interplay between bone and incretin hormones: a...

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Morphologie (2016) xxx, xxx—xxx

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GENERAL REVIEW

Interplay between bone and incretinhormones: A reviewInteraction entre l’os et les hormones incrétines : une revue

G. Mabilleaua,∗,b

a GEROM-LHEA, groupe d’études remodelage osseux et biomatériaux, institut de biologie en santé,université d’Angers, 4, rue Larrey, 49933 Angers cedex 09, Franceb SCIAM, institut de biologie en santé, université d’Angers, 4, rue Larrey, 49933 Angers cedex 09, France

KEYWORDSBone;GIP;GLP-1;Incretins;Digestive hormones

Summary Bone is a tissue with multiple functions that is built from the molecular to anatom-ical levels to resist and adapt to mechanical strains. Among all the factors that might controlthe bone organization, a role for several gut hormones called ‘‘incretins’’ has been suspected.The present review summarizes the current evidences on the effects of glucose-dependentinsulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) in bone physiology.© 2016 Elsevier Masson SAS. All rights reserved.

MOTS CLÉSRésumé Le tissu osseux est un tissu conjonctif avec de multiples fonctions qui est organisédepuis l’échelle moléculaire jusqu’à l’échelle anatomique pour résister et s’adapter aux con-

Os ;GIP ;GLP-1 ;Incrétines ;Hormones digestives

traintes mécaniques. Parmi tous les facteurs qui pourraient contrôler son organisation, le rôlede certaines hormones intestinales appelées « incrétines » a émergé. La présente revue résumeles connaissances actuelles sur les effets du polypeptide insulinotrope dépendant du glucose(GIP) et du glucagon-like peptide-1 (GLP-1) en physiologie osseuse.© 2016 Elsevier Masson SAS. Tous droits reserves.

•

•

Introduction

Bone is a tissue with multiple functions:

Please cite this article in press as: Mabilleau G. Interplay betw(2016), http://dx.doi.org/10.1016/j.morpho.2016.06.004

• it supports the body weight and protects essential organsfrom potential mechanical injuries (mechanical function);

∗ Correspondence.E-mail address: [email protected]

•

b

http://dx.doi.org/10.1016/j.morpho.2016.06.0041286-0115/© 2016 Elsevier Masson SAS. All rights reserved.

it acts as a calcium, phosphate and sodium reservoir(metabolic function);

it is a host tissue for hematopoietic bone marrow and; it is also an endocrine organ involved in the regulation

of glucose metabolism, energy expenditure, regulationof testosterone production and phosphate homeostasis

een bone and incretin hormones: A review. Morphologie

[1—4].

From the molecular to anatomical levels, bones areuilt to resist and adapt to mechanical strains according to

dx.doi.org/10.1016/j.morpho.2016.06.004


http://www.sciencedirect.com/science/journal/12860115

mailto:[email protected]


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ARTICLEORPHO-283; No. of Pages 10

ve different levels of organization [5]. First of all, bonesave a dual composition as the bone matrix is a complexanocomposite material made of mineral and organichases. The organic phase is mostly composed of type Iollagen (∼90% of total bone proteins) and non-collagenousroteins (∼10% bone proteins). The mineral phase is madef poorly crystalline hydroxyapatite tablets with hydrogenhosphate and carbonate groups substituting for phosphateons [6]. Bone texture represents a second degree of orga-ization. In lamellar bone, collagen is oriented in a preciseay with angular changes between each lamella, giving theharacteristic appearance of bone texture in polarizationicroscopy. Woven bone, also called non-lamellar bone,

an be found in zones where osteoblast activity is very highfracture callus, microfractures, metaplastic bone in boneetastasis, Paget’s disease. . .) and is characterized by an

narchic texture in which collagen microfibers have randomirections. Biomechanical properties of woven bone areeduced compared to those of lamellar bone. The thirdegree of organization is represented by the presence ofsteons and arch-like bone structure units. In the cortices,he bone structural units consist of osteons with a cylindri-al shape centred on a canal. Typically a complete osteons 200—300 �m in diameter with a central canal of ∼50 �mn diameter. Inside the canal, blood vessel and sympatheticerve fibres may be observed. Canals are intercommuni-ating and branched to ensure the communication betweeneriosteal and endosteal spaces. Between complete osteonsre incomplete remnants of old osteons, partially erodedhat constitute the interstitial bone. In trabecular bone,tructural units have an arch-like appearance. Theserch-like units are ∼40—45 �m in thickness and represent atack of lamellae. Trabecular bone (or cancellous bone) isometimes improperly termed ‘‘spongy bone’’; this term isow considered as improper since it underlies a biomechan-cal property that bone does not have [7]. New structuralnits are laid over the trabecular surfaces that have beenreviously eroded by osteoclasts. Between the newlypposed structural units, remnants of partially eroded unitsersist and constitute the interstitial trabecular bone. Theourth degree is represented by the bone microarchitecture5,7]. In the cortices, osteons are compacted so that thexes of the central canal run parallel with the resultingtress line exerted on bone. Trabecular bone tissue isomposed of structural units constituting two differentypes of trabeculae: large plates (arranged along the stressine) connected laterally by pillars or rods, which ensurehe cohesion of the network [8]. The role of trabecular bones to resist to compression loads and transfer the strains tohe cortices. Finally the fifth and last level of organizations represented by the bone macroarchitecture. Bones havepecial angulations and curvatures that are genetically andpigenetically determined and enable them to resist toechanical strains, including compression, tension or shear

tress loads [9,10]. As such, any modification of one of therganization level would affect the quality of the matrix,.e. an umbrella term representing microarchitectures,icrocrack propagation and tissue material properties.


To adapt to its mechanical and metabolic functions,one is remodeled permanently by a coupling betweensteoclasts, the bone-resorbing cells, and osteoblasts,he bone-forming cells responsible for the synthesis of

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ew structural units. Osteocytes (‘‘the third bone cell’’)re derived from osteoblasts and are found embed-ed in the bone matrix where their main role is toerve as a mechanosensor/mechanotransducer and tonform osteoclasts and osteoblasts about bone areas thatre damaged and should be remodeled. Bone remod-ling is traditionally considered to be regulated byormones (parathyroid hormone, calcitonin, estrogen. . .),utocrine/paracrine signals from the bone microenviron-ent (receptor activator of nuclear factor kappa-B ligand,

umor necrosis factor-alpha, cell-to-cell contact, etc.),echanical loading and the central and sympathetic nervous

ystems.In the quest of better understanding the different

ndocrine factors that may regulate bone remodeling, theole of several products from the gastrointestinal tract haseen suspected. Indeed, metabolic bone disease associ-ted with long-term parenteral feeding was first describedn the early 1980s. Klein et al. reported that, in patientseceiving long-term parenteral nutrition, bone physiologyas altered with evidence of bone pain, hypercalciuria, ele-ated serum alkaline phosphatase despite normal ranges oferum calcium, phosphorus and 25-hydroxyvitamin D [11].hese findings have then been confirmed by several boneroups and are reviewed in [12]. Although these effectsay be related to the composition of parenteral nutrition

tself (low calcium and phosphorus, aluminum, fluoride,tc.), a role for the gastrointestinal tract can also be sus-ected. Further evidences are brought by a reduction inone resorption after nutrient ingestion [13]. Indeed thelegant study of Henriksen et al. highlighted reductions of2%, 39% and 52% after oral intake of glucose, triglyceri-es, and protein, respectively, in healthy individuals agedetween 30 and 40 years old and with a body mass indexf 22.7 kg/m2 [13]. Furthermore, the experimental use ofood fractionation results in higher bone mineral density asompared with a matched nutrient load given once a day14].

The gastrointestinal (GI) tract is one of the largestndocrine organs with more than 12 different endocrineells [15]. Among the plethora of bioactive peptides thathe GI tract secretes, a class of peptides called incretins hasmerged as important modulators of energy metabolism.he term ‘‘incretin’’ was initially proposed by Creutzfeld

n 1979 and represents hormones that are secreted fromhe intestine in response to glucose and stimulate insulinelease in a glucose-dependent manner [16]. Althougheveral hormones with insulinotropic action are secretedy the gut, glucose-dependent insulinotropic polypeptideGIP) and glucagon-like peptide-1 (GLP-1) are the onlywo physiological incretins identified so far [17]. GIP andLP-1 are produced by the K- and L-enteroendocrineells, respectively, that are localized sparsely in thentestinal epithelium (Fig. 1A). Once released to thelood stream, these two hormones are rapidly degradedy an endopeptidase, the dipeptidylpeptidase-4 (DPP-) found in the vicinity of capillaries in the intestinalucosa or liver. DPP-4 is expressed widely as indicated


n Table 1. However, due to the wide list of DPP-4 sub-trates, the role of DPP-4 inhibitors in bone physiology isut of the scope of this review and will not be discussedurther.



Bone and incretins 3

Figure 1 GIP expression and schematic representation of GIP-producing enteroendocrine K-cells. A. GIP detection was made ona sample from a 68-year old woman using the HPA021612 antibody (Sigma Aldrich). GIP-positive cell localizations are indicated byarrows. This immunostaining is part of the human protein atlas [80] and full dataset can be found at http://www.proteinatlas.org.B. GIP-containing secretory granules are found at the basal pole of the open-type K-cells in close proximity with nerve endingsand capillaries in the lamina propria. Entry of nutrients at the apical pole of the K-cells results in a cascade of activation ofintracellular pathways leading to augmentations of protein kinase A and C activities (PKA and PKC, respectively) and ultimatelyrise in intracellular calcium responsible for GIP secretion. Several paracrine peptide (somatostatin) and neuromediators may alsomodulate GIP secretion.Expression du GIP et représentation schématique des cellules entéroendocrines K produisant le GIP. A. La détection du GIP aété effectuée dans un prélèvement chez une femme de 68 ans en utilisant l’anticorps HPA021612 (Sigma Aldrich). La localisationdes cellules positives au GIP est indiquée par des flèches. Cette détection immunologique fait partie de l’atlas des protéineshumaines [80] et l’intégralité des données est disponible à l’adresse http://www.proteinatlas.org. B. Les granules contenant duGIP sont retrouvées au pôle basal des cellules K en étroite association avec des terminaisons nerveuses et des capillaires sanguinsdu chorion. L’entrée de nutriments au pôle apical des cellules K provoque une cascade d’évènements intracellulaires conduisant àl’activation des protéines kinases A et C (PKA et PKC, respectivement) et finalement à l’augmentation du calcium intracellulaire

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qui est responsable de la sécrétion de GIP. D’autres peptides apeuvent également moduler la sécrétion de GIP.

The aim of the current review is to provide the readerwith a comprehensive overview of the effects of incretinhormones on bone physiology.

Glucose-dependent insulinotropic polypeptide(GIP)

GIP is produced and secreted mostly by intestinal K-cells,located primarily in proximal region of the small intestine.The K-cell is highly polarized with the GIP-containing secre-tory granules concentrated at the basal pole of the cell,ready to be released through the basolateral membrane[18,19] (Fig. 1B). Based on the morphological features, GIPsecretion from K-cells is regulated by neural stimuli, hor-mones and intraluminal contents [20]. K-cells are found inclose association with the capillary network running throughthe lamina propria allowing GIP to enter into the bloodstream rapidly after secretion. Furthermore, intraluminalcontents do not only affect GIP secretion but also GIP expres-


sion. Indeed, glucose and lipids are potent stimulator of GIPgene transcription [21,22].

The human proGIP is a 153-amino acid polypeptide thatis encoded by six exons representing a 459-bp open reading

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nt par voie paracrine (somatostatine) et des neuromédiateurs

rame and whose gene is localized in humans on chro-osome 17q [23,24]. The mature 42-amino acid bioactive

orm of GIP (GIP1-42), mainly encoded by exons 3 and 4,s released from its precursor via prohormone convertase/3-dependent post-translational cleavage at flanking sin-le arginine residues [25]. The peptides encoded within theemaining fragments of the proGIP have no known biologicalunctions [26].

To exert its biological actions, GIP binds to its recep-or, the GIPr. The human GIPR gene comprises 14 exonshat span approximately 14.2 kb and is localized on chro-osome 19q13.3 [27]. The GIPr belongs to the class

7-transmembrane-spanning G-protein coupled receptorGPCR) superfamily [28] and is composed of 466 aminocids. The GIPr is expressed in the endocrine pancreas, gas-rointestinal tract, adipose tissue, adrenal cortex, pituitaryland, vascular endothelium and several regions in the CNS26]. The principal physiological role of the GIP/GIPr is toncrease insulin secretion from the pancreatic beta-cellsn a glucose-dependent manner. However, extrapancreatic


ctions of this pathway have been reported. GIP acts onipid metabolism including augmentation of plasma triglyc-ride clearance, increased lipoprotein lipase activity andromotion of fat storage in adipocytes [29—31]. In animal


http://www.proteinatlas.org/



4 G. Mabilleau

Table 1 Expression profile of the dipeptidylpeptidase4 (DPP-4) in human. The original data are part of theHuman Proteome Atlas project and can be found athttp://www.proteinatlas.org. The mRNA has been detectedby RNA seq whereas the Origene TA500733 has been used todetect DPP-4 at the protein level by immunohistochemistry[80].Profil d’expression de la dipeptidylpeptidase 4 (DPP-4)chez l’homme. Les données originales font partie du pro-jet «Human Proteome Atlas» qui peut être consulté àhttp://www.proteinatlas.org. L’ARNm a été détecté parARN seq tandis que l’Origene TA500733 a été utilisé pourdétecter la DPP-4 au niveau protéique par immunohis-tochimie [80].

Tissue mRNA expression Protein expression

Placenta High HighProstate High HighSmall intestine High HighKidney Medium HighSalivary gland Medium MediumEndometrium Low HighColon Low LowLiver Low LowGallbladder Low Not detectedPancreas Low Not detectedUrinary bladder Low Not detectedTestis Low Not detectedOvary Low Not detectedEsophagus Low Not detectedStomach Low Not detectedSkin Low Not detectedSmooth muscle Low Not detectedHeart muscle Low Not detectedAdipose tissue Low Not detectedBone marrow Low Not detectedLymph node Low Not detectedSpleen Low Not detectedThyroid gland Low Not detectedAdrenal gland Low Not detectedLung Low Not detectedCerebral cortex Low Not detected

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Figure 2 Alterations of bone microarchitectures observed inGIPr-deficient mice. Bone microarchitectures were assessed byX-ray microcomputed tomography (Skyscan, Bruker MicroCT,Kontich, Belgium). Representative 3D models of wild-type (WT)and GIPr KO mice are presented here. Arrows point out at thedifferences in bone microarchitecture between the two models.Altérations des microarchitectures osseuses observées dansles souris invalidées pour le récepteur du GIP. Les microar-chitectures trabéculaires et corticales ont été étudiées parmicrotomographie à rayons X (Skyscan, Bruker MicroCT, Kon-tich, Belgique). Les modèles 3D représentatifs des sourissauvages (WT) ou invalidées (GIPr KO) sont présentés ici. Lesflèches indiquent les différences microarchitecturales entre lesdeux modèles.

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odel of GIP deficiency or chemically-induced GIPr antago-ism, interrupting GIP signaling appears to be beneficial ineducing high fat diet induced obesity [30,32—34]. GIP haslso been reported to play a role in neural progenitor cellroliferation and behavior [35].

The GIP/GIPr pathway is also an important regulatorf bone physiology. First of all, the GIPr is physiologicallyxpressed at the mRNA and protein level in osteoblastsnd osteoclasts [36—39]. In osteoblasts, GIP administrationesults in increases in intracellular calcium and cAMP thatead to augmentations in type I collagen expression, alka-ine phosphatase and lysyl oxidase activities, and higher


nzymatic collagen cross-linking [37,40]. GIP is also capa-le of reducing the extent of bone resorption by fullyature osteoclasts although the exact molecular mecha-

isms remain to be elucidated [39,41]. At the organ level,

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uch of our understanding of the actions of the GIP/GIPrathway arises from genetically-modified mice. Indeed, toate, two animal models of GIPr deletion exists with eithereletion of exons 4—5 or exons 1—6. These portions of theipr gene either code for several amino acids of the extra-ellular domains, involved in GIP binding (exons 4—5) or forhe totality of the extracellular domain and the first trans-embrane segment (exons 1—6). These animals, althoughith opposite trabecular bone phenotype, suggest that tra-ecular and cortical bone are dramatically compromisedn the absence of a functional GIPr as presented in Fig. 241—44]. Furthermore, these animal models also presentith alterations of tissue material properties represented by

educed mineralization degree (Fig. 3) and collagen matu-ity in the bone matrix [43]. Overall these modifications ofone mass, microarchitecture and tissue material propertiesesult in skeletal fragility.

However, lack of GIPr action leads to increased GLP-1 sen-itivity [45]. As such, to answer whether the observed abovelterations observed in GIPr KO mice resulted from the lackf a functional incretin receptor or by a compensatory mech-nism induced by elevated sensitivity to the other incretinormone, the bone phenotype of double incretin receptornock-out (DIRKO) animals, lacking both the GIPr and GLP-1r,as investigated [46]. DIRKO animals also exhibit profound

eductions in bone mass as well as alterations of trabecularnd cortical microarchitectures and tissue material proper-


ies demonstrating the important role of incretins in bonehysiology.




ARTICLE IN+ModelMORPHO-283; No. of Pages 10

Bone and incretins

Figure 3 Tissue mineral density in wild-type (WT) and GIPr-deficient animals. Calcium map was obtained by quantitativebackscattered electron imaging at the mid-diaphysis of thefemur. The calcium content was calculated with a lab-maderoutine in Matlab and clearly demonstrated lower tissue mineraldensity in GIPr-deficient mice.Densité minérale de la matrice osseuse dans les souris sauvages(WT) et invalidées pour le récepteur du GIP (GIPr KO). Lescartographies de concentration massique en calcium ont étéobtenues par microscopie quantitative en électrons rétrodif-fusés (sur microscopie électronique à balayage) au centre de ladiaphyse fémorale. La concentration massique en calcium a étécalculée avec une routine du logiciel Matlab et montre claire-

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ment la diminution de densité minérale de la matrice osseusedans les souris invalidées pour le récepteur du GIP.

Pre-clinical data on the use of GIP mimetic in animalmodels are scarce nevertheless, administration of N-acetyl-GIP (N-AcGIP) ameliorated tissue material properties whenadministered for 28 days in healthy rodents [47]. Fur-thermore, in streptozotocin-injected mice, developing aclassical picture of type 1 diabetes, with a compromisedbone mass and microarchitecture as well as tissue mate-rial properties, the administration of (D-ala2)GIP for 21 daysresulted in bone turnover almost similar to non-diabeticanimals, and reduction in matrix collagen destruction [48].These results are particularly interesting as insulin release


was unchanged in (D-ala2)-treated as compared with saline-treated mice, suggesting that these beneficial effects of GIPmimetic is independent of insulin secretion.

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In healthy humans, although initially GIP had been shownneffective in reducing bone resorption [13], recent evi-ences suggested that exogenous administration of GIPas effective in reducing circulating markers of bone

esorption [49]. However, whether these results reflectirect actions of GIP on osteoclast-mediated resorptionr indirect actions of other GIP-targeted tissues remaino be determined in the future. Furthermore, the linketween GIP/GIPr pathway and bone mass/strength is def-nitely established and represented by low mineral boneensity at the femoral neck and total hip, as well asigher incidence of non-vertebral fractures, in a cohort oferimenopausal women with a single-nucleotide polymor-hism (rs1800437), that results in decreased GIPr activity50,51].

lucagon-like peptide-1 (GLP-1)

lucagon-like peptide 1 is produced by post-translationalrocessing of the glucagon gene (Fig. 4). Indeed, in intesti-al L-cells, the glucagon gene (160-amino acids) gives rise tolicentin (amino acids 1—69), glicentin-related polypeptideamino acids 1—30), oxyntomodulin (amino acids 33—69),lucagon (amino acids 33—61), peptide 1 (amino acids4—69), GLP-1 (amino acids 72—107), peptide 2 (aminocids 111—123) and GLP-2 (amino acids 126—158) [52] [53].hus, processing of proglucagon in intestinal cells generatesquimolar concentration of GLP-1 and GLP-2. Until now, lit-le is known about the biological actions of glicentin andeptide 2.

Two forms of GLP-1 are produced in the intestine, GLP-7-36NH2 and GLP-17-37, although the major circulatingorm is GLP-17-36NH2 [54]. L-cells are also an open-typendocrine cells highly polarized with secretory granules atheir basolateral pole. GLP-1 secretion from L-cells is regu-ated by intraluminal contents, neural stimuli and hormones26]. L-cells are found in close association with the capil-ary network running through the lamina propria. GLP-1 haslso been suspected to act via the autonomous nerve systemnd vagal afference on specific hypothalamic and brainstemuclei to exert its action [55].

To act, GLP-1 engages its receptor, the GLP-1r that isoded by the human GLP1R gene comprises 13 exons thatpan approximately 13.8 kb [56] and is localized on chromo-ome 6p21 [27]. The GLP-1r is expressed in the endocrineancreas, gastrointestinal tract, lung, heart, kidney and sev-ral regions of the brain [26]. Recent evidences also suggesthat GLP-1 can bind in specific circumstances to the glucagoneceptor [57]. The principal physiological role of GLP-1 is tootentiate glucose-dependent insulin secretion [58]. Extra-ancreatic actions of GLP-1 result in reduction of food intakehrough the CNS, inhibition of gastric emptying, positivections on the cardiovascular system and a role in energyxpenditure [58].

Presence of the known GLP-1r in bone cells was con-roversial until recently. Indeed, this confusion was due tohe use of poorly-characterized cell lines [36], selection


nti-GLP-1r antibodies [60] that were not very specific ofhe mouse Glp1r gene. Recent evidences by Pereira et al.eem suggest the presence of the known GLP-1r in skeletal



6 G. Mabilleau

Exon 1 Exon 2 Exon 6Exon 5Exon 4Exon 3

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P1 P25’-UTR 3’-UTR

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Figure 4 Post-translational processing of proglucagon. Theproglucagon gene spans over 12.4 kb located in human on chro-mosome 2q36-q37. The proglucagon gene comprised 6 exonsthat encode several peptide: a signal peptide (SP, in blue),glicentin-related polypeptide (GRPP, in green), glucagon (GLG,in orange), peptide-1 (P1, in black), glicentin, oxyntomodulin(Oxm), glucagon-like peptide-1 (GLP-1, in pink), peptide-2 (P2,in violet), glucagon-like peptide-2 (GLP-2, in yellow) and themajor proglucagon fragment (MPGF). Depending on the tissue,post-translational processing by prohormone convertase 1 and2 may give rise to several combinations of these peptides.Modifications post-traductionnelles du proglucagon. Chezl’homme, le gène du proglucagon s’étend sur 12,4 kb aulocus 2q36-q37. Le gène du proglucagon comprend 6 exonsqui encodent plusieurs peptides : un peptide signal (SP,en bleu), le glicentin-related polypeptide (GRPP, en vert),le glucagon (GLG, en orange), le peptide-1 (P1, en noir),la glicentine, l’oxyntomoduline (Oxm), le glucagon-like pep-tide 1 (GLP-1, en rose), le peptide-2 (P2, en violet), leglucagon-like peptide-2 (GLP-2, en jaune) et le fragmentmajeur du proglucagon (MPGF). En fonction du tissu, lesmodifications post-traductionnelles par les prohormones con-vertases 1 et 2 peuvent donner plusieurs combinaisons de cesp

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Figure 5 Skeletal phenotype observed in GLP-1r-deficientanimals. Bone microarchitectures were assessed by X-raymicrocomputed tomography. Three-dimensional models of tibiaproximal metaphysis observed in wild-type (WT) and GLP-1r-KOmice.Phénotype osseux observé dans les souris invalidées pour lerécepteur du GLP-1. Les microarchitectures trabéculaires etcorticales ont été analysées par microtomographie à rayons X.Des modèles 3D de la métaphyse proximale du tibia des sourissauvages (WT) et invalidées pour le récepteur du GLP-1 sontp

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eptides.

issues [61]. However, other studies failed to demonstratehe presence of GLP-1r in MC3T3-E1 [62] or primary murinesteoblasts or osteoclasts [42] and this discrepancy needso be investigated in the future. Nevertheless, previoustudies in other tissues such as liver and skeletal muscleevealed the presence of a second GLP-1 receptor different


rom the known GLP-1r in function and/or structure.ndeed, this second GLP-1 receptor does not activate theAMP pathway as GLP-1r does but rather the productionf inositolphosphoglycan as a second messenger [63,64].

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résentés.

uche-Berenguer et al. revealed the presence of thisecond GLP-1 receptor in the MC3T3-E1 murine osteoblasticell line [62]. However, as recent evidences pointed outhat GLP-1 could bind to the glucagon receptor in certainircumstances [57], it would be compulsory to ascertainhether this second GLP-1r correspond to a modulation of

igand specificity at the glucagon receptor.Here again, our understanding of GLP-1 actions in skele-

al physiology arises from GLP-1r-KO mouse. At 10 weeks ofge, GLP-1r-KO animals exhibited a mild reduction in tra-ecular bone volume at the tibia, although not significant,ssociated with increased number of osteoclasts and erodedurfaces [65]. Unpublished observation from our laboratoryade in the same KO model at 16 weeks of age corrobo-

ated these findings (Fig. 5). On the other hand, the mineralpposition and bone formation rates appeared unaffectedy GLP-1r inactivation [65]. Taken together these resultsuggested a control of bone resorption (osteoclast differ-ntiation and/or action) by the GLP-1r. However, GLP-1 wasnable to directly control osteoclast formation and resorp-ion in osteoclast cultures [65], suggesting that the controlf bone resorption was indirect. Indeed, these authors found

reduction in calcitonin gene expression in GLP-1r-deficientnimals. Now, we know that in rodents, but not in non-uman primate or humans, GLP-1r is expressed in C-cellsf the thyroid gland, and responsible for a rise in calci-onin secretion [66]. However, in humans, administration ofxogenous GLP-1 does not result into lower CTx levels [13].he effect of GLP-1r deficiency in cortical bone has been


nvestigated in 16 week-old GLP-1r-deficient mice. Thesenimals presented with reduction in bone strength observedy 3-point bending [42]. In these animals, modification ofortical microarchitecture was evidenced with reduction in



Bone and incretins

Figure 6 Tissue mineral density in wild-type (WT) and GLP-1r-deficient animals. Calcium map was obtained by quantitativebackscattered electron imaging at the mid-diaphysis of thefemur. The calcium content distribution was also plotted anddid not show any sign of tissue mineral density alterations inGLP-1r-deficient mice [42].Densité minérale de la matrice osseuse dans les animauxsauvages (WT) ou invalidés pour le récepteur du GLP-1. Lescartographies de concentration massique de calcium ont étéobtenues par microscopie quantitative en électrons rétrodif-fusés au centre de la diaphyse fémorale. La distribution desconcentrations calciques a été représentée graphiquement etn’a pas montré d’altération de densité minérale de la matrice

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dmaromiiiebdatbmimetic. Nevertheless, it is also important to bear in mind

osseuse dans les souris invalidées pour le récepteur du GLP-1[42].

cortical thickness [42]. Tissue material properties are alsoreduced at cortical site in GLP-1r-deficient animals and areassociated with reduced collagen maturity with a normaldistribution of the degree of mineralization [42] (Fig. 6).These data highlight that a functional GLP-1r is not onlyrequired for the control of bone resorption but also for thepreservation of an optimal bone matrix quality.

Several stable GLP-1 analogues, resistant to enzymaticdegradation and with amelioration of renal clearance havebeen designed and already approved for the treatment oftype-2 diabetes mellitus. Several pre-clinical and proof-of-


concept studies have been undertaken in animal models withskeletal fragility (T1DM, T2DM and OVX-induced osteoporo-sis). As such, administration of GLP-17-36NH2 or exendin-4,

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GLP-1r agonist, results in a rapid augmentation ofsteocalcin gene expression and reductions in sclerostinxpression and in the balance RANKL/OPG (receptor acti-ator of nuclear factor kappa-B ligand/osteoprotegerin) inhe bones of normal, type 2 diabetic or insulino-resistantats [59,67,68].

In type-1 diabetes mellitus, administration of liraglu-ide for 21 days contributed to significant improvementn bone strength at the tissue levels and reduction inollagen degradation in the bone matrix [48]. However,o improvements in neither trabecular nor cortical boneicroarchitecture were observed. Nevertheless, these mild

meliorations occurred in the absence of insulin secretion.ue to the marketing of GLP-1 mimetic for type 2 diabetes,he efficacy of GLP-1 analogues has been performed in type

diabetic rodent models. In this condition, the adminis-ration of liraglutide ameliorated trabecular and corticalone microarchitectures [69]. It is however worth notinghat the administration of liraglutide has been performedt a dose regiment of 0.4 mg/kg/day as compared withhe 0.02 mg/kg/day used in human clinical trials [70,71].he effects of GLP-1 mimetic have also been conducted insteoporotic rodent models. Indeed, 16 weeks administra-ion of exendin-4 (dose regimen of 10 �g/kg/day similar tohe dose used in humans [72]) in OVX rats, is capable ofmproving trabecular bone mass and microarchitecture athe femur and lumbar vertebra, bone strength and revertyper-resorption observed after ovariectomy [61,73]. It islso noteworthy that at this dose regimen, exendin-4 wasncapable of reversing the observed deterioration of corti-al microarchitecture. Pereira et al., also reported positiveffects of liraglutide (0.3 mg/kg/day) in improving tra-ecular but not cortical microarchitecture [61]. However,dministration of liraglutide at a dose of 1.8 mg/kg/dayor 8 weeks, significantly improved trabecular and corti-al microarchitecture [74]. This dose regimen is 90 timesore elevated than the dose given to humans. Moreover,

ecently, evidences have been provided that undercarboxy-ated osteocalcin was capable of inducing GLP-1 releaserom the L-cells and as such this complementary mechanismeinforced the role of osteocalcin in energy expenditure75,76].

Now several GLP-1 analogues, enzymatically resistant toegradation by DPP-4, have been approved for the treat-ent of type-2 diabetes mellitus. Regarding the skeletal

lterations observed in animals deficient in GLP-1r and theapid and favorable effects of GLP-1 or GLP-1r agonistsn bone gene expression, one could expect an improve-ent in bone quality in type-2 diabetic patients. However,

n a recent meta-analysis conducted by our group on thencidence of bone fractures in type-2 diabetic patients tak-ng GLP-1 mimetic, we failed to evidence any beneficialffects of GLP-1 mimetic [77]. This was further confirmedy investigation of the British clinical practice researchatalink, where 216,816 diabetic patients were screenednd dichotomized depending on their anti-diabetic medica-ions [78,79]. No significant reduction in the occurrence ofone fractures have been evidenced under the use of GLP-1


hat no increase of fracture risk was noted with these med-cations and that GLP-1 mimetics in humans are neutral inerm of bone safety.



8

Figure 7 Summary of actions of incretin hormones on bonephysiology. Incretin hormones improved bone mass, microarchi-tecture and quality by acting on several targets. First of all,GIP is capable of direct modulation of osteoblast and osteoclastbiology as well as modulation of adipokines and insulin secre-tion. GLP-1 can also modulate osteoblast and osteocyte actionsalthough further studies would be required in ascertaining themolecular mechanisms involved in such process. Furthermore,GLP-1 in rodents, but not in humans, is capable of increasing cal-citonin from the thyroid gland and hence interferes with calciummetabolism and bone physiology.Résumé des actions des hormones incrétines en physiolo-gie osseuse. Les hormones incrétines améliorent la masse,la microarchitecture et la qualité osseuse en agissant surplusieurs cibles. Premièrement, le GIP est capable de modulerdirectement la biologie des ostéoblastes et des ostéo-clastes mais également de moduler la sécrétion d’adipokineset d’insuline. Le GLP-1 peut aussi moduler les actionsdes ostéoblastes et des ostéocytes bien que des étudescomplémentaires soient requises pour identifier les mécan-ismes moléculaires impliqués dans cette voie. De plus, chezles rongeurs, mais pas chez l’homme, le GLP-1 est capabled’augmenter la sécrétion de calcitonine par la glande thyroïdeeg

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t ainsi interférer avec le métabolisme calcique et la physiolo-ie osseuse.

onclusions

clear link between incretins and bone physiology has beenemonstrated and incretins appear as potent modulatorsf bone mass but also of bone quality and ultimately bonetrength. A summary of incretin actions is presented Fig. 7.owever, the major challenge to face in this field in the

uture will be to ascertain by which mechanisms incretinsxert their control on skeletal physiology and to demon-trate the efficacy of incretin therapies in bone diseases withompromised bone strength.

isclosure of interest

he author declares that he has no competing interest.


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