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Reversal of Diabetes in Mice by Islet Neogenesis in the Liver induced by Helper Dependant Adenoviral Delivery of Ngn3 Involves the Reprogramming of Albumin-expressing Cell Lineage
Vijay Yechoor, MD1, Victoria Liu, BS1, Hideto Kojima, MD PhD2, Mineko Fujimiya, MD PhD2, Benny Chang, PhD1, Lawrence Chan, MD1
1 Baylor College of Medicine, Houston, TX ; 2Shiga University, Shiga, Japan
Type 1 diabetes is characterized by hyperglycemia due to a deficiency of insulin resulting from a destruction of pancreatic beta-cells. Though exogenous insulin replacement corrects hyperglycemia, a restoration of physiological glucose responsiveness has been elusive until the advent of islet replacement therapy, which is limited by a significant donor shortage and a requirement for immunosuppression.
Our approach to circumvent this is to induce islet neogenesis which would restore physiological glucose responsiveness. We reported earlier the reversal of diabetes after helper dependent adenoviral (HDAd) mediated gene transfer of NeuroD, an islet transcription factor and betacellulin (Btc), an islet growth factor, in streptozotocin (STZ) diabetic mice by inducing insulin positive cells (IPC) in the liver (Kojima et. al. Nat Med 2003). Although hyperglycemia was reversed in 3 weeks, unlike normal pancreatic islets, the newly formed IPC produced multiple islet hormones in the same cell.
To improve on this, we decided to use Neurogenin 3 (Ngn3) instead of NeuroD because Ngn 3 is an islet lineage defining transcription factor. Its role in islet development was established by a complete absence of all islet endocrine cells in mice that lacked Ngn 3 function. This along with supporting in vitro studies indicating that Ngn 3 is sufficient and necessary for normal islet development prompted us to study reversal of diabetes with HDAd mediated delivery of Ngn 3 along with Btc.
In STZ-diabetic mice a single injection of HDAd-Ngn3-Btc restored euglycemia within 1 week and this persisted at least 9 weeks. An IP-GTT at 6 weeks revealed restoration of glucose tolerance and glucose-stimulated insulin secretion indistinguishable from non-diabetic controls. 72 hr fast did not produce hypoglycemia indicating glycemia-regulated insulin secretion. Q-RT-PCR revealed presence of Ins1, Ins2, Glucagon transcripts in the livers of treated mice. Immunostaining revealed clusters of IPC next to glucagon-positive cells, as seen in mature islets. To establish that these IPC in the liver are responsible for the glucose responsive insulin secretion, we then performed an in situ isolated liver perfusion with stepwise increase in glucose concentrations of buffer infused continuously via the portal vein and observed glucose concentration-responsive insulin secretion in the effusate from the hepatic veins collected via the IVC, from the livers of HDAd-Ngn3-Btc treated mice, but not from empty virus-treated diabetic controls.
We traced the lineage of these IPC using transgenic mice (Rosa-STOP-Lox-GFP mice x Albumin-Cre mice) in which only albumin-expressing cells were permanently marked by GFP. Insulin co-localized with GFP in most of the IPC, suggesting IPC originated from cells that had expressed albumin in their lineage history. However, insulin rarely co-localized with immunoreactive albumin, suggesting that IPC did not express albumin once they started producing insulin. Taken together, this suggests that the neurogenin 3 reprograms albumin-expressing-lineage cells into islet forming cells that not only give rise to mature insulin-producing cells but also separate glucagon-producing cells, thus largely recapitulating normal embryological islet development.
In conclusion, in vivo gene therapy with HDAd-Ngn3-Btc induces albumin-expressing liver cells to reprogram into mature islets containing glycemia-regulated insulin-producing beta cells that completely reverse diabetes in mice.
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