Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

Hohmeier, Hans E.; Mulder, Hindrik; Chen, Guoxun; Henkel-Rieger, Rosemarie; Prentki, Marc; Newgard, Christopher B.

doi:10.2337/diabetes.49.3.424

Cited by 832 publications

(820 citation statements)

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“…INS832/13 cells [42] (passages were grown in monolayer cultures as described previously [43]. This was done in a humidified (5% CO 2 , 95% air) atmosphere at 37°C in regular RPMI 1640 medium supplemented with 10 mmol/l HEPES, 10% FCS, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 50 µmol/l β-mercaptoethanol (referred to below as complete medium).…”

Section: Methodsmentioning

confidence: 99%

“…This was done in a humidified (5% CO 2 , 95% air) atmosphere at 37°C in regular RPMI 1640 medium supplemented with 10 mmol/l HEPES, 10% FCS, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 50 µmol/l β-mercaptoethanol (referred to below as complete medium). This clone (832/13) of the INS-1 cell was used because it shows better differentiation characteristics in terms of glucose-stimulated insulin secretion than the original INS-1 cell line [42].…”

Section: Methodsmentioning

confidence: 99%

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Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

et al. 2004

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Aims/hypothesis. We have provided evidence that glucagon-like peptide-1, a potential therapeutic agent in the treatment of diabetes, activates phosphatidylinositol-3 kinase/protein kinase B signalling in the pancreatic beta cell. Since this pathway promotes cell survival in a variety of systems, we tested whether glucagon-like peptide-1 protects beta cells against cell death induced by elevated glucose and/or non-esterified fatty acids. Methods. Human islets and INS832/13 cells were cultured at glucose concentrations of 5 or 25 mmol/l in the presence or absence of palmitate. Apoptosis was evaluated by monitoring DNA fragmentation and chromatin condensation. Wild-type and protein kinase B mutants were overexpressed in INS832/13 cells using adenoviruses. Nuclear factor-κB DNA binding was assayed by electrophoretic mobility shift assay. Results. In human pancreatic beta cells and INS832/13 cells, glucagon-like peptide-1 prevented beta cell apoptosis induced by elevated concentrations of (i) glucose (glucotoxicity), (ii) palmitate (lipotoxicity) and (iii) both glucose and palmitate (glucolipotoxicity). Overexpression of a dominant-negative protein kinase B suppressed the anti-apoptotic action of glucagonlike peptide-1 in INS832/13 cells, whereas a constitutively active protein kinase B prevented beta cell apoptosis induced by elevated glucose and palmitate. Glucagon-like peptide-1 enhanced nuclear factor-κB DNA binding activity and stimulated the expression of inhibitor of apoptosis protein-2 and Bcl-2, two antiapoptotic genes under the control of nuclear factor-κB. Inhibition of nuclear factor-κB by BAY 11-7082 abolished the prevention of glucolipotoxicity by glucagon-like peptide-1. Conclusions/interpretation. The results demonstrate a potent protective effect of glucagon-like peptide-1 on beta cell gluco-, lipo-and glucolipotoxicity. This effect is mediated via protein kinase B activation and possibly its downstream target nuclear factor-κB.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

et al. 2004

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“…Clonal INS-1 832/13 cells expressing the human insulin gene (a gift from Dr Christopher Newgard, Duke University), 14 were grown in culture at 37°C, 5% CO 2 in 75 cm 2 cell culture T-flasks (Corning Inc., Corning, NY). Standard RPMI-1640 culture medium (Sigma-Aldrich, St. Louis, Mo) was supplemented with 10% fetal calf serum (Mediatech, Herndon, Va), 10 mmol/L HEPES (Media-tech), 2 mmol/L L-glutamine (Sigma-Aldrich), 1 mmol/L sodium pyruvate (Sigma-Aldrich), and 50 μmol/L 2-mercaptoethanol (Sigma-Aldrich).…”

Section: Ins-1 Cell Culture and Human Islet Isolation And Culturementioning

confidence: 99%

“…After 2 minutes of incubation, trypsin was neutralized with 6 mL of standard serum-supplemented culture media. 14 The cell suspension was then transferred to a 15-mL conical tube and placed into a 60 °C water bath for 60 minutes. For time course experiments, aliquots of INS-1 cells were removed from the 15-mL conical tube at specified timepoints (1,3,5,25, and 47 hours).…”

Section: Heat Treatment Of Ins-1 Cells and Human Isletsmentioning

confidence: 99%

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Suszynski¹,

Wildey²,

Falde³

et al. 2008

Transplantation Proceedings

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Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/ DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.Islet cell transplantation is emerging as a promising therapy for the treatment of type 1 diabetes. [1][2][3][4][5] Despite recent advances, the transplantation of islets poses a unique challenge with respect to achieving a consistent clinical outcome. Part of this challenge is being able to reliably and rapidly assess clinical islet quality through the quantification of viability and function before transplantation. Current viability assays are limited in their ability to accurately predict transplantation outcomes in vivo. 6 Consequently, to improve the clinical islet transplantation outcomes, it is imperative to develop more accurate viability assays.A proposed method to assess islet viability and potency before transplantation is quantification of the ADP/ATP ratio. 6 -10 This ratio has been specifically applied in discriminating islet preparations that are suitable for clinical transplantation from those that are not. 6 However, this application may be problematic under certain conditions. Intracellular ADP and ATP levels fluctuate rapidly because these high-energy phosphate molecules are rapidly produced and consumed in many intracellular biochemical reactions. It is widely known that viable cells turn over entire ATP stores on the order of minutes, and that dead cells are incapable of replenishing To better account for dead cells and to more accurately assess the viability of an islet preparation, we suggest the use of an ATP/DNA ratio. DNA does not degrade as rapidly as ADP or ATP in a dead cell. Therefore, using direct measurements of DNA, dead cells in an islet preparation...

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“…Interestingly, in a recent study Neve et al [10] reported a high glucoseinduced stimulation of KLF11 mRNA expression in INS832/13 beta cells. This opposing observation may be explainable by possible differences between native INS-1E beta cells used in our study and the INS832/13 beta cell line, which is derived from a highly selected INS-1 subclone stably transfected with a plasmid containing the human proinsulin gene [16]. More importantly, Neve et al [10] demonstrated activation of hInsP by cotransfected FLAG-tagged KLF11 in beta-TC3 beta cells.…”

Section: Discussionmentioning

confidence: 67%

Human Krüppel-like factor 11 inhibits human proinsulin promoter activity in pancreatic beta cells

et al. 2007

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Aims/hypothesis The Krüppel-like factor 11 (KLF11; TIEG2), a pancreas-enriched Sp1-like transcription factor, is a known negative regulator of pancreatic exocrine cell growth. A recent study indicated KLF11-induced activation of the human proinsulin promoter (hInsP). Materials and methods We investigated the functional role of KLF11 in pancreatic beta cells. Results Endogenous KLF11 mRNA expression was found in whole rat pancreas, human pancreatic islets and INS-1E beta cells and was profoundly reduced by high glucose in INS-1E. Cotransfections of INS-1E and beta-TC3 beta cells with a human (h)KLF11 expression plasmid and an hInsP-driven reporter plasmid resulted in a substantial dose-dependent and glucose-independent inhibition of proinsulin promoter activity. 5′-deletion of hInsP demonstrated that hKLF11 acts via DNA sequences upstream of −173 and requires the beta cellspecific transcription machinery, since hKLF11-mediated inhibition of promoter activity was abolished in HEK293

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Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

Cited by 832 publications

References 27 publications

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Human Krüppel-like factor 11 inhibits human proinsulin promoter activity in pancreatic beta cells

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