Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice.
We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10-20 min of exposure to 25 mM glucose or 60 microM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes ( approximately 8 x 10(5) viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P< or =0.001) without fasting hypoglycemia and better glucose tolerance (P< or =0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for beta-galactosidase activity and with anti-human insulin antibody.
Chen
N K F
NK
Division of Medical Sciences, National Cancer Centre, Singapore, Republic of Singapore.
Sivalingam
J
J
Tan
S Y
SY
Kon
O L
OL
D000818:Animals; D001835:Body Weight; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D018274:Electroporation; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism* / Q000637:transplantation*; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D008099:Liver / Q000378:metabolism / Q000601:surgery; D008115:Liver Regeneration; D008297:Male; D008643:Mesentery; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D013194:Staining and Labeling; D019076:Transgenes; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
D007328:Insulin; D001616:beta-Galactosidase