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ScienceDirect Incretins and bone: friend or foe? Guillaume Mabilleau1,2 To adapt to its various functions, the bone tissue is remodeled permanently and is under the influence of hormonal, local, mechanical and nervous signals. Among them, a role for gut hormones in controlling bone mass and quality has emerged in the recent years. The aim of this review is to provide the reader with a summary of recent developments in the interaction between incretin hormones and bone physiology. Addresses 1 LUNAM Universite´, GEROM-LHEA, Institut de Biologie en Sante´, Angers, France 2 LUNAM Universite´, SCIAM, Institut de Biologie en Sante´, Angers, France Corresponding author: Mabilleau, Guillaume ([email protected])
Current Opinion in Pharmacology 2015, 22:72–78 This review comes from a themed issue on Musculoskeletal Edited by James Gallagher and Graham Russell
http://dx.doi.org/10.1016/j.coph.2015.03.007 1471-4892/# 2015 Elsevier Ltd. All rights reserved.
Introduction Bone is a tissue with multiple functions: (i) it has a strong biomechanical function by supporting the body weight and protects essential organs from potential mechanical injuries, (ii) it acts as a calcium, phosphate and sodium reservoir, (iii) it is a host tissue for hematopoietic bone marrow and (iv) it is also an endocrine organ that is involved in the regulation of glucose metabolism, energy expenditure, regulation of testosterone production and phosphate homeostasis [1–4]. To adapt to these functions, bone is remodeled permanently by a coupling between osteoclasts, the bone-resorbing cells, and osteoblasts, the bone-forming cells responsible for the synthesis of new structural units. Bone remodeling is traditionally considered to be regulated by hormones (parathyroid hormone, calcitonin, estrogen, among others), autocrine/paracrine signals from the microenvironment (receptor activator of nuclear factor kappa B ligand, tumor necrosis factor-alpha, among others), mechanical loading and the central and sympathetic nervous systems. From the molecular to anatomical levels, bones are built to resist and adapt to mechanical strain according to five Current Opinion in Pharmacology 2015, 22:72–78
different levels of organization (reviewed in review [5]). These levels of organization include the dual composition (organic vs. mineral phase) of the bone matrix, bone texture (lamellar vs. woven bone), bone structure (osteon vs. arch-like trabeculae), microarchitecture and macroarchitecture. As such, any modification of these degrees of organization may compromise bone strength that ultimately if not corrected result in bone fracture. Recently some evidences have emerged that a group of gut hormones may also be involved in the maintenance of bone strength and quality by acting on bone cell physiology. This body of evidence arises from the fact that (i) bone remodeling is reduced after parenteral feeding [6], (ii) that the pattern of bone remodeling is rapidly modified after a meal [7] and (iii) that food fractionation is capable of altering bone resorption and increasing bone mineral density with a greater extent as compared with a matched nutrient load given once a day [8]. The gastrointestinal tract produces and releases a plethora of bioactive peptides that act as neuromodulators/neurotransmitters, hormones or local regulators of cell function. Among them, a class of peptides called incretins have emerged as important modulator of energy metabolism. The term ‘incretin’ was initially proposed by Creutzfeld in 1979 and represents hormones that are secreted from the intestine in response to glucose and stimulate insulin release in a glucosedependent manner [9]. Although several hormones with insulinotropic action are secreted by the gut, glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 are the only two physiological incretins identified so far [10]. Once released to the blood stream, these two hormones are rapidly degraded by an endopeptidase, the dipeptidylpeptidase 4 (DPP-4). However, due to the wide list of DPP-4 substrates, the role of DPP-4 inhibitors in bone physiology is out of the scope of this review and will not be discussed further. The aim of the current review is to provide the reader with a comprehensive overview of the effects of incretin hormones on bone physiology.
Glucose-dependent insulinotropic polypeptide (GIP) GIP is produced by intestinal K-cells, located primarily in proximal regions of the small intestine. GIP gene transcription is increased in response to glucose and lipids and decreased in prolonged fasting periods, suggesting a nutrient-dependent transcriptional control of the GIP gene [11,12]. The human proGIP is a 153-amino acid polypeptide that is encoded by six exons representing a 459-bp open reading frame and whose gene is localized in www.sciencedirect.com
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humans on chromosome 17q [13,14]. The mature 42amino acid bioactive form of GIP (GIP1–42), mainly encoded by exons 3 and 4, is released from its precursor via prohormone convertase 1/3-dependent posttranslational cleavage at flanking single arginine residues [15]. The peptides encoded within the remaining fragments of the proGIP have no known biological functions [16]. The sequence of GIP is extremely conserved between species and more than 90% homology is found among human, porcine, bovine, mouse, and rat [17]. The K-cell is an open type endocrine cell and as such luminal content of the gut is thought to play a major role in GIP secretion. The K-cell is highly polarized with the GIP-containing secretory granules concentrated at the basal pole of the cell, ready to be released from secretory granules through the basolateral membrane [18,19]. In addition, K-cells are found in close association with the capillary network running through the lamina propria allowing GIP to enter into the blood stream. Based on the morphological features, GIP secretion from K-cells is regulated by intraluminal contents, neural stimuli and hormones. To exert its biological actions, GIP binds to its receptor, the GIPr. The human gipr gene comprises 14 exons that span approximately 13.8 kb [20] and is localized on chromosome 19q13.3 [21]. The GIPr belongs to the 7-transmembranespanning G-protein coupled receptor (GPCR) superfamily [22] and is composed of 430 amino acids. The GIPR is expressed in the endocrine pancreas, gastro-intestinal tract, adipose tissue, adrenal cortex, pituitary gland, vascular endothelium and several regions in the CNS [16]. The principal physiological role of GIP is to increase insulin secretion from the pancreatic beta-cells in a glucose-dependent manner. However, extrapancreatic actions of GIP have been observed. GIP acts on lipid metabolism including augmentation of plasma triglyceride clearance, increased lipoprotein lipase activity and promotion of fat storage in adipocytes [23–25]. In animal model of GIP deficiency or chemically-induced GIPr antagonism, interrupting GIP signaling appears to be beneficial in reducing high fat diet induced obesity [24,26–28]. GIP has also been reported to play a role in neural progenitor cell proliferation and behavior [29].
Bone action of the GIP/GIPr pathway The GIPr has been demonstrated to be expressed at the mRNA and protein level in several osteoblastic cell lines including MG-63, TE-85, Saos-2, Ros 17/2.8 and MC3T3 [30–32], primary murine osteoblasts [31] and murine osteoclast precursors [33], suggesting a role for the GIP/GIPr pathway in bone. A polymorphism of the gipr gene caused by substitution of a guanine by a cytosine at rs1800437 induces a substitution of glutamate to glutamine at position 354 in the sixth transmembrane domain of GIPr resulting in decreased receptor activity [34]. Recently, Torekov and colleagues reported that this polymorphism is associated, in a Danish-based perimenopausal www.sciencedirect.com
women cohort, with low mineral bone density at the femoral neck and total hip but not at the lumbar spine [35]. Furthermore, women carrying the lower frequency C allele also presented with higher incidence of nonvertebral fractures [35]. Taken together, these data suggest a role for the GIP/GIPr pathway in controlling bone density and strength. A summary of GIP/GIPr actions on bone is presented in Figure 1. Our understanding of the action of this pathway in bone has been greatly enhanced from studies conducted in GIPr knock-out mice. Two models of GIPr KO have been developed. The first model consists in a deletion of exons 4 and 5 of the gipr gene, which encode a portion, but not the totality, of the extracellular domain of this receptor [36]. The second model consists in the deletion of the first 6 exons, which encode the totality of the extracellular domain and a portion of the first transmembrane domain of the GIPr [37]. Both animal models result into an inactive GIP/GIPr pathway but surprisingly the consequences of this inactivation on trabecular bone are opposed. Indeed, in model 1, GIPr-deficient mice present with augmentation in fat mass and reduction of trabecular bone mass detectable as early as 4 week-old at the femur and 8 week-old at the tibia [38,39]. Histomorphometric analysis revealed a bone formation defect in these animals with decreased mineral apposition rate and bone formation rate and an increase in osteoid maturation
Figure 1
PANCREATIC β-CELL ↑ insulin secretion
OSTEOBLAST ↑ Type 1 collagen expression ↑ Maturation of collagen ↑ Alkaline phosphatase activity ↑ Mineralisation of the matrix ↑ TGF-β secretion
OSTEOCLAST ↓ Osteoclast formation ↓ Osteoclast activity
GIP
ADIPOCYTE Modification of adipokine secretion
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Mechanisms of action of GIP on bone cells. Upon entry of nutrients into the small intestine, GIP is secreted by duodenal K-cells into the blood stream and acts on target cells. When GIP binds the GIPr at the surface of osteoblasts, it induces expression of type 1 collagen and TGF-beta, maturation of collagen matrix, augments alkaline phosphatase activity and mineralization of the bone matrix. When GIP engages its receptor at the surface of osteoclast precursors, it results an augmentation into osteoclast formation and osteoclast resorption. GIP also stimulates insulin secretion from beta-cells of the pancreas that could potentially modify the pattern of osteoblast and osteoclast action. GIP also affects adipokine secretion from adipocytes that could potentially alter the activity of bone cells. Current Opinion in Pharmacology 2015, 22:72–78
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rate [39]. These animals exhibited also a reduction in circulating osteocalcin and an augmented leptin concentration in plasma [38]. In model 2, GIPr-deficient mice presented with decreased body weight and fat mass, increased trabecular bone mass at the tibia at 16 weeks of age and higher trabecular number [40]. This high bone phenotype was even more pronounced at 45 weeks of age suggesting that these animals were protected from ageing-induced bone loss. This phenotype was accompanied by higher values for the mineral apposition rate and the bone formation rate [40]. Plasma level of circulating osteocalcin was unchanged whilst leptin was reduced and adiponectin augmented [40]. To date, no satisfactory explanations have been formulated to explain the two opposed bone phenotype observed in the two GIPr KO models. However, in model 2, despite an apparent higher trabecular bone mass, the strength of the trabecular bone matrix was dramatically reduced and associated with altered maturation of the mineral and collagen component of the bone matrix [40]. To the best of our knowledge, the cortical bone phenotype has only been investigated in model 2. Nevertheless, augmentation of the marrow diameter with a normal outer bone diameter at the femur resulted in decreased cortical thickness and reduced bone strength at the whole bone level [41]. Investigation of tissue material properties by nanoindentation, quantitative backscattered electron imaging and Fourier-transform infrared microspectroscopy revealed alterations of bone strength at the tissue level associated with reduction in the degree of bone mineralization and collagen maturation [41]. Taken together these data suggest a positive role of the GIPr in order to gain optimal bone strength and quality. This hypothesis is further strengthened by the improved tissue material properties and bone strength in a rodent model treated with exogenous GIP [42]. However, as the GIPr is widely distributed, it is not clear whether the above skeletal effects result from a direct action of the bone GIPr or action of extraskeletal GIPr that indirectly contribute to the observed bone phenotype. Nevertheless, few studies have investigated the direct action of GIP on osteoblast and osteoclast cultures. Administration of GIP in osteoblast cultures results in augmentation in osteoblast proliferation, osteoblast-produced TGF-beta, alkaline phosphatase and type 1 collagen expression [43,44]. Furthermore, addition of GIP, at physiological concentration, in osteoblast cultures resulted in higher lysyl oxidase activity and collagen maturity [45]. Administration of exogenous GIP leads to reduction in osteoclast-mediated resorption as evidenced by reduction in circulating CTx levels [46], although conflicting results have been reported regarding a direct action of GIP to mediate this effect [33,39]. Current Opinion in Pharmacology 2015, 22:72–78
Glucagon-like peptide-1 (GLP-1) The proglucagon gene is expressed at high levels in intestinal L-cells [47]. L-cells can be found in the duodenum but occur at higher number in the ileum and colon [48]. The single proglucagon gene encodes a 160-amino acid peptide that undergoes posttranslational processing [49]. In intestinal L-cells, after processing by the prohormone convertase 1/3, the proglucagon gene gives rise to glicentin, corresponding to the first 69-amino acids of the precursor, GLP-1 (amino acids 78–107) composed of 31 amino acids, peptide 2 (amino acids 111–123) and GLP-2 (amino acids 126–158) comprising 33 amino acids [50]. Thus, processing of the proglucagon gene in intestinal cells generates equimolar concentration of GLP-1 and GLP-2. Until now, little is known about the biological actions of glicentin and peptide 2. Two forms of GLP-1 are produced in the intestine, GLP17–36NH2 and GLP-17–37 although the major circulating form is GLP-17–36NH2 [51]. As with K-cells, L-cells are an open type endocrine cells highly polarized with secretory granules at their basolateral pole, ready to release GLP-1and GLP-2 into the blood stream. L-cells are found in close association with the capillary network running through the lamina propria. Based on the morphological features, GLP-1 secretion from L-cells is regulated by intraluminal contents, neural stimuli and hormones. GLP-1 has also been suspected to act via the autonomous nerve system on specific hypothalamic and brainstem nuclei to exert its action [52]. To act, GLP-1 engages its receptor, the GLP-1r expressed on target tissue. The human glp1r gene comprises also 14 exons and is localized on chromosome 6p21. GLP-1r also belongs to the 7-transmembrane-spanning GPCR superfamily like the GIPr. It is composed of 463 amino acids [53] and is expressed in the endocrine pancreas, gastro-intestinal tract, lung, heart, kidney and several regions of the brain [16]. The principal physiological role of GLP-1 is to potentiate glucose-dependent insulin secretion [54]. Extrapancreatic actions of GLP-1 results in reduction of food intake through the CNS, inhibition of gastric emptying, positive actions on the cardiovascular system and a role in energy expenditure [54].
Bone action of the GLP-1/GLP-1r pathway Presence of the GLP-1r in bone cells is controversial. It has been evidenced at the mRNA level in MG-63 and TE-85 osteoblastic cell lines [30]; however, these cells are not very representative of osteoblast cells [55]. On the other hand, GLP-1r was not found in MC3T3-E1 [56] or primary murine osteoblasts or osteoclasts [57]. However, previous studies in other tissues such as liver and skeletal muscle revealed the presence of a GLP-1 receptor different from the known GLP-1r in function and/or structure. Indeed, this second GLP-1 receptor does not www.sciencedirect.com
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activate the cAMP pathway as GLP-1r does but rather the production of inositolphosphoglycan as a second messenger [58,59]. Recently, Nuche-Berenguer et al. revealed the presence of a second GLP-1 receptor in MC3T3-E1 cells different from the known GLP-1r [56]. Indeed, the binding of (125I)GLP-1 to this second receptor was displaced by addition of cold GLP-1 but not exendin-4, a known agonist of the GLP-1r, and resulted in increase in inositolphosphoglycan and activation of PI3 kinase [56]. However, no data is available regarding the presence of this second GLP-1 receptor on osteoclasts. Recently, Kim et al. evidenced the presence of GLP-1r at the mRNA and protein level in the MLO-Y4 osteocyte cell line [60]. However, when a search-for-specificity alignment is performed with the primer-BLAST tool of the National Centre for Biotechnology Information (NCBI), it appears that the primer pair used by Kim et al. was not specific of the glp1r mRNA but might also reveal the presence of timeless, a protein involved in the circadian clock (unpublished data). Furthermore, following a publication of Panjwani et al. in 2013 [61], a growing body of evidence suggests that most of commercially available antibodies used in immunohistochemistry and western blotting in published studies are not specific of the GLP-1r [62,63]. As such it is difficult to ascertain whether the known GLP-1r is expressed in bone cells. Until more convincing studies are performed, scientists may have to consider the possibility that the known GLP-1r may not be expressed in bone cells.
Figure 2
PANCREATIC β-CELLS ↑ insulin secretion
GLP-1
BONE CELLS
L-CELLS ILEUM
C-CELLS THYROID GLAND ↑ Calcitonin secretion
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Mechanisms of action of GLP-1 on bone cells. Upon entry of nutrients into the small intestine, GLP-1 is secreted by ileal L-cells into the blood stream and acts on target cells. The known GLP-1r receptor is not expressed in osteoblast or osteoclast, but on the other hand, GLP-1 might act directly on osteoblast physiology by binding to a second GLP-1 receptor. On the other hand, in rodent, the GLP-1r is expressed in C-cells of the thyroid and is involved in calcitonin secretion that might exert anti-resorptive actions in bone. Furthermore, as GLP-1 is an incretin hormone, it potentiates insulin secretion from pancreatic b-cells and as insulin is considered anabolic for bone, it is plausible that insulin mediates some of the observed bone effects. Osteocalcin produces by osteoblasts is capable of enhancing GLP-1 secretion from ileal L-cells. www.sciencedirect.com
A summary of GLP-1/GLP-1r actions on bone is presented in Figure 2. Here again, our understanding of GLP-1 actions in skeletal physiology arises from GLP1r KO mouse. At 10 weeks of age, GLP-1r KO animals exhibited a mild reduction in trabecular bone volume at the tibia, although not significant, associated with increased number of osteoclasts and eroded surfaces [64]. Unpublished observation from our lab made in the same KO model at 16 weeks of age corroborated these findings. On the other hand, the mineral apposition and bone formation rates appeared unaffected by GLP-1r inactivation [64]. Taken together these results suggested a control of bone resorption (osteoclast differentiation and/or action) by the GLP-1r. However, GLP-1 was unable to directly control osteoclast formation and resorption in osteoclast cultures [64], suggesting that the control of bone resorption was indirect. Indeed, these authors found a reduction in calcitonin gene expression in GLP-1rdeficient animals. Now, we know that in rodents, but not in non-human primate or humans, GLP-1r is expressed in C-cells of the thyroid gland, and responsible for a rise in calcitonin secretion [65]. However, in humans, administration of exogenous GLP-1 does not result into lower CTx levels [7]. The effect of GLP-1r deficiency in cortical bone has been investigated in 16 week-old GLP-1r-deficient mice. These animals presented with reduction in bone strength observed by 3-point bending [57]. In these animals, modification of cortical microstructure was evidenced with reduction in the bone outer diameter whilst the marrow diameter was conserved. These alterations led to a lower cortical thickness [57]. Tissue material properties are also reduced at cortical site in GLP-1r-deficient animals and are associated with reduced collagen maturity with a normal distribution of the degree of mineralization [57]. These data highlighted that a functional GLP1r is not only required for the control of bone resorption but also for the preservation of an optimal bone matrix quality. Administration of GLP-17–36NH2 or exendin-4, a GLP-1r agonist, results in a rapid augmentation of osteocalcin gene expression and a reduction in the balance RANKL/ OPG in the bones of normal, type 2 diabetic or insulinoresistant rats [66,67]. Furthermore, 16 weeks administration of exendin-4 in OVX rats, is capable of improving trabecular bone mass and microarchitecture at the femur and lumbar vertebra, bone strength and revert hyperresorption observed after ovariectomy [68]. Moreover, the administration of exendin-4 into type 2 diabetic animals was also capable of reducing sclerostin expression and improving bone mineral density [60]. Recently, evidences have been provided that undercarboxylated osteocalcin was capable of inducing GLP-1 release from the L-cells and as such this complementary mechanism reinforces the role of osteocalcin in energy expenditure [69,70]. However, as the GLP-1r is not expressed in Current Opinion in Pharmacology 2015, 22:72–78
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bone cells, the major challenge to face in the near future would be to ascertain how the GLP-1/GLP-1r pathway in other tissues may control bone physiology. Now several GLP-1 analogues, enzymatically resistant to DPP-4 degradation, have been approved for the treatment of type 2 diabetes mellitus. Regarding the skeletal alterations observed in animals deficient in GLP-1r and the rapid and favorable effects of GLP-1 or GLP-1r agonist on bone gene expression, one could expect an improvement in bone quality in type 2 diabetic patients. However, in a recent meta-analysis conducted by our group on the incidence of bone fractures in type 2 diabetic patients taking GLP-1 mimetic, we failed to evidence any beneficial effects of GLP-1 mimetic [71].
Conclusions A link between incretin and bone physiology has been highlighted in the past years suggesting that incretin are essential for the maintenance of bone mass but also to gain optimal bone quality and strength. However, the major challenge to face in this field in the future will be to ascertain by which mechanisms incretins exert their control on skeletal physiology.
Conflict of interest statement Nothing declared.
Acknowledgements GM is grateful to members of the GEROM-LHEA lab (LUNAM Universite´, Angers, France) and Diabetes group (Ulster University, Coleraine, UK) for their invaluable help in his researches in this field. This work was supported by grants from the University of Angers and Socie´te´ Franc¸aise de Rhumatologie.
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insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice. Bone 2013, 53:221-230. 41. Mieczkowska A, Irwin N, Flatt PR, Chappard D, Mabilleau G: Glucose-dependent insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength and quality. Bone 2013, 56:337-342. Demonstrates the important role of GIPr in cortical bone quality and strength. 42. Mabilleau G, Mieczkowska A, Irwin N, Simon Y, Audran M, Flatt PR, Chappard D: Beneficial effects of a N-terminally modified GIP agonist on tissue-level bone material properties. Bone 2014, 63:61-68. Effects of GIP mimetic on bone physiology. 43. Zhong Q, Ding KH, Mulloy AL, Bollag RJ, Isales CM: Glucosedependent insulinotropic peptide stimulates proliferation and TGF-beta release from MG-63 cells. Peptides 2003, 24:611-616. 44. Bollag RJ, Zhong Q, Ding KH, Phillips P, Zhong L, Qin F, Cranford J, Mulloy AL, Cameron R, Isales CM: Glucosedependent insulinotropic peptide is an integrative hormone with osteotropic effects. Mol Cell Endocrinol 2001, 177:35-41. 45. Mieczkowska A, Bouvard B, Chappard D, Mabilleau G: Glucosedependent insulinotropic polypeptide (GIP) directly affects collagen fibril diameter and collagen cross-linking in osteoblast cultures. Bone 2015, 74:29-36. 46. Nissen A, Christensen M, Knop FK, Vilsboll T, Holst JJ, Hartmann B: Glucose-dependent insulinotropic polypeptide inhibits bone resorption in humans. J Clin Endocrinol Metab 2014, 99:E2325-E2329. Shows the important role of GIP in the control of bone resorption. 47. Lee YC, Brubaker PL, Drucker DJ: Developmental and tissuespecific regulation of proglucagon gene expression. Endocrinology 1990, 127:2217-2222. 48. Habib AM, Richards P, Cairns LS, Rogers GJ, Bannon CA, Parker HE, Morley TC, Yeo GS, Reimann F, Gribble FM: Overlap of endocrine hormone expression in the mouse intestine revealed by transcriptional profiling and flow cytometry. Endocrinology 2012, 153:3054-3065. 49. Holst JJ: Gut glucagon, enteroglucagon, gut glucagonlike immunoreactivity, glicentin — current status. Gastroenterology 1983, 84:1602-1613. 50. Hartmann B, Johnsen AH, Orskov C, Adelhorst K, Thim L, Holst JJ: Structure, measurement, and secretion of human glucagonlike peptide-2. Peptides 2000, 21:73-80. 51. Orskov C, Rabenhoj L, Wettergren A, Kofod H, Holst JJ: Tissue and plasma concentrations of amidated and glycine-extended glucagon-like peptide I in humans. Diabetes 1994, 43:535-539. 52. Holst JJ, Deacon CF: Glucagon-like peptide-1 mediates the therapeutic actions of DPP-IV inhibitors. Diabetologia 2005, 48:612-615. 53. Thorens B: Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A 1992, 89:8641-8645. 54. McIntosh CH, Widenmaier S, Kim SJ: Pleiotropic actions of the incretin hormones. Vitam Horm 2010, 84:21-79. 55. Clover J, Gowen M: Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone 1994, 15:585-591. 56. Nuche-Berenguer B, Portal-Nunez S, Moreno P, Gonzalez N, Acitores A, Lopez-Herradon A, Esbrit P, Valverde I, VillanuevaPenacarrillo ML: Presence of a functional receptor for GLP-1 in osteoblastic cells, independent of the cAMP-linked GLP-1 receptor. J Cell Physiol 2010, 225:585-592. 57. Mabilleau G, Mieczkowska A, Irwin N, Flatt PR, Chappard D: Optimal bone mechanical and material properties require a functional GLP-1 receptor. J Endocrinol 2013, 219:59-68. Illustrates the role of the GLP-1r pathway in controlling cortical bone quality and strength. Current Opinion in Pharmacology 2015, 22:72–78
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58. Luque MA, Gonzalez N, Marquez L, Acitores A, Redondo A, Morales M, Valverde I, Villanueva-Penacarrillo ML: Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human myocytes. J Endocrinol 2002, 173:465-473.
GLP-1 receptor agonists and the thyroid: C-cell effects in mice are mediated via the GLP-1 receptor and not associated with RET activation. Endocrinology 2012, 153:1538-1547.
59. Villanueva-Penacarrillo ML, Delgado E, Trapote MA, Alcantara A, Clemente F, Luque MA, Perea A, Valverde I: Glucagon-like peptide-1 binding to rat hepatic membranes. J Endocrinol 1995, 146:183-189.
66. Nuche-Berenguer B, Moreno P, Esbrit P, Dapia S, Caeiro JR, Cancelas J, Haro-Mora JJ, Villanueva-Penacarrillo ML: Effect of GLP-1 treatment on bone turnover in normal, type 2 diabetic, and insulin-resistant states. Calcif Tissue Int 2009, 84:453-461.
60. Kim JY, Lee SK, Jo KJ, Song DY, Lim DM, Park KY, Bonewald LF, Kim BJ: Exendin-4 increases bone mineral density in type 2 diabetic OLETF rats potentially through the down-regulation of SOST/sclerostin in osteocytes. Life Sci 2013, 92:533-540. GLP-1 mimetic decreases sclerostin secretion in vivo. 61. Panjwani N, Mulvihill EE, Longuet C, Yusta B, Campbell JE, Brown TJ, Streutker C, Holland D, Cao X, Baggio LL et al.: GLP-1 receptor activation indirectly reduces hepatic lipid accumulation but does not attenuate development of atherosclerosis in diabetic male ApoE(S/S) mice. Endocrinology 2013, 154:127-139. First demonstration that commercially-available antibodies were not specific to the GLP-1r. 62. Pyke C, Heller RS, Kirk RK, Orskov C, Reedtz-Runge S, Kaastrup P, Hvelplund A, Bardram L, Calatayud D, Knudsen LB: GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody. Endocrinology 2014, 155:1280-1290. Shows GLP-1r localization in tissue. 63. Pyke C, Knudsen LB: The glucagon-like peptide-1 receptor — or not? Endocrinology 2013, 154:4-8. 64. Yamada C, Yamada Y, Tsukiyama K, Yamada K, Udagawa N, Takahashi N, Tanaka K, Drucker DJ, Seino Y, Inagaki N: The murine glucagon-like peptide-1 receptor is essential for control of bone resorption. Endocrinology 2008, 149:574-579. 65. Madsen LW, Knauf JA, Gotfredsen C, Pilling A, Sjogren I, Andersen S, Andersen L, de Boer AS, Manova K, Barlas A et al.:
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67. Nuche-Berenguer B, Moreno P, Portal-Nunez S, Dapia S, Esbrit P, Villanueva-Penacarrillo ML: Exendin-4 exerts osteogenic actions in insulin-resistant and type 2 diabetic states. Regul Pept 2010, 159:61-66. 68. Ma X, Meng J, Jia M, Bi L, Zhou Y, Wang Y, Hu J, He G, Luo X: Exendin-4, a glucagon-like peptide-1 receptor agonist, prevents osteopenia by promoting bone formation and suppressing bone resorption in aged ovariectomized rats. J Bone Miner Res 2013, 28:1641-1652. Beneficial effect of GLP-1 mimetic on bone mass in OVX model. 69. Mizokami A, Yasutake Y, Gao J, Matsuda M, Takahashi I, Takeuchi H, Hirata M: Osteocalcin induces release of glucagonlike peptide-1 and thereby stimulates insulin secretion in mice. PLoS One 2013, 8:e57375. 70. Mizokami A, Yasutake Y, Higashi S, Kawakubo-Yasukochi T, Chishaki S, Takahashi I, Takeuchi H, Hirata M: Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion. Bone 2014, 69:68-79. Shows that a peptide produced by osteoblasts, osteocalcin, is involved into GLP-1 secretion in vivo. 71. Mabilleau G, Mieczkowska A, Chappard D: Use of glucagon-like peptide-1 receptor agonists and bone fractures: a metaanalysis of randomized clinical trials. J Diabetes 2014, 6: 260-266. Analyzes the fracture risk in GLP-1Ra takers.
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ScienceDirect Incretins and bone: friend or foe? Guillaume Mabilleau1,2 To adapt to its various functions, the bone tissue is remodeled permanently and is under the influence of hormonal, local, mechanical and nervous signals. Among them, a role for gut hormones in controlling bone mass and quality has emerged in the recent years. The aim of this review is to provide the reader with a summary of recent developments in the interaction between incretin hormones and bone physiology. Addresses 1 LUNAM Universite´, GEROM-LHEA, Institut de Biologie en Sante´, Angers, France 2 LUNAM Universite´, SCIAM, Institut de Biologie en Sante´, Angers, France Corresponding author: Mabilleau, Guillaume ([email protected])
Current Opinion in Pharmacology 2015, 22:72–78 This review comes from a themed issue on Musculoskeletal Edited by James Gallagher and Graham Russell
http://dx.doi.org/10.1016/j.coph.2015.03.007 1471-4892/# 2015 Elsevier Ltd. All rights reserved.
Introduction Bone is a tissue with multiple functions: (i) it has a strong biomechanical function by supporting the body weight and protects essential organs from potential mechanical injuries, (ii) it acts as a calcium, phosphate and sodium reservoir, (iii) it is a host tissue for hematopoietic bone marrow and (iv) it is also an endocrine organ that is involved in the regulation of glucose metabolism, energy expenditure, regulation of testosterone production and phosphate homeostasis [1–4]. To adapt to these functions, bone is remodeled permanently by a coupling between osteoclasts, the bone-resorbing cells, and osteoblasts, the bone-forming cells responsible for the synthesis of new structural units. Bone remodeling is traditionally considered to be regulated by hormones (parathyroid hormone, calcitonin, estrogen, among others), autocrine/paracrine signals from the microenvironment (receptor activator of nuclear factor kappa B ligand, tumor necrosis factor-alpha, among others), mechanical loading and the central and sympathetic nervous systems. From the molecular to anatomical levels, bones are built to resist and adapt to mechanical strain according to five Current Opinion in Pharmacology 2015, 22:72–78
different levels of organization (reviewed in review [5]). These levels of organization include the dual composition (organic vs. mineral phase) of the bone matrix, bone texture (lamellar vs. woven bone), bone structure (osteon vs. arch-like trabeculae), microarchitecture and macroarchitecture. As such, any modification of these degrees of organization may compromise bone strength that ultimately if not corrected result in bone fracture. Recently some evidences have emerged that a group of gut hormones may also be involved in the maintenance of bone strength and quality by acting on bone cell physiology. This body of evidence arises from the fact that (i) bone remodeling is reduced after parenteral feeding [6], (ii) that the pattern of bone remodeling is rapidly modified after a meal [7] and (iii) that food fractionation is capable of altering bone resorption and increasing bone mineral density with a greater extent as compared with a matched nutrient load given once a day [8]. The gastrointestinal tract produces and releases a plethora of bioactive peptides that act as neuromodulators/neurotransmitters, hormones or local regulators of cell function. Among them, a class of peptides called incretins have emerged as important modulator of energy metabolism. The term ‘incretin’ was initially proposed by Creutzfeld in 1979 and represents hormones that are secreted from the intestine in response to glucose and stimulate insulin release in a glucosedependent manner [9]. Although several hormones with insulinotropic action are secreted by the gut, glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 are the only two physiological incretins identified so far [10]. Once released to the blood stream, these two hormones are rapidly degraded by an endopeptidase, the dipeptidylpeptidase 4 (DPP-4). However, due to the wide list of DPP-4 substrates, the role of DPP-4 inhibitors in bone physiology is out of the scope of this review and will not be discussed further. The aim of the current review is to provide the reader with a comprehensive overview of the effects of incretin hormones on bone physiology.
Glucose-dependent insulinotropic polypeptide (GIP) GIP is produced by intestinal K-cells, located primarily in proximal regions of the small intestine. GIP gene transcription is increased in response to glucose and lipids and decreased in prolonged fasting periods, suggesting a nutrient-dependent transcriptional control of the GIP gene [11,12]. The human proGIP is a 153-amino acid polypeptide that is encoded by six exons representing a 459-bp open reading frame and whose gene is localized in www.sciencedirect.com
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humans on chromosome 17q [13,14]. The mature 42amino acid bioactive form of GIP (GIP1–42), mainly encoded by exons 3 and 4, is released from its precursor via prohormone convertase 1/3-dependent posttranslational cleavage at flanking single arginine residues [15]. The peptides encoded within the remaining fragments of the proGIP have no known biological functions [16]. The sequence of GIP is extremely conserved between species and more than 90% homology is found among human, porcine, bovine, mouse, and rat [17]. The K-cell is an open type endocrine cell and as such luminal content of the gut is thought to play a major role in GIP secretion. The K-cell is highly polarized with the GIP-containing secretory granules concentrated at the basal pole of the cell, ready to be released from secretory granules through the basolateral membrane [18,19]. In addition, K-cells are found in close association with the capillary network running through the lamina propria allowing GIP to enter into the blood stream. Based on the morphological features, GIP secretion from K-cells is regulated by intraluminal contents, neural stimuli and hormones. To exert its biological actions, GIP binds to its receptor, the GIPr. The human gipr gene comprises 14 exons that span approximately 13.8 kb [20] and is localized on chromosome 19q13.3 [21]. The GIPr belongs to the 7-transmembranespanning G-protein coupled receptor (GPCR) superfamily [22] and is composed of 430 amino acids. The GIPR is expressed in the endocrine pancreas, gastro-intestinal tract, adipose tissue, adrenal cortex, pituitary gland, vascular endothelium and several regions in the CNS [16]. The principal physiological role of GIP is to increase insulin secretion from the pancreatic beta-cells in a glucose-dependent manner. However, extrapancreatic actions of GIP have been observed. GIP acts on lipid metabolism including augmentation of plasma triglyceride clearance, increased lipoprotein lipase activity and promotion of fat storage in adipocytes [23–25]. In animal model of GIP deficiency or chemically-induced GIPr antagonism, interrupting GIP signaling appears to be beneficial in reducing high fat diet induced obesity [24,26–28]. GIP has also been reported to play a role in neural progenitor cell proliferation and behavior [29].
Bone action of the GIP/GIPr pathway The GIPr has been demonstrated to be expressed at the mRNA and protein level in several osteoblastic cell lines including MG-63, TE-85, Saos-2, Ros 17/2.8 and MC3T3 [30–32], primary murine osteoblasts [31] and murine osteoclast precursors [33], suggesting a role for the GIP/GIPr pathway in bone. A polymorphism of the gipr gene caused by substitution of a guanine by a cytosine at rs1800437 induces a substitution of glutamate to glutamine at position 354 in the sixth transmembrane domain of GIPr resulting in decreased receptor activity [34]. Recently, Torekov and colleagues reported that this polymorphism is associated, in a Danish-based perimenopausal www.sciencedirect.com
women cohort, with low mineral bone density at the femoral neck and total hip but not at the lumbar spine [35]. Furthermore, women carrying the lower frequency C allele also presented with higher incidence of nonvertebral fractures [35]. Taken together, these data suggest a role for the GIP/GIPr pathway in controlling bone density and strength. A summary of GIP/GIPr actions on bone is presented in Figure 1. Our understanding of the action of this pathway in bone has been greatly enhanced from studies conducted in GIPr knock-out mice. Two models of GIPr KO have been developed. The first model consists in a deletion of exons 4 and 5 of the gipr gene, which encode a portion, but not the totality, of the extracellular domain of this receptor [36]. The second model consists in the deletion of the first 6 exons, which encode the totality of the extracellular domain and a portion of the first transmembrane domain of the GIPr [37]. Both animal models result into an inactive GIP/GIPr pathway but surprisingly the consequences of this inactivation on trabecular bone are opposed. Indeed, in model 1, GIPr-deficient mice present with augmentation in fat mass and reduction of trabecular bone mass detectable as early as 4 week-old at the femur and 8 week-old at the tibia [38,39]. Histomorphometric analysis revealed a bone formation defect in these animals with decreased mineral apposition rate and bone formation rate and an increase in osteoid maturation
Figure 1
PANCREATIC β-CELL ↑ insulin secretion
OSTEOBLAST ↑ Type 1 collagen expression ↑ Maturation of collagen ↑ Alkaline phosphatase activity ↑ Mineralisation of the matrix ↑ TGF-β secretion
OSTEOCLAST ↓ Osteoclast formation ↓ Osteoclast activity
GIP
ADIPOCYTE Modification of adipokine secretion
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Mechanisms of action of GIP on bone cells. Upon entry of nutrients into the small intestine, GIP is secreted by duodenal K-cells into the blood stream and acts on target cells. When GIP binds the GIPr at the surface of osteoblasts, it induces expression of type 1 collagen and TGF-beta, maturation of collagen matrix, augments alkaline phosphatase activity and mineralization of the bone matrix. When GIP engages its receptor at the surface of osteoclast precursors, it results an augmentation into osteoclast formation and osteoclast resorption. GIP also stimulates insulin secretion from beta-cells of the pancreas that could potentially modify the pattern of osteoblast and osteoclast action. GIP also affects adipokine secretion from adipocytes that could potentially alter the activity of bone cells. Current Opinion in Pharmacology 2015, 22:72–78
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rate [39]. These animals exhibited also a reduction in circulating osteocalcin and an augmented leptin concentration in plasma [38]. In model 2, GIPr-deficient mice presented with decreased body weight and fat mass, increased trabecular bone mass at the tibia at 16 weeks of age and higher trabecular number [40]. This high bone phenotype was even more pronounced at 45 weeks of age suggesting that these animals were protected from ageing-induced bone loss. This phenotype was accompanied by higher values for the mineral apposition rate and the bone formation rate [40]. Plasma level of circulating osteocalcin was unchanged whilst leptin was reduced and adiponectin augmented [40]. To date, no satisfactory explanations have been formulated to explain the two opposed bone phenotype observed in the two GIPr KO models. However, in model 2, despite an apparent higher trabecular bone mass, the strength of the trabecular bone matrix was dramatically reduced and associated with altered maturation of the mineral and collagen component of the bone matrix [40]. To the best of our knowledge, the cortical bone phenotype has only been investigated in model 2. Nevertheless, augmentation of the marrow diameter with a normal outer bone diameter at the femur resulted in decreased cortical thickness and reduced bone strength at the whole bone level [41]. Investigation of tissue material properties by nanoindentation, quantitative backscattered electron imaging and Fourier-transform infrared microspectroscopy revealed alterations of bone strength at the tissue level associated with reduction in the degree of bone mineralization and collagen maturation [41]. Taken together these data suggest a positive role of the GIPr in order to gain optimal bone strength and quality. This hypothesis is further strengthened by the improved tissue material properties and bone strength in a rodent model treated with exogenous GIP [42]. However, as the GIPr is widely distributed, it is not clear whether the above skeletal effects result from a direct action of the bone GIPr or action of extraskeletal GIPr that indirectly contribute to the observed bone phenotype. Nevertheless, few studies have investigated the direct action of GIP on osteoblast and osteoclast cultures. Administration of GIP in osteoblast cultures results in augmentation in osteoblast proliferation, osteoblast-produced TGF-beta, alkaline phosphatase and type 1 collagen expression [43,44]. Furthermore, addition of GIP, at physiological concentration, in osteoblast cultures resulted in higher lysyl oxidase activity and collagen maturity [45]. Administration of exogenous GIP leads to reduction in osteoclast-mediated resorption as evidenced by reduction in circulating CTx levels [46], although conflicting results have been reported regarding a direct action of GIP to mediate this effect [33,39]. Current Opinion in Pharmacology 2015, 22:72–78
Glucagon-like peptide-1 (GLP-1) The proglucagon gene is expressed at high levels in intestinal L-cells [47]. L-cells can be found in the duodenum but occur at higher number in the ileum and colon [48]. The single proglucagon gene encodes a 160-amino acid peptide that undergoes posttranslational processing [49]. In intestinal L-cells, after processing by the prohormone convertase 1/3, the proglucagon gene gives rise to glicentin, corresponding to the first 69-amino acids of the precursor, GLP-1 (amino acids 78–107) composed of 31 amino acids, peptide 2 (amino acids 111–123) and GLP-2 (amino acids 126–158) comprising 33 amino acids [50]. Thus, processing of the proglucagon gene in intestinal cells generates equimolar concentration of GLP-1 and GLP-2. Until now, little is known about the biological actions of glicentin and peptide 2. Two forms of GLP-1 are produced in the intestine, GLP17–36NH2 and GLP-17–37 although the major circulating form is GLP-17–36NH2 [51]. As with K-cells, L-cells are an open type endocrine cells highly polarized with secretory granules at their basolateral pole, ready to release GLP-1and GLP-2 into the blood stream. L-cells are found in close association with the capillary network running through the lamina propria. Based on the morphological features, GLP-1 secretion from L-cells is regulated by intraluminal contents, neural stimuli and hormones. GLP-1 has also been suspected to act via the autonomous nerve system on specific hypothalamic and brainstem nuclei to exert its action [52]. To act, GLP-1 engages its receptor, the GLP-1r expressed on target tissue. The human glp1r gene comprises also 14 exons and is localized on chromosome 6p21. GLP-1r also belongs to the 7-transmembrane-spanning GPCR superfamily like the GIPr. It is composed of 463 amino acids [53] and is expressed in the endocrine pancreas, gastro-intestinal tract, lung, heart, kidney and several regions of the brain [16]. The principal physiological role of GLP-1 is to potentiate glucose-dependent insulin secretion [54]. Extrapancreatic actions of GLP-1 results in reduction of food intake through the CNS, inhibition of gastric emptying, positive actions on the cardiovascular system and a role in energy expenditure [54].
Bone action of the GLP-1/GLP-1r pathway Presence of the GLP-1r in bone cells is controversial. It has been evidenced at the mRNA level in MG-63 and TE-85 osteoblastic cell lines [30]; however, these cells are not very representative of osteoblast cells [55]. On the other hand, GLP-1r was not found in MC3T3-E1 [56] or primary murine osteoblasts or osteoclasts [57]. However, previous studies in other tissues such as liver and skeletal muscle revealed the presence of a GLP-1 receptor different from the known GLP-1r in function and/or structure. Indeed, this second GLP-1 receptor does not www.sciencedirect.com
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activate the cAMP pathway as GLP-1r does but rather the production of inositolphosphoglycan as a second messenger [58,59]. Recently, Nuche-Berenguer et al. revealed the presence of a second GLP-1 receptor in MC3T3-E1 cells different from the known GLP-1r [56]. Indeed, the binding of (125I)GLP-1 to this second receptor was displaced by addition of cold GLP-1 but not exendin-4, a known agonist of the GLP-1r, and resulted in increase in inositolphosphoglycan and activation of PI3 kinase [56]. However, no data is available regarding the presence of this second GLP-1 receptor on osteoclasts. Recently, Kim et al. evidenced the presence of GLP-1r at the mRNA and protein level in the MLO-Y4 osteocyte cell line [60]. However, when a search-for-specificity alignment is performed with the primer-BLAST tool of the National Centre for Biotechnology Information (NCBI), it appears that the primer pair used by Kim et al. was not specific of the glp1r mRNA but might also reveal the presence of timeless, a protein involved in the circadian clock (unpublished data). Furthermore, following a publication of Panjwani et al. in 2013 [61], a growing body of evidence suggests that most of commercially available antibodies used in immunohistochemistry and western blotting in published studies are not specific of the GLP-1r [62,63]. As such it is difficult to ascertain whether the known GLP-1r is expressed in bone cells. Until more convincing studies are performed, scientists may have to consider the possibility that the known GLP-1r may not be expressed in bone cells.
Figure 2
PANCREATIC β-CELLS ↑ insulin secretion
GLP-1
BONE CELLS
L-CELLS ILEUM
C-CELLS THYROID GLAND ↑ Calcitonin secretion
Current Opinion in Pharmacology
Mechanisms of action of GLP-1 on bone cells. Upon entry of nutrients into the small intestine, GLP-1 is secreted by ileal L-cells into the blood stream and acts on target cells. The known GLP-1r receptor is not expressed in osteoblast or osteoclast, but on the other hand, GLP-1 might act directly on osteoblast physiology by binding to a second GLP-1 receptor. On the other hand, in rodent, the GLP-1r is expressed in C-cells of the thyroid and is involved in calcitonin secretion that might exert anti-resorptive actions in bone. Furthermore, as GLP-1 is an incretin hormone, it potentiates insulin secretion from pancreatic b-cells and as insulin is considered anabolic for bone, it is plausible that insulin mediates some of the observed bone effects. Osteocalcin produces by osteoblasts is capable of enhancing GLP-1 secretion from ileal L-cells. www.sciencedirect.com
A summary of GLP-1/GLP-1r actions on bone is presented in Figure 2. Here again, our understanding of GLP-1 actions in skeletal physiology arises from GLP1r KO mouse. At 10 weeks of age, GLP-1r KO animals exhibited a mild reduction in trabecular bone volume at the tibia, although not significant, associated with increased number of osteoclasts and eroded surfaces [64]. Unpublished observation from our lab made in the same KO model at 16 weeks of age corroborated these findings. On the other hand, the mineral apposition and bone formation rates appeared unaffected by GLP-1r inactivation [64]. Taken together these results suggested a control of bone resorption (osteoclast differentiation and/or action) by the GLP-1r. However, GLP-1 was unable to directly control osteoclast formation and resorption in osteoclast cultures [64], suggesting that the control of bone resorption was indirect. Indeed, these authors found a reduction in calcitonin gene expression in GLP-1rdeficient animals. Now, we know that in rodents, but not in non-human primate or humans, GLP-1r is expressed in C-cells of the thyroid gland, and responsible for a rise in calcitonin secretion [65]. However, in humans, administration of exogenous GLP-1 does not result into lower CTx levels [7]. The effect of GLP-1r deficiency in cortical bone has been investigated in 16 week-old GLP-1r-deficient mice. These animals presented with reduction in bone strength observed by 3-point bending [57]. In these animals, modification of cortical microstructure was evidenced with reduction in the bone outer diameter whilst the marrow diameter was conserved. These alterations led to a lower cortical thickness [57]. Tissue material properties are also reduced at cortical site in GLP-1r-deficient animals and are associated with reduced collagen maturity with a normal distribution of the degree of mineralization [57]. These data highlighted that a functional GLP1r is not only required for the control of bone resorption but also for the preservation of an optimal bone matrix quality. Administration of GLP-17–36NH2 or exendin-4, a GLP-1r agonist, results in a rapid augmentation of osteocalcin gene expression and a reduction in the balance RANKL/ OPG in the bones of normal, type 2 diabetic or insulinoresistant rats [66,67]. Furthermore, 16 weeks administration of exendin-4 in OVX rats, is capable of improving trabecular bone mass and microarchitecture at the femur and lumbar vertebra, bone strength and revert hyperresorption observed after ovariectomy [68]. Moreover, the administration of exendin-4 into type 2 diabetic animals was also capable of reducing sclerostin expression and improving bone mineral density [60]. Recently, evidences have been provided that undercarboxylated osteocalcin was capable of inducing GLP-1 release from the L-cells and as such this complementary mechanism reinforces the role of osteocalcin in energy expenditure [69,70]. However, as the GLP-1r is not expressed in Current Opinion in Pharmacology 2015, 22:72–78
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bone cells, the major challenge to face in the near future would be to ascertain how the GLP-1/GLP-1r pathway in other tissues may control bone physiology. Now several GLP-1 analogues, enzymatically resistant to DPP-4 degradation, have been approved for the treatment of type 2 diabetes mellitus. Regarding the skeletal alterations observed in animals deficient in GLP-1r and the rapid and favorable effects of GLP-1 or GLP-1r agonist on bone gene expression, one could expect an improvement in bone quality in type 2 diabetic patients. However, in a recent meta-analysis conducted by our group on the incidence of bone fractures in type 2 diabetic patients taking GLP-1 mimetic, we failed to evidence any beneficial effects of GLP-1 mimetic [71].
Conclusions A link between incretin and bone physiology has been highlighted in the past years suggesting that incretin are essential for the maintenance of bone mass but also to gain optimal bone quality and strength. However, the major challenge to face in this field in the future will be to ascertain by which mechanisms incretins exert their control on skeletal physiology.
Conflict of interest statement Nothing declared.
Acknowledgements GM is grateful to members of the GEROM-LHEA lab (LUNAM Universite´, Angers, France) and Diabetes group (Ulster University, Coleraine, UK) for their invaluable help in his researches in this field. This work was supported by grants from the University of Angers and Socie´te´ Franc¸aise de Rhumatologie.
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