Lynda Elghazi

Conditional gene targeting in mouse pancreatic β-cells: Analysis of ectopic Cre transgene expression in the brain

OBJECTIVE Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. CONCLUSIONS Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.

Disruption of Tsc2 in pancreatic β cells induces β cell mass expansion and improved glucose tolerance in a TORC1-dependent manner

Regulation of pancreatic β cell mass and function is a major determinant for the development of diabetes. Growth factors and nutrients are important regulators of β cell mass and function. The signaling pathways by which these growth signals modulate these processes have not been completely elucidated. Tsc2 is an attractive candidate to modulate these processes, because it is a converging point for growth factor and nutrient signals. In these experiments, we generated mice with conditional deletion of Tsc2 in β cells (βTsc2−/−). These mice exhibited decreased glucose levels and hyperinsulinemia in the fasting and fed state. Improved glucose tolerance in these mice was observed as early as 4 weeks of age and was still present in 52-week-old mice. Deletion of Tsc2 in β cells induced expansion of β cell mass by increased proliferation and cell size. Rapamycin treatment reversed the metabolic changes in βTsc2−/− mice by induction of insulin resistance and reduction of β cell mass. The reduction of β cell mass in βTsc2−/− mice by inhibition of the mTOR/Raptor (TORC1) complex with rapamycin treatment suggests that TORC1 mediates proliferative and growth signals induced by deletion of Tsc2 in β cells. These studies uncover a critical role for the Tsc2/mTOR pathway in regulation of β cell mass and carbohydrate metabolism in vivo.

Neurofibromatosis-1 Regulates Neuronal and Glial Cell Differentiation from Neuroglial Progenitors In Vivo by Both cAMP- and Ras-Dependent Mechanisms

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Role for FGFR2IIIb-mediated signals in controlling pancreatic endocrine progenitor cell proliferation

Pancreatic development is a classic example of epithelium–mesenchyme interaction. During embryonic life, signals from the mesenchyme control the proliferation of precursor cells within the pancreatic epithelium and their differentiation into endocrine or acinar cells. It has been shown that signals from the mesenchyme activate epithelial cell proliferation but repress development of the pancreatic epithelium into endocrine cells. Here, experiments with specific inhibitors established that mesenchymal effects on epithelial cell development depended on the mitogen-activated protein kinase pathway. Then we demonstrated that these effects of the mesenchyme were mimicked by fibroblast growth factor 7 (FGF7), a specific ligand of FGFR2IIIb, which is a tyrosine kinase receptor of the FGF-receptor family. When pancreatic epithelium expressing FGFR2IIIb was grown with FGF7, epithelial cell growth occurred in a concentration-dependent manner, whereas endocrine tissue development was repressed. The epithelial cells that proliferated in response to FGF7 were endocrine pancreatic precursor cells, as shown by their differentiation en masse into endocrine cells on FGF7 removal. Thus, efficient propagation of pancreatic progenitor cells can be achieved in vitro by exposure to FGF7, which does not affect their ability to differentiate en masse into endocrine cells on FGF7 removal.

mTORC1 Activation Regulates β-Cell Mass and Proliferation by Modulation of Cyclin D2 Synthesis and Stability

Growth factors, insulin signaling, and nutrients are important regulators of β-cell mass and function. The events linking these signals to the regulation of β-cell mass are not completely understood. The mTOR pathway integrates signals from growth factors and nutrients. Here, we evaluated the role of the mTOR/raptor (mTORC1) signaling in proliferative conditions induced by controlled activation of Akt signaling. These experiments show that the mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclin D2, D3, and Cdk4 activity. The regulation of cell cycle progression by mTORC1 signaling resulted from modulation of the synthesis and stability of cyclin D2, a critical regulator of β-cell cycle, proliferation, and mass. These studies provide novel insights into the regulation of cell cycle by the mTORC1, provide a mechanism for the antiproliferative effects of rapamycin, and imply that the use of rapamycin could negatively impact the success of islet transplantation and the adaptation of β-cells to insulin resistance.

Cyclooxygenase-2 Inhibition Sensitizes Human Colon Carcinoma Cells to TRAIL-Induced Apoptosis through Clustering of DR5 and Concentrating Death-Inducing Signaling Complex Components into Ceramide-Enriched Caveolae

Cyclooxygenase-2 (COX-2) is up-regulated in human colon carcinomas, and its inhibition is associated with a reduction in tumorigenesis and a promotion of apoptosis. However, the mechanisms responsible for the antitumor effects of COX-2 inhibitors and how COX-2 modulates apoptotic signaling have not been clearly defined. We have shown that COX-2 inhibition sensitizes human colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inducing clustering of the TRAIL receptor DR5 at the cell surface and the redistribution of the death-inducing signaling complex components (DR5, FADD, and procaspase-8) into cholesterol-rich and ceramide-rich domains known as caveolae. This process requires the accumulation of arachidonic acid and sequential activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet. The current study highlights a novel mechanism to circumvent colorectal carcinoma cell resistance to TRAIL-mediated apoptosis using COX-2 inhibitors to manipulate the lipid metabolism within the plasma membrane.

Akt and PTEN: β-cell mass and pancreas plasticity

The capacity of pancreatic beta-cells to adapt to insulin resistance is crucial for glucose homeostasis and is a factor in the development of type 2 diabetes. The insulin receptor substrate (insulin receptor 2/phosphoinositide 3-kinase [PI3K]) pathway plays a crucial part in regulating beta-cell mass and function. The serine-threonine kinase Akt, also known as protein kinase B, is one of the major downstream targets of the PI3K pathway and is negatively regulated by phosphatase and tensin homologue deleted on chromosome 10. This Akt signaling pathway has recently been implicated in cell-cycle progression and survival of pancreatic beta-cells. Understanding the mechanisms that link Akt to modulation of beta-cell mass, function and plasticity will positively affect treatment of human diabetes.

Decreased IRS Signaling Impairs β-Cell Cycle Progression and Survival in Transgenic Mice Overexpressing S6K in β-Cells

OBJECTIVE The purpose of this study was to evaluate the role of the S6K arm of mammalian target of rapamycin complex 1 (mTORC1) signaling in regulation of β-cell mass and function. Additionally, we aimed to delineate the importance of in vivo S6K activation in the regulation of insulin signaling and the extent to which alteration of insulin receptor substrate (IRS) signaling modulates β-cell mass and function. RESEARCH DESIGN AND METHODS The current experiments describe the phenotype of transgenic mice overexpressing a constitutively active form of S6K under the control of the rat insulin promoter. RESULTS Activation of S6K signaling in these mice improved insulin secretion in the absence of changes in β-cell mass. The lack of β-cell mass expansion resulted from decreased G1-S progression and increased apoptosis. This phenotype was associated with increased p16 and p27 and decreased Cdk2 levels. The changes in cell cycle were accompanied by diminished survival signals because of impaired IRS/Akt signaling. CONCLUSIONS This work defines the importance of S6K in regulation of β-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces β-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in β-cell failure and diabetes in conditions of nutrient overload.

Regulation of β‐cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate‐2/phosphoinositide 3‐kinase (PI3K) pathway plays a critical role in the regulation of β‐cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of β cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of β‐cell mass and function by Akt.

Regulation of Pancreas Plasticity and Malignant Transformation by Akt Signaling

BACKGROUND & AIMS Extensive evidence suggests that Akt signaling plays an important role in beta-cell mass and function, although its function in the regulation of the different pancreatic fates has not been adequately investigated. The goal of these studies was to assess the role of Akt signaling in the pancreatic differentiation programs. METHODS For these experiments, we have generated a double reporter mouse model that provides activation of Akt signaling in a cell type-specific manner. This mouse model conditionally overexpresses a constitutively active form of Akt upon Cre-mediated recombination. Activation of Akt signaling in pancreatic progenitors and acinar and beta-cells was achieved by crossing this animal model to specific Cre-lines. RESULTS We showed that overexpression of a constitutively active Akt in pancreatic and duodenal homeobox 1 (Pdx1) progenitors induced expansion of ductal structures expressing progenitor markers. This expansion resulted in part from increased proliferation of the ductal epithelium. Lineage-tracing experiments in mice with activation of Akt signaling in mature acinar and beta-cells suggested that acinar-to-ductal and beta-cell-to-acinar/ductal transdifferentiation also contributed to the expansion of the ductal compartment. In addition to the changes in cell plasticity, these studies demonstrated that chronic activation of Akt signaling in Pdx1 progenitors induced the development of premalignant lesions and malignant transformation in old mice. CONCLUSIONS The current work unravels some of the molecular mechanisms of cellular plasticity and reprogramming, and demonstrates for the first time that activation of Akt signaling regulates the fate of differentiated pancreatic cells in vivo.