Exendin-4 regulates glucokinase expression by CaMKK/CaMKIV pathway in pancreatic beta-cell line.
OBJECTIVE
Glucokinase (GK) in pancreatic beta cells is thought to be involved in insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, mediates stimulatory effects on GK gene expression through the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade.


METHODS
GK expression was examined by real-time PCR, western blot analysis and reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation loop phosphorylation (Thr(196)) of CaMKIV. We investigated the effect of the constitutively active form (CaMKIVc) of CaMKIV on GK promoter activity.


RESULTS
Increased expression level of GK protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Real-time PCR analysis showed a significant increase in the amount of GK mRNA in response to rising concentrations of exendin-4. Exendin-4 also stimulated GK promoter activity but failed to do so in the presence of STO-609, a CaMKK inhibitor. This result is consistent with the observations that the upregulation of CaMKIV phosphorylation (at Thr(196)) peaked after 15 min of exposure to exendin-4 and that CaMKIVc enhanced or upregulated GK promoter activity in INS-1 cells. Furthermore, STO-609 significantly suppressed the exendin-4 - upregulated the expression of the GK protein.


CONCLUSIONS
Activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GK gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta cells.
Murao
K
K
Division of Endocrinology and Metabolism, Department of Internal Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. mkoji@med.kagawa-u.ac.jp
Li
J
J
Imachi
H
H
Muraoka
T
T
Masugata
H
H
Zhang
G X
GX
Kobayashi
R
R
Ishida
T
T
Tokumitsu
H
H
D000818:Animals; D015153:Blotting, Western; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000378:metabolism; D002460:Cell Line; D000077270:Exenatide; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D050417:Insulin-Secreting Cells / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014688:Venoms / Q000494:pharmacology*
D007004:Hypoglycemic Agents; D010455:Peptides; D014688:Venoms; D000077270:Exenatide; D005941:Glucokinase; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; C517686:Camk4 protein, rat