Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

Nicotinic acid inhibits glucose-stimulated insulin secretion via the G protein-coupled receptor PUMA-G in murine islet ß cells.

OBJECTIVES: Chronic administration of nicotinic acid (NA), a potent antilipidemic compound, aggravates glycemic control in diabetic patients. It is not known if NA has direct effects on islet ß cells. METHODS: Real-time reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunofluorescence techniques were used to examine the expression of NA receptor PUMA-G, a member of the G protein-coupled receptor (G-PCR) family, in murine islet ß cells. Calcium transient was measured using confocal microscopy, whereas the intracellular cyclic adenosine monophosphate and glucose-stimulated insulin secretion (GSIS) from isolated islets were determined by the enzyme-linked immunosorbent assay. RESULTS: High levels of PUMA-G transcripts and protein were detected in all ß cells, and about 40% of α cells. PUMA-G transcripts increased more than 3-fold in islets incubated with interferon γ. Cyclic adenosine monophosphate accumulation, induced by IBMX/forskolin, was inhibited by NA; however, the inhibition was completely abolished by pretreatment of the culture with pertussis toxin. No calcium transient was detected in islet cells in the presence of NA. Static incubation of islets with NA led to an approximately 30% reduction of GSIS. CONCLUSIONS: The results indicated that PUMA-G stimulation by NA in islet ß cells inhibited GSIS likely via activation of the Gi signaling pathway.

Intra-arterial targeted islet-specific expression of Sirt1 protects ß cells from streptozotocin-induced apoptosis in mice.

Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of ß cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of ß-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.

Heterogeneity in mitotic activity and telomere length implies an important role of young islets in the maintenance of islet mass in the adult pancreas.

Understanding the mechanisms of beta-cell dynamics in postnatal animals is central to cure diabetes. A major obstacle in evaluating the status of pancreatic cells is the lack of surface markers. Here we performed quantitative measurements of two internal markers to follow the developmental history of islets. One marker, cell-cycle activity, was established by measuring expression of Ki67 and the incorporation of 5-bromodeoxyuridine. The other marker, the aging process, was delineated by the determination of telomere length. Moreover, islet neogenesis, possibly from ductal precursors, was monitored by pancreatic duct labeling with an enhanced green fluorescence protein (EGFP) transgene. We found that islets from younger animals, on average, expressed higher Ki67 transcripts, displayed elevated 5-bromodeoxyuridine incorporation, and had longer telomeres. However, significant heterogeneity of these parameters was observed among islets from the same mouse. In contrast, the levels of proinsulin-1 transcripts in islets of different ages did not change significantly. Moreover, mitotic activities correlated significantly with telomere lengths of individual islets. Lastly, after 5.5 d pancreatic duct labeling, a few EGFP-positive islets could be identified in neonatal but not from adult pancreases. Compared with unlabeled control islets, EGFP-positive islets had higher mitotic activities and longer telomeres. The results suggest that islets are born at different time points during the embryonic and neonatal stages and imply that young islets might play an important role in the maintenance of islet mass in the adult pancreas.