Effects of carboxypeptidase E overexpression on insulin mRNA levels, regulated insulin secretion, and proinsulin processing of pituitary GH3 cells transfected with a furin-cleavable human proinsulin cDNA.
We recently developed two rat pituitary GH3 cell clones engineered to secrete human insulin (InsGH3). InsGH3 cells convert proinsulin into mature insulin, which is partially stored into a readily releasable pool of secretory granules. The efficiency of these processes, however, is relatively low in these cells, either in vitro or in vivo. This study was aimed at determining whether carboxypeptidase E (Cpe) overexpression can increase proinsulin processing and regulated secretion by InsGH3 clones. Indeed, in its membrane-bound form Cpe works as sorting receptor for the regulated secretory pathway of many hormones while, in its soluble form, Cpe takes part to the late step of insulin maturation. We obtained two Cpe-overexpressing cell lines from two different InsGH3 clones (InsGH3/C1 and C7). In the Cpe-overexpressing cell lines, derived from InsGH3 of clone 1 (InsGH3/C1-HACpe), in which the membrane-bound form of exogenous Cpe is accounted for by 90% of total Cpe immunoreactivity, we observed an increase in proinsulin gene expression, and in basal and stimulated insulin secretion compared with the original clone. In contrast, in the Cpe-overexpressing cell line derived from InsGH3 of clone 7 (InsGH3/C7-HACpe), where the exogenous membrane-bound form was only 60% of total Cpe, we detected a decrease in basal insulin release and a modest, albeit significant, increase in intracellular proinsulin processing. In conclusion, Cpe overexpression can increase regulated insulin secretion and proinsulin processing in InsGH3 cells; however, such improvements appear quantitatively and qualitatively modest.
Polastri
Luca
L
Department of Medicine, San Raffaele Scientific Institute, Milan, Italy.
Galbiati
Francesca
F
Folli
Franco
F
Davalli
Alberto M
AM
D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics*; D002460:Cell Line; D017690:Cell Transplantation / Q000379:methods*; D005576:Colforsin / Q000494:pharmacology; D018076:DNA, Complementary; D045683:Furin; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D010902:Pituitary Gland / Q000166:cytology* / Q000502:physiology; D011384:Proinsulin / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013381:Subtilisins; D023822:Tissue Engineering; D014162:Transfection
D018076:DNA, Complementary; D007328:Insulin; D012333:RNA, Messenger; D005576:Colforsin; D011384:Proinsulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D013381:Subtilisins; D045683:Furin