Lentivirus-mediated transduction of connexin cDNAs shows level- and isoform-specific alterations in insulin secretion of primary pancreatic beta-cells.
We have generated novel lentiviral vectors to integrate various connexin cDNAs into primary, non-dividing cells. We have used these vectors to test whether proper control of insulin secretion depends on a specific connexin isoform and/or on its level of expression. We have observed that transduced connexin32, connexin36 and connexin43 were expressed by primary adult beta-cells at membrane interfaces, were packed into typical gap junction plaques and formed functional channels that allowed a variable coupling, depending on the type and level of connexin expressed. The infected cells spontaneously reaggregated into three-dimensional pseudo-islet organs that could be maintained in culture. We have found that pseudo-islets made by cells transduced with either GFP- or connexin43-expressing lentivirus released insulin in response to various secretagogues similarly to controls. By contrast, pseudo-islets made by cells expressing connexin32, a connexin exogenous to pancreatic islets, or over-expressing connexin36, the endogenous islet connexin, featured a marked decrease in the secretory response to glucose. The data show: (1) that lentiviral vectors allow stable modulation of various connexin in primary, non-proliferating cells; (2) that specific connexin isoforms affect insulin secretion differently; and (3) that adequate levels of coupling via connexin36 channels are required for proper beta-cell function.
Caton
David
D
Department of Morphology, University of Geneva Medical School, 1211 Geneva 4, Switzerland. david.caton@medecine.unige.ch
Calabrese
Alessandra
A
Mas
Christophe
C
Serre-Beinier
Véronique
V
Charollais
Anne
A
Caille
Dorothée
D
Zufferey
Romain
R
Trono
Didier
D
Meda
Paolo
P
D000818:Animals; D002449:Cell Aggregation / Q000502:physiology; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics; D017630:Connexins / Q000235:genetics*; D018076:DNA, Complementary; D017629:Gap Junctions / Q000502:physiology; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014161:Transduction, Genetic / Q000379:methods*; D000097002:Gap Junction beta-1 Protein; D000097004:Gap Junction delta-2 Protein
D018031:Connexin 43; D017630:Connexins; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D007328:Insulin; D008164:Luminescent Proteins; D000097002:Gap Junction beta-1 Protein; D000097004:Gap Junction delta-2 Protein