Norman Fleischer

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 Calcium/Calmodulin-dependent Phosphodiesterase PDE1C Down-regulates Glucose-induced Insulin Secretion

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic β-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 βTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from βTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of βTC3 cells is a novel isozyme possessing aK m of 0.47 μm for cAMP and 0.25 μm for cGMP. The PDE1C isozyme of βTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC50 = 7.5 and 4.5 μm, respectively) and resistant to vinpocetine (IC50 > 100 μm). 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.

Murine Insulinoma Cell Line With Normal Glucose-Regulated Insulin Secretion

Pancreatic βTC lines derived from insulinomas arising in transgenic mice expressing SV40 Tag under control of the insulin promoter manifest a differentiated β-cell phenotype and secrete insulin in response to glucose. Previously reported βTC lines respond to subphysiological extracellular glucose levels compared with normal β-cells. Recently, several βTC 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 βTC lines, βTC7, was characterized in detail. βTC7 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 β-cell function. On continuous propagation in culture, βTC7 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 β-cell insulin secretory response to glucose.

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

Pancreatic β cell lines may offer an abundant source of cells for β-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 β cells, we have shown that reversible immortalization is an efficient approach for regulated β-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 β 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.

Disorders of Glucose Metabolism among HIV-Infected Women

BACKGROUND Abnormal glucose metabolism in HIV-infected patients has largely been attributed to the use of protease inhibitors. However, most studies of glucose metabolism in HIV-infected patients have focused on men or have lacked appropriate control groups. METHODS We assessed the factors associated with previously diagnosed diabetes among 620 middle-aged women with or at risk for HIV infection. For a subset of 221 women without previously diagnosed diabetes, we performed an oral glucose tolerance test (OGTT) to measure glucose and insulin levels, and we assessed factors associated with abnormal glucose tolerance, insulin resistance, and insulin secretion. RESULTS Thirteen percent of the women in the present study had previously diagnosed diabetes. Among women without previously diagnosed diabetes who underwent an OGTT, 6% had previously undiagnosed diabetes, and 12% had impaired glucose tolerance (IGT). According to multivariate analysis, factors that were associated with previously diagnosed diabetes included current methadone treatment, body mass index of > or =25, family history of diabetes, and physical inactivity. Factors that were independently associated with an abnormal result of an OGTT (i.e., a result consistent with IGT or diabetes) included age > or =50 years, family history of diabetes, physical inactivity, and a high number of pack-years of smoking. Factors independently associated with insulin resistance included waist circumference, Hispanic ethnicity, physical inactivity, and, among HIV-infected women, use of HAART that did not include protease inhibitors. Factors associated with lower levels of insulin secretion included current opiate use (i.e., methadone or heroin) and older age. CONCLUSIONS Abnormal glucose metabolism is highly prevalent among middle-aged women with or at risk for HIV infection, particularly women who use opiates. Screening for diabetes in the HIV primary care setting should occur for women who have classic risk factors for diabetes, rather than solely for women who are taking PIs. Interventions that target modifiable risk factors, including obesity and physical inactivity, are also warranted.

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.

Pancreatic β-cell overexpression of the glucagon receptor gene results in enhanced β-cell function and mass

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, beta-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT.

Abnormal glucose metabolism among older men with or at risk of HIV infection

To determine factors associated with diabetes, insulin resistance, and abnormal glucose tolerance in older men with or at risk of HIV infection.

Diabetes Susceptibility of BALB/cBOM Mice Treated with Streptozotocin: Inhibition by Lethal Irradiation and Restoration by Splenic Lymphocytes

In genetically susceptible strains of mice, repeated injections of a subdiabetogenic dose of streptozotocin induces the development of progressive insulin-dependent hyperglycemia. We showed previously that host T-cell functions play an obligatory etiologic role in this experimental disease by demonstrating that the athymic nude mouse is resistant to diabetes induction unless its T-cell functions are reconstituted by thymus graft (Paik et al., Proc. Natl. Acad. Sci. USA 77:6129–33, 1980). Here we show that lethal irradiation of euthymic (+/nu) mice of BALB/cBOM background causes selective resistance of the mice to the diabetogenic effects of the multiple low doses of streptozotocin without affecting their sensitivity to a high pharmacologic dose of the toxin. We also show that reconstitution of the irradiated mice with splenic lymphocytes causes the restoration of diabetes susceptibility. Lethally irradiated mice thus represent a useful experimental model for analyzing the host functions involved in the development of this disease. These results provide an additional support for the hypothesis that the induction of diabetes in this model system is mediated by an autoimmune amplification mechanism.

cAMP-Dependent Phosphorylation of the Cardiac-Type α1 Subunit of the Voltage-Dependent Ca2+ channel in a murine pancreatic β-cell line

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the β-cell/endocrine-brain and cardiac subtypes. The cardiac-type α1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic β-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type α 1 subunit as evidenced by a 40–60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [γ-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type α 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.