Given that absolute and relative insulin deficiencies, respectively, form the basis of type 1 and 2 diabetes, the identification of a means for restoring functional β-cell mass would hold immense promise as a means for curing these disorders. In this study, we made several novel findings: 1) in vivo administration of recombinant Pdx1 ameliorates hyperglycemia in diabetic mice, 2) amelioration of hyperglycemia is attended by both pancreatic β-cell regeneration and liver cell transdifferentiation, and 3) the observed therapeutic effect is likely to require Pdx1 to have an intact protein transduction domain. Our experiments therefore constitute a proof-of-principle demonstration that protein therapy in the form of in vivo Pdx1 delivery to whole animals can be a highly effective therapeutic strategy, one that exploits intrinsic properties of a naturally occurring pancreatic transcription factor and that avoids undesirable effects typically associated with viral vector-mediated gene therapies.
Specifically, our results indicate that in vivo delivery of rPdx1 can promote both β-cell regeneration as well as liver cell transdifferentiation into IPCs. Pdx1-mediated pancreatic islet β-cell regeneration appears to be the dominant effect on glucose homeostasis, since marked hyperglycemia was observed in mice receiving nearly total pancreatectomy. Although the exact cellular and molecular events responsible for rPdx1-mediated β-cell regeneration and liver cell transdifferentiation remain to be defined, we believe that, based on our findings, rPdx1 protein enters the circulation via terminal veins and capillaries and penetrates cellular membranes to gain nucleus entry into target cells in the liver and pancreas (), resulting in activation of rPdx1-dependent transcription factor cascade. The notion that rPdx1 promotes pancreatic β-cell regeneration is supported by the presence of numerous pancreatic large islets (), an increasing number of proliferating islet cells (), an ensuing increase in the level of pancreatic tissue insulin (), and the significant upregulation of several key genes related to pancreatic cell regeneration in rPdx1-treated mice (). The fact that rPdx1 vigorously promoted pancreatic islet cell proliferation and regeneration raises intriguing questions about the type of pancreatic cells that are the targets of rPdx1 (e.g., what is the cell origin for the newly regenerated islets?). Possible mechanisms include residual islet cell proliferation, exocrine acinar cell transdifferentiation, and pancreatic ductal/stem cell neogenesis. Presently, our approach limits us from tracking the cells of origin into/within the target tissue, resulting in the newly formed islets.
Several genes (INGAPrP, Reg-3γ
, and PAP
) upregulated by rPdx1 treatment are members of the pancreatic regenerating (Reg
) gene family originally identified in animal models of β-cell regeneration (33
). Their gene products play important roles in the maintenance of progenitors in the process of pancreas regeneration (10
). Despite variation between individual samples, expression levels of the Reg
genes determined by RT-PCR correlated well with an earlier study involving conditional expression of PDX1
in a transgenic mouse model (14
The observed functional effects of rPdx1 on the liver are consistent with the published results of ectopic expression of PDX1
gene via adenovirus vectors resulting in liver cell transdifferentiation (17
). In our experimental models, liver and pancreas appear to work in a sequential, compensatory manner to ameliorate hyperglycemia and ultimately to restore euglycemia in rPdx1-treated mice. The kinship between the liver and pancreas in controlling glucose homeostasis is also supported by a recent study using liver and pancreas double-injury animal models (34
). The early phase of rPdx1-induced hepatic insulin production is supported by an intense expression of the insulin I gene () and a nearly 18-fold increase in liver tissue insulin in comparison with that of control liver (). While the precise mechanism underlying the rPdx1-mediated surge in hepatic insulin during early-stage glucose homeostasis remains to be elucidated, possible explanations include the following: 1
) action of hepatic insulin on pancreatic progenitor cells via the insulin signaling pathway to promote β-cell regeneration via IRS2-Akt-Pdx1–mediated signal transduction (35
) insulin-mediated facilitation of β-cell neogenesis, involving amelioration of hyperglycemic toxic effects on residual β-cell regeneration (36
); and 3
) rPdx1-mediated hepatic insulin production, resulting in an increased rate of glucose clearance by the liver, perhaps by promoting glucokinase expression and/or by insulin’s stimulatory action on glycogen synthase, thereby lowering blood glucose levels (37
Pdx1 expression has also been reported to be associated with β-cell neogenesis in rodent pancreas injury models (10
). Although our studies showed roughly a 2 to 4× increase of PDX1
gene expression in the pancreas of rPdx1- over PTD-GFP–treated mice, pancreatic β-cell function appears to be exquisitely sensitive to small changes in PDX1
gene expression levels in both humans and mice (38
). The rPdx1 protein can positively regulate PDX1
gene expression, as evidenced by upregulation of endogenous PDX1
gene expression in the livers of rPdx1-treated mice. These observations are consistent with the findings of others indicating that Pdx1 binds to its own promoter and positively regulates its own gene expression (40
). These results suggest that rPdx1-based protein therapy may not require a large-dose or long-term treatment and, thus, may reduce or eliminate potential dosage-related systemic toxicity.
Although we have shown that rPdx1 effectively reverses hyperglycemia in diabetic mice, there are potential obstacles to clinical translation. One concern is that rPdx1 could be partially degraded by serum proteases. To assess this possibility, further studies on rPdx1 stability in whole blood and plasma would be helpful. Moreover, detailed pharmacokinetic studies are needed to optimize dosages, routes of delivery, and the interval between treatments. The polyclonal anti-Pdx1 antiserum used in this study precludes distinguishing between intact and any partially degraded rPdx1 protein. Significantly, the ability of rPdx1 to ameliorate hyperglycemia in diabetic mice, along with its nuclear localization in liver and pancreatic acinar cells (), indicates in vivo availability of a sufficient amount of intact rPdx1 protein or biologically active degradation products capable of translocation into cells. Another concern is the potential toxicity of rPdx1 to off-target organs via its PTD, since the rPdx1 protein has the potential to enter almost any tissue or cell type. We can, however, exclude the activation of rPdx1 target genes in tissues other than liver and pancreas at day 14 posttreatment. Moreover, the animals appeared normal, without evidence of weight loss or abnormal organ morphology. In fact, the diabetic mice treated with rPdx1 gained body weight, showed an improved IPGTT, and exhibited markedly reduced blood glucose levels. Nonetheless, a full toxicity profile of rPdx1, especially at earlier time points, is required to address this question. These studies are beyond the scope of the present manuscript and will be pursued in the future.
Interestingly, the distribution of rPdx1 in the kidney is quite different from that in the liver and pancreas at the early 1-h time point (). Instead of being present in the cell nuclei, rPdx1 was localized near the brush border of the proximal tubular cells (). Such a distribution was not observed at 24 h (). As the rPdx1 protein rapidly enters the bloodstream () after intraperitoneal injection, it may be filtered through glomerular capillaries into the urinary spaces via the fenestrated capillary endothelial cells and glomerular basement membrane. Alternatively, rPdx1 may gain entry via cells by virtue of its on-board PTD. Once in the urinary spaces, it would not be surprising if the cationic rPdx1 protein interacts electrostatically with polyanions (e.g., sialic acid and phopholipids) present on the apical surface of proximal tubular epithelial cells.
In conclusion, this demonstration that in vivo rPdx1 delivery into diabetic mice rapidly restores euglycemia exploits the intrinsic properties of this key pancreatic transcription factor (e.g., its built-in antennapedia-like PTD, its positive autoregulation, [28, 41], its vital role in pancreatic cell development and regeneration [10, 14], and its role in maintaining pancreatic β-cell function) (14
). Indeed, we believe that Pdx1-based protein therapy should allow for a redirection (or reactivation) of pancreatic stem/precursor cell differentiation and transdifferentiation (or reprogramming) from nonpancreatic cells along pancreatic β-cell developmental pathways, a feature that could prove beneficial in treating patients with diabetes.