Shouhong Xuan

Pancreatic β Cell Dedifferentiation as a Mechanism of Diabetic β Cell Failure

Diabetes is associated with β cell failure. But it remains unclear whether the latter results from reduced β cell number or function. FoxO1 integrates β cell proliferation with adaptive β cell function. We interrogated the contribution of these two processes to β cell dysfunction, using mice lacking FoxO1 in β cells. FoxO1 ablation caused hyperglycemia with reduced β cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of β cell mass was due to β cell dedifferentiation, not death. Dedifferentiated β cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient β cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of β cell failure and suggest that treatment of β cell dysfunction should restore differentiation, rather than promoting β cell replication.

Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, maintains bone mass in adulthood. We now report that IGF-1 released from the bone matrix during bone remodeling stimulates osteoblastic differentiation of recruited mesenchymal stem cells (MSCs) by activation of mammalian target of rapamycin (mTOR), thus maintaining proper bone microarchitecture and mass. Mice with knockout of the IGF-1 receptor (Igf1r) in their pre-osteoblastic cells showed lower bone mass and mineral deposition rates than wild-type mice. Further, MSCs from Igf1rflox/flox mice with Igf1r deleted by a Cre adenovirus in vitro, although recruited to the bone surface after implantation, were unable to differentiate into osteoblasts. We also found that the concentrations of IGF-1 in the bone matrix and marrow of aged rats were lower than in those of young rats and directly correlated with the age-related decrease in bone mass. Likewise, in age-related osteoporosis in humans, we found that bone marrow IGF-1 concentrations were 40% lower in individuals with osteoporosis than in individuals without osteoporosis. Notably, injection of IGF-1 plus IGF binding protein 3 (IGFBP3), but not injection of IGF-1 alone, increased the concentration of IGF-1 in the bone matrix and stimulated new bone formation in aged rats. Together, these results provide mechanistic insight into how IGF-1 maintains adult bone mass, while also providing a further rationale for its therapeutic targeting to treat age-related osteoporosis.

Defective insulin secretion in pancreatic β cells lacking type 1 IGF receptor

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

Insulin-Like Growth Factor (IGF) Signaling through Type 1 IGF Receptor Plays an Important Role in Remyelination

We examined the role of IGF signaling in the remyelination process by disrupting the gene encoding the type 1 IGF receptor (IGF1R) specifically in the mouse brain by Cre-mediated recombination and then exposing these mutants and normal siblings to cuprizone. This neurotoxicant induces a demyelinating lesion in the corpus callosum that is reversible on termination of the insult. Acute demyelination and oligodendrocyte depletion were the same in mutants and controls, but the mutants did not remyelinate adequately. We observed that oligodendrocyte progenitors did not accumulate, proliferate, or survive within the mutant mice, compared with wild type, indicating that signaling through the IGF1R plays a critical role in remyelination via effects on oligodendrocyte progenitors.

Conditional mutagenesis in mice with heat shock promoter-driven cre transgenes

Abstract. To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntingtons disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a ``deleter strain, since the majority of progeny (∼70–85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as ``mosaicking strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (∼30–60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.

Generation of functional insulin-producing cells in the gut by Foxo1 ablation

Restoration of regulated insulin secretion is the ultimate goal of therapy for type 1 diabetes. Here, we show that, unexpectedly, somatic ablation of Foxo1 in Neurog3+ enteroendocrine progenitor cells gives rise to gut insulin-positive (Ins+) cells that express markers of mature β cells and secrete bioactive insulin as well as C-peptide in response to glucose and sulfonylureas. Lineage tracing experiments showed that gut Ins+ cells arise cell autonomously from Foxo1-deficient cells. Inducible Foxo1 ablation in adult mice also resulted in the generation of gut Ins+ cells. Following ablation by the β-cell toxin streptozotocin, gut Ins+ cells regenerate and produce insulin, reversing hyperglycemia in mice. The data indicate that Neurog3+ enteroendocrine progenitors require active Foxo1 to prevent differentiation into Ins+ cells. Foxo1 ablation in gut epithelium may provide an approach to restore insulin production in type 1 diabetes.

Igf1r as a therapeutic target in a mouse model of basal-like breast cancer

Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for targeted anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, histopathologically heterogeneous invasive carcinomas exhibiting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant KrasG12D). Immunophenotyping data and expression profiling analyses showed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no targeted therapy currently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (≈11-fold in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropodophyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was effective against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRASG13D mutation.

GATA4 and GATA6 regulate pancreatic endoderm identity through inhibition of hedgehog signaling

GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4fl/fl; Gata6fl/fl; Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube. Summary: Gata4 and Gata6 regulate mouse foregut endoderm patterning via control of hedgehog signaling. In their absence, the pancreatic anlage is respecified to intestinal and stomach fates.

Genetic Analysis of Type-1 Insulin-like Growth Factor Receptor Signaling through Insulin Receptor Substrate-1 and -2 in Pancreatic β Cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.