Mechanisms of adult human ß-cell in vitro dedifferentiation and redifferentiation.

Recent studies in animal models and human pathological specimens suggest the involvement of ß-cell dedifferentiation in ß-cell dysfunction associated with type 2 diabetes. Dedifferentiated ß-cells may be exploited for endogenous renewal of the ß-cell mass. However, studying human ß-cell dedifferentiation in diabetes presents major difficulties. We have analysed mechanisms involved in human ß-cell dedifferentiation in vitro, under conditions that allow cell proliferation. Although there are important differences between the two cellular environments, ß-cell dedifferentiation in the two conditions is likely to share a number of common pathways. Insights from the in vitro studies may lead to development of approaches for redifferentiation of endogenous dedifferentiated ß-cells.

Correction of hyperglycemia in diabetic mice transplanted with reversibly immortalized pancreatic beta cells controlled by the tet-on regulatory system.

Pancreatic beta cell lines may offer an abundant source of cells for beta-cell replacement in type I diabetes. Using regulatory elements of the bacterial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse beta cells, we have shown that reversible immortalization is an efficient approach for regulated beta-cell expansion, accompanied by enhanced cell differentiation upon growth arrest. The original system employed the tet-off approach, in which the cells proliferate in the absence of tet ligands and undergo growth arrest in their presence. The disadvantage of this system is the need for continuous treatment with the ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate beta cell lines in which proliferation is regulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [3H]thymidine and BrdU incorporation assays. These cell lines were derived from insulinomas, which heritably developed in transgenic mice continuously treated with the tet derivative doxycycline (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozotocin-induced diabetic mice in the absence of dox. Such a system is more suitable for transplantation, compared with cells regulated by the tet-off approach, because ligand treatment is limited to cell expansion in culture and is not required for long-term maintenance of growth arrest in vivo.

Cell therapy approaches for the treatment of diabetes.

Pancreatic beta-cell replacement represents an attractive approach for treatment of type 1 and insulin-requiring type 2 diabetic patients. This prospect is currently restricted by the limited availability of donor cells. Recent developments, including beta-cell expansion by reversible immortalization, and generation of beta-cells by differentiation from embryonic and adult tissue progenitor cells, may provide abundant sources of cultured human beta-cells. Such cells could be genetically modified, as well as encapsulated in semi-permeable membranes, to increase their resistance to recurring autoimmunity (in type 1 diabetics) and to beta-cell degenerative agents (in type 2 diabetics).

Inducible and reversible beta-cell autoimmunity and hyperplasia in transgenic mice expressing a conditional oncogene.

Expression of the SV40 T antigen (Tag) in pancreatic beta-cells in transgenic mice has been shown to induce beta-cell tumorigenesis. We generated transgenic mice in which Tag expression is inducible and reversible by the tet-on gene regulation system. These mice develop beta-cell tumors only when treated with the inducer doxycycline (dox). Tag expression in vivo is reversible upon dox withdrawal. As a result, beta-cell proliferation is greatly reduced, indicating that genetic changes, which may occur in the transformed cells, do not allow Tag-independent proliferation. Induction of Tag expression after immune recognition of self-antigens has been established triggers an autoimmune response against beta-cells, as evidenced by insulitis. Shut-off of Tag expression results in elimination of insulitis, suggesting that this process depends on continuous expression of the target antigen. In addition, the reversibility of autoimmunity suggests that beta-cell damage caused by the anti-Tag immune response does not elicit secondary responses to other newly exposed beta-cell antigens, which would have persisted after Tag elimination. beta-Cell proliferation in this model is accompanied by cell apoptosis. Apoptosis persisted for several weeks in the islets after dox removal. In close to 40% of the mice analyzed, this process reduced the islet size back to normal, suggesting the existence of a homeostatic mechanism that maintains beta-cell mass within the normal range.

Adenovirus early region 3(E3) immunomodulatory genes decrease the incidence of autoimmune diabetes in NOD mice.

The early three (E3) region of the adenovirus (Ad) encodes a number of immunomodulatory proteins that interfere with class I major histocompatibility-mediated antigen presentation and confer resistance to cytokine-induced apoptosis in cells infected by the virus. Transgenic expression of Ad E3 genes under the rat insulin II promoter (RIP-E3) in beta-cells in nonobese diabetic (NOD) mice decreases the incidence and delays the onset of autoimmune diabetes. The immune effector cells of RIP-E3/NOD mice maintain the ability to infiltrate the islets and transfer diabetes into NOD-scid recipients, although at a significantly reduced rate compared with wild-type littermates. The islets of RIP-E3/ NOD mice can be destroyed by adoptive transfer of splenocytes from wild-type NOD mice; however, the time to onset of hyperglycemia is delayed significantly, and 40% of these recipients were not diabetic at the end of the experiment. These findings suggest that expression of E3 genes in beta-cells affects both the activation of immune effector cells and the intrinsic resistance of beta-cells to autoimmune destruction.

Genes induced by growth arrest in a pancreatic beta cell line: identification by analysis of cDNA arrays.

Pancreatic beta cell lines are a potentially attractive source of material for cell therapy of insulin-dependent diabetes mellitus. However, induction of proliferation in post-mitotic, differentiated beta cells is likely to affect the expression of multiple genes associated with cell function, resulting in dedifferentiation. We have developed a murine beta cell line by conditional transformation with the SV40 T antigen oncoprotein. These cells can undergo reversible induction of proliferation and growth arrest. Here we utilized this model to identify differences in gene expression between proliferating and quiescent beta cells, by analyzing known beta cell genes and differentially secreted proteins, as well as by a systematic survey of a mouse cDNA array. Our findings demonstrate that growth arrest stimulates expression of the insulin gene and genes encoding components of the insulin secretory vesicles. Screening of the cDNA array revealed the activation of multiple genes following growth arrest, many of them novel genes which may be related to beta cell function. Characterization of these genes is likely to contribute to our understanding of beta cell function and the ability to employ beta cell lines in cell therapy of diabetes.

Genetically engineered pancreatic beta-cell lines for cell therapy of diabetes.

The optimal treatment of insulin-dependent diabetes mellitus (IDDM), which is caused by the autoimmune destruction of pancreatic islet beta cells, would require the regulated delivery of insulin by transplantation of functional beta cells. beta-cell transplantation has so far been restricted by the scarcity of human islet donors. This shortage would be alleviated by the development of differentiated beta-cell lines, which could provide an abundant and well-characterized source of beta cells for transplantation. Using conditional transformation approaches, our laboratory has generated continuous beta-cell lines from transgenic mice. These cells produce insulin amounts comparable to those of normal islets and release insulin in response to physiological stimuli. Cell replication in these beta cells can be tightly controlled both in culture and in vivo, allowing regulation of cell number and cell differentiation. Another challenge to cell therapy of IDDM is the protection of transplanted cells from immunological rejection and recurring autoimmunity. By employing adenovirus genes which downregulate antigen presentation and increase cell resistance to cytokines, beta-cell transplantation across allogeneic barriers was achieved without immunosuppression. In principle, similar beta-cell lines can be derived from isolated human islets using viral vectors to deliver conditionally regulated transforming and immunomodulatory genes into beta cells. The combination of these approaches with immunoisolation devices holds the promise of a widely available cell therapy for treatment of IDDM in the near future.

Prospects for gene therapy of insulin-dependent diabetes mellitus.

The application of gene therapy to Type I (insulin-dependent) diabetes mellitus awaits improvements in gene transfer technologies and the development of better tools for accurate diagnosis of pre-diabetic people. Identification of the most promising candidate genes for gene transfer requires further elucidation of the molecular events involved in beta-cell autoimmune destruction, islet ontogeny and differentiation, and beta-cell function. This review outlines a number of possible targets for gene therapy in Type I diabetes, which could help prevent the autoimmune damage to islets, induce islet regeneration, and restore insulin production through engineering of self non-beta cells or beta-cell transplantation. It also evaluates their potential merits and drawbacks.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

Functional analysis of a conditionally transformed pancreatic beta-cell line.

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.

Cell-based therapy for insulin-dependent diabetes mellitus.

Insulin-secreting pancreatic beta-cell lines represent a promising approach for treatment of insulin-dependent diabetes mellitus. Such cell lines can provide an abundant and reproducible source of beta-cell material for transplantation. A number of highly differentiated beta-cell lines have been developed using transgenic mice. These cells produce insulin amounts comparable to normal pancreatic islets and release it in response to physiological insulin secretagogues. Our laboratory has employed a reversible transformation approach to tightly regulate cell replication in these beta-cell lines, both in culture and in vivo. Beta-cell lines can be modulated by gene transfer to improve their function and survival. We have utilized adenovirus genes, which downregulate antigen presentation and increase cell resistance to cytokines, to facilitate transplantation of mouse beta cells across allogeneic barriers. These approaches could be applied to the development of human beta-cell lines by genetic engineering of isolated human islets.

Development of engineered pancreatic beta-cell lines for cell therapy of diabetes.

Insulin-secreting pancreatic beta-cell lines represent a promising approach for treatment of insulin-dependent diabetes mellitus (IDDM). Our laboratory has developed a number of highly-differentiated beta-cell lines in transgenic mice. These cells produce insulin amounts comparable to normal pancreatic islets and release it in response to physiological insulin secretagogues. Using a reversible transformation system it has become possible to tightly regulate cell replication in these beta-cell lines both in culture and in vivo. By employing adenovirus genes which downreguate antigen presentation and increase cell resistance to cytokines mouse beta cells could be transplanted across allogeneic barriers. These approaches could be applied to the development of human beta-cell lines by genetic engineering of isolated human islets.

Abnormal regulation of HGP by hyperglycemia in mice with a disrupted glucokinase allele.

Glucokinase (GK) catalyzes the phosphorylation of glucose in beta-cells and hepatocytes, and mutations in the GK gene have been implicated in a form of human diabetes. To investigate the relative role of partial deficiencies in the hepatic vs. pancreatic GK activity, we examined insulin secretion, glucose disposal, and hepatic glucose production (HGP) in response to hyperglycemia in transgenic mice 1) with one disrupted GK allele, which manifest decreased GK activity in both liver and beta-cells (GK+/-), and 2) with decreased GK activity selectively in beta-cells (RIP-GKRZ). Liver GK activity was decreased by 35-50% in the GK+/- but not in the RIP-GKRZ compared with wild type (WT) mice. Hyperglycemic clamp studies were performed in conscious mice with or without concomitant pancreatic clamp. In all studies [3-(3)H]glucose was infused to measure the rate of appearance of glucose and HGP during 80 min of euglycemia (Glc approximately 5 mM) followed by 90 min of hyperglycemia (Glc approximately 17 mM). During hyperglycemic clamp studies, steady-state plasma insulin concentration, rate of glucose infusion, and rate of glucose disappearance (Rd) were decreased in both GK+/- and RIP-GKRZ compared with WT mice. However, whereas the basal HGP (at euglycemia) averaged approximately 22 mg x kg(-1) x min(-1) in all groups, during hyperglycemia HGP was suppressed by only 48% in GK+/- compared with approximately 70 and 65% in the WT and RIP-GKRZ mice, respectively. During the pancreatic clamp studies, the ability of hyperglycemia per se to increase Rd was similar in all groups. However, hyperglycemia inhibited HGP by only 12% in GK+/-, vs. 42 and 45%, respectively, in the WT and RIP-GKRZ mice. We conclude that, although impaired glucose-induced insulin secretion is common to both models of decreased pancreatic GK activity, the marked impairment in the ability of hyperglycemia to inhibit HGP is due to the specific decrease in hepatic GK activity.

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Expression of adenoviral E3 transgenes in beta cells prevents autoimmune diabetes.

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistence in vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor alpha (TNF-alpha). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-alpha, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of beta cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the beta cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the beta-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in these in vivo studies, the relative importance of this process and the control of TNF-alpha cytolysis must await further genetic dissection of the introduced Ad genes.

Time-sensitive reversal of hyperplasia in transgenic mice expressing SV40 T antigen.

The role of viral oncoprotein expression in the maintenance of cellular transformation was examined as a function of time through controlled expression of simian virus 40 T antigen (TAg). Expression of TAg in the submandibular gland of transgenic mice from the time of birth induced cellular transformation and extensive ductal hyperplasia by 4 months of age. The hyperplasia was reversed when TAg expression was silenced for 3 weeks. When TAg expression was silenced after 7 months, however, the hyperplasia persisted even though TAg was absent. Although the polyploidy of ductal cells could be reversed at 4 months of age, cells at 7 months of age remained polyploid even in the absence of TAg. These results support a model of time-dependent multistep tumorigenesis, in which virally transformed cells eventually lose their dependence on the viral oncoprotein for maintenance of the transformed state.