Incretins and bone: friend or foe?
Guillaume Mabilleau
1,2Toadapttoitsvariousfunctions,thebonetissueisremodeled permanentlyandisundertheinfluenceofhormonal,local, mechanicalandnervoussignals.Amongthem,aroleforgut hormonesincontrollingbonemassandqualityhasemergedin therecentyears.Theaimofthisreviewistoprovidethereader withasummaryofrecentdevelopmentsintheinteraction betweenincretinhormonesandbonephysiology.
Addresses
1LUNAMUniversite´,GEROM-LHEA,InstitutdeBiologieenSante´, Angers,France
2LUNAMUniversite´,SCIAM,InstitutdeBiologieenSante´,Angers, France
Correspondingauthor:Mabilleau,Guillaume (guillaume.mabilleau@univ-angers.fr)
CurrentOpinioninPharmacology2015,22:72–78 ThisreviewcomesfromathemedissueonMusculoskeletal EditedbyJamesGallagherandGrahamRussell
http://dx.doi.org/10.1016/j.coph.2015.03.007 1471-4892/#2015ElsevierLtd.Allrightsreserved.
Introduction
Boneisatissuewithmultiplefunctions:(i)ithasastrong biomechanicalfunctionbysupportingthebodyweightand protectsessentialorgansfrompotentialmechanical inju- ries,(ii)itactsasacalcium,phosphateandsodiumreser- voir,(iii)itisahosttissueforhematopoieticbonemarrow and(iv)itisalsoanendocrineorganthatisinvolvedinthe regulation of glucose metabolism, energy expenditure, regulationof testosterone productionand phosphate ho- meostasis [1–4]. To adapt to these functions, bone is remodeled permanently by a coupling between osteo- clasts, the bone-resorbing cells, and osteoblasts, the bone-forming cells responsible for the synthesis of new structuralunits.Bone remodelingis traditionallyconsid- eredto beregulatedbyhormones(parathyroidhormone, calcitonin, estrogen, among others), autocrine/paracrine signals fromthemicroenvironment (receptoractivator of nuclearfactorkappaBligand,tumornecrosisfactor-alpha, among others), mechanical loading and the central and sympatheticnervoussystems.
Fromthemoleculartoanatomicallevels,bonesarebuilt toresistandadapttomechanicalstrainaccordingtofive
differentlevelsoforganization(reviewedin review[5]).
Theselevelsoforganizationincludethedualcomposition (organic vs. mineral phase) of the bone matrix, bone texture(lamellarvs.wovenbone),bonestructure(osteon vs. arch-like trabeculae), microarchitecture and macro- architecture.Assuch,anymodificationofthesedegreesof organization may compromise bone strength that ulti- matelyifnotcorrectedresultinbone fracture.
Recentlysomeevidences haveemergedthatagroup of guthormonesmayalsobeinvolvedinthemaintenanceof bonestrengthandqualitybyactingonbonecellphysiol- ogy.This bodyof evidencearises fromthe factthat (i) boneremodelingisreducedafterparenteralfeeding[6], (ii)thatthepatternof boneremodelingisrapidlymodi- fied after a meal [7] and (iii) that food fractionation is capable of altering bone resorption and increasing bone mineraldensitywithagreaterextentascomparedwitha matchednutrientloadgivenonceaday[8].Thegastroin- testinaltractproducesandreleasesaplethoraofbioactive peptides that act as neuromodulators/neurotransmitters, hormonesorlocalregulatorsofcellfunction.Amongthem, aclassofpeptidescalledincretinshaveemergedasimpor- tantmodulatorofenergymetabolism.Theterm‘incretin’
wasinitiallyproposedbyCreutzfeldin1979andrepresents hormonesthataresecretedfromtheintestineinresponse to glucose and stimulate insulin release in a glucose- dependentmanner [9]. Althoughseveral hormones with insulinotropic action are secreted by the gut, glucose- dependent insulinotropic polypeptide and glucagon-like peptide-1aretheonlytwophysiologicalincretinsidenti- fiedsofar[10].Oncereleased tothebloodstream,these twohormonesarerapidlydegradedbyanendopeptidase, thedipeptidylpeptidase4 (DPP-4).However,dueto the widelistofDPP-4substrates,theroleofDPP-4inhibitors inbonephysiologyisoutofthescopeofthisreviewand willnot bediscussedfurther.
The aim of thecurrent review is to provide thereader withacomprehensiveoverviewoftheeffectsofincretin hormonesonbonephysiology.
Glucose-dependentinsulinotropic polypeptide(GIP)
GIPisproducedbyintestinalK-cells,locatedprimarilyin proximalregions of thesmall intestine. GIP genetran- scriptionisincreasedinresponsetoglucoseandlipidsand 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 polypeptidethatisencodedbysixexonsrepresentinga 459-bpopenreadingframeandwhosegeneislocalizedin
humans on chromosome 17q [13,14]. The mature 42- amino acid bioactive form of GIP (GIP1–42), mainly encodedbyexons3and4,isreleasedfromitsprecursor via prohormone convertase 1/3-dependent posttransla- tional cleavageatflanking single arginineresidues [15].
Thepeptidesencodedwithintheremainingfragmentsof theproGIPhavenoknownbiologicalfunctions[16].The sequenceofGIPisextremelyconservedbetweenspecies and more than 90% homology is found among human, porcine, bovine, mouse, and rat [17]. The K-cell is an opentypeendocrinecellandassuchluminalcontentof thegutisthoughttoplayamajorroleinGIPsecretion.
The K-cell is highlypolarized with the GIP-containing secretory granules concentratedatthebasalpole of the cell,readytobereleasedfromsecretorygranulesthrough thebasolateralmembrane[18,19].Inaddition,K-cellsare found in close association with the capillary network runningthroughthelaminapropriaallowingGIPtoenter into thebloodstream. Based onthemorphological fea- tures, GIPsecretion from K-cells is regulated by intra- luminal contents,neuralstimuliand hormones.
Toexertitsbiologicalactions,GIPbindstoitsreceptor,the GIPr.Thehumangiprgenecomprises14exonsthatspan approximately13.8kb[20]andislocalizedonchromosome 19q13.3[21].TheGIPrbelongstothe7-transmembrane- spanningG-proteincoupledreceptor(GPCR)superfamily [22]andis composedof430aminoacids.TheGIPRis expressed in the endocrine pancreas, gastro-intestinal tract, adipose tissue, adrenal cortex, pituitary gland, vascular endothelium and several regions in the CNS [16].TheprincipalphysiologicalroleofGIPistoincrease insulin secretion from the pancreatic beta-cells in a glucose-dependent manner. However, extrapancreatic actions of GIP have been observed. GIP acts on lipid metabolismincludingaugmentationofplasmatriglycer- ide clearance, increased lipoprotein lipase activity and promotionoffatstorageinadipocytes[23–25].Inanimal 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 neuralprogenitorcellproliferationandbehavior[29].
Bone actionoftheGIP/GIPr pathway
TheGIPrhasbeendemonstratedtobeexpressedatthe mRNAandproteinlevelinseveralosteoblasticcelllines includingMG-63,TE-85,Saos-2,Ros17/2.8andMC3T3 [30–32], primary murine osteoblasts [31] and murine osteoclast precursors [33], suggesting a role for the GIP/GIPrpathwayinbone.Apolymorphism ofthegipr genecausedbysubstitutionofaguaninebyacytosineat rs1800437 induces a substitution of glutamate to gluta- mineatposition354inthesixthtransmembranedomain of GIPr resulting in decreased receptor activity [34].
Recently,Torekovandcolleaguesreportedthatthispoly- morphismisassociated,inaDanish-basedperimenopausal
women cohort, with low mineral bone density at the femoral neck and total hip but notat the lumbar spine [35].Furthermore,womencarryingthelowerfrequency C allele also presented with higher incidence of non- vertebral fractures [35]. Taken together, these data suggest a role for the GIP/GIPr pathway in controlling bonedensityandstrength.
AsummaryofGIP/GIPractionsonboneispresentedin Figure1.Ourunderstandingoftheactionofthispathway in bone has been greatly enhanced from studies con- ductedinGIPrknock-outmice.TwomodelsofGIPrKO have been developed. The first model consists in a deletionofexons4and5ofthegiprgene,whichencode aportion,butnotthetotality,oftheextracellulardomain of this receptor [36].The second modelconsists in the deletionofthefirst6exons,whichencodethetotalityof theextracellular domainandaportionofthefirsttrans- membranedomainoftheGIPr[37].Bothanimalmodels resultintoaninactiveGIP/GIPrpathwaybutsurprisingly theconsequencesofthisinactivationontrabecularbone are opposed. Indeed, in model 1, GIPr-deficient mice present withaugmentation infatmassandreductionof trabecularbonemassdetectableasearlyas4week-oldat thefemurand8week-oldatthetibia[38,39].Histomor- phometric analysis revealed aboneformation defectin theseanimalswithdecreasedmineralappositionrateand boneformationrateandanincreaseinosteoidmaturation
Figure1
GIP OSTEOBLAST
↑ Type 1 collagen expression
↑ Maturation of collagen
↑ Alkaline phosphatase activity
↑ Mineralisation of the matrix
↑ TGF-β secretion
PANCREATIC β-CELL
↑ insulin secretion
OSTEOCLAST
↓ Osteoclast formation
↓ Osteoclast activity
ADIPOCYTE Modification of adipokine
secretion
Current Opinion in Pharmacology
MechanismsofactionofGIPonbonecells.Uponentryofnutrients intothesmallintestine,GIPissecretedbyduodenalK-cellsintothe bloodstreamandactsontargetcells.WhenGIPbindstheGIPratthe surfaceofosteoblasts,itinducesexpressionoftype1collagenand TGF-beta,maturationofcollagenmatrix,augmentsalkaline
phosphataseactivityandmineralizationofthebonematrix.WhenGIP engagesitsreceptoratthesurfaceofosteoclastprecursors,itresults anaugmentationintoosteoclastformationandosteoclastresorption.
GIPalsostimulatesinsulinsecretionfrombeta-cellsofthepancreas thatcouldpotentiallymodifythepatternofosteoblastandosteoclast action.GIPalsoaffectsadipokinesecretionfromadipocytesthat couldpotentiallyaltertheactivityofbonecells.
rate[39].These animals exhibited alsoa reductionin circulating osteocalcin and an augmented leptin con- centration inplasma [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].
Thishighbonephenotypewasevenmorepronounced at45weeksofage suggestingthattheseanimals were protectedfromageing-inducedboneloss.Thispheno- typewasaccompaniedbyhighervaluesforthemineral appositionrateandthe boneformationrate[40].Plas- ma level of circulating osteocalcin was unchanged whilstleptinwasreducedandadiponectinaugmented [40]. To date, no satisfactory explanations have been formulatedtoexplainthetwoopposedbonephenotype 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 dramaticallyreducedandassociatedwithalteredmat- urationofthe mineralandcollagencomponent ofthe bonematrix [40].
Tothebestofourknowledge,thecorticalbonepheno- typehasonlybeeninvestigatedinmodel2.Nevertheless, augmentationofthemarrowdiameterwithanormalouter bonediameteratthefemurresultedindecreasedcortical thicknessandreducedbonestrengthatthewhole bone level[41].Investigationoftissuematerialpropertiesby nanoindentation,quantitativebackscatteredelectronim- aging andFourier-transform infrared microspectroscopy revealedalterations of bone strength atthetissue level associatedwithreductioninthedegreeofboneminerali- zation and collagen maturation [41]. Taken together thesedatasuggestapositiveroleoftheGIPrinorderto gainoptimalbonestrengthandquality.Thishypothesisis further strengthened by the improved tissue material propertiesand bonestrength in arodent modeltreated withexogenousGIP[42].
However,astheGIPriswidelydistributed,itisnotclear whetherthe aboveskeletal effects result from adirect actionoftheboneGIProractionofextraskeletalGIPr thatindirectly contributetotheobserved bonepheno- type. Nevertheless, few studies have investigated the direct action of GIP on osteoblast and osteoclast cul- tures. Administration of GIP in osteoblast cultures resultsinaugmentationinosteoblastproliferation,oste- oblast-produced TGF-beta, alkaline phosphatase and type1 collagenexpression [43,44]. Furthermore,addi- tionofGIP,atphysiologicalconcentration,inosteoblast cultures resulted in higher lysyl oxidase activity and collagen maturity [45]. Administration of exogenous GIPleadstoreductioninosteoclast-mediatedresorption as evidenced by reduction in circulating CTx levels [46], although conflicting results have been reported regardinga direct actionof GIPtomediate thiseffect [33,39].
Glucagon-likepeptide-1 (GLP-1)
The proglucagon gene is expressed at high levels in intestinalL-cells[47].L-cells canbefoundin theduo- denumbutoccurathighernumberintheileumandcolon [48].The singleproglucagongeneencodesa160-amino acidpeptidethatundergoes posttranslationalprocessing [49].InintestinalL-cells,afterprocessingbytheprohor- moneconvertase1/3,theproglucagongenegivesrise to glicentin,correspondingtothefirst69-aminoacidsofthe precursor, GLP-1 (amino acids 78–107) composed of 31 amino acids, peptide 2 (amino acids 111–123) and GLP-2(aminoacids126–158)comprising33aminoacids [50].Thus,processingoftheproglucagongeneinintes- tinal cells generates equimolar concentration of GLP-1 andGLP-2.Untilnow,littleisknownaboutthebiological actionsofglicentinandpeptide2.
TwoformsofGLP-1areproducedintheintestine,GLP- 17–36NH2 and GLP-17–37 although the major circulating formisGLP-17–36NH2[51].AswithK-cells,L-cellsarean opentypeendocrinecellshighlypolarizedwithsecretory granulesattheirbasolateralpole,readytoreleaseGLP-1- and GLP-2 intothe blood stream.L-cells are found in close association with the capillary network running throughthelaminapropria.Basedonthemorphological features, GLP-1 secretion from L-cells is regulated by intraluminal contents, neural stimuli and hormones.
GLP-1hasalsobeensuspectedtoactviatheautonomous nerve system on specific hypothalamic and brainstem nucleitoexert itsaction[52].
To act, GLP-1 engages its receptor, the GLP-1r expressedontargettissue.The humanglp1rgenecom- prisesalso14exonsandislocalizedonchromosome6p21.
GLP-1r also belongs to the 7-transmembrane-spanning GPCR superfamily like the GIPr. It is composed of 463 aminoacids[53] and isexpressed in theendocrine pancreas, gastro-intestinaltract,lung, heart,kidney and several regionsof the brain [16]. The principalphysio- logicalroleofGLP-1istopotentiateglucose-dependent insulinsecretion[54].Extrapancreaticactionsof GLP-1 results in reduction of food intake through the CNS, inhibition of gastric emptying, positive actions on the cardiovascular systemand a role in energy expenditure [54].
Boneactionofthe GLP-1/GLP-1rpathway PresenceoftheGLP-1rin bonecellsiscontroversial.It hasbeen evidenced atthe mRNAlevel in MG-63 and TE-85osteoblasticcelllines[30];however,thesecellsare notvery representative of osteoblast cells [55].On the otherhand,GLP-1rwasnotfoundinMC3T3-E1[56]or primarymurineosteoblastsorosteoclasts[57].Howev- er, previous studies in other tissues such as liver and skeletalmusclerevealedthepresenceofaGLP-1recep- tordifferentfromtheknownGLP-1rinfunctionand/or structure.Indeed, thissecond GLP-1receptor doesnot
activatethecAMPpathwayasGLP-1rdoesbutratherthe productionofinositolphosphoglycanasasecondmessen- ger [58,59]. Recently, Nuche-Berenguer et al. revealed thepresenceofasecondGLP-1receptorinMC3T3-E1 cellsdifferentfromtheknownGLP-1r[56].Indeed,the binding of (125I)GLP-1to this secondreceptor was dis- placed byaddition of coldGLP-1 but not exendin-4,a knownagonistoftheGLP-1r,andresultedinincreasein inositolphosphoglycan andactivationofPI3kinase[56].
However,nodataisavailableregardingthepresenceof thissecondGLP-1receptoronosteoclasts.Recently,Kim etal.evidencedthepresenceofGLP-1ratthemRNAand protein level in the MLO-Y4 osteocyte cell line [60].
However,whenasearch-for-specificityalignmentisper- formed with the primer-BLAST tool of the National CentreforBiotechnologyInformation(NCBI),itappears thattheprimerpairusedbyKimetal.wasnotspecificof the glp1rmRNA but might also reveal thepresence of timeless, a protein involvedin thecircadian clock (un- publisheddata).Furthermore,followingapublicationof Panjwanietal.in2013[61],agrowingbodyofevidence suggests thatmostof commercially availableantibodies used in immunohistochemistry and western blotting in publishedstudiesarenotspecificoftheGLP-1r[62,63].
As such it is difficult to ascertain whether the known GLP-1risexpressedinbonecells.Untilmoreconvincing studiesareperformed,scientistsmayhavetoconsiderthe possibilitythattheknownGLP-1rmaynotbeexpressed in bonecells.
A summary of GLP-1/GLP-1r actions on bone is pre- sented in Figure 2. Here again, our understanding of GLP-1 actions in skeletalphysiology arises from GLP- 1r KO mouse.At10 weeksofage, GLP-1r KOanimals exhibitedamildreductionintrabecularbonevolumeat the tibia, although not significant, associated with in- creased numberofosteoclastsand erodedsurfaces[64].
Unpublishedobservationfromourlabmadeinthesame KOmodelat16weeksofagecorroboratedthesefindings.
On the other hand, the mineral apposition and bone formationratesappearedunaffectedbyGLP-1rinactiva- tion[64].Takentogethertheseresultssuggestedacon- trolof boneresorption (osteoclast differentiation and/or action) bytheGLP-1r.However, GLP-1was unableto directly control osteoclast formation and resorption in osteoclast cultures [64], suggesting that the control of boneresorptionwasindirect.Indeed,theseauthorsfound a reduction in calcitonin gene expression in GLP-1r- deficient animals. Now, we know that in rodents, but not in non-human primate or humans, GLP-1r is expressedinC-cellsofthethyroidgland,andresponsible forariseincalcitoninsecretion[65].However,inhumans, administration ofexogenousGLP-1doesnotresultinto lowerCTxlevels[7].
TheeffectofGLP-1rdeficiencyincorticalbonehasbeen investigated in 16 week-old GLP-1r-deficient mice.
Theseanimalspresentedwithreductioninbonestrength 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 toalowercorticalthickness[57].Tissuematerialprop- ertiesarealsoreducedatcorticalsiteinGLP-1r-deficient animalsandareassociatedwithreducedcollagenmaturi- tywithanormaldistributionofthedegreeofmineraliza- tion[57].ThesedatahighlightedthatafunctionalGLP- 1risnotonlyrequiredforthecontrolofboneresorption but also for thepreservation of an optimal bonematrix quality.
AdministrationofGLP-17–36NH2orexendin-4,aGLP-1r agonist, results in a rapid augmentation of osteocalcin geneexpressionandareductioninthebalanceRANKL/
OPGinthebonesofnormal,type2diabeticorinsulino- resistantrats[66,67].Furthermore,16weeksadministra- tion of exendin-4 in OVX rats, iscapable of improving trabecularbonemassandmicroarchitectureatthefemur and lumbar vertebra, bone strength and revert hyper- resorption observedafter ovariectomy [68].Moreover, theadministrationofexendin-4intotype2diabeticani- mals was also capable of reducing sclerostinexpression and improving bone mineral density [60]. Recently, evidences have been provided that undercarboxylated osteocalcinwascapableofinducingGLP-1releasefrom theL-cells andas suchthiscomplementarymechanism reinforces therole of osteocalcinin energyexpenditure [69,70]. However, as the GLP-1r is not expressed in
Figure2
C-CELLS THYROID GLAND
↑ Calcitonin secretion PANCREATIC β-CELLS
↑ insulin secretion
L-CELLS ILEUM
GLP-1
BONE CELLS
Current Opinion in Pharmacology
MechanismsofactionofGLP-1onbonecells.Uponentryofnutrients intothesmallintestine,GLP-1issecretedbyilealL-cellsintothe bloodstreamandactsontargetcells.TheknownGLP-1rreceptoris notexpressedinosteoblastorosteoclast,butontheotherhand, GLP-1mightactdirectlyonosteoblastphysiologybybindingtoa secondGLP-1receptor.Ontheotherhand,inrodent,theGLP-1ris expressedinC-cellsofthethyroidandisinvolvedincalcitonin secretionthatmightexertanti-resorptiveactionsinbone.Furthermore, asGLP-1isanincretinhormone,itpotentiatesinsulinsecretionfrom pancreaticb-cellsandasinsulinisconsideredanabolicforbone,itis plausiblethatinsulinmediatessomeoftheobservedboneeffects.
OsteocalcinproducesbyosteoblastsiscapableofenhancingGLP-1 secretionfromilealL-cells.
bonecells,themajorchallengetofaceinthenearfuture wouldbetoascertainhowtheGLP-1/GLP-1rpathwayin othertissuesmaycontrolbone physiology.
NowseveralGLP-1analogues,enzymaticallyresistantto DPP-4 degradation, have been approved for the treat- mentoftype2diabetesmellitus.Regardingtheskeletal alterationsobservedin animalsdeficientin GLP-1rand the rapid and favorable effects of GLP-1 or GLP-1r agonist on bone gene expression, one could expect an improvementinbonequalityintype2diabeticpatients.
However, in a recent meta-analysis conducted by our groupontheincidenceofbonefracturesintype2diabetic patientstakingGLP-1mimetic,wefailedtoevidenceany beneficialeffects ofGLP-1mimetic[71].
Conclusions
A linkbetween incretin and bone physiology has been highlightedinthepastyearssuggestingthatincretinare essential for the maintenanceof bone massbut also to gain optimal bone quality and strength. However, the majorchallengetofaceinthisfieldinthefuturewillbeto ascertainbywhichmechanismsincretinsexerttheircon- trolonskeletalphysiology.
Conflictofintereststatement Nothingdeclared.
Acknowledgements
GMisgratefultomembersoftheGEROM-LHEAlab(LUNAM Universite´,Angers,France)andDiabetesgroup(UlsterUniversity, Coleraine,UK)fortheirinvaluablehelpinhisresearchesinthisfield.This workwassupportedbygrantsfromtheUniversityofAngersandSocie´te´
Franc¸aisedeRhumatologie.
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