Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.
OBJECTIVE
The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis.


METHODS
Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst 33342-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-3beta, activated caspase-3 and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors.


RESULTS
Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-3 (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05).


CONCLUSIONS
The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.
Li
L
L
Department of Physiology, University of Toronto, Room 3366, Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
El-Kholy
W
W
Rhodes
C J
CJ
Brubaker
P L
PL
D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D000077270:Exenatide; D000067757:Glucagon-Like Peptide-1 Receptor; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D008297:Male; D009336:Necrosis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010455:Peptides / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D018027:Receptors, Glucagon / Q000819:agonists / Q000502:physiology*; D014688:Venoms / Q000494:pharmacology*
D016207:Cytokines; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010455:Peptides; D011518:Proto-Oncogene Proteins; D018027:Receptors, Glucagon; D014688:Venoms; D000077270:Exenatide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases