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Objective: Low birth weight is associated with diabetes in adult life. Accelerated or "catch-up" postnatal growth in response to small birth size is thought to presage disease years later. Whether adult disease is caused by intrauterine beta-cell-specific programming or by altered metabolism associated with catch-up growth is unknown.Research Design and Methods: We generated a new model of intrauterine growth restriction due to fatty acid synthase (FAS) haploinsufficiency (FAS deletion [FASDEL]). Developmental programming of diabetes in these mice was assessed from in utero to 1 year of age.Results: FASDEL mice did not manifest catch-up growth or insulin resistance. beta-Cell mass and insulin secretion were strikingly increased in young FASDEL mice, but beta-cell failure and diabetes occurred with age. FASDEL beta-cells had altered proliferative and apoptotic responses to the common stress of a high-fat diet. This sequence appeared to be developmentally entrained because beta-cell mass was increased in utero in FASDEL mice and in another model of intrauterine growth restriction caused by ectopic expression of uncoupling protein-1. Increasing intrauterine growth in FASDEL mice by supplementing caloric intake of pregnant dams normalized beta-cell mass in utero.Conclusions: Decreased intrauterine body size, independent of postnatal growth and insulin resistance, appears to regulate beta-cell mass, suggesting that developing body size might represent a physiological signal that is integrated through the pancreatic beta-cell to establish a template for hyperfunction in early life and beta-cell failure with age. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism by which GLP-1 agonists enhance β-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation. Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of mTOR, the effects of intra- and extracellular Ca[sup2+] were examined. Glyburide, an inhibitor of ATP-sensitive K[sup+] channels (K[subATP] channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca[sup2+], and diazoxide, an activator of K[subATP] channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca[sup+] stores by cAMP. BAPTA, a chelator of intracellular Ca[sup+] resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca[sup2+] concentrations. Selective blockade of glucose-stimulated Ca[sup2+] influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca[sup2+] stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP participates in the metabolic activation of mTOR by mobilizing intracellular Ca[sup2+] stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate K[subATP] channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation. Diabetes 53 (Suppl. 3):S225-S232, 2004 [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) is a serine and threonine protein kinase that regulates numerous cellular functions, in particular, the initiation of protein translation. mTOR-mediated phosphorylation of both the translational repressor eukaryotic initiation factor 4E binding protein-1 and p70 S6 kinase are early events that control the translation initiation process. Rapamycin, an inhibitor of mTOR, is a potent immunosuppressant due, in part, to its ability to interfere with T-cell activation at the level of translation, and it has gained a prominent role in preventing the development and progression of rejection in pancreatic islet transplant recipients. The characterization of the insulin signaling cascade that modulates mTOR in insulin-sensitive tissues has been a major focus of investigation. Recently, the ability of nutrients, in particular the branched-chain amino acid leucine, to activate mTOR independent of insulin by a process designated as nutrient signaling has been identified. The beta-cell expresses components of the insulin signaling cascade and utilizes the metabolism of nutrients to affect insulin secretion. These combined transduction processes make the beta-cell an unique cell to study metabolic and autocrine regulation of mTOR signaling. Our studies have described the ability of insulin and IGFs in concert with the nutrients leucine, glutamine, and glucose to modulate protein translation through mTOR in beta-cells. These findings suggest that mitochondria-derived factors, ATP in particular, may be responsible for nutrient signaling. The significance of these findings is that the optimization of mitochondrial function is not only important for insulin secretion but may significantly impact the growth and proliferation of beta-cells through these mTOR signaling pathways. [ABSTRACT FROM AUTHOR]

Recent findings have demonstrated that the branched-chain amino acid leucine can activate the translational regulators, phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I) and p70 S6 kinase (p70[s6k), in an insulin-independent and rapamycin-sen-sitive manner through mammalian target of rapamycin (mTOR), although the mechanism for this activation is undefined. It has been previously established that leucine-induced insulin secretion by β-cells involves increased mitochondrial metabolism by oxidative decar-boxylation and allosteric activation of glutamate dehy-drogenase (GDH). We now show that these same intramitochondrial events that generate signals for leucine-induced insulin exocytosis are required to activate the mTOR mitogenic signaling pathway by β-cells. Thus, a minimal model consisting of leucine and glutamine as substrates for oxidative decarboxylation and an activator of GDH, respectively, confirmed the requirement for these two metabolic components and mimicked closely the synergistic interactions achieved by a complete complement of amino acids to activate p70[s6k] in a rapamycin-sensitive manner. Studies using various leucine analogs also confirmed the close association of mitochondrial metabolism and the ability of leucine analogs to activate p70[s6k]. Furthermore, selective inhibitors of mitochondrial function blocked this activation in a reversible manner, which was not associated with a global reduction in ATP levels. These findings indicate that leucine at physiological concentrations stimulates p70[s6k] phosphoryla-tion via the mTOR pathway, in part, by serving both as a mitochondrial fuel and an allosteric activator of GDH. Leucine-mediated activation of protein translation through mTOR may contribute to enhanced β-cell function by stimulating growth-related protein synthesis and proliferation associated with the maintenance of β-cell mass. Diabetes 50:353-360, 2001 [ABSTRACT FROM AUTHOR]

Stimulating islet β-cells to proliferate is a clinical goal that could have a beneficial impact on both T1 and T2DM. The nutrient- and energy-sensing protein kinase, mammalian Target of Rapamycin (mTOR), plays a prominent role in regulating protein and DNA synthesis and entry into the cell cycle from the quiescent state, and we have searched for ways to promote the activation of this centrally positioned kinase in hopes of inducing adult β-cell regeneration. Although glucose, leucine, free fatty acids, and the K[sub ATP] channel inhibitor glyburide showed great potential for activating mTOR in rodent islets, none of these agents translated into consistent activators of human islet mTOR. A recent report linked the inactivation of glycogen synthase kinase-3 (GSK3) to inactivation of the mTOR repressor protein Tuberous Sclerosis 2 in non-islet cells, and we examined the existent of this pathway in islets. When isolated rat islets are cultured for 4d at 8 mM glucose with an inhibitor of GSK-3, lithium chloride (LiCl), we observe a 2-fold increase in DNA synthesis, over that seen with 8 mM glucose alone, that is completely reversed by the mTOR inhibitor rapamycin (25 nM). Treatment of rat islets for 4d with a more specific inhibitor of GSK-3, SB-216763 (5 µM), also yields a significant increase in DNA synthesis that is blocked by rapamycin. Acute (2h) LiCl treatment of rat islets increases the phosphorylation of GSK-3 on serine residues that results in its inactivation. Steady state levels β-catenin, which is normally targeted for rapid proteolytic degradation when GSK-3 is active, also increase in rat islets upon acute LiCl treatment. LiCl in the presence of basal (5 mM) or intermediate (8 mM) glucose concentrations promotes a significant entry of rot islet cells into the cell cycle as indicated by flow cytometry. Most significantly, with cadaver-derived human islets we observe that 4d LiCl treatment consistently promotes a greater than 2-fold stimulation of rapamycin-sensitive DNA synthesis and 2h LiCl exposure increases phospho-Ser-GSK-3 levels. Collectively, these studies identify a novel GSK-3/mTOR signaling pathway that is operative in both rodent and human islets and which has the potential to be modulated to promote β-cell proliferation. ADA-Funded Research [ABSTRACT FROM AUTHOR]

Low birth weight predicts the development of type 2 diabetes in adulthood. The underlying mechanisms, including the fundamental question of how body size impacts metabolism, are not understood. We have identified a new genetic model of intrauterine growth restriction--fatty acid synthase (FAS) haploin-sufficiency--that allows us to test the hypothesis that body size and growth in early life program the pancreatic beta cell to determine risk for type 2 diabetes. Mice with heterozygous deficiency of FAS, the rate limiting enzyme for de novo lipogenesis, had decreased intrauterine body size and increased fetal beta cell mass compared to their wildtype littermates. The increased beta cell mass in utero persisted postnatally and was associated with hypersecretion of insulin and increased serum branched chain amino acid levels at 3-mo of age. However, by 18-mo, these mice with whole body FAS haploinsufficiency developed marked beta cell atrophy and insulin deficiency. Remarkably, complete knockout of FAS targeted to beta cells resulted in no beta cell phenotype, suggesting that intrauterine body size itself programs pancreatic beta cell development. These findings were confirmed in another model of intrauterine growth restriction--transgenic mice overexpressing the mitochondrial uncoupling protein 1 (UCP1) in skeletal muscle--which also showed increased fetal beta cell mass. Growth stimulatory branched chain amino acids appear to mediate the beta cell hypertrophy via phosphorylated ribosomal protein S6, phosphorylated Akt, and PDX1. Each was increased in 3-mo-old FAS haploinsufficient mice but decreased in 18-mo-old mice, mirroring the insulin hypersecretion at 3-mo and insulin deficiency at 18-mo. Taken together, these results implicate intrauterine body size as a novel regulator of beta cell mass, suggesting that a possible body size sensor is integrated through the pancreatic beta cell. [ABSTRACT FROM AUTHOR]

This perspective delineates approaches to develop therapeutic strategies to stimulate the proliferative potential of adult human β-cells in vitro. Previous findings demonstrated that nutrients, through regulation of mTOR signaling, promote regenerative processes including DNA synthesis, cell cycle progression and β-cell proliferation in rodent islets but rarely in human islets. Recently, we discovered that regulation of the Wnt/GSK-3/β-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 β-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 β-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.