P-glycoprotein regulating biphasic insulin secretion in rat pancreatic beta cells.
BACKGROUND
A 65-kD mdr1 (multi-drug resistance protein 1, P-glycoprotein)-like protein has been suggested to be the regulatory protein to the chloride channel protein 3 (ClC-3) mediating insulin granules acidification and release in mouse pancreatic beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions.


METHODS
Total RNAs of rat kidneys served as positive controls, and pancreas, islets and INS-1 cells were extracted for reverse-transcript PCR (RT-PCR), respectively. The cDNAs were run with specific primers selected from the mRNA of abcb1b encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody, C219. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay.


RESULTS
Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose (16.7 mmol/L), and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A (CsA), specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced.


CONCLUSIONS
The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release.
Tang
Yun-zhao
YZ
Key Lab of Hormone and Development (Tianjin and Ministry of Health of the People's Republic of China), Institution of Endocrinology, Tianjin Metabolic Diseases Hospital, Tianjin Medical University, Tianjin 300070, China.
Li
Dai-qing
DQ
Sun
Fu-jun
FJ
Li
Li
L
Yu
De-min
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007328:Insulin