C-peptide stimulates nitrites generation via the calcium-JAK2/STAT1 pathway in murine macrophage Raw264.7 cells.
OBJECTIVE
C-peptide is a product of pro-insulin cleavage. Numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper was to present a novel function of C-peptide, focusing on its role in nitric oxide (NO) generation.


METHODS
Murine macrophage Raw264.7 cells and primary peritoneal macrophages were incubated under control conditions, or with C-peptide. Expression level of iNOS and phosphorylation status of JAK2/STAT1 were analyzed by Western blot. Fluorometric NO assay kit was used to assess the concentration of nitrite in culture medium. Intracellular calcium concentration was measured with calcium indicator dyes, such as Fura-2 and Fluo-3 AM.


RESULTS
C-peptide increased the level of nitrites in murine macrophage Raw264.7 cells. The nitrites production induced by lipopolysaccharide (LPS) was further enhanced by co-treatment of C-peptide. This up-regulation of nitrites generation also correlated with the induction of inducible nitric oxide synthase (iNOS), a prominent marker of macrophage activation. In addition, C-peptide increased the intracellular concentration of calcium levels. Moreover, C-peptide-induced nitrites generation and increase in calcium was observed in freshly isolated primary peritoneal macrophages. In addition, C-peptide specifically affected the Janus activated kinase (JAK)/signal transducer and activated transcription (STAT) pathway. Finally, C-peptide-mediated nitrites generation and JAK2/STAT1 phosphorylation were not detected in the presence of the intracellular calcium chelator, BAPTA-AM.


CONCLUSIONS
These results suggest that C-peptide may elicit immune modulatory function via modulation of the calcium/JAK-STAT pathway.
Lee
Soo Kyung
SK
Department of Anatomy, Korea University College of Medicine, Seoul 136-701, Republic of Korea.
Lee
Jung Ok
JO
Kim
Ji Hae
JH
Jung
Jin Hee
JH
You
Ga Young
GY
Park
Sun Hwa
SH
Kim
Hyeon Soo
HS
D000814:Aniline Compounds; D000818:Animals; D002096:C-Peptide / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D016257:Fura-2; D015151:Immunoblotting; D053614:Janus Kinase 2 / Q000187:drug effects / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D010766:Phosphorylation; D050794:STAT1 Transcription Factor / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014966:Xanthenes
D000814:Aniline Compounds; D002096:C-Peptide; D008070:Lipopolysaccharides; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D014966:Xanthenes; C059715:Fluo-3; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D002118:Calcium; D016257:Fura-2