Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus.
The enigmatic process of beta-cell maturation has significant implications for diabetes pathogenesis, and potential diabetes therapies. This study examined the dynamics and heterogeneity of insulin and pancreatic duodenal homeobox (Pdx)-1 gene expression in adult beta-cells. Insulin and Pdx1 expression were monitored in human and mouse islet cells and MIN6 cells using a Pdx1-monomeric red fluorescent protein/insulin-enhanced green fluorescent protein dual-reporter lentivirus. The majority of fluorescent cells were highly positive for both Pdx1 and insulin. Cells expressing Pdx1 but little or no insulin (Pdx1(+)/Ins(low)) comprised 15-25% of the total population. Time-lapse imaging demonstrated that Pdx1(+)/Ins(low) primary beta-cells and MIN6 cells could convert to Pdx1(+)/Ins(+) cells without cell division. Genes involved in the mature beta-cell phenotype (Glut2, MafA) were expressed at higher levels in Pdx1(+)/Ins(+) cells relative to Pdx1(+)/Ins(low) cells. Conversely, genes implicated in early beta-cell development (MafB, Nkx2.2) were enriched in Pdx1(+)/Ins(low) cells. Sorted Pdx1(+)/Ins(low) MIN6 cells had a higher replication rate and secreted less insulin relative to double-positive cells. Long-term phenotype tracking of Pdx1(+)/Ins(low) cells showed two groups, one that matured into Pdx1(+)/Ins(+) cells and one that remained immature. These results demonstrate that adult beta-cells pass through distinct maturation states, which is consistent with previously observed heterogeneity in insulin and Pdx1 expression in adult beta-cells. At a given time, a proportion of adult beta-cells share similar characteristics to functionally immature embryonic beta-cell progenitors. The maturation of adult beta-cells recapitulates development in that Pdx1 expression precedes the robust expression of insulin and other mature beta-cell genes. These results have implications for harnessing the maturation process for therapeutic purposes.
Szabat
Marta
M
Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.
Luciani
Dan S
DS
Piret
James M
JM
Johnson
James D
JD
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007328:Insulin / Q000235:genetics*; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors
D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; C000715628:Nkx2-2 protein, rat; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins; D011189:Potassium Chloride; D005947:Glucose