[16] A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of langerhans

McDaniel, Michael L.; Colca, Jerry R.; Kotagal, Nirmala; Lacy, Paul E.

doi:10.1016/0076-6879(83)98149-1

Cited by 140 publications

(95 citation statements)

References 21 publications

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“…Pancreatic islets were isolated from adult male Wistar rats, as described previously (McDaniel et al 1983). Isolated islets were cultured in RPMI medium (Sigma) supplemented with 2 mM -glutamine (Gibco), 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Pen-Strep; Bio-Whittaker Europe, Verviers, Belgium), and containing either 5·5 or 24·4 mmol/l glucose (Ling et al 1994).…”

Section: Isolation and Culture Of Rat Isletsmentioning

confidence: 99%

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

Mellado-Gil¹,

Diosdado²

2004

Journal of Endocrinology

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Pancreatic -cell apoptosis is known to participate in the -cell destruction process that occurs in diabetes. A better understanding of how it takes place is essential for future development of therapeutic strategies aimed at preventing -cell loss and diabetes. In this study we determine the possible role that high glucose concentration might play as an enhancer of cytokine-and streptozotocin (STZ)-mediated rat islet cell apoptosis in vitro and its relationship with potential changes in the expression of pro-and anti-apoptotic proteins. Rat islets treated with a cytokine combination (interleukin (IL)-1 , tumor necrosis factor (TNF)-and interferon (IFN)-) displayed a significant increase in islet cell apoptosis when the islets were incubated in 24·4 mM glucose compared with untreated islets at the same glucose concentration (13·07 1·78% vs 6·09 0·78%; P<0·01) or islets incubated in 5·5 mM glucose concentration and cytokines (13·07 1·78% vs 8·04 1·56%; P<0·05). IL-1 alone did not induce a significant increase in the apoptotic rates in islet cells cultured at normal or high glucose concentrations. STZ significantly increased islet cell apoptosis when islets were cultured in 24·4 mM glucose concentration compared with untreated islets at the same glucose concentration (6·02 0·62% vs 4·44 0·63%; P<0·05). High glucose induced an increase in Fas expression in the islet cells, and this increase was maintained after cytokine or STZ treatment. However, the expression of anti-apoptotic mediators such as bcl-2 and bcl-xL did not show any significant change. These results suggest that cytokine-and STZmediated apoptotic effects on islet cells might be mediated by a glucose-induced hyperfunctional status and associated with an increase in Fas (Apo-1, CD-95) expression and no changes in the expression of the anti-apoptotic proteins bcl-xL and bcl-2.

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Section: Isolation and Culture Of Rat Isletsmentioning

confidence: 99%

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

Mellado-Gil¹,

Diosdado²

2004

Journal of Endocrinology

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“…The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Islet Isolation-Islets were isolated from male Sprague-Dawley rats (200 -250 g) by collagenase digestion as described previously (22). Briefly, pancreases were inflated with Hanks' balanced salt solution, and the tissue was isolated, minced, and digested with 20 mg of collagenase/ pancreas for 6 min at 39°C.…”

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Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by KATP Channels

Kwon

Marshall

Liu

et al. 2006

Journal of Biological Chemistry

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The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) 3 H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.Both types 1 and 2 diabetes result from the inability of pancreatic ␤-cells to secrete sufficient amounts of insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate ␤-cell mass. Increased metabolic demands or stress responses that exert a positive effect on ␤-cell mass include obesity, pregnancy, partial pancreatectomy, or chronic glucose exposure. ␤-Cell mass is regulated by cellular mechanisms that include replication, neogenesis, hypertrophy, and apoptosis (1, 2). Recent studies have emphasized the importance of the proliferative capacity of existing adult ␤-cells as a major source of new ␤-cells during adult life that may significantly contribute to the maintenance of ␤-cell mass (3).Mammalian target of rapamycin (mTOR) 2 is a serine/threonine protein kinase that integrates signals derived from growth factors and nutrients to regulate cell growth and proliferation through the regulatory proteins 70-kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein-1 (4EBP1). This signaling cascade stimulates protein translation and increases the capacity of the ribosomal protein machinery necessary for the onset of DNA synthesis (4, 5). Our previous studies have demonstrated that glucose robustly activates mTOR/S6K1/4EBP1 in an amino acid-dependent manner via its metabolism in both rodent and human islets. Glucose and amino acids, especially leucine and glutamate, are the most prominent activators of mTOR in islets, possibly through ATP production mediated by mitochondrial metabolism (6 -9). Insulin secreted by the ␤-cell and growth factors also provide input to mTOR through the insulin signaling cascade to Akt (6). Akt may directly activate mTOR but also has been shown to inhibit the tumor suppressor proteins TSC1/2. These proteins are activated by AMP-dependent protein kinase that is regulated by the ATP/AMP ratio. Rapamycin specifically inhibits mTOR activation and signaling to 4EBP1 and S6K1. Recent reports have demonstrated a negative feedback pathway from chronically stimulated mTOR to IRS2 that inhibits the insulin signaling pathway (10). Apparently, this negative feedback to IRS2 does not reduce nutrient-stimulated mTOR activation in the ␤-cell as S6K1 remains fully activated during a 4-or 6-day exposure to elevated glucose (7), although other IRS2-dependent pathways may be inhibited.Our previous studies demonstrated that the majority of glucose-stimulated [3 H]thymidine incorporation by rodent islets...

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“…Islet isolation. Islets were isolated from 250-to 300-g male Sprague-Dawley rats by collagenase digestion as previously described (28). Islets were cultured overnight in complete CMRL-1066 (containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37°C under an atmosphere of 95% air-5% CO 2 before experimentation.…”

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confidence: 99%

Inhibition of IFN-γ-induced STAT1 activation by 15- deoxy-Δ^12,14-prostaglandin J₂

Weber

Scarim

Corbett

2003

American Journal of Physiology-Endocrinology and Metabolism

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bett. Inhibition of IFN-␥-induced STAT1 activation by 15-deoxy-⌬ 12,14 -prostaglandin J2. Am J Physiol Endocrinol Metab 284: E883-E891, 2003. First published January 7, 2003 10.1152/ajpendo.00515.2002The inhibitory actions of 15-deoxy-⌬ 12,14 -prostaglandin J2 (PGJ2) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-B. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ 2. We show that PGJ2 inhibits interferon (IFN)-␥-stimulated phosphorylation and DNAbinding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1-to 2-h incubation and are maximal after a 6-h incubation with PGJ 2, and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ2 on inducible nitric oxide synthase (iNOS) expression and NF-B activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ2 on IL-1-induced NF-B or IFN-␥-induced STAT1 activation or cytokine-stimulated iNOS expression by ␤-cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ2 on IL-1 plus IFN-␥-stimulated iNOS expression and NO production by ␤-cells are persistent for extended periods (Յ48 h). These findings suggest that PGJ2 inhibits multiple cytokine-signaling pathways (IL-1 and IFN-␥), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ2 on IL-1 and IFN-␥ signaling in ␤-cells.interferon-␥; signal transducer and activator of transcription-1; peroxisome proliferator-activated receptor-␥ AUTOIMMUNE DIABETES IS CHARACTERIZED by islet inflammation followed by the selective destruction of insulinsecreting ␤-cells found in pancreatic islets of Langerhans (14). Cytokines released from inflammatory macrophages and T cells are believed to participate in ␤-cell damage during diabetes development (3, 35). Treatment of rat islets with IL-1 results in a timeand concentration-dependent inhibition of glucosestimulated insulin secretion that is followed by islet degeneration (8,22,29). The inhibitory and destructive actions of IL-1 on ␤-cell function correlate with the time-dependent expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide and can be prevented by inhibitors of iNOS (10,12,36). Alone, interferon (IFN)-␥ does not appear to modulate ␤-cell function; however, IFN-␥ has been shown to both prime and potentiate the stimulatory actions of IL-1 on iNOS expression by rat islets (19,21). In addition, a combination of IL-1 and IFN-␥ is required to stimulate iNOS expression by mouse and human ␤-cells (6, 41). Nitric oxide, produced following iNOS expression, impairs ␤-cell function by inhibiting mitochondrial oxidative metabolism, a...

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[16] A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of langerhans

Cited by 140 publications

References 21 publications

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by KATP Channels

Inhibition of IFN-γ-induced STAT1 activation by 15- deoxy-Δ^12,14-prostaglandin J₂

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[16] A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of langerhans

Cited by 140 publications

References 21 publications

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes

Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by KATP Channels

Inhibition of IFN-γ-induced STAT1 activation by 15- deoxy-Δ12,14-prostaglandin J2

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Inhibition of IFN-γ-induced STAT1 activation by 15- deoxy-Δ^12,14-prostaglandin J₂