Modulation of Glucose-Induced Insulin Secretion from a Rat Clonal β-Cell Line*

Clark, Sam; Burnham, Brenda; Chick, William L.

doi:10.1210/endo-127-6-2779

Cited by 86 publications

(41 citation statements)

References 48 publications

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“…RIN 1046±38 cells were plated (3´10 4 cells per well) into 24-well microtitre plates (Falcon, Fortworth, Tex., USA) and grown [15,16]. Endogenous expression of the long leptin receptor, IRS-1, IRS-2, JAK-2, SHP1 and SHP2 could be shown for RINr 1046±38 cells by RT-PCR analysis (data not shown).…”

Section: Methodsmentioning

confidence: 93%

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance

et al. 2001

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The role of leptin in the pathogenesis of obesity has been intensively studied in recent years [1]. Many studies in obese patients found an apparent discrepancy between circulating leptin levels and leptin effects, suggesting that leptin resistance might be a common phenomenon in obese individuals [1]. The mechanisms causing leptin resistance are not, however, clear. Structural defects of the leptin receptor, associated with a loss of function which might cause cellular leptin resistance, do not seem to be a common cause in human beings [2] indicating that abnormalities in postreceptor signalling elements, either genetically determined or caused by regulatory mecha- Diabetologia (2001) Abstract Aims/hypothesis. Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain.Methods. This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046±38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain.Results. The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. Conclusion/interpretation. In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance. [Diabetologia (2001

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Section: Methodsmentioning

confidence: 93%

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance

et al. 2001

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“…The SV40-transformed murine beta-cell lines, MIN6 [18] and bTC3 [19] were cultured in 90 % DMEM (22.5 mmol/l glucose) supplemented with 10 % fetal bovine serum (FBS, Life Technologies, Gaithersburg, MD., USA). Hamster HIT-T15 [20], rat RINm5F [4] and INS-1 [19] insulinoma cell lines were maintained in 90 % RPMI 1640 (11 mmol/l glucose), 10 % FBS with necessary additives [19]. 3T3 fibroblasts (ATCC) were grown in 90 % DMEM (22.5 mmol/l glucose), 10 % donor calf serum (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

“…Immediately following glucose stimulation, synthesis is primarily augmented through enhanced translation [2] and decreased degradation of existing insulin mRNAs [3]. Glucose also induces delayed responses that are manifested several hours after stimulation [4] by increasing insulin gene transcription [5] and expression of other genes involved in beta-cell function. Increased gene expression of glucose transporter 2 (GLUT-2) [6], pyruvate kinase [7], acetyl-coenzyme A-carboxylase [8], and 64 kDa glutamic acid decarboxylase (GAD65) [9] have been observed in primary rat islets in vitro and in rodent insulin-producing tumour cell lines following exposure to glucose.…”

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confidence: 99%

Glucose induces early growth response gene (Egr-1) expression in pancreatic beta cells

et al. 1999

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Glucose homeostasis is maintained by beta cells of pancreatic islets through precisely regulated release of insulin from preformed granules. To maintain constant intracellular insulin stores while meeting wide physiological variations in demand, insulin synthesis is modulated in parallel with secretion rates [1]. Immediately following glucose stimulation, synthesis is primarily augmented through enhanced translation [2] and decreased degradation of existing insulin mRNAs [3]. Glucose also induces delayed responses that are manifested several hours after stimulation [4] by increasing insulin gene transcription [5] and expression of other genes involved in beta-cell function. Increased gene expression of glucose transporter 2 (GLUT-2) [6], pyruvate kinase [7], acetyl-coenzyme A-carboxylase [8], and 64 kDa glutamic acid decarboxylase (GAD65) [9] have been observed in primary rat islets in vitro and in rodent insulin-producing tumour cell lines following exposure to glucose. Together these changes comprise a delayed adaptive response that could be necessary to meet increased metabolic and secretory demands during extended or repeated periods of hyperglycaemia. Diabetologia (1999) Summary A copy deoxyribonucleic acid (cDNA) clone of the immediate early growth response gene, egr-1 (Krox-24, Zif268, NGFI-1), was isolated through subtractive hybridization screening to identify glucose-induced genes in pancreatic beta cells. Glucose rapidly and transiently induced egr-1 mRNA in the SV40-transformed murine beta-cell line, MIN6. Glucose also increased egr-1 mRNA expression in INS-1, bTC3 and RINm5F beta-cell lines, although with different kinetics. Expression of the 82 kDa Egr-1 protein was induced both in MIN6 cells stimulated with glucose in vitro and in primary rat islet cells stimulated in vivo or in vitro. This response is unique to beta cells since glucose did not affect egr-1 expression in NIH-3T3 fibroblasts or glucosesensitive hepatocytes. In beta cells egr-1 induction is specifically associated with insulin secretion, as it was not observed after stimulation with serum or insulin but was elicited by insulin secretagogues, including membrane depolarizing agents and cAMP agonists. Moreover, induction of egr-1 by glucose was inhibited by EDTA, indicating dependence on influx of extracellular Ca 2+. Other immediate early response genes, c-fos and junB, were also induced following glucose stimulation with kinetics similar to egr-1, whereas c-jun and junD expression were not affected. Since the zinc-finger protein encoded by egr-1 is highly homologous to transcription factors that control expression of glucose-regulated genes in yeast, Egr-1 could mediate delayed adaptive responses of beta cells to sustained glucose stimulation through transcriptional regulation. [Diabetologia (1999) 42: 195±203]

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“…Recently, our attention has turned to the rodent insulinoma cell line RIN1046-38, which is able to respond to glucose at low passage numbers, but loses both glucose sensing and expression of GLUT2 and glucokinase with time in culture [11,12]. Stable transfection of these or other glucose unresponsive insulinoma lines such as RINm5F with GLUT2 results in restoration of glucose sensing [11,13].…”

Section: : S 42-s 47]mentioning

confidence: 99%

Engineered cell lines for insulin replacement in diabetes: current status and future prospects

Newgard

Clark²,

BeltrandelRio

et al. 1997

Diabetologia

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The recently concluded diabetes control and complications trial (DCCT) [1] has emphatically underscored the potential for improvement in current treatment protocols for diabetes mellitus. 'Conventional' insulin therapy, involving 1-2 injections of the hormone per day and intermittent glucose monitoring is clearly not as effective at slowing the progression of the debilitating and costly secondary complications of the disease as 'tight control', in which insulin pumps or multiple injections are combined with frequent glucose monitoring to limit hyperglycaemic excursions. Unfortunately, improvement in glycaemic control through increased insulin dosing requires a new level of compliance and discipline, and places the patient at significantly enhanced risk for dangerous hypoglycaemic episodes. These concerns have produced a surprising reticence among both physicians and patients towards implementation of tight control therapy. This reaction has served to place a new emphasis on the design of better methods for insulin replacement in insulin-dependent diabetes mellitus (IDDM). A primary consideration in such work is to develop a 'closed loop' system in which the hormone is delivered in response to metabolic demand, thereby eliminating the need for self monitoring by the patient. Three basic approaches to achieving this end have been contemplated, and are being investigated with ever increasing vigour. The first involves development of mechanical devices consisting of a pump or other mechanism for insulin delivery linked to a detector system for blood glucose. The second involves transplantation of insulin-producing cells from human or large mammal donors into IDDM patients. This can be achieved by transplantation of the entire pancreas or isolated pancreatic islets. Because of the difficulty and cost associated with islet isolation and pancreas transplantation, a third approach has emerged, in which the tools of molecular biology are used to fashion insulin-secreting cell lines with fuelmediated insulin secretion responses resembling Diabetologia (1997) Summary The recently completed diabetes complications and control trial has highlighted the need for improvement of insulin delivery systems for treatment of insulin-dependent diabetes mellitus. Despite steady improvement in methods for islet and whole pancreas transplantation over the past three decades, the broad-scale applicability of these approaches remains uncertain due in part to the difficulty and expense associated with procurement of functional tissue. To address this concern, we and others have been using the tools of molecular biology to develop cell lines with regulated insulin secretion that might serve as a surrogate for primary islets or pancreas tissue in transplantation therapy. This article seeks to provide a brief summary of the current status of this growing field, with a particular emphasis on progress in producing cell lines with appropriate glucose-stimulated insulin secretion. [Diabetologia (1997) 40: S 42-S 47]

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Modulation of Glucose-Induced Insulin Secretion from a Rat Clonal β-Cell Line*

Cited by 86 publications

References 48 publications

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance

Glucose induces early growth response gene (Egr-1) expression in pancreatic beta cells

Engineered cell lines for insulin replacement in diabetes: current status and future prospects

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