40 The Open Endocrinology Journal, 2010, 4, 40-54 Open Access Therapeutic Strategies to Increase Human -Cell Growth and Proliferation by Regulating mTOR and GSK-3/-Catenin Pathways Nidhi Rohatgi1, Maria S. Remedi2, Guim Kwon3, Kirk L. Pappan1, Connie A. Marshall1 and *,1 Michael L. McDaniel 1Department of Pathology and Immunology, 2Department of Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri, USA and 3Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University Edwardsville, Edwardsville, IL, USA Abstract: This perspective delineates approaches to develop therapeutic strategies to stimulate the proliferative potential of adult human -cells in vitro. Previous findings demonstrated that nutrients, through regulation of mTOR signaling, promote regenerative processes including DNA synthesis, cell cycle progression and -cell proliferation in rodent islets but rarely in human islets. Recently, we discovered that regulation of the Wnt/GSK-3/-catenin pathway by directly inhibiting GSK-3 with pharmacologic agents, in combination with nutrient activation of mTOR, was required to increase growth and proliferation in human islets. Studies also revealed that nuclear translocation of -catenin in response to GSK- 3 inhibition regulated these processes and was rapamycin sensitive, indicating a role for mTOR. Human islets displayed a high level of insulin resistance consistent with the inability of exogenous insulin to activate Akt and engage the Wnt pathway by GSK-3 inhibition. This insulin resistance in human islets is not present in rodent islets and may explain the differential requirement in human islets to inhibit GSK-3 to enhance these regenerative processes. Human islets exhibited normal insulin secretion but a loss of insulin content, which was independent of all treatment conditions. The loss of insulin content may be related to insulin resistance, the isolation process or culture conditions. In this perspective, we provide strategies to enhance the proliferative capacity of adult human -cells and highlight important differences between human and rodent islets: the lack of a nutrient response, requirement for direct GSK-3 inhibition, insulin resistance and loss of insulin content that emphasize the physiological significance of conducting studies in human islets. Keywords: Human -cell, mTOR, GSK-3, -catenin, proliferation, Exenatide. Globally, the number of people with diabetes mellitus is on glucose-dependent insulin secretion [4]. These drugs have expected to rise from the current estimate of 190 to 330 also been shown to increase -cell proliferation in rodent million in the next 20 years [1]. This striking increase models. In postnatal human islets their efficacy to stimulate suggests that diabetes is now at epidemic proportions and proliferation of -cells is yet to be ascertained [5]. However, constitutes a significant socioeconomic problem. A marked Movassat et al. have reported the proliferation of human reduction of -cell mass plays an important role in the fetal islets when treated with Exendin-4 [6]. In addition, pathogenesis of both Type 1 (T1DM) and Type 2 (T2DM) studies in both human and rodent islets using GLP-1 or Diabetes. Autoimmune-mediated destruction of pancreatic - analogs have also shown anti-apoptotic effects that are cells results in T1DM with absolute insulin deficiency and necessary to maintain -cell mass [7,8,9]. constitutes 5-10% of cases [2]. T2DM is characterized by CELLULAR MECHANISMS CONTROLLING - insulin resistance, as well as -cell dysfunction with a CELL MASS relative insulin deficiency and constitutes 90-95% of cases [3]. Pancreatic -cell mass is not firmly set but is regulated There are several forms of insulin and synthetic amylin by several mechanisms including self-duplication, -cell available for the treatment of T1DM. Various therapeutic size, neogenesis (new -cells from other cell types), and agents are used to treat hyperglycemia associated with apoptosis [10]. A balance between these mechanisms T2DM that act by increasing insulin secretion, decreasing regulates -cell mass. The contribution made by each production of glucose from the liver or increasing insulin mechanism is variable, and depends on the stage of life, sensitization in peripheral tissues. However, these agents are metabolic demands and animal species. The disruption of known to lose their efficacy over time, resulting in a loss of this balance results in rapid and marked changes in -cell glycemic control. The newly available incretin and mass. Analyses of the regulatory parameters described above dipeptidyl peptidase-4 (DPP-IV) drugs have positive effects have shown that there is continual loss and renewal of - cells throughout life [11,12]. Nutrients, growth factors and secondary messengers *Address correspondence to this author at the Department of Pathology and through multiple pathways regulate -cell growth and Immunology, Washington University in St. Louis, Campus Box 8118, 660 proliferation, and there are significant differences in the S. Euclid Avenue, St. Louis, Missouri 63110, USA; Tel: 314-362-7435; Fax: 314-362-4096; E-mail: [email protected] regulation of these pathways between humans and rodents. 1874-2165/10 2010 Bentham Open Human -Cell Growth and Proliferation The Open Endocrinology Journal, 2010, Volume 4 41 Important pathways include PI-3K, Akt, mTOR, Wnt, al. [26] employing a novel DNA analog-based lineage calcineurin/NFAT [13,14] and G-protein coupled receptor tracking technique reported that the growth and regeneration [5,15]. Growth Hormone (GH), prolactin (PRL) and of adult -cells did not involve specialized progenitors or placental lactogen (PL) are potent growth factors that stem cells from adult mice in vivo. Rather, adult -cells are increase -cell mass through the Jak/Stat pathway [16,17]. In derived by self-replication from a large pool of mature - rodent models, the atrial natriuretic peptide (ANP) [18], cells. HGF [19], PDGF [13], and EGF [20] have also been shown In response to the novel observations highlighting the to contribute to increased -cell mass. Studies in rodents, importance of the proliferative capacity of existing adult - have also demonstrated that chronic infusion of elevated cells to duplicate [24-28], the goal of our research is to glucose results in increased -cell mass, through develop therapeutic strategies targeting growth-related proliferation and/or the neogenesis of -cells [21-23]. In 2+ signaling pathways to increase adult human -cell growth addition, Ca and cAMP are important secondary and proliferation while maintaining insulin biosynthesis and messengers integrating mitogenic pathways in -cells. secretory function. Although there are multiple pathways involved in -cell growth and proliferation, this perspective will focus on two mTOR SIGNALING PATHWAY major growth-related pathways, mammalian target of mTOR, a member of the phosphatidylinositol 3-kinase rapamycin (mTOR) and glycogen synthase kinase-3 (GSK- (PI3K)-related kinase (PIKK) family of protein kinases, is 3)/-catenin, that are regulated by nutrients and growth conserved from yeast to humans [29,30]. Fig. (1) depicts factors. An additional focus will be to identify significant schematically pathways involved in mTOR signaling. mTOR differences that exist between human and rodent islets regulates cell growth and metabolism by functioning as part regarding these two signaling pathways. of two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 SOURCE OF NEW -CELLS DURING ADULT LIFE contains the kinase, target of rapamycin (mTOR, also known Recently, Dor et al. [24] reported the significance of the as FRAP1), raptor (regulatory associated protein of mTOR), proliferative capacity of existing adult -cells as a source of GL (also known as LST8), Deptor and PRAS-40 and new -cells during adult life [25]. This investigation regulates protein synthesis in mammals [31]. The emphasized by genetic lineage tracing that adult pancreatic phosphorylation of eukaryotic initiation factor 4E binding -cells are formed by self duplication rather than by protein 1 (4EBP1) by mTOR results in the release of differentiation of adult stem cells. In similar studies, Teta et eukaryotic initiation factor 4E (eIF-4E) and allows its Fig. (1). General schematic of the mTOR and Wnt signaling pathways. 42 The Open Endocrinology Journal, 2010, Volume 4 Rohatgi et al. participation in the initiation of cap-dependent mRNA phosphorylation of eukaryotic initiation factor, eIF-4E [43]. translation. mTOR-mediated phosphorylation of ribosomal Our initial studies in rat islets established that glucose protein S6 kinase 1 (S6K1, also known as p70s6k) results in metabolism and insulin signaling, in an amino acid the phosphorylation of ribosomal protein S6 (rpS6), that dependent manner, resulted in phosphorylated 4EBP1 and correlates with the translation of mRNAs encoding both S6K1 [44,45]. The phosphorylation of 4EBP1 and S6K1 was ribosomal proteins and translational elongation factors [32]. prevented by rapamycin. Rapamycin also inhibited glucose- mTORC1 signaling is potently and specifically inhibited by stimulated 35S-methionine incorporation into islet protein rapamycin that prevents the phosphorylation of these [44]. Overall these studies demonstrated that the production translational regulators [32,33]. of energy derived from glucose metabolism and insulin signaling, in an amino acid dependent manner, regulates mTORC2 consists of mTOR, Rictor,