Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice

Abstract

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 mM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes (B8 Â 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 postimplantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (Pp0.001) without fasting hypoglycemia and better glucose tolerance (Pp0.0003) 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 b-galactosidase activity and with anti-human insulin antibody. Gene Therapy (2005) 12, 655-667.

Cell transplantation for diabetes

“…(iii) Transfecting adult cells with the human insulin gene with a glucose sensing promoter This approach can use non-viral electroporition to introduce the insulin gene plasmid into cells in vitro or in vivo, with encouraging experimental results using adult liver cells (Chen et al 2005). Alternatively viral vectors can be used which are more efficient, but some viruses have the danger of unmasking oncogenes (McCormack & Rabbitts 2004).…”

Glucose regulates secretion of exogenously expressed insulin from HepG2 cells in vitro and in a mouse model of diabetes mellitus in vivo

“…Previous attempts to mimic pancreatic function in regulating insulin have used nonislet cells, including hepatocytes, to express and secrete insulin (Lu et al 1998, Chen et al 2005, Wilson et al 2005, Hsu et al 2008. The general approach involved use of a glucose-responsive promoter or motif to control insulin gene transcription , Chen et al 2001, Alam & Sollinger 2002, Hsu et al 2008).…”

Insulin expressed from endogenously active glucose-responsive EGR1 promoter in bone marrow mesenchymal stromal cells as diabetes therapy

“…Intraperitoneal glucose tolerance tests were performed on days À10, À5, 7 and 21 as described previously. 4 …”

“…Transplantation of plasmid-electroporated syngeneic primary hepatocytes corrected murine streptozotocin (STZ)-induced diabetes. 4 Similarly, intrahepatic (IH) implantation of autologous primary hepatocytes in diabetic pigs showed durable (X47 weeks) correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities, which correlated significantly with the number of hepatocytes implanted. 5 Importantly, we observed no hypoglycemia, regardless of feeding behavior, that is, the 'nibbling' behavior of mice and 'humanized' mealtime feeding of pigs, and even under fasting conditions in both animal models.…”