Gene transfer of active Akt1 by an infectivity-enhanced adenovirus impacts β-cell survival and proliferation differentially in vitro and in vivo

Abstract

Type 1 Diabetes is characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing β-cells in the pancreatic islets. Akt1/Protein Kinase B is the direct downstream target of PI3 Kinase activation, and has shown potent anti-apoptotic and proliferation-inducing activities. This study was designed to explore whether gene transfer of constitutively active Akt1 (CA-Akt1) would promote β-cell survival and proliferation, thus be protective against experimental diabetes. In the study, a fiber-modified infectivity-enhanced adenoviral vector, Ad5RGDpK7, was used to deliver rat insulin promoter (RIP)-driven CA-Akt1 into β-cells. Our data showed this vector efficiently delivered CA-Akt1 into freshly isolated pancreatic islets, and promoted islet cell survival and β-cell proliferation in vitro. The therapeutic effect of the vector in vivo was assessed using streptozotocin (STZ)-induced diabetes mice. Two means of vector administration were explored: intravenous and intra-bile ductal injections. While direct vector administration into pancreas via bile-ductal injection resulted in local adverse effect, intravenous injection of the vectors offered therapeutic benefits. Further analysis suggests systemic vector administration caused endogenous Akt expression and activation in islets, which may be responsible, at least in part, for the protective effect of the infectivity-enhanced CA-Akt1 gene delivery vector. Taken together, our data suggest CA-Akt1 is effective in promoting β-cell survival and proliferation in vitro, but direct in vivo use is compromised by the efficacy of transgene delivery into β-cells. Nonetheless, the vector evoked the expression and activation of endogenous Akt in the islets, thus offering beneficial bystander effect against STZ-induced diabetes.

Activation of AKT by O-Linked N-Acetylglucosamine Induces Vascular Calcification in Diabetes Mellitus

“…To determine a direct effect of increased O-GlcNAcylation on vascular calcification, we induced O-GlcNAcylation in cultured VSMC using Thiamet-G, a highly potent and selective inhibitor of OGA that has been shown to increase O-GlcNAc modification 24 . We found that Thiamet-G dose-dependently increased O-GlcNAcylation, independent of OGT (Fig 2A, and 2B).…”

“…The distinct function of O-GlcNAcylation in chronic and acute disease model may be related to differential activation of unknown signaling cascades. While the STZ model has its limitation due to its toxicity in vitro and its inhibitory effect on OGA also, its diabetogenic mechanism of action has been shown to be independent of these side effects since STZ has a very short half-life 24 . Using Thiamet-G, a potent and selective OGA inhibitor, our studies have provided the first evidence that increased vascular O-GlcNAcylation enhanced vascular calcification in diabetic mice in vivo (Fig 4).…”

“…Briefly, C57BL/6 mice were intraperitoneally injected with STZ (50 mg/kg) for 5 consecutive days, and blood glucose was monitored weekly for 4 months using the AlphaTrak glucose meter and strips (Abbott, Abbott Park, IL). For Thiamet-G treatment, mice were injected intravenously with Thiamet-G 24 (20 mg/kg) one week after STZ treatment and weekly for 2 months. Both food and fluid intake were given ad libitum .…”

“…O-GlcNAcylation was induced by inhibition of OGA, using a pharmacological inhibitor, Thiamet-G 24 , or OGA knockdown by lentivirus-mediated short hairpin RNA specific targeting OGA (GenBank NC_000085.6, shRNA, Thermo Scientific, Waltham, MA) as we have described previously 26 .…”

Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

“…We place such importance on cell adhesion because many molecular biological techniques for cells are performed under cell adhesion conditions. Regenerative medicine associated with the establishment of pluripotent stem cells [9][10][11][12] and gene therapy [13][14][15] may offer new treatments for severe pancreatic diseases, such as diabetes mellitus.…”