Exendin-4 inhibits interleukin-1beta-induced iNOS expression at the protein level, but not at the transcriptional and posttranscriptional levels, in RINm5F beta-cells.
Cytokines such as interleukin-1beta (IL-1beta) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to beta-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for beta-cell proliferation. However, the protective mechanisms of GLP-1 in beta-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1beta-induced iNOS gene expression were investigated employing RINm5F beta-cells. EX-4 inhibited IL-1beta-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1beta-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-kappaB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1beta-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1beta-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1beta-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.
Kang
Jung-Hoon
JH
Departments of Physiology, College of Medicine, The Catholic University of Korea, Seoul, South Korea.
Chang
Seo-Yoon
SY
Jang
Hyun-Jong
HJ
Kim
Dong-Bin
DB
Ryu
Gyeong Ryul
GR
Ko
Seung Hyun
SH
Jeong
In-Kyung
IK
Jo
Yang-Hyeok
YH
Kim
Myung-Jun
MJ
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D000077270:Exenatide; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014688:Venoms / Q000494:pharmacology*
D007004:Hypoglycemic Agents; D053583:Interleukin-1beta; D016328:NF-kappa B; D010455:Peptides; D012333:RNA, Messenger; D014688:Venoms; D009569:Nitric Oxide; D000077270:Exenatide; D000242:Cyclic AMP; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat