mTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice.

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

mTORC1 and mTORC2 expression in inner retinal neurons and glial cells.

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

The Absence of Indoleamine 2,3-Dioxygenase Inhibits Retinal Capillary Degeneration in Diabetic Mice.

Purpose: Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods: IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results: The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions: The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR.

Role of nutrients and mTOR signaling in the regulation of pancreatic progenitors development.

OBJECTIVE: Poor fetal nutrition increases the risk of type 2 diabetes in the offspring at least in part by reduced embryonic ß-cell growth and impaired function. However, it is not entirely clear how fetal nutrients and growth factors impact ß-cells during development to alter glucose homeostasis and metabolism later in life. The current experiments aimed to test the impact of fetal nutrients and growth factors on endocrine development and how these signals acting on mTOR signaling regulate ß-cell mass and glucose homeostasis. METHOD: Pancreatic rudiments in culture were used to study the role of glucose, growth factors, and amino acids on ß-cell development. The number and proliferation of pancreatic and endocrine progenitor were assessed in the presence or absence of rapamycin. The impact of mTOR signaling in vivo on pancreas development and glucose homeostasis was assessed in models deficient for mTOR or Raptor in Pdx1 expressing pancreatic progenitors. RESULTS: We found that amino acid concentrations, and leucine in particular, enhance the number of pancreatic and endocrine progenitors and are essential for growth factor induced proliferation. Rapamycin, an mTORC1 complex inhibitor, reduced the number and proliferation of pancreatic and endocrine progenitors. Mice lacking mTOR in pancreatic progenitors exhibited hyperglycemia in neonates, hypoinsulinemia and pancreatic agenesis/hypoplasia with pancreas rudiments containing ductal structures lacking differentiated acinar and endocrine cells. In addition, loss of mTORC1 by deletion of raptor in pancreatic progenitors reduced pancreas size with reduced number of ß-cells. CONCLUSION: Together, these results suggest that amino acids concentrations and in particular leucine modulates growth responses of pancreatic and endocrine progenitors and that mTOR signaling is critical for these responses. Inactivation of mTOR and raptor in pancreatic progenitors suggested that alterations in some of the components of this pathway during development could be a cause of pancreatic agenesis/hypoplasia and hyperglycemia.

Overexpression of Kinase-Dead mTOR Impairs Glucose Homeostasis by Regulating Insulin Secretion and Not ß-Cell Mass.

Regulation of glucose homeostasis by insulin depends on ß-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in ß-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in ß-cells. We examined glucose homeostasis and ß-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in ß-cell function without alterations in ß-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their ß-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and ß-cell insulin secretion defect but showed no changes in ß-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to ß-cell growth but is involved in regulating ß-cell function in normal and diabetogenic conditions.

Noninvasive in vivo imaging of embryonic ß-cell development in the anterior chamber of the eye.

The fetal environment plays a decisive role in modifying the risk for developing diabetes later in life. Developing novel methodology for noninvasive imaging of ß-cell development in vivo under the controlled physiological conditions of the host can serve to understand how this environment affects ß-cell growth and differentiation. A number of culture models have been designed for pancreatic rudiment but none match the complexity of the in utero or even normal physiological environment. Speier et al. recently developed a platform of noninvasive in vivo imaging of pancreatic islets using the anterior chamber of the eye where islets get vascularized, grow and respond to physiological changes. The same methodology was adapted for the study of pancreatic development. E13.0, still undifferentiated rudiments with fluorescent lineage tracing were implanted in the AC of the eye, allowing the longitudinal study of their growth and differentiation. Within 48 h the anlages get vascularized and grow but their mesenchyme displays a selective growth advantage. The resulting imbalance leads to alteration in the differentiation pattern of the progenitors. Reducing the mesenchyme to its bare minimum before implantation allows the restoration of a proper balance and a development that mimics the normal pancreatic development. These groundbreaking observations demonstrate that the anterior chamber of the eye provides a good system for noninvasive in vivo fluorescence imaging of the developing pancreas under the physiology of the host and can have important implications for designing strategies to prevent or reverse the deleterious effects of hyperglycemia on altering ß-cell function later in life.

S6K1 controls pancreatic ß cell size independently of intrauterine growth restriction.

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of ß cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased ß cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic ß cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore ß cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic ß cell lesion. Consistent with this hypothesis, reexpression of S6K1 in ß cells of S6K1-/- mice restored embryonic ß cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic ß cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced ß cell growth and eventual development of T2DM later in life.

Exposure of mouse embryonic pancreas to metformin enhances the number of pancreatic progenitors.

AIMS/HYPOTHESIS: Developing beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for treating diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signalling. This study examined the effect of metformin on developing pancreas and beta cells. METHODS: Pancreatic rudiments from CD-1 mice at embryonic day 13.0 (E13.0) were cultured with metformin, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR, an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant C57BL/6 mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreases were then analysed for their morphometry. RESULTS: In vitro metformin treatment led to an increase in the proliferation and number of pancreatic duodenal homeobox 1-positive (PDX1(+)) progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, metformin administration to pregnant dams induced an increase in both PDX1(+) and neurogenin 3-positive progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates. CONCLUSIONS/INTERPRETATION: These results indicate that exposure to metformin during gestation modulates the early steps of beta cell development (prior to E14.0) towards an increase in the number of pancreatic and endocrine progenitors. These changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.

Long-lived crowded-litter mice exhibit lasting effects on insulin sensitivity and energy homeostasis.

The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first 3 wk of life [by increasing the number of pups in the crowded-litter (CL) model] leads to extension of mean and maximal lifespan in genetically normal mice. In this study, we aimed to characterize the impact of early-life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study, we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to eight pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 mo of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 mo, CL female mice also have smaller adipocytes compared with controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 mo old CL male mice, and females become more insulin sensitive later in life. Furthermore, ß-cell mass was significantly reduced in the CL male mice and was associated with reduction in ß-cell proliferation rate in these mice. Together, these data show that early-life nutrient intervention has a significant lifelong effect on metabolic characteristics that may contribute to the increased lifespan of CL mice.

Importance of ß-Catenin in glucose and energy homeostasis.

In settings of increased insulin demand, failure to expand pancreatic ß-cells mass leads to diabetes. Genome-wide scans of diabetic populations have uncovered several genes associated with susceptibility to type 2 diabetes and a number of them are part of the Wnt signaling. ß-Catenin, a Wnt downstream effector participates in pancreatic development, however, little is known about its action in mature ß-cells. Deletion of ß-Catenin in Pdx1 pancreatic progenitors leads to a decreased ß-cell mass and impaired glucose tolerance. Surprisingly, loss of ß-catenin made these mice resistant to high fat diet because of their increased energy expenditure and insulin sensitivity due to hyperactivity. The complexity of this phenotype was also explained in part by ectopic expression of Cre recombinase in the hypothalamus. Our data implicates ß-Catenin in the regulation of metabolism and energy homeostasis and suggest that Wnt signaling modulates the susceptibility to diabetes by acting on different tissues.

mTORC1 signaling and regulation of pancreatic ß-cell mass.

The capacity of ß cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of ß cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for ß-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of ß-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in ß cells. This review will discuss recent advances in the understanding of how this pathway regulates ß-cell mass and present data on the role of TSC1 in modulation of ß-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic ß cells results in improved glucose tolerance, hyperinsulinemia and expansion of ß-cell mass that persists with aging.

Conditional gene targeting in mouse pancreatic ß-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 ß-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate ß-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 ß-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in ß-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 ß-cells.

Decreased IRS signaling impairs beta-cell cycle progression and survival in transgenic mice overexpressing S6K in beta-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 ß-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 ß-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 ß-cell mass. The lack of ß-cell mass expansion resulted from decreased G(1)-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 ß-cell cycle, cell size, function, and survival. These experiments also demonstrate that in vivo downregulation of IRS signaling by TORC1/S6K induces ß-cell insulin resistance, and that this mechanism could explain some of the abnormalities that ultimately result in ß-cell failure and diabetes in conditions of nutrient overload.

Islet beta-cell-specific MafA transcription requires the 5'-flanking conserved region 3 control domain.

MafA is a key transcriptional activator of islet beta cells, and its exclusive expression within beta cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs -8118 to -7750 relative to the transcription start site), one of six conserved 5' cis domains of the MafA promoter, is capable of directing beta-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs -10428 to +230), and R1-6 lacking R3 (R1-6(DeltaR3)) were generated. Only the R1-6 transgene was active in MafA(+) insulin(+) cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6(DeltaR3) line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6(DeltaR3) lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the beta-cell-specific expression of MafA.

Akt and PTEN: beta-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.

mTORC1 activation regulates beta-cell mass and proliferation by modulation of cyclin D2 synthesis and stability.

Growth factors, insulin signaling, and nutrients are important regulators of beta-cell mass and function. The events linking these signals to the regulation of beta-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 beta-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 beta-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 beta-cells to insulin resistance.

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.

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

Regulation of pancreatic beta cell mass and function is a major determinant for the development of diabetes. Growth factors and nutrients are important regulators of beta 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 beta cells (betaTsc2(-/-)). 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 beta cells induced expansion of beta cell mass by increased proliferation and cell size. Rapamycin treatment reversed the metabolic changes in betaTsc2(-/-) mice by induction of insulin resistance and reduction of beta cell mass. The reduction of beta cell mass in betaTsc2(-/-) 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 beta cells. These studies uncover a critical role for the Tsc2/mTOR pathway in regulation of beta cell mass and carbohydrate metabolism in vivo.

Generation of a reporter mouse line expressing Akt and EGFP upon Cre-mediated recombination.

The serine-threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre-mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage-tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues.