Calcium/calmodulin-dependent protein kinase IV involvement in the pathophysiology of glucotoxicity in rat pancreatic β-cells.
Glucotoxicity is a critical component of the pathophysiology of type 2 diabetes mellitus; however, the molecular mechanisms of glucotoxicity are still not fully understood. We have attempted to determine the protein kinases involved in glucotoxicity in pancreatic β-cells by the use of a new technique. Using Multi-PK antibodies, which are capable of detecting a wide variety of protein kinases, we analyzed the protein kinase that correlated with insulin synthesis in INS-1 cells under glucotoxic conditions. When expression patterns of protein kinases in INS-1 cells were analyzed by Western blotting with Multi-PK antibodies, a kinase of 63 kd was significantly reduced concomitant with the decrease of insulin secretion under glucotoxic conditions. To identify the 63-kd kinase, we used a unique 2-dimensional gel electrophoretic technique and MicroRotofor (Bio-Rad Laboratories, Tokyo, Japan) electrophoresis. From the molecular size of a native kinase/cyanogen bromide fragment and pI value, the 63-kd protein kinase was deduced to be CaMKIV. This was confirmed by Western blotting analysis using anti-CaMKIV antibodies. The decreased CaMKIV levels under glucotoxic conditions recovered to original levels after changing the medium to a normal glucose concentration. Recombinant CaMKIV was degraded in a Ca²+-dependent manner by incubation with cell lysates from INS-1 cells under glucotoxic conditions, and degradation was protected by calpain inhibitor. Furthermore, CaMKIV was reduced in the pancreatic islets of diabetic Otsuka Long-Evans Tokushima fatty rats, whereas that of nondiabetic Long-Evans Tokushima Otsuka rats was not. This study suggests that the abnormal regulation of CaMKIV is a component of β-cell dysfunction caused by high glucose.
Sugiyama
Yasunori
Y
Department of Life Sciences, Faculty of Agriculture, Kagawa University, 2393 Ikenobe Miki-cho, Kita-gun, Kagawa 761-0795, Japan.
Murao
Koji
K
Imachi
Hitomi
H
Sueyoshi
Noriyuki
N
Ishida
Toshihiko
T
Kameshita
Isamu
I
D000818:Animals; D002118:Calcium / Q000378:metabolism; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000032:analysis / Q000502:physiology*; D002154:Calpain / Q000502:physiology; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D005947:Glucose / Q000633:toxicity*; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects*; D051381:Rats
D007328:Insulin; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D002154:Calpain; D005947:Glucose; D002118:Calcium