High Glucose Is Necessary for Complete Maturation of Pdx1-VP16-Expressing Hepatic Cells into Functional Insulin-Producing Cells

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, USA.
Diabetes (Impact Factor: 8.1). 01/2005; 53(12):3168-78. DOI: 10.2337/diabetes.53.12.3168
Source: PubMed


Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.

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    • "Based on our zebrafish and mouse data we hypothesize that one conserved function for pre-pancreatic insulin expression may be to suppress glucose production, as it can be teratogenic to embryos ([4] Akazawa, 2005; 23] Eriksson et al., 1991; 24] Freinkel, 1988; 44] Moley, 1999). Significantly, human, rodent, and apparently zebrafish preferentially utilize non-glucose substrates at early stages ([9] Biggers et al., 1967), further supporting this idea. "
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    ABSTRACT: Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not a genetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.
    General and Comparative Endocrinology 10/2010; 170(2):334-45. DOI:10.1016/j.ygcen.2010.10.010 · 2.47 Impact Factor
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    • "Fusion of Pdx-1 to the VP16 activation domain from Herpes simplex virus (Pdx-1-VP16) leads to more efficient liver-to-pancreas conversion than Pdx-1 alone [8-12]. Stable mouse Pdx-1 or Pdx-1-VP16 gene transfection initiates conversion of rat epithelial liver stem-like WB cell line into pancreatic endocrine precursor cells [13,14]. In these cells, long-term high glucose (HG) culture in vitro is necessary for further pancreatic endocrine differentiation. "
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    ABSTRACT: Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction. WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. beta-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into beta-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and beta-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein. These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for beta-cell engineering for diabetes cell therapy.
    BMC Research Notes 02/2009; 2(1):3. DOI:10.1186/1756-0500-2-3
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    • "Apart from Pdx1, Hnf6, and Ngn3, BMEL cells do not express any of the downstream effectors of more advanced pancreatic commitment. Moreover, expression of pancreatic markers in aggregates remains unchanged following culture in high glucose medium, which is known to stimulate maturation of pancreatic progenitors (25 mM glucose, data not shown) [19]. Hes1, a direct repressor of Ngn3, is expressed in BMEL cells regardless of culture conditions, albeit quantitative real-time PCR shows a decrease with time in aggregate culture. "
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    BMC Research Notes 01/2009; 1(1):136. DOI:10.1186/1756-0500-1-136
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