglycemia (30). Moreover, no controlled strategy for restoration of ␤-cell mass has been identified for the type 2 disease. Thus, a more complete understanding of the mechanisms that control islet ␤-cell growth and function is required in order to develop more effective therapies for both major forms of diabetes.Several members of the homeodomain family of transcription factors, including Pdx1, Hb9/Hlxb9, Nkx2.2, Nkx6.1, Isl-1, Pax6, an...