Shimon Efrat

Clonal Insulinoma Cell Line That Stably Maintains Correct Glucose Responsiveness

A number of pancreatic β-tumor cell (β 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 β 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 β TC6-F7, was characterized in detail. β 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 β-cells. These results demonstrate that transformed β-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous β-cell lines for transplantation therapy of diabetes.

The pancreatic β-cell glucose sensor

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

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

Glucokinase (GK) catalyzes the phosphorylation of glucose in β-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 β-cells (GK+/-), and 2) with decreased GK activity selectively in β-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 [3-3H]glucose was infused to measure the rate of appearance of glucose and HGP during 80 min of euglycemia (Glc ∼5 mM) followed by 90 min of hyperglycemia (Glc ∼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 ∼22 mg ⋅ kg-1 ⋅ min-1in all groups, during hyperglycemia HGP was suppressed by only 48% in GK+/- compared with ∼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.

Genetically Engineered Pancreatic β-Cell Lines for Cell Therapy of Diabetes

ABSTRACT: The optimal treatment of insulin‐dependent diabetes mellitus (IDDM), which is caused by the autoimmune destruction of pancreatic islet β cells, would require the regulated delivery of insulin by transplantation of functional β cells. β‐cell transplantation has so far been restricted by the scarcity of human islet donors. This shortage would be alleviated by the development of differentiated β‐cell lines, which could provide an abundant and well‐characterized source of β cells for transplantation. Using conditional transformation approaches, our laboratory has generated continuous β‐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 β 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, β‐cell transplantation across allogeneic barriers was achieved without immunosuppression. In principle, similar β‐cell lines can be derived from isolated human islets using viral vectors to deliver conditionally regulated transforming and immunomodulatory genes into β 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.

Development of engineered pancreatic β-cell lines for cell therapy of diabetes

Abstract Insulin-secreting pancreatic β-cell lines represent a promising approach for treatment of insulin-dependent diabetes mellitus (IDDM). Our laboratory has developed a number of highly-differentiated β-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 β-cell lines both in culture and in vivo. By employing adenovirus genes which downreguate antigen presentation and increase cell resistance to cytokines mouse β cells could be transplanted across allogeneic barriers. These approaches could be applied to the development of human β-cell lines by genetic engineering of isolated human islets.

Role of glucokinase in regulation of insulin secretion: Lessons from transgenic mice

Impairments in the insulin secretory response to glucose in both type II and early type I diabetes have prompted studies into the mechanisms that allow pancreatic B-cells to sense and respond to physiological changes in blood glucose. Glucose-induced insulin secretion requires the metabolism of glucose in B-cells,1 and the phosphorylation of glucose to glucose-6-phosphate, which determines the rate of glycolysis, is considered the major glucose-sensing step for regulating insulin secretion.2 B-cells and hepatocytes express a high-Km member of the hexokinase family, glucokinase (GK), which is responsible for the majority of glucose phosphorylation activity in B-cells.2 While in the liver transcription of the GK gene is induced by insulin,3,4 GK expression in B-cells is primarily regulated by glucose at the translational and post-translational levels.3,5