Doris A. Stoffers

Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1

Intrauterine growth retardation (IUGR) has been linked to the onset of diseases in adulthood, including type 2 diabetes, and has been proposed to result from altered gene regulation patterns due to epigenetic modifications of developmental genes. To determine whether epigenetic modifications may play a role in the development of adult diabetes following IUGR, we used a rodent model of IUGR that expresses lower levels of Pdx1, a pancreatic and duodenal homeobox 1 transcription factor critical for beta cell function and development, which develops diabetes in adulthood. We found that expression of Pdx1 was permanently reduced in IUGR beta cells and underwent epigenetic modifications throughout development. The fetal IUGR state was characterized by loss of USF-1 binding at the proximal promoter of Pdx1, recruitment of the histone deacetylase 1 (HDAC1) and the corepressor Sin3A, and deacetylation of histones H3 and H4. Following birth, histone 3 lysine 4 (H3K4) was demethylated and histone 3 lysine 9 (H3K9) was methylated. During the neonatal period, these epigenetic changes and the reduction in Pdx1 expression could be reversed by HDAC inhibition. After the onset of diabetes in adulthood, the CpG island in the proximal promoter was methylated, resulting in permanent silencing of the Pdx1 locus. These results provide insight into the development of type 2 diabetes following IUGR and we believe they are the first to describe the ontogeny of chromatin remodeling in vivo from the fetus to the onset of disease in adulthood.

Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates

Epithelial metaplasia occurs when one predominant cell type in a tissue is replaced by another, and is frequently associated with an increased risk of subsequent neoplasia. In both mouse and human pancreas, acinar-to-ductal metaplasia has been implicated in the generation of cancer precursors. We show that pancreatic epithelial explants undergo spontaneous acinar-to-ductal metaplasia in response to EGFR signaling, and that this change in epithelial character is associated with the appearance of nestin-positive transitional cells. Lineage tracing involving Cre/lox-mediated genetic cell labeling reveals that acinar-to-ductal metaplasia represents a true transdifferentiation event, mediated by initial dedifferentiation of mature exocrine cells to generate a population of nestin-positive precursors, similar to those observed during early pancreatic development. These results demonstrate that a latent precursor potential resides within mature exocrine cells, and that this potential is regulated by EGF receptor signaling. In addition, these observations provide a novel example of rigorously documented transdifferentiation within mature mammalian epithelium, and suggest that plasticity of mature cell types may play a role in the generation of neoplastic precursors.

Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise through a multistep model comprised of putative precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). Recent genetically engineered mouse models of PDAC demonstrate a comparable morphologic spectrum of murine PanIN (mPanIN) lesions. The histogenesis of PanIN and PDAC in both mice and men remains controversial. The most faithful genetic models activate an oncogenic KrasG12D knockin allele within the pdx1- or ptf1a/p48-expression domain of the entire pancreatic anlage during development, thus obscuring the putative cell(s)-of-origin from which subsequent mPanIN lesions arise. In our study, activation of this knockin KrasG12D allele in the Elastase- and Mist1-expressing mature acinar compartment of adult mice resulted in the spontaneous induction of mPanIN lesions of all histological grades, although invasive carcinomas per se were not seen. We observed no requirement for concomitant chronic exocrine injury in the induction of mPanIN lesions from the mature acinar cell compartment. The acinar cell derivation of the mPanINs was established through lineage tracing in reporter mice, and by microdissection of lesional tissue demonstrating Cre-mediated recombination events. In contrast to the uniformly penetrant mPanIN phenotype observed following developmental activation of KrasG12D in the Pdx1-expressing progenitor cells, the Pdx1-expressing population in the mature pancreas (predominantly islet β cells) appears to be relatively resistant to the effects of oncogenic Kras. We conclude that in the appropriate genetic context, the differentiated acinar cell compartment in adult mice retains its susceptibility for spontaneous transformation into mPanIN lesions, a finding with potential relevance vis-à-vis the origins of PDAC.

On the origin of the β cell

The major forms of diabetes are characterized by pancreatic islet beta-cell dysfunction and decreased beta-cell numbers, raising hope for cell replacement therapy. Although human islet transplantation is a cell-based therapy under clinical investigation for the treatment of type 1 diabetes, the limited availability of human cadaveric islets for transplantation will preclude its widespread therapeutic application. The result has been an intense focus on the development of alternate sources of beta cells, such as through the guided differentiation of stem or precursor cell populations or the transdifferentiation of more plentiful mature cell populations. Realizing the potential for cell-based therapies, however, requires a thorough understanding of pancreas development and beta-cell formation. Pancreas development is coordinated by a complex interplay of signaling pathways and transcription factors that determine early pancreatic specification as well as the later differentiation of exocrine and endocrine lineages. This review describes the current knowledge of these factors as they relate specifically to the emergence of endocrine beta cells from pancreatic endoderm. Current therapeutic efforts to generate insulin-producing beta-like cells from embryonic stem cells have already capitalized on recent advances in our understanding of the embryonic signals and transcription factors that dictate lineage specification and will most certainly be further enhanced by a continuing emphasis on the identification of novel factors and regulatory relationships.

Preexisting pancreatic acinar cells contribute to acinar cell, but not islet β cell, regeneration

It has been suggested that pancreatic acinar cells can serve as progenitors for pancreatic islets, a concept with substantial implications for therapeutic efforts to increase insulin-producing beta cell mass in patients with diabetes. We report what we believe to be the first in vivo lineage tracing approach to determine the plasticity potential of pancreatic acinar cells. We developed an acinar cell-specific inducible Cre recombinase transgenic mouse, which, when mated with a reporter strain and pulsed with tamoxifen, resulted in permanent and specific labeling of acinar cells and their progeny. During various time periods of observation and using several models to provoke injury, we failed to observe any chase of the labeled cells into the endocrine compartment, indicating that acinar cells do not normally transdifferentiate into islet beta cells in vivo in adult mice. In contrast, we observed a substantial role for replication of preexisting acinar cells in the regeneration of new acinar cells after partial pancreatectomy. These results indicate that mature acinar cells harbor a facultative acinar but not endocrine progenitor capacity.

On the origin of the beta cell.

The major forms of diabetes are characterized by pancreatic islet beta-cell dysfunction and decreased beta-cell numbers, raising hope for cell replacement therapy. Although human islet transplantation is a cell-based therapy under clinical investigation for the treatment of type 1 diabetes, the limited availability of human cadaveric islets for transplantation will preclude its widespread therapeutic application. The result has been an intense focus on the development of alternate sources of beta cells, such as through the guided differentiation of stem or precursor cell populations or the transdifferentiation of more plentiful mature cell populations. Realizing the potential for cell-based therapies, however, requires a thorough understanding of pancreas development and beta-cell formation. Pancreas development is coordinated by a complex interplay of signaling pathways and transcription factors that determine early pancreatic specification as well as the later differentiation of exocrine and endocrine lineages. This review describes the current knowledge of these factors as they relate specifically to the emergence of endocrine beta cells from pancreatic endoderm. Current therapeutic efforts to generate insulin-producing beta-like cells from embryonic stem cells have already capitalized on recent advances in our understanding of the embryonic signals and transcription factors that dictate lineage specification and will most certainly be further enhanced by a continuing emphasis on the identification of novel factors and regulatory relationships.

Vertical Sleeve Gastrectomy Is Effective in Two Genetic Mouse Models of Glucagon-Like Peptide 1 Receptor Deficiency

Glucagon-like peptide 1 (GLP-1) is a peptide hormone that is released from the gut in response to nutrient ingestion and that has a range of metabolic effects, including enhancing insulin secretion and decreasing food intake. Postprandial GLP-1 secretion is greatly enhanced in rats and humans after some bariatric procedures, including vertical sleeve gastrectomy (VSG), and has been widely hypothesized to contribute to reduced intake, weight loss, and the improvements in glucose homeostasis after VSG. We tested this hypothesis using two separate models of GLP-1 receptor deficiency. We found that VSG-operated GLP-1 receptor–deficient mice responded similarly to wild-type controls in terms of body weight and body fat loss, improved glucose tolerance, food intake reduction, and altered food selection. These data demonstrate that GLP-1 receptor activity is not necessary for the metabolic improvements induced by VSG surgery.

Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein.

The homeodomain transcription factor insulin promoter factor-1 (IPF-1) is required for development of the pancreas and also mediates glucose-responsive stimulation of insulin gene transcription. Earlier we described a human subject with pancreatic agenesis attributable to homozygosity for a cytosine deletion in codon 63 of the IPF-1 gene (Pro63fsdelC). Pro63fsdelC resulted in the premature truncation of an IPF-1 protein which lacked the homeodomain required for DNA binding and nuclear localization. Subsequently, we linked the heterozygous state of this mutation with type 2 diabetes mellitus in the extended family of the pancreatic agenesis proband. In the course of expressing the mutant IPF-1 protein in eukaryotic cells, we detected a second IPF-1 isoform, recognized by COOH- but not NH2-terminal-specific antisera. This isoform localizes to the nucleus and retains DNA-binding functions. We provide evidence that internal translation initiating at an out-of-frame AUG accounts for the appearance of this protein. The reading frame crosses over to the wild-type IPF-1 reading frame at the site of the point deletion just carboxy proximal to the transactivation domain. Thus, the single mutated allele results in the translation of two IPF-1 isoproteins, one of which consists of the NH2-terminal transactivation domain and is sequestered in the cytoplasm and the second of which contains the COOH-terminal DNA-binding domain, but lacks the transactivation domain. Further, the COOH-terminal mutant IPF-1 isoform does not activate transcription and inhibits the transactivation functions of wild-type IPF-1. This circumstance suggests that the mechanism of diabetes in these individuals may be due not only to reduced gene dosage, but also to a dominant negative inhibition of transcription of the insulin gene and other beta cell-specific genes regulated by the mutant IPF-1.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Hedgehog Signaling Is Required for Effective Regeneration of Exocrine Pancreas

Although both endocrine and the exocrine pancreas display a significant capacity for tissue regeneration and renewal, the existence of progenitor cells in the adult pancreas remains uncertain. Using a model of cerulein-mediated injury and repair, we demonstrate that mature exocrine cells, defined by expression of an Elastase1 promoter, actively contribute to regenerating pancreatic epithelium through formation of metaplastic ductal intermediates. Acinar cell regeneration is associated with activation of Hedgehog (Hh) signaling, as assessed by up-regulated expression of multiple pathway components, as well as activation of a Ptch-lacZ reporter allele. Using both pharmacologic and genetic techniques, we also show that the ability of mature exocrine cells to accomplish pancreatic regeneration is impaired by blockade of Hh signaling. Specifically, attenuated regeneration in the absence of an intact Hh pathway is characterized by persistence of metaplastic epithelium expressing markers of pancreatic progenitor cells, suggesting an inhibition of redifferentiation into mature exocrine cells. Given the known role of Hh signaling in exocrine pancreatic cancer, these findings may provide a mechanistic link between injury-induced activation of pancreatic progenitors and subsequent pancreatic neoplasia.