research article loss of insulin receptor in...

Research ArticleLoss of Insulin Receptor in Osteoprogenitor Cells ImpairsStructural Strength of Bone

Kathryn Thrailkill12 R Clay Bunn1 Charles Lumpkin Jr13 Elizabeth Wahl13

Gael Cockrell1 Lindsey Morris14 C Ronald Kahn5 John Fowlkes1 and Jeffry S Nyman678

1 Department of Pediatrics University of Arkansas for Medical Sciences Little Rock AR USA2Arkansas Childrenrsquos Hospital 1 Childrenrsquos Way Slot 512-6 Little Rock AR 72202 USA3Arkansas Childrenrsquos Hospital Research Institute Little Rock AR USA4Division of Diabetes Endocrinology amp Metabolism Department of Medicine Vanderbilt University School of MedicineNashville TN USA

5 Joslin Diabetes Center and Harvard Medical School Boston MA USA6VA Tennessee Valley Health Care System Vanderbilt University Medical Center Nashville TN USA7Department of Orthopaedic Surgery amp Rehabilitation Vanderbilt University Medical Center Nashville TN USA8Center for Bone Biology Vanderbilt University Medical Center Nashville TN USA

Correspondence should be addressed to KathrynThrailkill thrailkillkathrynmuamsedu

Received 7 February 2014 Accepted 30 April 2014 Published 18 May 2014

Academic Editor Md Shahidul Islam

Copyright copy 2014 KathrynThrailkill et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Type 1 diabetes mellitus (T1D) is associated with decreased bone mineral density a deficit in bone structure and subsequently anincreased risk of fragility fracture These clinical observations paralleled by animal models of T1D suggest that the insulinopeniaof T1D has a deleterious effect on bone To further examine the action of insulin signaling on bone development we generatedmicewith an osteoprogenitor-selective (osterix-Cre) ablation of the insulin receptor (IR) designated OIRKO OIRKOmice exhibited an80 decrease in IR in osteoblasts Prenatal elimination of IR did not affect fetal survival or gross morphology However loss of IRin mouse osteoblasts resulted in a postnatal growth-constricted phenotype By 10ndash12 weeks of age femurs of OIRKO mice weremore slender with a thinner diaphyseal cortex and consequently a decrease in whole bone strength when subjected to bendingIn male mice alone decreased metaphyseal trabecular bone with thinner and more rodlike trabeculae was also observed OIRKOmice did not however exhibit abnormal glucose tolerance The skeletal phenotype of the OIRKO mouse appeared more severethan that of previously reported bone-specific IR knockdown models and confirms that insulin receptor expression in osteoblastsis critically important for proper bone development and maintenance of structural integrity

1 IntroductionSkeletal health is compromised in personswith type 1 diabetesmellitus (T1D) as characterized by decreased bone mineraldensity (BMD) a deficit in bone structure and subsequentlyan increased risk of fragility fractures [1 2] Indeed T1Dhas been listed among the top 10 factors associated with thehighest risk of fracture [3] Because T1D is characterizedin humans and rodents as a state of insulin deficiency dueto destruction of pancreatic beta cells (either autoimmune-mediated or chemically induced) a role for insulinopenia in

the pathogenesis of osteoporosis and increased fracture risknow categorized as diabetic bone disease has been postulated[4]

Insulin has been demonstrated in vitro and in vivo to be anosteogenic hormone Primary osteoblasts and osteoblast-likecells in culture bind insulin and respond to insulin treatment[5ndash7] as is evidenced by increased cell proliferation rates [89] enhanced collagen synthesis [6 10ndash12] secretion of boneformation markers [13] and increased uptake of glucose [1415] In vivo systemic insulin therapy can prevent or reverse

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2014 Article ID 703589 9 pageshttpdxdoiorg1011552014703589

2 Journal of Diabetes Research

skeletal deficits which occur in rodentmodels of T1D includ-ing improving bone strength and biomechanical integrity[16ndash19] Even local insulin delivery to fracture sites in diabeticrodents can ameliorate poor fracture healing which occursin T1D [20] Taken together these data suggest that insulinmay play a critical role in normal osteoblastogenesis andthat conditions of insulin deficiency may lead to abnormalskeletogenesis

A direct link between insulin signaling and bone for-mation in vivo is supported by transgenic mouse modelsKnockdown of the insulin receptor (IR) in osteoblasts resultsin altered bone formation [21] and in abnormal trabeculararchitecture [22] These studies support a role for insulin inmature osteoblasts Ferron et al [21] report a 60knockdownof the Insr using the Col1a1-Cre mouse while Fulzele et al[22] have eliminated the Insr in mature osteoblasts using theosteocalcin- (OC-) Cre construct

To examine further the specific action of insulin signalingon bone we generated mice lacking the insulin receptor inosteoprogenitor cells Unlike previous reports that deletedIR in mature osteoblasts [21 22] we assessed the effect ofIR deletion at the early stages of osteoblast differentiation(osterix) on the architectural structural and biomechanicalproperties of bone We hypothesized that the ablation ofinsulin signaling prenatally in cells of the osteoblast lineagevia a loss of IR would impair the fracture resistance of mousebones independent of body size

2 Materials and Study Design

21 Ethics Statement This research protocol was approvedby the Institutional Animal Care and Use Committee of theUniversity of Arkansas for Medical Sciences

22 OIRKO Mice Our experimental objective was to gen-erate mice lacking the insulin receptor in osteoblast lineagecells considered as precursors to previously reported models(ie Col1a1-Cre or osteocalcin- (OC-) Cre) Therefore togenerate mice with an osteoprogenitor-selective ablation ofthe insulin receptor (OIRKO) we first crossed mice thatwere homozygous for a floxed insulin receptor (IR) allelein which loxP sites flank exon 4 of the IR gene (designatedIRloxlox) [23] with heterozygous osterix- (Osx-) Cre transgenicmice (B6Cg-Tg(Sp7-tTAtetO-EGFPcre)1AmcJ The Jack-son Laboratory Bar Harbor ME) F1 progeny were crossedto produce IRloxloxCre+minus(OIRKO) and IRloxloxCreminusminus micewhich were then bred to produce experimental animals TheIRloxloxCreminusminus mouse (IR flox) was designated as the controlfor this study in keeping with previous reports [21 22] Cretransgene expression is repressed when mice receive chowcontaining doxycycline (Diet S3888 Bio-Serv FrenchtownNJ) Upon switching mice to a regular chow diet (Diet 8640Harlan Indianapolis IN) the Cre transgene is expressed inthe osteoblast lineage resulting in disruption of the floxedIR alleles in the osteoblast lineage In this study pregnantfemales were switched to regular chow approximately 1 weekprior to birth of experimental pups Nursing dams weremaintained on regular chow and experimental mice were

weaned to a regular chow diet Genotyping was performedusing published procedures [23]

Postnatal growth as assessed by weight gain was moni-tored in both male (119899 = 15 IR flox (6) + OIRKO (9)) andfemale (119899 = 15 IR flox (6) + OIRKO (9)) mice at 4 6 8 and10ndash12 weeks of age (Figure 1) Detailed metabolic and skeletalassessments were then completed

23 Metabolic Assessment Between 10-11 weeks of age inmales and 12-13 weeks of age in females a 150-minute glucosetolerance test (GTT) was performed Following an overnightfast blood glucosemeasurements were obtained from all ani-mals before intraperitoneal injection of a 2 gkg body weightglucose bolus Blood glucose levels were then measured at15 30 45 60 and 150 minutes following the glucose bolusIn males only insulin levels were also measured at 0 1530 45 60 and 150 minutes Blood glucose was measuredvia glucometer (OneTouchUltra2 BloodGlucoseMonitoringSystem Lifescan Inc Milpitas CA average intra-assay coef-ficient of variation of 17 across a range of 40ndash300mgdLtarget glucose concentrations) Serum insulin levels weremeasured using a mouse insulin ultrasensitive ELISA (80-INSMSU-E01 ALPCO Immunoassays SalemNH sensitivity0115 ngmL intra-assay coefficient of variation 63 inter-assay coefficient of variation 1061) Glucose (males andfemales) and insulin (males only) area-under-curve (AUC)an integrated measure of glucose tolerance was determinedusing GraphPad PRISM software (version 502) Serumtotal osteocalcin (OC) was measured using the MILLIPLEXMAP Mouse Osteocalcin-Single Plex assay (MBN-41 K-10CMillipore Corp Billerica MA sensitivity 87 pgmL intra-assay coefficient of variation 112 inter-assay coefficient ofvariation 75) Serum amino-terminal propeptide of type 1procollagen (PINP)wasmeasured using theRatMouse PINPEIA (AC-33F1 Immunodiagnostics Systems Ltd FountainHills AZ sensitivity 07 ngmL) Serum c-terminal telopep-tide of type 1 collagen a marker of bone resorption was mea-sured using the RatLAPs ELISA (AC-06F1 Immunodiagnos-tics Systems Inc Scottsdale AZ sensitivity 17 ngmL intra-assay coefficient of variation 58 inter-assay coefficient ofvariation 104)

24 Skeletal Assessment After euthanasia (males sim10-11weeks females sim12-13 weeks) the left tibia and femur wereharvested from IR flox (6 male and 6 female) and OIRKO(9 male and 9 female) littermates and stored in phosphatebuffered saline (PBS) at minus20∘C until assessment H amp E stain-ing of the tibial growth plate was performed as previouslydescribed [24] Femur length was measured using calipersFemurs were immersed in PBS at room temperature and thelong axis of the bone was aligned with the scanning axisof the 120583CT40 (Scanco Medical Bruttisellen Switzerland)All scans were acquired with an isotropic voxel size of12 120583m using the same energy settings (70 kVp0114mA)acquisition parameters (integration time of 300ms with 1000projections per full rotation) and calibration to a hydrox-yapatite (HA) phantom with a beam hardening correctionfrom Scanco Medical as we have previously described [25]

Journal of Diabetes Research 3

minus + minus + minus + minus + minus + minus +

minus + minus + minus + minus +

Cre

Cre

IRΔIR

IRΔIR

Calv

aria

Tibi

a

Live

r

S M

uscle

Panc

reas

Kidn

ey

WAT BA

T

Brai

n

Inte

stine

(a)

IR 120573-subunit

GAPDH

OIRKOIR flox

(b)

80100120140160180200220240260

7-8 11-12

80100120140160180200220240260280300

64Weeks of age

Wei

ght (

g)W

eigh

t (g) lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

IR floxOIRKO

(c)

OIRKOIR flox

(d)

Female

Male

OIRKOIR flox

(e)

10 mm

Figure 1 OIRKO mice are smaller in size with slighter bones PCR of the IR locus revealed that recombination of floxed IR alleles occursonly in bone ((a) ΔIR calvaria tibia) OIRKOmice exhibited an sim80 decrease in IR in whole bone byWestern blot (119899 = 3 IR flox 3 OIRKOtibiae (b)) Loss of the insulin receptor in osteoblasts reduced the size of male and female mice Postnatal growth curves for male ((c) top)and female ((c) bottom) IR flox and OIRKOmice are shown (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) Disruption in the growth plate wasnot apparent (a representative H amp E stain is shown (d)) 120583CT images of the femur midshaft demonstrated that femurs from OIRKO miceare narrower than from comparably aged IR flox mice (e)

The regions of interest (ROI) included the trabecular boneof the metaphysis (024ndash140mm above the growth plate)and the cortical bone of the midshaft (119mm in lengthspanning the midpoint between the condyles and femoralneck) Each ROI had a unique threshold (406mgHAcm3and 774mgHAcm3 for trabecular and cortical bone resp)and Gaussian noise filter (sigma of 02 with support of 2for trabecular bone and sigma of 08 with support of 2

for cortical bone) that were used for all scans Standardevaluation scripts from the manufacturer were used to deter-mine the architectural and structural properties of trabecularand cortical bone respectively (Table 1) Moreover in thecalculations of tissue mineral density (TMD) partial volumeeffects were suppressed by peeling the first voxel from allsurfaces following segmentation of the boneThe slendernessof the femur was calculated as the caliper-derived length of


Table 1 Selected properties of bone as determined by 120583CT and three-point bending for males (top) and females (bottom) comparingcontrol (IR flox) and OIRKO mice

Property (male) Units IR flox (119899 = 6) OIRKO (119899 = 9) 119875-valueMetaphysis (trabecular bone)

BVTV 1171 plusmn 094 895 plusmn 204 0009TbN mmminus1 515 plusmn 033 495 plusmn 061 047TbTh mm 00392 plusmn 0002 00344 plusmn 0002 lt0001TbSp mm 0195 plusmn 0013 0206 plusmn 0028 038SMIa mdash 233 plusmn 008 261 plusmn 020 0006TbTMD mgHAcm3

901 plusmn 8 888 plusmn 17 010Diaphysis (cortical bone)

MaV mm3122 plusmn 012 103 plusmn 011 0005

CtTh mm 0148 plusmn 0011 0124 plusmn 0011 0001Imin mm4

0102 plusmn 0007 0066 plusmn 0009 lt0001CtAr mm2

0640 plusmn 0033 04892 plusmn 0044 lt0001Length mm 1370 plusmn 035 1259 plusmn 036 lt0001Slendernessb mmmm2

208 plusmn 07 251 plusmn 22 lt0001CtTMD mgHAcm3

1185 plusmn 27 1176 plusmn 25 049Stiffness Nmm 407 plusmn 85 369 plusmn 126 057Modulus GPa 42 plusmn 08 61 plusmn 26 0137Bending strength MPa 147 plusmn 17 172 plusmn 36 0177Property (female) Units IR flox (119899 = 6) OIRKO (119899 = 9) 119875-value

Metaphysis (trabecular bone)BVTV 438 plusmn 092 484 plusmn 152 052TbN mmminus1 337 plusmn 033 335 plusmn 059 095TbTh mm 0037 plusmn 0002 0036 plusmn 0004 053TbSp mm 0302 plusmn 0032 0316 plusmn 0084 069SMIa mdash 319 plusmn 017 280 plusmn 018 0001TbTMD mgHAcm3



CtTh mm 0162 plusmn 0005 0138 plusmn 0011 lt0001Imin mm4



213 plusmn 0687 235 plusmn 1236 0002CtTMD mgHAcm3

1251 plusmn 15 1209 plusmn 13 lt0001Stiffness Nmm 806 plusmn 115 573 plusmn 67 lt0001Modulus GPa 94 plusmn 15 85 plusmn 12 026Bending strength MPa 166 plusmn 10 161 plusmn 13 050aStructuralmodel index characterizes the shape of trabecular bone (1 platelike 3 rodlike) bSlenderness is the ratio of the length to the total cross-sectional areaof the femurmidshaft Abbreviations BVTV bone volumetissue volume TbN trabecular number TbTh trabecular thickness TbSp trabecular separationSMI structural model index TbTMD trabecular tissue mineral density MaV marrow volume CtTh cortical thickness Imin moment of inertia CtArcortical area CtTMD cortical tissue mineral density Significant differences are highlighted in bold font

the bone divided by the 120583CT-derived total cross-sectionalarea of the midshaft (ie average cross-sectional area includ-ing medullary canal)

25 BiochemicalMolecular Testing Tissues were harvestedfrom animals and immediately frozen in liquid nitrogenApproximately 20 milligrams of tissue was digested at 58∘Covernight in 200120583L of buffer (50mM Tris-HCl pH 8005 Triton X-100) containing Proteinase K (1mgmL) One

microliter of digested tissue was used in PCR reactionsRecombination of the floxed insulin receptor allele was eval-uated by the polymerase chain reaction using the followingprimer pairs that amplify exons 3ndash5 51015840-GATGTGCACCCC-ATGTCTG-31015840 51015840-CTGAATAGCTGAGACCACAG-31015840 and51015840-ACGCCTACACATCACATGC-31015840 PCR reactions werehot starting at 95∘C for 15 minutes followed by 35 cyclesof 94∘C for 45 seconds 60∘C for 1 minute and 72∘C for 1minute with a final extension step of 72∘C for 5 minutes


All reactions were performed on an MJ Research PTC-200thermal cycler Western blots were performed with anti-bodies directed against insulin receptor beta subunit (SantaCruz Biotechnology Dallas TX catalog sc-711 1 200) andGAPDH (EMD Millipore Billerica MA catalog MAB3741 10000) as described [26]

26 Biomechanical Testing To determine the differences instrength between OIRKO and IR flox mice a preload (1N)held each hydrated femur in place on the lower supportpoints of a three-point bending fixture with the anteriorside down (ie bending about the medial-lateral plane)The span between the lower supports was 8mm and theload rate was 30mmmin Then forces from a 100N loadcell (Honeywell Morristown NJ) and displacements fromthe LVDT (Dynamight 8841 Instron Canton OH) wererecorded at 50Hz during a monotonic load-to-failure testMeasured differences in biomechanical properties includedstiffness and the peak force endured by the bone Materialproperties of modulus and strength of the cortex were alsoestimated using standard beam theory [27] The 120583CT scansprovided the moment of inertia and the distance betweenthe neutral axis of bending and the outermost point in theanterior-posterior direction (119862min)

To ascertain if any skeletal differences in the OIRKOmice could be attributed to the presence of the Cre trans-gene alone 120583CT and three-point bending were also carriedout in 8-week-old male mice that were Creminusminus or Cre+minus(Supplemental Table 1 in Supplementary Material availableonline at httpdxdoiorg1011552014703589) on a wild-type background (These mice were generated from breedingof IR++Osx-Creminusminus and IR++Osx-Cre+minus animals)

27 Statistical Analysis Statistically significant differencesbetween groups (ie IR flox versus OIRKO) within eachgender were detected using the unpaired Studentrsquos t-testData are reported as the mean plusmn the standard deviation (SD)unless otherwise specified and differences were consideredstatistically significant at 119875 lt 005

3 Results

Prenatal elimination of the IR did not appear to affectfetal survival OIRKO mice and IR flox control mice wereborn with the expected Mendelian frequencies PCR of theIR locus indicated that recombination of floxed IR alleles(ΔIR) occurred only in bone (Figure 1(a) calvaria tibia)Assessments of the efficiency and specificity of the IR knock-out revealed an sim80 decrease (range 44ndash97) in IR inwhole bone by Western blot (Figure 1(b)) Both male andfemale OIRKOmice appeared grossly normal however bothmale and female OIRKO mice displayed a postnatal growthconstriction evident by significant reductions in weightgain (Figure 1(c)) and by shorter femur length (Table 1)This occurred despite normally appearing growth plates(Figure 1(d)) Micro-CT images of the femur midshaft alsodemonstrated that femurs from OIRKO mice were narrowerthan those from IR flox mice (Figure 1(e))

Not surprising given their overall size reduction OIRKOmice demonstrated a striking skeletal phenotype but in anotably gender-specific manner For male mice the loss ofIR in osteoprogenitors resulted in a lower trabecular bonevolume fraction (BVTV) with thinner (TbTh) more rodliketrabeculae in the femur metaphysis (Table 1) In contrast forfemale OIRKO mice trabecular bone was largely unaffectedwith less rodlike trabeculae In the diaphyseal compartmenthowever the cortices of femurs from both male and femaleOIRKO mice were thinner and exhibited lower structuralparameters (eg bone cross-sectional area or CtAr) com-pared with the cortices from IR flox mice With a smallermoment of inertia (119868min Table 1) loss of IR in osteoblastscaused a decrease in the structural strength of the femurin bending compared with IR flox bones (Figure 2 peakforce) A decrease in cortical tissuemineral density (CtTMDTable 1) was also seen in female OIRKOmice However therewas no difference in the estimated bending strength betweengenotypes

As determined by the ratio of bone length to total cross-sectional area (TtAR) the femur slenderness for OIRKOmice was considerably greater than the femur slendernessfor IR flox bones This structural impairment in the OIRKOmice was decoupled from body weight because the ratioof slenderness to body weight (LengthTtAR per BW) washigher for knockout than for control mice of both genders(Figures 2(a) and 2(b))That is bones fromOIRKOmiceweremore slender (ie more susceptible to failure) than expectedfor their reduced body weight

While Cre expression alone in the (Osx)-Cre+minus malemouse has been shown in one study to negatively affectweight at 4 weeks of age the effect of Cre was not evidentby 12 weeks of age [28] We also observed no differences inweight between (Osx)-Creminusminus and (Osx)-Cre+minusmalemice at 8weeks of age (Supplemental Table 1)Moreover those skeletalparameters that were significantly affected in the OIRKOphenotype (Table 1) were not significantly different betweenCreminusminus and Cre+minus genotypes suggesting that the skeletalphenotype observed in male and female OIRKO mice isaccounted for principally through the diminished expressionof the IR

Because insulin signaling in bone has been linked byothers to a feed-forward mechanism to activate osteocalcinwhich in turn regulates glucose homeostasis [21 22] wealso examined the metabolic phenotype of the OIRKO mice(Table 2) In this model there were no significant abnormal-ities in fasting blood glucose or insulin levels or in glucoseor insulin dynamics after a glucose challenge (Table 2) inOIRKO mice compared with IR flox mice Moreover totalosteocalcin levels were not significantly different betweengroups in either male or female mice While undercarboxy-lated osteocalcin has been reported to be lower in micein which the IR has been eliminated in mature osteoblasts[21 22] this was not measured in our study due to thelack of a validated and commercially available assay forundercarboxylated osteocalcin in mice

Osteoclast activity was assessed by examining systemicconcentrations of c-terminal telopeptide of type 1 collagen


00

05

10

15

20

IR flox OIRKO

P = 00006

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(a)

00

05

10

15

20

IR flox OIRKO

P = 0002

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(b)

10

15

IR flox OIRKO

Peak

forc

e (N

)

P = 0012

(c)

10

15

Peak

forc

e (N

)

IR flox OIRKO

P = 00003

(d)

Figure 2 OIRKO femurs are weaker and more slender Loss of IR in osteoblasts caused the bone to be more slender (ratio of length to totalcross-sectional area) relative to body weight (LengthTtAR per BW (a) male (b) female) Loss of IR in osteoblasts also caused a decreasein the structural strength of the femur in bending (peak force (c) male (d) female) Data is displayed as a scatter-plot with mean and SEMindicated

Table 2 Metabolic phenotype of males (top) and females (bottom) comparing control (IR flox) and OIRKO mice

Units IR flox (119899 = 6) OIRKO (119899 = 9) 119875 valueMeasurements (male)

Final body weight g 247 plusmn 22 193 plusmn 20 lt0001Fasting insulin ngmL 014 plusmn 004 026 plusmn 022 024Fasting blood glucose mgdL 678 plusmn 114 6811 plusmn 114 097Insulin AUC mdash 438 plusmn 170 405 plusmn 89 063Glucose AUC mdash 46660 plusmn 10660 43450 plusmn 10328 057OC ngmL 1096 plusmn 97 995 plusmn 387 054RatLAPS ngmL 251 plusmn 23 213 plusmn 38 005

Measurements (female)Final body weight g 231 plusmn 26 176 plusmn 24 0001Fasting blood glucose mgdL 527 plusmn 76 526 plusmn 116 098Glucose AUC mdash 27781 plusmn 3985 30695 plusmn 5300 027OC ngmL 1207 plusmn 381 925 plusmn 350 016RatLAPS ngmL 255 plusmn 51 238 plusmn 76

a 064Abbreviations AUC Area under the curve OC osteocalcin RatLAPS a marker of bone resorption a119899 = 8 for this measurement Significant differences arehighlighted in bold font


(ie RatLAPS) C-terminal telopeptide of type 1 collagen wasmodestly lower in male OIRKO mice (Table 2) comparedwith IR flox mice but not different when genders werecombined (119875 = 015) Osteogenesis was further assessed bymeasuring systemic concentrations of PINP these concen-trations were not different between OIRKO mice and IR floxmice (119875 = 097)

4 Discussion

Elimination of the insulin receptor specifically in osteopro-genitor cells affords the opportunity to study the impact ofthe IR and its ligand in vivo on bone development withoutthe metabolic consequences associated with eliminating theIR in all tissues which consistently leads to hyperinsu-linemia OIRKO mice also provide a model of osteoblast-specific decreased insulin action as expected in patientswith suboptimally controlled T1D but without concurrenthyperglycemia As such loss of IR in the osteoblast lineageresulted in a growth-constricted phenotype Although thefemur length indicative of appendicular bone growth wasreduced in the OIRKO the thickness of the diaphyseal cortexwas more greatly affected resulting in a more slender femurwhich manifested a decrease in the structural strength Inmale mice alone decreased metaphyseal trabecular bonewith thinner andmore rodlike trabeculae was also observedMoreover the skeletal phenotype of the OIRKO mouseappears more severe than that reported using the Col1a1-Cre or the OC-Cre construct [21 22] suggesting that earlierelimination of the IR in osteoblast development may providefurther insights into its overall role in osteoblastogenesis

The reduced body size of both male and female OIRKOmice was evident as early as at 4 weeks of age and persistedthroughout this study By 10ndash12weeks of age amicroarchitec-tural skeletal phenotype was clearly evident It remains to beseen how elimination of IR in this model will impact skeletalintegrity bone strength and bone healing asOIRKOmice agefurther thoughmore profound deficitsmay become apparentin older mice Studies are ongoing to address this question

In mice made insulin deficient insulin treatment canrestore trabecular architecture suggesting that insulin maybe involved in trabecular homeostasis [19] Fulzele et al haveshown that lack of insulin receptor signaling in osteoblastsalters trabecular acquisition as evidenced by decreasedBVTV TbN and TbTh and increased TbSp in youngmale mice (3ndash6 weeks of age) lacking IR expression inmature osteoblasts [22] However these differences appear todissipate as the mice age [22] In the current study BVTVand TbTh were decreased in male OIRKO mice consistentwith the previous findings by Fulzele et al [22] howeverwe did not study older OIRKO mice to see if the trabecularphenotype persists Indeed in slightly older female OIRKOmice no major differences were noted in the trabecularcompartment however these differences between OIRKOmale and female mice might not simply be attributable toage but may reflect the impact of gender as estrogen has aprotective effect on metaphyseal bone [29]

As in previous studies investigating osteoblast-specificdeletion of IR in male mice [21 22] we too observed amodest reduction in a serummarker of bone resorption in themale OIRKO mouse (but not in the female OIRKO mouse)Nevertheless decreased resorption in male OIRKO mice didnot translate into elevated glucose levels or changes in insulindynamics in theOIRKOmodelThis lack of glucose or insulindysregulation varies from the observations of others in malemice wherein IR expression in osteoblasts was reducedusing OC-Cre and Col1a1-Cre [21 22] It was anticipated thateliminating the IR in osteoprogenitors rather than in moremature osteoblasts might cause an evenmore profound effecton glucose homeostasis more consistent with frank diabetesHowever within each gender of OIRKO mice no significantdifferences in glucose tolerance were seen suggesting amore complex connection between osteoblasts and energymetabolism than previously appreciated Additionally totalosteocalcin levels were not significantly different betweenIR flox and OIRKO mice Total osteocalcin levels havebeen reported to be suppressed in male mice in which theIR was eliminated from mature osteoblasts using the OC-Cre construct yet when the IR in osteoblasts is eliminatedusing the Col1a1-Cre no difference in osteocalcin levels wasobserved [21 22] similarly to the OIRKO mouse

At present little is known about the specific mecha-nisms underlying diabetic bone disease While the currentstudy does not yet elucidate mechanistic pathways by whichinsulin signaling is critical to osteoblastogenesis we caninfer that insulin receptor expression in osteoblasts is quiteimportant for proper bone development and maintenanceof structural integrity Further mechanistic analysis of theOIRKO genotype in future studies will be necessary toexplain the observed phenotype and studies using thesegeneticallymodifiedmice should broaden our understandingof diabetic bone disease From the present studies howeverwe hypothesize that in T1D insulin deficiency leadingto long-term decreased insulin receptor signaling in thegrowing bones of children and adolescents with this diseasemight suppress periosteal bone expansion and alter the bonemicroarchitecture increasing susceptibility to fracture inlater years Validating this hypothesis in humans also requiresfurther investigation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Kathryn Thrailkill researched data contributed to dis-cussion wrote paper and reviewededited paper ClayBunn researched data contributed to discussion wrotepaper and reviewededited paper Charles Lumpkin con-tributed to discussion and reviewededited paper Eliza-beth Wahl researched data contributed to discussion and


reviewededited paper Gael Cockrell researched data con-tributed to discussion and reviewededited paper Lind-sey Morris researched data contributed to discussion andreviewededited paper Ronald Kahn provided necessaryIR flox animals and reviewededited paper John Fowlkesresearched data contributed to discussion wrote paper andreviewededited paper Jeffry S Nyman researched datacontributed to discussion wrote paper and reviewededitedpaper All authors have approved the final version of thispaper Clay Bunn John Fowlkes and Jeffry Nyman accept theresponsibility for the integrity of the data analysis

Acknowledgments

This work was supported by grants from the NationalInstitutes of Health (R01 DK084045 to John Fowlkes andC06RR16517 to ACHRI) the Martha Ann Pugh DiabetesResearch Fund (to Kathryn Thrailkill) the Arkansas Bio-sciences Institute (to John Fowlkes) and the ChildrenrsquosUniversity Medical Group Grant Program (no 36112 to JohnFowlkes)This data was presented in part at the 42nd AnnualInternational Sun Valley Workshop onMusculoskeletal Biol-ogy of the International Bone and Mineral Society (SunValley Idaho 2012)

References

[1] R T Loder ldquoThe influence of diabetes mellitus on the healingof closed fracturesrdquo Clinical Orthopaedics and Related Researchno 232 pp 210ndash216 1988

[2] M Janghorbani D Feskanich W C Willett and F HuldquoProspective study of diabetes and risk of hip fracture thenursesrsquo health studyrdquoDiabetes Care vol 29 no 7 pp 1573ndash15782006

[3] M Espallargues L Sampietro-Colom M D Estrada et alldquoIdentifying bone-mass-related risk factors for fracture to guidebone densitometry measurements a systematic review of theliteraturerdquoOsteoporosis International vol 12 no 10 pp 811ndash8222001

[4] K MThrailkill C K Lumpkin Jr R C Bunn S F Kemp andJ L Fowlkes ldquoIs insulin an anabolic agent in bone Dissectingthe diabetic bone for cluesrdquoAmerican Journal of Physiology vol289 no 5 pp E735ndashE745 2005

[5] J R Levy E Murray S Manolagas and J M OlefskyldquoDemonstration of insulin receptors and modulation of alka-line phosphatase activity by insulin in rat osteoblastic cellsrdquoEndocrinology vol 119 no 4 pp 1786ndash1792 1986

[6] K K Pun P Lau and P W M Ho ldquoThe characterizationregulation and function of insulin receptors on osteoblast-like clonal osteosarcoma cell linerdquo Journal of Bone and MineralResearch vol 4 no 6 pp 853ndash862 1989

[7] D M Thomas D K Hards S D Rogers K W Ng and J DBest ldquoInsulin receptor expression in bonerdquo Journal of Bone andMineral Research vol 11 pp 1312ndash1320 1996

[8] M Hashizume and M Yamaguchi ldquoStimulatory effect of 120573-alanyl-L-histidinato zinc on cell proliferation is dependent onprotein synthesis in osteoblastic MC3T3-E1 cellsrdquo Molecularand Cellular Biochemistry vol 122 no 1 pp 59ndash64 1993

[9] J E Wergedal and D J Baylink ldquoCharacterization of cellsisolated and cultured from human bonerdquo Proceedings of the

Society for Experimental Biology and Medicine vol 176 no 1pp 60ndash69 1984

[10] E M Canalis J W Dietrich D M Maina and L G RaiszldquoHormonal control of bone collagen synthesis in vitro Effectsof insulin and glucagonrdquo Endocrinology vol 100 no 3 pp 668ndash674 1977

[11] D M Rosen and R A Luben ldquoMultiple hormonal mechanismsfor the control of collagen synthesis in an osteoblast-like cellline MMB-1rdquo Endocrinology vol 112 no 3 pp 992ndash999 1983

[12] B E Kream M D Smith E Canalis and L G RaiszldquoCharacterization of the effect of insulin on collagen synthesis infetal rat bonerdquo Endocrinology vol 116 no 1 pp 296ndash302 1985

[13] E Canalis ldquoEffect of hormones and growth factors on alkalinephosphatase activity and collagen synthesis in cultured ratcalvariaerdquoMetabolism vol 32 no 1 pp 14ndash20 1983

[14] T J Hahn S L Westbrook T L Sullivan W G Goodmanand L R Halstead ldquoGlucose transport in osteoblast-enrichedbone explants characterization and insulin regulationrdquo Journalof Bone and Mineral Research vol 3 no 3 pp 359ndash365 1988

[15] E A Ituarte L R Halstead A Iida-Klein H G Ituarteand T J Hahn ldquoGlucose transport system in UMR-106-01osteoblastic osteosarcoma cells regulation by insulinrdquo CalcifiedTissue International vol 45 no 1 pp 27ndash33 1989

[16] N Follak I Kloting and H Merk ldquoInfluence of diabeticmetabolic state on fracture healing in spontaneously diabeticratsrdquo DiabetesMetabolism Research and Reviews vol 21 no 3pp 288ndash296 2005

[17] N Follak I Kloting E Wolf and H Merk ldquoDelayed remod-elling in the early period of fracture healing in spontaneouslydiabetic BBOK rats depending on the diabetic metabolic staterdquoHistology and Histopathology vol 19 no 2 pp 473ndash486 2004

[18] J C-H Hou R F Zernicke and R J Barnard ldquoEffects of severediabetes and insulin on the femoral neck of the immature ratrdquoJournal of Orthopaedic Research vol 11 no 2 pp 263ndash271 1993

[19] K M Thrailkill L Liu E C Wahl et al ldquoBone formation isimpaired in a model of type 1 diabetesrdquoDiabetes vol 54 no 10pp 2875ndash2881 2005

[20] A Gandhi H A Beam J P OrsquoConnor J R Parsons and S SLin ldquoThe effects of local insulin delivery on diabetic fracturehealingrdquo Bone vol 37 no 4 pp 482ndash490 2005

[21] M Ferron J Wei T Yoshizawa et al ldquoInsulin signal-ing in osteoblasts integrates bone remodeling and energymetabolismrdquo Cell vol 142 no 2 pp 296ndash308 2010

[22] K Fulzele R C Riddle D J DiGirolamo et al ldquoInsulin receptorsignaling in osteoblasts regulates postnatal bone acquisition andbody compositionrdquo Cell vol 142 no 2 pp 309ndash319 2010

[23] J C Bruning M D Michael J N Winnay et al ldquoA muscle-specific insulin receptor knockout exhibits features of themetabolic syndrome of NIDDM without altering glucose tol-erancerdquoMolecular Cell vol 2 no 5 pp 559ndash569 1998

[24] E CWahl J Aronson L Liu et al ldquoRestoration of regenerativeosteoblastogenesis in agedmice modulation of TNFrdquo Journal ofBone and Mineral Research vol 25 no 1 pp 114ndash123 2010

[25] J S Nyman J L Even C-H Jo et al ldquoIncreasing duration oftype 1 diabetes perturbs the strength-structure relationship andincreases brittleness of bonerdquo Bone vol 48 no 4 pp 733ndash7402011

[26] R C Bunn G E Cockrell Y Ou K MThrailkill C K Lump-kin Jr and J L Fowlkes ldquoPalmitate and insulin synergisticallyinduce IL-6 expression in human monocytesrdquo CardiovascularDiabetology vol 9 article 73 2010


[27] C H Turner and D B Burr ldquoBasic biomechanical measure-ments of bone a tutorialrdquo Bone vol 14 no 4 pp 595ndash608 1993

[28] R A Davey M V Clarke S Sastra J P Skinner C Chiang etal ldquoDecreased body weight in young Osterix-Cre transgenicmice results in delayed cortical bone expansion and accrualrdquoTransgenic Research vol 21 pp 885ndash893 2012

[29] MMartin-Millan M Almeida E Ambrogini et al ldquoThe estro-gen receptor-120572 in osteoclasts mediates the protective effectsof estrogens on cancellous but not cortical bonerdquo MolecularEndocrinology vol 24 no 2 pp 323ndash334 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


skeletal deficits which occur in rodentmodels of T1D includ-ing improving bone strength and biomechanical integrity[16ndash19] Even local insulin delivery to fracture sites in diabeticrodents can ameliorate poor fracture healing which occursin T1D [20] Taken together these data suggest that insulinmay play a critical role in normal osteoblastogenesis andthat conditions of insulin deficiency may lead to abnormalskeletogenesis

A direct link between insulin signaling and bone for-mation in vivo is supported by transgenic mouse modelsKnockdown of the insulin receptor (IR) in osteoblasts resultsin altered bone formation [21] and in abnormal trabeculararchitecture [22] These studies support a role for insulin inmature osteoblasts Ferron et al [21] report a 60knockdownof the Insr using the Col1a1-Cre mouse while Fulzele et al[22] have eliminated the Insr in mature osteoblasts using theosteocalcin- (OC-) Cre construct

To examine further the specific action of insulin signalingon bone we generated mice lacking the insulin receptor inosteoprogenitor cells Unlike previous reports that deletedIR in mature osteoblasts [21 22] we assessed the effect ofIR deletion at the early stages of osteoblast differentiation(osterix) on the architectural structural and biomechanicalproperties of bone We hypothesized that the ablation ofinsulin signaling prenatally in cells of the osteoblast lineagevia a loss of IR would impair the fracture resistance of mousebones independent of body size

2 Materials and Study Design

21 Ethics Statement This research protocol was approvedby the Institutional Animal Care and Use Committee of theUniversity of Arkansas for Medical Sciences

22 OIRKO Mice Our experimental objective was to gen-erate mice lacking the insulin receptor in osteoblast lineagecells considered as precursors to previously reported models(ie Col1a1-Cre or osteocalcin- (OC-) Cre) Therefore togenerate mice with an osteoprogenitor-selective ablation ofthe insulin receptor (OIRKO) we first crossed mice thatwere homozygous for a floxed insulin receptor (IR) allelein which loxP sites flank exon 4 of the IR gene (designatedIRloxlox) [23] with heterozygous osterix- (Osx-) Cre transgenicmice (B6Cg-Tg(Sp7-tTAtetO-EGFPcre)1AmcJ The Jack-son Laboratory Bar Harbor ME) F1 progeny were crossedto produce IRloxloxCre+minus(OIRKO) and IRloxloxCreminusminus micewhich were then bred to produce experimental animals TheIRloxloxCreminusminus mouse (IR flox) was designated as the controlfor this study in keeping with previous reports [21 22] Cretransgene expression is repressed when mice receive chowcontaining doxycycline (Diet S3888 Bio-Serv FrenchtownNJ) Upon switching mice to a regular chow diet (Diet 8640Harlan Indianapolis IN) the Cre transgene is expressed inthe osteoblast lineage resulting in disruption of the floxedIR alleles in the osteoblast lineage In this study pregnantfemales were switched to regular chow approximately 1 weekprior to birth of experimental pups Nursing dams weremaintained on regular chow and experimental mice were

weaned to a regular chow diet Genotyping was performedusing published procedures [23]

Postnatal growth as assessed by weight gain was moni-tored in both male (119899 = 15 IR flox (6) + OIRKO (9)) andfemale (119899 = 15 IR flox (6) + OIRKO (9)) mice at 4 6 8 and10ndash12 weeks of age (Figure 1) Detailed metabolic and skeletalassessments were then completed

23 Metabolic Assessment Between 10-11 weeks of age inmales and 12-13 weeks of age in females a 150-minute glucosetolerance test (GTT) was performed Following an overnightfast blood glucosemeasurements were obtained from all ani-mals before intraperitoneal injection of a 2 gkg body weightglucose bolus Blood glucose levels were then measured at15 30 45 60 and 150 minutes following the glucose bolusIn males only insulin levels were also measured at 0 1530 45 60 and 150 minutes Blood glucose was measuredvia glucometer (OneTouchUltra2 BloodGlucoseMonitoringSystem Lifescan Inc Milpitas CA average intra-assay coef-ficient of variation of 17 across a range of 40ndash300mgdLtarget glucose concentrations) Serum insulin levels weremeasured using a mouse insulin ultrasensitive ELISA (80-INSMSU-E01 ALPCO Immunoassays SalemNH sensitivity0115 ngmL intra-assay coefficient of variation 63 inter-assay coefficient of variation 1061) Glucose (males andfemales) and insulin (males only) area-under-curve (AUC)an integrated measure of glucose tolerance was determinedusing GraphPad PRISM software (version 502) Serumtotal osteocalcin (OC) was measured using the MILLIPLEXMAP Mouse Osteocalcin-Single Plex assay (MBN-41 K-10CMillipore Corp Billerica MA sensitivity 87 pgmL intra-assay coefficient of variation 112 inter-assay coefficient ofvariation 75) Serum amino-terminal propeptide of type 1procollagen (PINP)wasmeasured using theRatMouse PINPEIA (AC-33F1 Immunodiagnostics Systems Ltd FountainHills AZ sensitivity 07 ngmL) Serum c-terminal telopep-tide of type 1 collagen a marker of bone resorption was mea-sured using the RatLAPs ELISA (AC-06F1 Immunodiagnos-tics Systems Inc Scottsdale AZ sensitivity 17 ngmL intra-assay coefficient of variation 58 inter-assay coefficient ofvariation 104)

24 Skeletal Assessment After euthanasia (males sim10-11weeks females sim12-13 weeks) the left tibia and femur wereharvested from IR flox (6 male and 6 female) and OIRKO(9 male and 9 female) littermates and stored in phosphatebuffered saline (PBS) at minus20∘C until assessment H amp E stain-ing of the tibial growth plate was performed as previouslydescribed [24] Femur length was measured using calipersFemurs were immersed in PBS at room temperature and thelong axis of the bone was aligned with the scanning axisof the 120583CT40 (Scanco Medical Bruttisellen Switzerland)All scans were acquired with an isotropic voxel size of12 120583m using the same energy settings (70 kVp0114mA)acquisition parameters (integration time of 300ms with 1000projections per full rotation) and calibration to a hydrox-yapatite (HA) phantom with a beam hardening correctionfrom Scanco Medical as we have previously described [25]




Cre

Cre

IRΔIR

IRΔIR

Calv

aria

Tibi

a

Live

r

S M

uscle

Panc

reas

Kidn

ey

WAT BA

T

Brai

n

Inte

stine

(a)

IR 120573-subunit

GAPDH

OIRKOIR flox

(b)

80100120140160180200220240260

7-8 11-12

80100120140160180200220240260280300

64Weeks of age

Wei

ght (

g)W

eigh

t (g) lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowast

IR floxOIRKO

(c)

OIRKOIR flox

(d)

Female

Male

OIRKOIR flox

(e)

10 mm































3 Results








00

05

10

15

20

IR flox OIRKO

P = 00006

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(a)

00

05

10

15

20

IR flox OIRKO

P = 0002

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(b)

10

15

IR flox OIRKO

Peak

forc

e (N

)

P = 0012

(c)

10

15

Peak

forc

e (N

)

IR flox OIRKO

P = 00003

(d)









4 Discussion












Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Cre

Cre

IRΔIR

IRΔIR

Calv

aria

Tibi

a

Live

r

S M

uscle

Panc

reas

Kidn

ey

WAT BA

T

Brai

n

Inte

stine

(a)

IR 120573-subunit

GAPDH

OIRKOIR flox

(b)

80100120140160180200220240260

7-8 11-12

80100120140160180200220240260280300

64Weeks of age

Wei

ght (

g)W

eigh

t (g) lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowast

IR floxOIRKO

(c)

OIRKOIR flox

(d)

Female

Male

OIRKOIR flox

(e)

10 mm































3 Results








00

05

10

15

20

IR flox OIRKO

P = 00006

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(a)

00

05

10

15

20

IR flox OIRKO

P = 0002

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(b)

10

15

IR flox OIRKO

Peak

forc

e (N

)

P = 0012

(c)

10

15

Peak

forc

e (N

)

IR flox OIRKO

P = 00003

(d)









4 Discussion












Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































3 Results








00

05

10

15

20

IR flox OIRKO

P = 00006

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(a)

00

05

10

15

20

IR flox OIRKO

P = 0002

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(b)

10

15

IR flox OIRKO

Peak

forc

e (N

)

P = 0012

(c)

10

15

Peak

forc

e (N

)

IR flox OIRKO

P = 00003

(d)









4 Discussion












Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20

IR flox OIRKO

P = 00006

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(a)

00

05

10

15

20

IR flox OIRKO

P = 0002

Slen

dern

ess p

er B

W (m

mminus1gminus

1)

(b)

10

15

IR flox OIRKO

Peak

forc

e (N

)

P = 0012

(c)

10

15

Peak

forc

e (N

)

IR flox OIRKO

P = 00003

(d)









4 Discussion












Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Discussion












Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article loss of insulin receptor in...

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