This model determined if pellet type affected the outcome measurements, if PTH affected the outcome measurements, and if there was a significant interaction between these two variables that would suggest that rapamycin is affecting PTH action

This model determined if pellet type affected the outcome measurements, if PTH affected the outcome measurements, and if there was a significant interaction between these two variables that would suggest that rapamycin is affecting PTH action. PTH doses, small effects in a minority of anabolic measures at moderate doses (30 g), but the anabolic effects of high dose PTH (90 g) were consistently and significantly suppressed by rapamycin (~4-36% reduction). Serum levels of Trap5b, a marker of resorption, were significantly enhanced by rapamycin, but these effects were observed whether PTH was absent or present. Our data suggest that intermittent PTH, particularly at lower doses, is effective in building bone mass in the presence of rapamycin. However, the full anabolic effects of higher doses of PTH are significantly suppressed by rapamycin, suggesting that PTH might normally activate additional pathways (including mTOR) for its enhanced high-dose anabolic effects. Clinical doses of intermittent PTH could be an effective treatment for maintaining or increasing bone mass among patients taking rapamycin analogs for unrelated health issues. Keywords:parathyroid hormone, mTOR, bone anabolism, Akt, osteoporosis == Introduction == Bone loss due to osteoporosis is a major health concern with an estimated 10 million people affected by the disease in the United States alone. Currently, the only FDA-approved anabolic therapy for osteoporosis is teriparatide, the 1-34 fragment of human parathyroid hormone (PTH). Intermittent PTH is known to increase bone mass and strength, and to decrease the risk of fractures (Neer et al., 2001;Verhaar and Lems, 2009). Despite its well-documented anabolic effects on bone, little ARP 101 is known regarding how intermittent PTH works to activate cells, though some key signaling components have been identified. An important finding that has emerged recently, regarding the signal transduction pathways used by anabolic PTH treatment, is the requirement of insulin-like growth factor-1 (IGF-I) and its receptor IGF-IR, for the bone-building effects of PTH (Bikle et al., 2002;Wang et al., 2007;Yamaguchi et al., 2005). A major downstream target of activated IGF-IR is the phosphatidylinositol-3-kinase (PI3k) pathway, which results in the activation of Akt and subsequently, themammaliantargetofrapamycin (mTOR), a key enzyme in protein synthesis and cell proliferation (Hay and Sonenberg, 2004). Activated mTOR phosphorylates p70S6-kinase 1 (p70S6k1) and 4E binding protein 1 (4E-BP1), leading to translation of proteins involved in cell proliferation and growth (Mamane et al., 2006;Manning and Cantley, 2007). Because PTH effects in bone NOTCH2 involve increased protein synthetic activity (bone matrix production) and enhanced cell (osteoblast) proliferationtwo functions mediated by mTORand because mTOR is downstream of a known modulator (IGF-IR) of anabolic PTH signaling, we sought to determine whether the anabolic effects of intermittent PTH, which are known to work through IGF-IR, are subsequently channeled through mTOR. To address this issue, we treated mice with daily PTH injections for 6 weeks, in the ARP 101 presence or absence of a compound (rapamycin) that selectively inhibits mTOR, and measured the effects on bone metabolism. We hypothesized that the anabolic actions of intermittent PTH are mediated by the mTOR pathway. We found that low dose PTH (10 g/kg/day) was fully anabolic whether rapamycin (3 mg/kg/day) was present or not. However, trabecular bone mass accrual induced by high-dose PTH (90 g/kg/day) was significantly impaired by rapamycin, suggesting that some of the anabolic effects of high dose PTH are dependent on additional pathways (beyond those activated at lower yet anabolic doses), including mTOR. == Materials and Methods == == Animals and pellet implantation == Eighty two female C57BL/6J mice were purchased from the Jackson Laboratory. The mice were 8 weeks old at arrival. Sixty-four ARP 101 mice were selected for the PTH experiment, and 18 mice were used to verify the efficacy of the rapamycin pellets used to inhibit mTOR signaling (see below). All mice were subcutaneously implanted with either a 45 day sustained-release rapamycin pellet (rapamycin: LC Laboratories, Woburn, MA; pellets: Innovative Research of America, Sarasota, FL) or a placebo pellet (methylcellulose). Pellet implantation was performed under isoflurane anesthesia (2% @1.5 L/min) using a 10 gauge trochar to position the pellet subcutaneously in the right dorsal shoulder/cervical region. Rapamycin pellets were designed to deliver 3 mg/kg/day based on a 20 g mouse (2.7 mg per 45 day pellet). All procedures performed in ARP 101 accordance with guidelines set by the Institutional Animal Care and Use Committee. == Verification of rapamycin efficacy in vivo == In order to test whether the rapamycin pellets delivered a sustained, pharmacologically potent dose of the compound into the general circulation, we performed a standard insulinskeletal muscle test. This is a reliablein vivoassay to assess rapamycin-induced blockade of mTOR function. Insulin normally stimulates the PI3k-Akt-mTOR-p70s6K pathway, but in the presence of rapamycin, which binds and.