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. 2005:7:162-71.
doi: 10.1251/bpo113. Epub 2005 Nov 7.

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Jose M Mellado-Gil  1 , Manuel Aguilar-Diosdado
Affiliations

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Jose M Mellado-Gil et al. Biol Proced Online. 2005.

Abstract

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).

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Figures

Fig. 1
Fig. 1. Glucose dose–response curve.
The islets were cultured with 2, 5.5, 11.1, 24.4 and 33.3 mM glucose for 48 h. Apoptotic cells were assessed by the TUNEL method under fluorescence microscopy. Data are expressed as the percentage of apoptotic cells. *P < 0.05 vs control 5.5 mM and P < 0.05 vs control 11.1 mM.
Fig. 2
Fig. 2. Influence of different glucose concentrations (5.5 or 24.4 mM) on islet cell apoptosis induced by STZ (1.5 mM).
The islets were cultured either with 5.5 or 24.4 mM glucose and exposed to STZ. Apoptotic cells were assessed by the TUNEL method under fluorescence microscopy. Data are expressed as the percentage of apoptotic cells. Open bars, islets cultured with 5.5 mM glucose; filled bars, islets cultured with 24.4 mM glucose. *P < 0.05 vs control 24.4 mM glucose.
Fig. 3
Fig. 3. Influence of different glucose concentrations (5.5 or 24.4 mM) on islet cell apoptosis induced by IL-1β and combined cytokines (IL-1β +IFN-γ +TNF-α).
A. Representative microscopy images of apoptotic cells stained with TUNEL (green) and Propidium Iodide (red). 5.5 mM glucose control (upper left); 5.5 mM glucose with cytokines (upper right); 24.4mM glucose control (bottom left) and 24.4mM glucose exposed to cytokines (bottom right). Arrows indicate apoptotic cells (co-location of red fluorescence of all cells plus green fluorescence of apoptotic cells). B. Percentage of apoptotic cells cultured with 5.5 or 24.4 mM glucose and exposed to IL-1β and combined cytokines. Open bars, islets cultured with 5.5 mM glucose; filled bars, islets cultured with 24.4 mM glucose. *P < 0.01 vs control 24.4 mM glucose and P < 0.05 vs. cytokines with 5.5 mM glucose.
Fig. 4
Fig. 4. Fas (CD95) expression in rat islets after exposure to IL-1β and combined cytokines (IL-1β, TNF-γ, IFN-α).
(A) Immunoblotting of Fas. Islets were cultured overnight with 5.5 or 24.4 mM glucose and exposed to IL-1β alone or combined with TNF-α plus IFN-γ, for 24 h. Antibodies against Fas and actin were blotted in the same filter after stripping. One of at least three experiments is shown. Each experiment gave similar results. C, control; IL-1β, interleukin-1β; CTK, cytokines (IL-1β + TNF-α + IFN-γ). (B) Densitometric quantitation of Fas-to-actin ratio showed a significant difference between 5 mM and 24.4 mM in the three groups (control, IL-1 and CTK). Y-axis represents arbitrary units. Open bars, islets cultured with 5.5 mM glucose; filled bars, islets cultured with 24.4 mM glucose. *P< 0.05 vs control, 5.5 mM glucose; **P < 0.05 vs. interleukin-1β, 5.5 mM glucose; *** P < 0.05 vs. cytokines, 5.5 mM glucose.
Fig. 5
Fig. 5. Expression of Fas (CD95) in rat islets after exposure to STZ.
(A) Immunoblotting of Fas. Islets were cultured for 48 h with 5.5 or 24.4 mM glucose and exposed to STZ for 24 h. Antibodies against Fas and actin were blotted in PVDF after stripping. One of at least three experiments is shown. Each experiment gave similar results. (B) Densitometric quantitation of Fas-to-actin ratio showed a significant difference between 5 mM and 24.4 mM. Y-axis represents arbitrary units. Open bars, islets cultured with 5.5 mM glucose; filled bars, islets cultured with 24.4 mM glucose. *P < 0.05 vs control, 5.5 mM glucose.
Fig. 6
Fig. 6. Expression of Bcl-2 and Bcl-xL in rat islets after exposure to IL-1β and combined cytokines (IL-1β + TNF-γ + IFN-α).
Islets were cultured overnight with 5.5 or 24.4 mM glucose and exposed to IL-1β alone or combined cytokines for 24 h. Immunoblottings were performed using antibodies against Bcl-2 and Bcl-xL. Actin levels were determined as loading control. Densitometric quantitation of both Bcl-2 and Bcl-xL-to-actin ratio did not generate significant differences (data not shown). One of at least three experiments is shown. Each experiment gave similar results. An amount of lymphocytes of chronic lymphoid leukemia (CLL) equivalent to number of islet cells per condition were used as positive control for Bcl-2. C, control; IL-1β, interleukin-1β; CTK, cytokines (IL-1β + TNF-α + IFN-γ).
Fig. 7
Fig. 7. Expression of Bcl-2 and Bcl-xL in rat islets after exposure to STZ.
(A) Immunoblotting of Bcl-2, Bcl-xL and Fas. Islets were cultured for 48 h with 5.5 or 24.4 mM glucose and exposed to STZ for 24 h. The antibodies were blotted in different PVDF filters and antiactin after stripping was used for loading control. Densitometric quantitation of both Bcl-2 and Bcl-xL-to-actin ratio did not generate significant differences (data not shown). One of at least three experiments is shown. Each experiment gave similar results. An amount of lymphocytes of chronic lymphoid leukemia (CLL) equivalent to number of islet cells per condition were used as positive control for Bcl-2.
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