Glucokinase catalyzes a rate-limiting step in glucose metabolism in hepatocytes and pancreatic ␤ cells and is considered the "glucose sensor" for regulation of insulin secretion. Patients with maturity-onset diabetes of the young (MODY) have heterozygous point mutations in the glucokinase gene that result in reduced enzymatic activity and decreased insulin secretion. However, it remains unclear whether abnormal liver glucose metabolism contributes to the MODY disease. Here we show that disruption of the glucokinase gene results in a phenotype similar to MODY in heterozygous mice. Reduced islet glucokinase activity causes mildly elevated fasting blood glucose levels. Hyperglycemic clamp studies reveal decreased glucose tolerance and abnormal liver glucose metabolism. These findings demonstrate a key role for glucokinase in glucose homeostasis and implicate both islets and liver in the MODY disease.Pancreatic ␤ cells and hepatocytes are the two major cell types responsible for maintaining glucose homeostasis. ␤ cells respond to changes in plasma glucose levels by regulating their insulin release, whereas hepatocytes adjust their glucose uptake and glucose production to changes in plasma insulin. Both cell types express a specialized high K m member of the hexokinase family of enzymes, glucokinase (GK), 1 which catalyzes a rate-limiting step in glucose metabolism, the phosphorylation of glucose to glucose 6-phosphate. In ␤ cells glucose metabolism generates signals for insulin secretion, and GK is considered the major "glucose sensor" that couples the extracellular glucose levels to insulin secretion (1). Recent DNA polymorphism studies of patients with maturity-onset diabetes of the young (MODY), a form of non-insulin-dependent (type II) diabetes mellitus, have established that heterozygous point mutations in the GK gene are associated with the development of diabetes (2, 3). The mutations result in reduced enzymatic activity (4, 5), which causes abnormal glucose sensing and decreased insulin secretion (6 -8). In ␤ cells the GK promoter is constitutively active, and GK activity is regulated by glucose at post-transcriptional levels (9). The dominance of the GK mutations in MODY has therefore been explained as a gene dosage effect. In contrast, hepatocytes utilize a different GK promoter, which is regulated by insulin (10,11). This promoter has the potential to up-regulate the expression of the normal allele in hepatocytes of MODY patients to compensate for the reduced GK activity caused by the mutant allele. However, this transcriptional regulation depends on plasma insulin levels, which in MODY patients are reduced due to the islet abnormality. Hence, it remains unclear whether abnormal liver glucose metabolism contributes to the MODY disease. Previously we have attenuated GK function specifically in ␤ cells in transgenic mice using an antisense approach (12). These mice manifested a decreased insulin secretory response to glucose; however, they did not show changes in fasting plasma glucose levels or glucose tol...

Development of beta-cell lines for cell therapy of diabetes is hindered by functional deviations of the replicating cells from the normal beta-cell phenotype. In a recently developed cell line, denoted betaTC-tet, derived from transgenic mice expressing the SV40 T antigen (Tag) under control of the tetracycline (Tc) gene regulatory system, growth arrest can be induced by shutting off Tag expression in the presence of Tc. Here, we compared differentiated cell functions in dividing and growth-arrested betaTC-tet cells, both in culture and in vivo. Proliferating cells stably maintained normal glucose responsiveness for >60 passages in culture. Growth-arrested cells survived for months in culture and in vivo and maintained normal insulin production and secretion. After growth arrest, the cells gradually increased their insulin content three- to fourfold. This occurred without significant changes in insulin biosynthetic rates. At high passage numbers, proliferating betaTC-tet cells exhibited an abnormal increase in hexokinase expression. However, the upregulation of hexokinase was reversible upon growth arrest. Growth-arrested cells transplanted intraperitoneally into syngeneic recipients responded to hyperglycemia by a significant increase in insulin secretion. These findings demonstrate that transformed beta-cells maintain function during long periods of growth arrest, suggesting that conditional transformation of beta-cells may be a useful approach for developing cell therapy for diabetes.

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic ␤-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in ␤TC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmentedglucose-stimulatedinsulinsecretioninadosedependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from ␤TC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of ␤TC3 cells is a novel isozyme possessing a K m of 0.47 M for cAMP and 0.25 M for cGMP. The PDE1C isozyme of ␤TC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC 50 ‫؍‬ 7.5 and 4.5 M, respectively) and resistant to vinpocetine (IC 50 > 100 M). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.

Insulin secretory physiology has been characterized in tumor cell lines derived by primary culture of insulinomas that developed in transgenic mice expressing the large T-antigen of SV40 in pancreatic islet beta-cells. Cells in one of these lines, beta TC-3, contain large amounts of insulin (3100 +/- 294 ng/100 micrograms cellular protein). Constitutive release of insulin over 2 h in static incubation was low at 31.9 ng/100 micrograms protein and was increased 2-fold by glucose (16.7 mM) and 8-fold by depolarizing concentrations of potassium (45 mM). Isobutylmethylxanthine (IBMX; 0.5 mM) and forskolin (5 and 50 microM), which elevated cellular levels of cAMP, were ineffective as secretagogues, but dramatically potentiated glucose and potassium effects on insulin release (6.5- and 4-fold, respectively). A variety of other known insulin secretagogues stimulated insulin release in a manner analogous to their effects in normal islets. The sulfonylurea glipizide (1 microM) and the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) stimulated insulin release 3.4- and 13.7-fold, respectively. The cholinergic agonist carbachol (2 microM) was ineffective alone, but potentiated glucose-induced insulin release 2.8-fold. Comparable stimulation of insulin release by glucose (16.7 mM) and glucose (16.7 mM) plus IBMX (0.5 mM) was noted with several other beta TC lines, which were derived independently from separate transgenic mice. Glucose- and glucose- plus IBMX (0.5 mM)-induced insulin release occurred progressively from 0.15-16.7 mM, indicating that insulin release from beta TC-3 cells occurred at much lower levels than that from normal islets. However, as in the normal islet, the glucose concentration dependency for insulin release was highly correlated (r = 0.93) with the glucose concentration dependency for glucose utilization (measured by 3H2O formation from [5-3H]glucose). This suggests that glucose induces insulin release from beta TC-3 cells by a mechanism similar to that in the normal islet. The high insulin content, the multifold stimulation of insulin release by a variety of secretagogues, their convenient propagation in culture, and the renewable source of these cell lines make the beta TC cells a convenient model for studies of beta-cell function.

Understanding the biological potential of fetal stem/progenitor cells will help define mechanisms in liver development and homeostasis. We isolated epithelial fetal human liver cells and established phenotype-specific changes in gene expression during continuous culture conditions. Fetal human liver epithelial cells displayed stem cell properties with multilineage gene expression, extensive proliferation and generation of mesenchymal lineage cells, although the initial epithelial phenotype was rapidly supplanted by meso-endodermal phenotype in culture. This meso-endodermal phenotype was genetically regulated through cytokine signaling, including transforming growth factor β, bone morphogenetic protein, fibroblast growth factor and other signaling pathways. Reactivation of HNF3α (FOXA1) transcription factor, a driver of hepatic specification in the primitive endoderm, indicated that the meso-endodermal phenotype represented an earlier developmental stage of cells. We found that fetal liver epithelial cells formed mature hepatocytes in vivo, including after genetic manipulation using lentiviral vectors, offering convenient assays for analysis of further cell differentiation and fate. Taken together, these studies demonstrate plasticity in fetal liver epithelial stem cells, offer paradigms for defining mechanisms regulating lineage switching in stem cells, and provide potential avenues for regulating cell phenotypes for applications of stem cells, such as for cell therapy.