Endocrinol Metab.  2011 Jun;26(2):142-149. 10.3803/EnM.2011.26.2.142.

GLP-1 Can Protect Proinflammatory Cytokines Induced Beta Cell Apoptosis through the Ubiquitination

• Lim DM
• Kim JY
• Lee KW
• Park KY
• Kim BJ

• KMID: 1497721
• DOI: http://doi.org/10.3803/EnM.2011.26.2.142

Abstract

BACKGROUND
Proinflammatory cytokines are one of the causes of diabetes mellitus. However, the exact molecular mechanism by which proinflammatory cytokines induce beta-cell death remains to be clearly elucidated. Glucagon-like peptide-1 (GLP-1) affects the stimulation of insulin secretion and the preservation of beta-cells. Additionally, it may exert an antiapoptotic effect on beta cells; however, the mechanism underlying this effect has yet to be demonstrated. Therefore, we investigated the protective effects of GLP-1 in endoplasmic reticulum (ER)-mediated beta-cell apoptosis using proinflammatory cytokines.
METHODS
To induce ER stress, hamster insulin-secreting tumor (HIT)-T15 cells were treated using a mixture of cytokines. Apoptosis was evaluated via MTT assay, Hoechst 33342 staining, and annexin/propidium iodide (PI) flow cytometry. The mRNA and protein expression levels of ER stress-related molecules were determined via PCR and Western blotting, respectively. Nitric oxide was measured with Griess reagent. The levels of inducible nitric oxide synthase (iNOS) mRNA and protein were analyzed via real-time PCR and Western blot, respectively. iNOS protein degradation was evaluated via immunoprecipitation. We pretreated HIT-T15 cells with exendin (Ex)-4 for 1 hour prior to the induction of stress.
RESULTS
We determined that Ex-4 exerted a protective effect through nitric oxide and the modulation of ER stress-related molecules (glucose-regulated protein [GRP]78, GRP94, and CCAAT/enhancer-binding protein homologous protein [CHOP]) and that Ex-4 stimulates iNOS protein degradation via the ubiquitination pathway. Additionally, Ex-4 also induced the recovery of insulin2 mRNA expression in beta cells.
CONCLUSION
The results of this study indicate that GLP-1 may protect beta cells against apoptosis through the ubiquitination pathway.

Keyword

Beta cell; ER stress; Incretin; Proinflammatory cytokine

MeSH Terms

Animals
Apoptosis
Benzimidazoles
Blotting, Western
Cricetinae
Cytokines
Diabetes Mellitus
Endoplasmic Reticulum
Ethylenediamines
Flow Cytometry
Glucagon-Like Peptide 1
HSP70 Heat-Shock Proteins
Immunoprecipitation
Incretins
Insulin
Membrane Proteins
Nitric Oxide
Nitric Oxide Synthase Type II
Polymerase Chain Reaction
Proteolysis
Real-Time Polymerase Chain Reaction
RNA, Messenger
Sulfanilamides
Ubiquitin
Ubiquitination
Benzimidazoles
Cytokines
Ethylenediamines
Glucagon-Like Peptide 1
HSP70 Heat-Shock Proteins
Incretins
Insulin
Membrane Proteins
Nitric Oxide
Nitric Oxide Synthase Type II
RNA, Messenger
Sulfanilamides
Ubiquitin

Figure

• 

  Fig. 1 After exposure to mixture of cytokines, HIT-T15 cells apoptosis increased by doses of cytokines mixture. A. Cells viability was measured with the MTT assay. HIT-T15 cells were pretreated Ex-4 for 1 hour before mixture of cytokines treatment. B. After treated of mixture of cytokine, effects of the Ex-4 on cell viability were measured by MTT assay. C. Proinflammatory cytokines induced apoptotic nuclei reduced via Ex-4. Fixed cells were stained with hoechst 33342 D. Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to 72 hours. *** < 0.001 vs. control cells; ### < 0.001 vs. CTYs alone.

• 

  Fig. 2 HIT-T15 cells were pretreated with Ex-4, forskolin, H89. After 1 hour, HIT-T15 cells were treated with mixture of cytokines for 48 hours. A. After treated of mixture of cytokines, effect of the Ex-4, forskolin, H89 on GRP 78, 94 and CHOP were determined by densitometry analysis. B. Western blotting of GRP78,94 and CHOP. * < 0.05, ** < 0.01, *** < 0.001 vs. control cells; # < 0.05, ## < 0.01 vs. CTYs alone; $ < 0.05, $$ < 0.01, $$$ < 0.001 vs. Ex-4 in treated CYTs.

• 

  Fig. 3 HIT-T15 cells were pretreated with Ex-4, forskolin, H89. After 1 hour, HIT-T15 cells were treated with mixture of cytokines for 48 hours. A. After treated of mixture of cytokines, effect of the Ex-4, foskolin, H89 on nitric oxide were determined by densitometry analysis. B. Western blotting of iNOS protein. C. After treated of mixture of cytokines, effect of the Ex-4, forskolin, H89 on iNOS mRNA were determined by densitometry analysis. *** < 0.001 vs. control cells; # < 0.05, ### < 0.001 vs. CTYs alone; $$$ < 0.001 vs. Ex-4 in treated CYTs.

• 

  Fig. 4 HIT-T15 cells were pretreated with Ex-4. After 1 hour, HIT-T15 cells were treated with mixture of cytokines for 48 hours. A. iNOS protein ubiquitination was increased by Ex-4. B. Expression levels of deubiquitnation enzyme were examined by real-time PCR and western blot. C. real-time PCR of ubiquitin enzyme after treatment cytokines or Ex-4. D. Expression levels of insulin2 mRNA were examined by real-time PCR. ** < 0.01, *** < 0.001 vs. control cells; ## < 0.01, ### < 0.001 vs. CTYs alone; $$ < 0.01, $$$ < 0.001 vs. Ex-4 in treated CYTs.

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