Biomedical subjects

S Efrat

Publications and source records attributed to S Efrat.

At least 37 records · Page 2Linked to original sources

PubMed1995

Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

Adenovirus E3 Proteins↗

PubMed1995

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Animals↗

PubMed1995

Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion.

Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic beta-cell line (beta TC cells). In vitro assays showed that carboxylmethylation of a membrane 23-kDa protein was induced by guanine nucleotides, best demonstrated by the nonhydrolyzable GTP analog, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). GTP gamma S also induced translocation of this 23-kilodalton (kDa) protein from cytosol to particulate fractions. Immunoblotting with antiserum sc-65 raised against Rap1 identified the carboxyl-methylated 23-kDa protein as Rap1. 1) The 23-kDa carboxyl-methylated protein separated by two-dimensional electrophoresis overlapped with the 23-kDa protein detected by immunoblotting. 2) GTP gamma S, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact beta TC cells demonstrated the carboxylmethylation of the 23-kDa protein in response to glucose and depolarizing concentrations of potassium, an effect that was abolished by the calcium channel inhibitor, D600. Similarly, N-acetyl-S-trans,trans-farnesyl-L-cysteine, an inhibitor of in vivo carboxylmethylation at COOH-terminal S-farnesylcysteine by methyltransferase, inhibited carboxylmethylation of the 23-kDa protein in intact cells and reduced insulin secretion in response to glucose and potassium. These data establish a correlation between insulin secretion and carboxylmethylation of a 23-kDa protein that comigrates with Rap1.

Acetylcysteine↗

PubMed1995

The role of the insulin control element and RIPE3b1 activators in glucose-stimulated transcription of the insulin gene.

The most important regulator of insulin expression in islet beta-cells is glucose, which stimulates insulin gene transcription, protein synthesis, and secretion. Glucose-induced insulin gene transcription is regulated by cis-acting elements found within the 5'-flanking region of the insulin gene. We previously demonstrated that the insulin control element (ICE, -100 to -91) and RIPE3b1 (-115 to -107) elements mediated this response in the HIT T-15 beta-cell line. In this study, we examined more closely how these insulin gene control elements regulate glucose-induced transcription. RIPE3b1 element binding was shown to be induced by glucose in both mouse beta TC-6 and beta TC-3 cell lines, although higher glucose concentrations were necessary in the beta-cells (beta TC-6) that responded to physiological glucose concentrations. RIPE3b1 binding was also regulated in glucose-stimulated beta- cells by various effectors of this response. The RIPE3b1 or ICE elements were shown to independently direct glucose-stimulated expression from minimal heterologous promoter constructs. We conclude that the RIPE3b1 and ICE elements are the principal mediators of glucose-stimulated transcription of the insulin gene.

Animals↗

PubMed1994

Ribozyme-mediated attenuation of pancreatic beta-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion.

Phosphorylation of glucose to glucose 6-phosphate by glucokinase (GK; EC 2.7.1.2) serves as a glucose-sensing mechanism for regulating insulin secretion in beta cells. Recent findings of heterozygous GK gene mutations in patients with maturity-onset diabetes of the young (MODY), a form of type II (non-insulin-dependent) diabetes characterized by autosomal dominant inheritance, have raised the possibility that a decrease in beta-cell GK activity may impair the insulin secretory response of these cells to glucose. To generate an animal model for MODY we have expressed in transgenic mice a GK antisense RNA with a ribozyme element under control of the insulin promoter. Mice in two independent lineages had about 30% of the normal islet GK activity. Insulin release in response to glucose from in situ-perfused pancreas was impaired; however, the plasma glucose and insulin levels of the mice remained normal. These mice are likely to be predisposed to type II diabetes and may manifest increased susceptibility to genetic and environmental diabetogenic factors. They provide an animal model for studying the interaction of such factors with the reduced islet GK activity.

Animals↗

PubMed1994

The pancreatic beta-cell glucose sensor.

Pancreatic beta cells secrete insulin in response to an increase in the level of blood glucose above 5 mM, which is characteristic of the fasting state. Glucose metabolism is essential for glucose sensing, and both the high-Km glucose transporter GLUT2 and the high-Km glucose phosphorylating enzyme glucokinase have been implicated in coupling insulin secretion to extracellular glucose levels. Experiments in isolated islets, immortalized beta-cell lines and transgenic animals, together with findings in humans with maturity-onset diabetes of the young, indicate that the primary beta-cell glucose sensor is glucokinase. Although the level of GLUT2 is frequently reduced in animal models of type II diabetes, GLUT2 does not limit glucose metabolism in beta cells and does not appear to regulate glucose induction of insulin secretion.

Animals↗

PubMed1994

In situ glucose uptake and glucokinase activity of pancreatic islets in diabetic and obese rodents.

The present study evaluated the involvement of glucose transport and phosphorylation in glucose-stimulated insulin release from pancreatic islets. Using quantitative histochemical techniques, we investigated basal islet glucose content, islet glucose uptake in situ during acute extreme experimental hyperglycemia, and islet glucokinase activity in several animal models of diabetes and obesity. The basal islet glucose content in anaesthetized diabetic or obese rodents was either the same or higher than that in their relevant controls. The rate of glucose uptake of islet tissue in these animals after an i.v. glucose injection was different. The db+/db+ mouse and the obese Zucker rat exhibited significantly reduced islet glucose uptake rates. RIP-cHras transgenic mice, BHE/cdb rats and partially pancreatectomized rats showed normal islet glucose uptake rates. The activity of islet glucokinase was increased to a different degree related to the blood glucose level. All five animal models of diabetes or obesity exhibited either a delay or a reduction of insulin release in response to supra maximal glucose stimulation. Our results indicate that the impairment of glucose-induced insulin release in diabetes is not consistently associated with a reduction of islet glucose uptake nor a change of glucokinase activity.

Animals↗

PubMed1994

Clonal insulinoma cell line that stably maintains correct glucose responsiveness.

A number of pancreatic beta-tumor cell (beta TC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a beta TC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for > 55 passages (over a year) in culture. One of these clonal lines, denoted beta TC6-F7, was characterized in detail. beta TC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal beta-cells. These results demonstrate that transformed beta-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous beta-cell lines for transplantation therapy of diabetes.

Animals↗

PubMed1993

Murine insulinoma cell line with normal glucose-regulated insulin secretion.

Pancreatic beta TC lines derived from insulinomas arising in transgenic mice expressing SV40 Tag under control of the insulin promoter manifest a differentiated beta-cell phenotype and secrete insulin in response to glucose. Previously reported beta TC lines respond to subphysiological extracellular glucose levels compared with normal beta-cells. Recently, several beta TC lines were developed with normal glucose-regulated insulin secretion from insulinomas obtained by breeding of the RIP-Tag transgene from the original C57BI/6 mouse strain into the C3HeB/FeJ strain. One of these beta TC lines, beta TC7, was characterized in detail. Beta TC7 cells express GLUT2 and have levels of glucokinase and hexokinase activity similar to those of normal islets. As a result these cells exhibit a normal glucose concentration dependency for glycolysis and insulin secretion, thus representing an accurate model of beta-cell function. On continuous propagation in culture, beta TC7 cells acquired a response to lower extracellular glucose levels. This change was associated with a fourfold increase in hexokinase activity, without significant changes in glucokinase activity and glucose uptake rates. These findings suggest an important role for glucose phosphorylation rates in regulation of the beta-cell insulin secretory response to glucose.

Animals↗

PubMed1992

[Val12] HRAS downregulates GLUT2 in beta cells of transgenic mice without affecting glucose homeostasis.

Glucose-induced insulin release from pancreatic beta cells depends on the beta-cell metabolism of glucose, which generates intracellular signals for secretion. The beta-cell glucose transporter isotype GLUT2 and the glucose phosphorylating enzyme glucokinase have both been implicated in coupling insulin secretion to extracellular glucose levels. Here we present evidence that a pronounced decrease in beta-cell GLUT2 has no immediate effect on glucose homeostasis. Analysis of transgenic mice overexpressing human [Val12]HRAS oncoprotein under control of the insulin promoter reveals a great reduction in plasma-membrane GLUT2 levels. These mice are nonetheless able to maintain normal fed and fasting plasma glucose and insulin levels for a period of several months. Insulin secretion studied in isolated islets and the perfused pancreas is characterized by a normal incremental response to increasing glucose concentrations. Glucose metabolism, as measured by glucose phosphorylation and oxidation in isolated islets, shows a normal dose dependence on extracellular glucose concentrations. These findings suggest that normal GLUT2 expression in beta cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role of glucose phosphorylation in regulation of insulin release in the absence of GLUT2.

Animals↗

PubMed1992

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.

Animals↗

PubMed1991

Glucose induces insulin gene transcription in a murine pancreatic beta-cell line.

The ability of insulin secretagogues to stimulate insulin gene transcription was analyzed in the murine insulinoma cell line beta TC3, which had been derived from a transgenic mouse expressing SV40 T antigen under control of the rat insulin II gene regulatory region. Glucose induced a 3-fold increase in the transcription of both the endogenous mouse insulin genes and the transgene. This effect was inhibited by D600, a calcium channel blocker, which also inhibited glucose-induced insulin secretion in these cells. This suggests that similar signals may be involved in glucose-stimulated insulin secretion and insulin gene transcription. Agents that increase intracellular levels of cAMP did not have a significant effect on the transcription of either the insulin genes or the transgene. Stimulation of transcription of the RIP-Tag transgene by glucose suggests that the 695-base pair fragment of the insulin gene regulatory region that is included in the transgene contains the cis elements required for response to the glucose-induced signal.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Glucagonomas of transgenic mice express a wide range of general neuroendocrine markers and bioactive peptides.

Pancreatic tumours of transgenic mice carrying a glucagon-promoted simian virus 40 (SV40) T antigen oncogene have been analysed by histological, histochemical, ultrastructural and radioimmunological means. Seven transgenic mice were examined revealing dysplastic and neoplastic lesions in the endocrine pancreas. Four tumours were identified, one of which metastasized to periadrenal spaces and paravertebral lymph nodes. Benign tumours were composed of argyrophilic, endocrine cells reactive to a range of antibodies against neuroendocrine markers (neuron-specific enolase, protein gene product 9.5, chromogranin A, synaptophysin and protein 7B2) and different fragments of the proglucagon molecule (glucagon, glicentin, glucagon-like polypeptides 1 and 2). A few tumour cells expressed pancreatic polypeptide, somatostatin or insulin. Conventional ultrastructural analysis and immunogold labelling revealed typical glucagon-immunoreactive alpha granules which co-stored glicentin and glucagon-like polypeptides 1 and 2. The malignant primary tumour and its metastases were composed mainly of cells which did not show immunoreactivity for neuroendocrine markers or peptides. Atypical, glucagon-immunogold labelled granules were detected at electron microscopy in differentiated tumour cells and C-type retroviral particles in the largest tumour population of degranulated cells. The transgene-encoded oncoprotein SV40 large T-antigen was detected in the nuclei of well-differentiated tumour cells and in alpha cells of some dysplastic islets. All tumour-bearing mice showed high levels of circulating glucagon-like immunoreactivity. Transgenic mice harbouring the glucagon-promoted SV40 T antigen oncogene may provide a model for human glucagonoma.

Animals↗

PubMed1991

Transplantation of beta cells from transgenic mice into nude athymic diabetic rats restores glucose regulation.

We have shown that elevated plasma D-glucose levels in experimentally-induced diabetic nude athymic rats can be reduced by intraperitoneal transplantation of microcarrier-attached insulin producing beta cells from the mouse pancreatic beta cell line, beta TC-1. The reduction in the level of hyperglycemia was observed as early as two days following cell transplantation and was associated with a concomitant increase in plasma insulin levels. beta TC-1 cell transplanted diabetic rats had plasma D-glucose levels similar to those found in non-diabetic control animals and remained normoglycemic throughout the 39 day experimental period. The beta TC-1 cell transplanted diabetic rats also had near normalization of body weight, food and water intake and of urine output when compared to control diabetic and non-diabetic rats. Similarly, they exhibited improved blood glucose clearance following intravenous D-glucose administration. These results suggest that beta TC-1 cells regulate D-glucose homeostasis following transplantation into diabetic rat recipients in a manner similar to that of endogenous pancreatic beta cells.

Animals↗

PubMed1991

Regulation of human interleukin-2 and interferon-gamma gene expression by suppressor T lymphocytes.

Concomitant with induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in human tonsil cells, mitogenic stimulation induces a transient activation of cells able to effectively suppress expression of these genes. Induction of IL-2 and IFN-gamma genes largely precedes appearance of suppressor cell activity, allowing expression of both genes to occur before strong down-regulation is exerted by activated suppressor cells. Suppressive activity induced in one cell population can inhibit IL-2 and IFN-gamma gene expression in another population from the same donor. The distinct nature of suppressor cells is supported by the absence of down-regulation of IL-2 gene expression in a helper cell line, MLA-144; yet, in these cells, negative control can be expressed when active suppressor cells are introduced. Our findings support the concept that actual levels of IL-2 and IFN-gamma gene activity are regulated to a large extent by the differential kinetics of activation of suppressor cells on one hand and of cells expressing the IL-2 and IFN-gamma genes on the other.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1991

Sexual dimorphism of pancreatic beta-cell degeneration in transgenic mice expressing an insulin-ras hybrid gene.

The human H-ras oncogene induces cell degeneration and diabetes when expressed in pancreatic beta-cells in transgenic mice. The disease develops predominantly in male mice between 5-8 months of age. Most transgenic female mice do not manifest this phenotype, even at much greater ages. However, ovariectomy induces female beta-cell degeneration similar to that in the males. In contrast, castration or the presence of the testicular feminization mutation do not alter the course of the disease in males. Treatment of males and ovariectomized females with estrogen prevents the development of diabetes. These results suggest that testicular androgens and a functional testosterone receptor are not required for the increased susceptibility of male beta-cells to the effects of the Ras oncoprotein, and that the relative resistance of female beta-cells is mediated by estrogen. In addition, a genetic component of female beta-cell resistance to Ras is revealed by crossing the transgenic mice with C3HeB/FeJ mice, which results in a pronounced increase in the incidence of female diabetes.

Animals↗

PubMed1990

Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells.

Transgenic mice expressing an insulin-promoted H-ras hybrid gene in pancreatic beta cells developed beta-cell degeneration and diabetes. The disease was manifested in male mice by hyperglycemia, glycosuria, and reduced plasma insulin levels, which appeared around 5 months of age and led to premature death. Histological analyses revealed large holes within the islets of Langerhans and a reduced number of beta cells. The destruction of the islets was not associated with an obvious inflammatory activity. Ultrastructural analysis showed extensive engorgement in the endoplasmic reticulum of the residual beta cells from diabetic males. The females carrying the insulin-promoted ras gene did not manifest any of the physiological abnormalities observed in males and showed only minor histological and ultrastructural changes, even at much greater ages.

Animals↗

PubMed1990

Regulation of insulin secretion from beta-cell lines derived from transgenic mice insulinomas resembles that of normal beta-cells.

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.

1-Methyl-3-isobutylxanthine↗