NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH

PTH induces phosphorylation of the transcriptional coregulator NACA on serine 99 through Gαs and PKA. This leads to nuclear translocation of NACA and expression of the target gene Lrp6, encoding a coreceptor of the PTH receptor (PTH1R) necessary for full anabolic response to intermittent PTH (iPTH) treatment. We hypothesized that maintaining enough functional PTH1R/LRP6 coreceptor complexes at the plasma membrane through NACA-dependent Lrp6 transcription is important to ensure maximal response to iPTH. To test this model, we generated compound heterozygous mice in which one allele each of Naca and Lrp6 is inactivated in osteoblasts and osteocytes, using a knock-in strain with a Naca99 Ser-to-Ala mutation and an Lrp6 floxed strain (test genotype: Naca99S/A; Lrp6+/fl;OCN-Cre). Four-month-old females were injected with vehicle or 100 μg/kg PTH(1-34) once daily, 5 days a week for 4 weeks. Control mice showed significant increases in vertebral trabecular bone mass and biomechanical properties that were abolished in compound heterozygotes. Lrp6 expression was reduced in compound heterozygotes vs. controls. The iPTH treatment increased Alpl and Col1a1 mRNA levels in the control but not in the test group. These results confirm that NACA and LRP6 form part of a common genetic pathway that is necessary for the full anabolic effect of iPTH.

191 Pulsatile Administration of Leucine Promotes mTOR Signaling and Protein Synthesis in Skeletal Muscle of Continuously Fed Preterm Pigs

Continuous feeding does not elicit an optimal anabolic response but is required for some premature infants. We reported previously that intermittent intravenous pulses of Leu (800 μmol Leu·kg-1·h-1 every 4 h) to continuously fed pigs born at term enhanced skeletal muscle mTOR signaling and protein synthesis (PS). The objective of this study was to determine if the anabolic response of skeletal muscle to intermittent Leu pulses is altered following premature birth. Pigs delivered 10 d preterm by cesarean section were advanced to full oral feeding over four days (195 kcal·kg-1·d-1; 13.5 g protein·kg-1·d-1). Pigs were assigned to 1 of 4 treatments: 1) ALA (continuous feeding; 800 μmol Ala·kg-1·h-1 every 4 h; n = 7); 2) L1× (continuous feeding; 800 μmol Leu·kg-1·h-1 every 4 h; n = 6); 3) L2× (continuous feeding; 1600 μmol Leu·kg-1·h-1 every 4 h; n = 6); and 4) INT (intermittent feeding every 4 h; n = 5). Pigs received a Phe tracer 30 min following the amino acid pulse or intermittent feeding to measure PS in longissimus dorsi muscle. Anabolic signaling downstream of mTOR was determined by immunoblot.ResultsPS was higher in L2× and INT compared to ALA (P < 0.05) but was not different between L2× and INT; PS in L1× was intermediate and not different from other groups. Phosphorylation of 4EBP1 and S6K1 was higher in INT compared to L1× and ALA groups (P < 0.05) but was not different compared to L2×. Phosphorylation of 4EBP1, but not S6K1, was higher in L2× compared to ALA (P < 0.05); phosphorylation of 4EBP1 and S6K1 was not different between L1× and L2×.ConclusionsThese results demonstrate that intravenous Leu enhances mTOR activation and PS in LD muscle of continuously fed preterm pigs. However, the amount required may be higher than in pigs born at term.Source of Research SupportNIH and USDA.

Osteoblast-Specific Wnt Secretion is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in adult bone homeostasis and the anabolic response to mechanical loading is not well documented. We inhibited Wnt ligand secretion in adult (5-mo) mice using a systemic (drug) and a bone-targeted (genetic) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the porcupine inhibitor WNT974 exhibited a decrease in bone formation in non-loaded limbs as well as a 54% decline in the periosteal bone formation response to tibial loading. Similarly, within 1-2 weeks of Wls deletion in osteoblasts (Osx-CreERT2;WlsF/F mice), skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (WlsF/F). These findings establish a requirement for Wnt ligand secretion by osteoblasts for adult bone homeostasis and the anabolic response to mechanical loading.

An Unanticipated Role for Sphingosine Kinase-2 in Bone and in the Anabolic Effect of Parathyroid Hormone

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. SPHK is the rate-limiting enzyme in S1P production and has two isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc -/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2 -/-). SPHK1-Oc -/- had normal bone mass. By contrast, SPHK2 -/- female mice had a 14% lower spinal BMD (p<0.01) and males a 22% lower BMD at the same site (p<0.001). SPHK2 -/- and control mice were subsequently treated either with daily PTH(1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2 -/-mice. The mean femoral BV/TV increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (p <0.01). In contrast, in the SPHK2 -/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs.13.3%, PTH vs. vehicle, p = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2 -/- mice (29.7% vs. 23.1%, PTH vs. vehicle, in controls, p <0.05; 26.9% vs. 19.5% PTH vs. vehicle in SPHK2 -/- mice, p = NS). The spine responded normally in the SPHK2 -/- female mice. Interestingly, suppression of Sost was blunted in the SPHK2 -/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.

The Anabolic Response to Dietary Protein Is Not Limited by the Maximal Stimulation of Protein Synthesis in Healthy Older Adults: A Randomized Crossover Trial

We have recently demonstrated in young adults that an anabolic response with mixed meal protein intake above ~35 g/meal, previously recognized as an “optimal” protein dose, was further stimulated. However, it is unknown if this applies to older adults. We therefore examined anabolic response to a mixed meal containing either 35 g (MOD, moderate amount of protein) or 70 g (HIGH, high amount of protein) in a randomized cross-over metabolic study in older adults (n = 8). Primed continuous infusions of L-[2H5] phenylalanine and L-[2H2]tyrosine were performed to determine whole-body protein kinetics and muscle protein fractional synthesis rate (MPS) in basal fasted and fed states. Whole-body protein kinetics (NB, net protein balance; PS, protein synthesis; PB, protein breakdown) and MPS was expressed as changes from the baseline post-absorptive state. Consistent with our previous findings in young adults, both feedings resulted in a positive NB, with HIGH being more positive than MOD. Furthermore, NB (expressed as g protein∙240 min) increased linearly with an increasing amount of protein intake, expressed relative to lean body mass. The positive NB was achieved due mainly to the suppression of PB in both MOD and to a greater extent HIGH, while PS was only increased in HIGH. Consistent with the whole-body data, MPS was significantly higher in HIGH than MOD. Plasma concentrations of essential amino acids and insulin were greater in HIGH vs. MOD. We conclude that in the context of mixed meals, whole-body anabolic response linearly increases with increasing protein intake primarily through the suppression of PB, and MPS was further stimulated with protein intake above the previously considered “optimal” protein dose in older adults.

Author Correction: Bone Anabolic Response in the Calvaria Following Mild Traumatic Brain Injury is Mediated by the Cannabinoid-1 Receptor

An amendment to this paper has been published and can be accessed via a link at the top of the paper.