Conditional ablation of Gsk-3ß in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice.

AIMS/HYPOTHESIS: Glycogen synthase kinase 3ß (GSK-3ß) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3ß in mice would result in enhanced beta cell mass and function. METHODS: Mice with beta cell deficiency of GSK-3ß (ß-Gsk-3ß [also known as Gsk3b](-/-)) were generated by breeding Gsk-3ß (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting. RESULTS: On a normal diet ß-Gsk-3ß ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet ß-Gsk-3ß (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation. CONCLUSIONS/INTERPRETATION: These results are consistent with a mechanism whereby endogenous GSK-3ß activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.

Regulation of beta-cell mass and function by the Akt/protein kinase B signalling pathway.

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of beta-cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of beta cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of beta-cell mass and function by Akt.

An expression profile of human pancreatic islet mRNAs by Serial Analysis of Gene Expression (SAGE).

AIMS/HYPOTHESIS: The Human Genome Project seeks to identify all genes with the ultimate goal of evaluation of relative expression levels in physiology and in disease states. The purpose of the current study was the identification of the most abundant transcripts in human pancreatic islets and their relative expression levels using Serial Analysis of Gene Expression. METHODS: By cutting cDNAs into small uniform fragments (tags) and concatemerizing them into larger clones, the identity and relative abundance of genes can be estimated for a cDNA library. Approximately 49,000 SAGE tags were obtained from three human libraries: (i) ficoll gradient-purified islets (ii) islets further individually isolated by hand-picking, and (iii) pancreatic exocrine tissue. RESULTS: The relative abundance of each of the genes identified was approximated by the frequency of the tags. Gene ontology functions showed that all three libraries contained transcripts mostly encoding secreted factors. Comparison of the two islet libraries showed various degrees of contamination from the surrounding exocrine tissue (11 vs 25%). After removal of exocrine transcripts, the relative abundance of 2180 islet transcripts was determined. In addition to the most common genes (e.g. insulin, transthyretin, glucagon), a number of other abundant genes with ill-defined functions such as proSAAS or secretagogin, were also observed. CONCLUSION/INTERPRETATION: This information could serve as a resource for gene discovery, for comparison of transcript abundance between tissues, and for monitoring gene expression in the study of beta-cell dysfunction of diabetes. Since the chromosomal location of the identified genes is known, this SAGE expression data can be used in setting priorities for candidate genes that map to linkage peaks in families affected with diabetes.

Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation.

During embryonic life, the development of a proper mass of mature pancreatic tissue is thought to require the proliferation of precursor cells, followed by their differentiation into endocrine or acinar cells. We investigated whether perturbing the proliferation of precursor cells in vitro could modify the final mass of endocrine tissue that develops. For that purpose, we used activators or inhibitors of signals mediated by receptor tyrosine kinases. We demonstrated that when embryonic day 13.5 rat pancreatic epithelium is cultured in the presence of PD98059, an inhibitor of the mitogen-activated protein (MAP) kinase, epithelial cell proliferation is decreased, whereas endocrine cell differentiation is activated. On the other hand, in the presence of epidermal growth factor (EGF), an activator of the MAP kinase pathway, the mass of tissue that develops is increased, whereas the absolute number of endocrine cells that develops is decreased. Under this last condition, a large number of epithelial cells proliferate but remain undifferentiated. In a second step, when EGF is removed from the pool of immature pancreatic epithelial cells, the cells differentiate en masse into insulin-expressing cells. The total number of insulin-expressing cells that develop can thus be increased by first activating the proliferation of immature epithelial cells with growth factors, thus allowing an increase in the pool of precursor cells, and next allowing their differentiation into endocrine cells by removing the growth factor. This strategy suggests a possible tissue engineering approach to expanding beta-cells.

No evidence for linkage or for diabetes-associated mutations in the activin type 2B receptor gene (ACVR2B) in French patients with mature-onset diabetes of the young or type 2 diabetes.

Activins are members of the transforming growth factor-beta superfamily. They have a wide range of biological effects on cell growth and differentiation. For transmembrane signaling, activins bind directly to activin receptor type 2A (ACVR2A) or 2B (ACVR2B). Transgenic and knock-out mice for the ACVR2B gene display various endocrine pancreas-related abnormalities, including islet hypoplasia and glucose intolerance, demonstrating the crucial role of ACVR2B in the regulation of pancreas development. We have thus examined the contribution of this factor to the development of mature-onset diabetes of the young (MODY) and type 2 diabetes. No evidence of linkage at the ACVR2B locus has been detected in MODY families with unknown etiology for diabetes or found in affected sib pairs from families with type 2 diabetes. Mutation screening of the coding sequence in MODY probands and in a family with severe type 2 diabetes, including a case of pancreatic agenesis, showed single nucleotide polymorphisms that did not cosegregate with MODY and were not associated with type 2 diabetes. Our results indicate that ACVR2B does not represent a common cause of either MODY or type 2 diabetes in the French Caucasian population.

Pancreatic pattern of expression of thyrotropin-releasing hormone during rat embryonic development.

In rodents, the first insulin-producing cells appear in the pancreas at mid-gestation around embryonic day 11 (E11). However, on the basis of various features, such as morphology or hormonal coexpression, it is apparent that these initial insulin-expressing cells are different from those that develop after E15. In the present study, the pancreatic expression of both thyrotropin-releasing hormone (TRH) mRNA and insulin was studied during embryonic and fetal life. We report here that in the rat, while insulin mRNA is detected in the pancreas as early as E12, TRH mRNA cannot be detected before E16. At that stage and later on during fetal and early postnatal life, TRH mRNA is detected in insulin-producing cells, no signal being detected in other endocrine cell types or in exocrine tissue. It was also noted, by means of triple staining performed at E17, that the expression of TRH mRNA was restricted to insulin-expressing cells negative for glucagon, whereas the few insulin-expressing cells present at that stage, which coexpress insulin and glucagon, did not express TRH mRNA. Taken together, these data indicate that TRH is a marker of insulin-expressing cells, which develop after E15.