ABSTRACT
Retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an amplified, glucose-regulated secretion of insulin. We constructed LhI*TFSN virus to encode a glucose-regulatable transforming growth factor alpha promoter controlling furin expression with a viral LTR promoter driving constitutive expression of furin-cleavable human proinsulin. Autologous BB rat vascular smooth muscle cells transduced with LhI*TFSN virus and cultured in 1.7 and 16.7 mM glucose secreted 50.7 +/- 3.2 and 136.0 +/- 11.0 microU (mean +/- SD) of insulin per 10(6) cells per day, respectively. After the onset of diabetes spontaneously diabetic congenic DR lyp/lyp BB rats received stomach implants containing 2 x 10(6) LhI*TFSN-transduced primary rat vascular smooth muscle cells. In eight treated rats there was a major reduction in insulin requirement to as low as 25% of pretreatment level for up to 3 months and one rat became insulin free without hypoglycemia. Intraperitoneal glucose tolerance tests (IPGTTs) in diabetic rats receiving control implants did not show the characteristic decline in blood glucose of normal rats after glucose administration. In contrast, diabetic rats receiving LhI*TFSN-transduced cells showed significant clearances of blood glucose. These data suggest clinically significant levels of glucose-regulated insulin delivery from implanted vascular smooth muscle cells transduced with LhI*TFSN vector.
Subject(s)
Diabetes Mellitus, Type 1/metabolism , Glucose/metabolism , Insulin/biosynthesis , Animals , Cells, Cultured , Diabetes Mellitus, Type 1/therapy , Erythropoietin/metabolism , Furin , Glucose Tolerance Test , Humans , Male , Muscle, Smooth, Vascular/physiology , Promoter Regions, Genetic , RNA, Messenger/genetics , Rats , Rats, Inbred BB , Subtilisins/metabolism , Transduction, Genetic , Transforming Growth Factor alpha/genetics , Weight GainABSTRACT
Platelet-activating factor (PAF) has been implicated in the development of type 1 diabetes. Our previous studies have suggested that PAF inhibitors reduce insulitis and the frequency of diabetes in BB rats. In this study, serum PAF levels were reduced to address the hypothesis that PAF is important for the development of insulitis. From the age of 35 days on, DP-BB rats were treated with human recombinant PAF acetylhydrolase (rPAF-AH), which efficiently inactivates PAF. Our data indicate that intraperitoneal injections of rPAF-AH reduce the incidence of diabetes in the DP-BB rat. Daily intraperitoneal injections of 6.0 mg/kg body wt rPAF-AH reduced the frequency of diabetes in saline-injected rats from 90% (27/30) to 57% (17/30) (P = 0.004). As found by morphometric analysis on pancreatic islets, DP-BB rats protected from diabetes had less severe degrees of insulitis in a dose-dependent manner. DP-BB rats protected by rPAF-AH also had a higher percentage of insulin-positive cells in pancreas sections compared with those from diabetic animals. We therefore speculated that the beta-cells were protected from insulitis by rPAF-AH.