Ryan C. Riddle

Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice

Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism.

From streaming-potentials to shear stress: 25 years of bone cell mechanotransduction.

Mechanical loads are vital regulators of skeletal mass and architecture as evidenced by the increase in bone formation following the addition of exogenous loads and loss of bone mass following their removal. While our understanding of the molecular mechanisms by which bone cells perceive changes in their mechanical environment has increased rapidly in recent years, much remains to be learned. Here, we outline the effects of interstitial fluid flow, a potent biophysical signal induced by the deformation of skeletal tissue in response to applied loads, on bone cell behavior. We focus on the molecular mechanisms by which bone cells are hypothesized to perceive interstitial fluid flow, the cell signaling cascades activated by fluid flow, and the use of this signal in tissue engineering protocols.

Role of hypoxia-inducible factor-1α in angiogenic―osteogenic coupling

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate mesenchymal cell specification to promote bone formation. Recent studies suggest that the hypoxia-inducible factors (HIFs) are required for the initiation of the angiogenic–osteogenic cascade. Genetic manipulation of individual components of the HIF/vascular endothelial growth factor (VEGF) pathway in mice has provided clues to how coupling is achieved. In this article, we review the current understanding of the cellular and molecular mechanisms responsible for angiogenic–osteogenic coupling. We also briefly discuss the therapeutic manipulation of HIF and VEGF in skeletal repair. Such discoveries suggest promising approaches for the development of novel therapies to improve bone accretion and repair.

ATP Release Mediates Fluid Flow–Induced Proliferation of Human Bone Marrow Stromal Cells

Oscillatory fluid flow induced the vesicular release of ATP from human BMSCs that directly contributes to the induction of BMSC proliferation. Degrading extracellular nucleotides prevents fluid flow–induced increases in intracellular calcium concentration, the activation of calcineurin, and the nuclear translocation of NFAT.

ptena and ptenb Genes Play Distinct Roles in Zebrafish Embryogenesis

PTEN is a tumor suppressor gene associated with multiple tumor types. PTEN function is essential for early embryonic development and is involved in the regulation of cell size, number, and survival. By dephosphorylating PIP3, PTEN normally acts to inhibit the PI3‐Kinase/AKT pathway. Here we have identified two zebrafish orthologs, ptena and ptenb, of the single mammalian PTEN gene and analyzed the role of these genes in zebrafish development. Ptena transcripts were expressed throughout the embryo at early somitogenesis. By 24 hpf, expression was predominant in the central nervous system, axial vasculature, retina, branchial arches, ear, lateral line primordium, and pectoral fin bud. Ptenb was also ubiquitously expressed early in somitogenesis, but transcripts became more restricted to the somites and central nervous system as development progressed. By 48 hpf, ptena and ptenb were expressed predominantly in the central nervous system, branchial arches, pectoral fins, and eye. Antisense morpholinos were used to knock down translation of ptena and ptenb mRNA in zebrafish embryos. Knockdown of either pten gene caused increased levels of phosphorylated Akt in morphant embryos, indicating that Ptena and Ptenb each possess PIP3 lipid phosphatase activity. Ptena morphants had irregularities in notochord shape (73%), vasculogenesis (83%), head shape (72%), and inner ear development (59%). The most noticeable defects in ptenb morphants were upward hooked tails (73%), domed heads (83%), and reduced yolk extensions (90%). These results indicate that ptena and ptenb encode functional enzymes and that each pten gene plays a distinct role during zebrafish embryogenesis. Developmental Dynamics 234:911–921, 2005.

Chemotransport contributes to the effect of oscillatory fluid flow on human bone marrow stromal cell proliferation

Mechanical loads produce a diverse set of biophysical signals that may regulate bone cell activity, but accumulating evidence suggests that interstitial fluid flow is the primary signal that bone cells perceive. Because we previously demonstrated that oscillatory fluid flow increases human bone marrow stromal cell proliferation, we investigated the contribution of fluid shear stress and chemotransport, two stimuli induced by interstitial fluid flow. Alterations in flow rate at a constant peak shear stress were associated with decreases in oscillatory fluid flow‐induced marrow stromal cell proliferation, while variations in peak fluid shear stress had no significant effect. Modulation of marrow stromal cell proliferation by flow rate may be attributed to changes in the release of ATP and intracellular calcium signaling. We found that if the flow rate is decreased while maintaining a constant peak fluid shear stress, marrow stromal cells release less ATP into the extracellular environment. Moreover, as the flow rate decreased fewer cells respond to fluid flow with an increase in intracellular calcium concentration. These data suggest that chemotransport is a prerequisite for marrow stromal cells to respond to interstitial fluid flow.

In vivo radiometric analysis of glucose uptake and distribution in mouse bone

Bone formation and remodeling occurs throughout life and requires the sustained activity of osteoblasts and osteoclasts, particularly during periods of rapid bone growth. Despite increasing evidence linking bone cell activity to global energy homeostasis, little is known about the relative energy requirements or substrate utilization of bone cells. In these studies, we measured the uptake and distribution of glucose in the skeleton in vivo using positron-emitting 18F-fluorodeoxyglucose ([18F]-FDG) and non-invasive, high-resolution positron emission tomography/computed tomography (PET/CT) imaging and ex vivo autoradiography. Assessment of [18F]-FDG uptake demonstrated that relative to other tissues bone accumulated a significant fraction of the total dose of the glucose analog. Skeletal accumulation was greatest in young mice undergoing the rapid bone formation that characterizes early development. PET/CT imaging revealed that [18F]-FDG uptake was greatest in the epiphyseal and metaphyseal regions of long bones, which accords with the increased osteoblast numbers and activity at this skeletal site. Insulin administration significantly increased skeletal accumulation of [18F]-FDG, while uptake was reduced in mice lacking the insulin receptor specifically in osteoblasts or fed a high-fat diet. Our results indicated that the skeleton is a site of significant glucose uptake and that its consumption by bone cells is subject to regulation by insulin and disturbances in whole-body metabolism.

Hypoxia-inducible Factor-1α Protein Negatively Regulates Load-induced Bone Formation

Background: The mechanisms by which bone responds to changes in its loading environment are poorly understood. Results: Mechanical signals induce Hif-1α expression, and mice lacking Hif-1α in bone are more responsive to loading. Conclusion: Hif-1α is a novel regulator of skeletal mechanotransduction that impinges on Wnt signaling. Significance: Understanding skeletal mechanotransduction may lead to the development of therapies designed to enhance bone formation. Mechanical loads induce profound anabolic effects in the skeleton, but the molecular mechanisms that transduce such signals are still poorly understood. In this study, we demonstrate that the hypoxia-inducible factor-1α (Hif-1α) is acutely up-regulated in response to exogenous mechanical stimuli secondary to prostanoid signaling and Akt/mTOR (mammalian target of rapamycin) activation. In this context, Hif-1α associates with β-catenin to inhibit Wnt target genes associated with bone anabolic activity. Mice lacking Hif-1α in osteoblasts and osteocytes form more bone when subjected to tibia loading as a result of increased osteoblast activity. Taken together, these studies indicate that Hif-1α serves as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation by altering the sensitivity of osteoblasts and osteocytes to mechanical signals.

Insulin, osteoblasts, and energy metabolism: Why bone counts calories

Recent studies have demonstrated that insulin stimulates bone cells to produce and activate osteocalcin, an endocrine hormone that increases the efficiency of glucose metabolism through its actions on the pancreas and other peripheral tissues. In this issue of the JCI, Wei and colleagues directly explore the contribution of insulin signaling in osteoblasts to the disturbances in whole-body glucose metabolism associated with a high-fat diet. In mice fed a high-fat diet, increased uptake of saturated fatty acids by the osteoblast accelerates the ubiquitination and degradation of the insulin receptor. In this setting, impairments in osteoblast insulin signaling reduce serum levels of undercarboxylated osteocalcin, which in turn exacerbate insulin resistance in muscle and white adipose tissue. These findings underscore the importance of insulin-responsive skeletal cells as components of a newly appreciated endocrine network critical for regulating global energy homeostasis.

Bone Cell Bioenergetics and Skeletal Energy Homeostasis

The rising incidence of metabolic diseases worldwide has prompted renewed interest in the study of intermediary metabolism and cellular bioenergetics. The application of modern biochemical methods for quantitating fuel substrate metabolism with advanced mouse genetic approaches has greatly increased understanding of the mechanisms that integrate energy metabolism in the whole organism. Examination of the intermediary metabolism of skeletal cells has been sparked by a series of unanticipated observations in genetically modified mice that suggest the existence of novel endocrine pathways through which bone cells communicate their energy status to other centers of metabolic control. The recognition of this expanded role of the skeleton has in turn led to new lines of inquiry directed at defining the fuel requirements and bioenergetic properties of bone cells. This article provides a comprehensive review of historical and contemporary studies on the metabolic properties of bone cells and the mechanisms that control energy substrate utilization and bioenergetics. Special attention is devoted to identifying gaps in our current understanding of this new area of skeletal biology that will require additional research to better define the physiological significance of skeletal cell bioenergetics in human health and disease.