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Biomedical subjects

N Fleischer

Publications and source records attributed to N Fleischer.

At least 19 recordsLinked to original sources
PubMed2006

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

OBJECTIVES: To determine factors associated with diabetes, insulin resistance, and abnormal glucose tolerance in older men with or at risk of HIV infection. METHODS: Diabetes was assessed by self-report in 643 men >or=49 years old with or at risk of HIV infection. In a subset of 216 men without previously diagnosed diabetes [including 90 HIV-uninfected men, 28 HIV-infected, antiretroviral-naive men, 28 HIV-infected men taking non-protease inhibitor (PI)-containing highly active antiretroviral therapy (HAART), and 70 HIV-infected men taking PI-containing HAART], an oral glucose tolerance test with insulin levels was performed. HIV serology, CD4 cell count, weight, height and waist circumference were measured. Antiretroviral use, drug use, family history of diabetes, physical activity and sociodemographic data were obtained using standardized interviews. RESULTS: Of 643 participants, 116 (18%) had previously diagnosed diabetes. With the oral glucose tolerance test, 15 of 216 men (7%) were found to have undiagnosed diabetes and 40 (18%) impaired glucose tolerance. Factors independently associated with previously diagnosed diabetes included use of non-PI-containing HAART, methadone treatment, positive CAGE test for alcoholism, obesity and family history of diabetes. Factors independently associated with greater insulin resistance included waist circumference and heroin use. Factors independently associated with abnormal glucose tolerance (impaired glucose tolerance or diabetes) included age >or=55 years and Hispanic ethnicity. CONCLUSIONS: HIV-infected men with diabetes risk factors should undergo screening for diabetes regardless of HAART use. Interventions targeting modifiable risk factors, including overweight and physical inactivity, are warranted. The potential impact of opiate and alcohol abuse on glucose metabolism should be recognized in clinical care, and addressed in future research studies of HIV-infected persons.

Antiretroviral Therapy, Highly Active↗
PubMed2001

Physiological increase in plasma leptin markedly inhibits insulin secretion in vivo.

The demonstration of leptin receptors on the pancreatic beta-cells suggests the possibility of direct actions of leptin on insulin secretion. In vitro studies on islets or perfused pancreas and beta-cell lines produced inconsistent results. We performed an in vivo study to distinctly examine whether leptin has an effect on glucose-stimulated insulin secretion. Young chronically catheterized Sprague-Dawley rats (n = 28) were subjected to a 4-h hyperglycemic clamp study (approximately 11 mmol/l). At minute 120 to 240, rats were assigned to receive either saline or leptin (0.1, 0.5, and 5 microg x kg(-1) x min) infusion. Leptin decreased plasma insulin levels abruptly, and an approximately twofold decrease in plasma insulin levels compared with saline control was sustained over the 2 h of the study (14.8 +/- 5.8 vs. 34.8 +/- 2.6 ng/ml with leptin and saline infusion, respectively, P < 0.001). Moreover, a dose-dependent decrease in plasma insulin levels was noted (r = -0.731, P < 0.01). Since milrinone, an inhibitor of cAMP phosphodiesterase (PDE) 3, did not reverse the effect of leptin on glucose-induced insulin secretion, its action may be independent of PDE3. These findings suggest that acute physiological increase in plasma leptin levels acutely and significantly inhibits glucose-stimulated insulin secretion in vivo. The site of leptin effects on insulin secretion remains to be determined.

Animals↗
PubMed2001

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.

Animals↗
PubMed1999

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines.

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

1-Methyl-3-isobutylxanthine↗
PubMed1999

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 beta-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 betaTC3 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 betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). 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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

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.

Animals↗
PubMed1996

cAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line.

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-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 alpha 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 [gamma-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 alpha 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.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Animal model for maturity-onset diabetes of the young generated by disruption of the mouse glucokinase gene.

Glucokinase catalyzes a rate-limiting step in glucose metabolism in hepatocytes and pancreatic beta 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.

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↗
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

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↗
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↗