Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes ...

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1 Pathophysiological mechanism of bone loss in type 2 diabetes involves inverse 1 regulation of osteoblast function by PPARγ coactivator-1α and skeletal muscle 2 atrogenes: adiponectin receptor 1 as a potential target for reversing diabetes-induced 3 osteopenia 4 5 Running title: Adiponectin receptor-1 and PGC-1α protect against type-2 diabetes-induced 6 osteopenia 7 8 Mohd. Parvez Khan , Abhishek Kumar Singh , Amit Arvind Joharapurkar 3 , Manisha 9 Yadav 2 , Sonal Shree 4 , Harish Kumar 2 , Anagha Gurjar 2 , Jay Sharan Mishra 2 , Mahesh 10 Chandra Tiwari 1 , Geet Kumar Nagar 1 , Sudhir Kumar 5 , Ravishankar Ramachandran 4 , 11 Anupam Sharan 6 , Mukul Rameshchandra Jain 3 , Arun Kumar Trivedi 2 , Rakesh Maurya 5 , 12 Madan Madhav Godbole 7 , Jiaur Rahaman Gayen 8 , Sabyasachi Sanyal 2 * and Naibedya 13 Chattopadhyay 1 * 14 15 Authors’ affiliations: 16 1 Division of Endocrinology, 2 Division of Biochemistry, 4 Division of Molecular and 17 Structural Biology, 5 Division of Medicinal and Process Chemistry, 8 Division of 18 Pharmacokinetics, CSIR-Central Drug Research Institute, 10 Janakipuram Extn, Sitapur 19 Road, Lucknow 226031, UP, India 20 3 Zydus Research Center, Sarkhej-Bavla NH#8A, Moraiya, Ahmedabad 382210, Gujarat, 21 India 22 6 Vinayak Cosmetic surgery clinic, I-127, Vipul Khand, Gomatinagar, Lucknow 226010, UP, 23 India 24 7 Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, 25 Lucknow 226014, UP, India 26 27 § These authors contributed equally 28 29 30 *Address Correspondences to: [email protected] or [email protected] 31 CSIR-Central Drug Research Institute, 10 Janakipuram Extn, Sitapur Road, Lucknow 32 226031, UP, India ; Tel: (+91)-9792366322,9455279614; Fax: (+91)-(522)-2771941 33 34 Word count 5314 35 36 Number of figures 8 37 38 39 40 41 Page 1 of 49 Diabetes Diabetes Publish Ahead of Print, published online January 29, 2015

Transcript of Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes ...

Page 1: Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes ...

1

Pathophysiological mechanism of bone loss in type 2 diabetes involves inverse 1

regulation of osteoblast function by PPARγγγγ coactivator-1α and skeletal muscle 2

atrogenes: adiponectin receptor 1 as a potential target for reversing diabetes-induced 3

osteopenia 4

5

Running title: Adiponectin receptor-1 and PGC-1α protect against type-2 diabetes-induced 6

osteopenia 7

8

Mohd. Parvez Khan1§

, Abhishek Kumar Singh2§

, Amit Arvind Joharapurkar3, Manisha 9

Yadav2, Sonal Shree

4, Harish Kumar

2, Anagha Gurjar

2, Jay Sharan Mishra

2, Mahesh 10

Chandra Tiwari1, Geet Kumar Nagar

1, Sudhir Kumar

5, Ravishankar Ramachandran

4, 11

Anupam Sharan6, Mukul Rameshchandra Jain

3, Arun Kumar Trivedi

2, Rakesh Maurya

5, 12

Madan Madhav Godbole7, Jiaur Rahaman Gayen

8, Sabyasachi Sanyal

2* and Naibedya 13

Chattopadhyay1* 14

15

Authors’ affiliations: 16 1Division of Endocrinology,

2Division of Biochemistry,

4Division of Molecular and 17

Structural Biology, 5Division of Medicinal and Process Chemistry,

8Division of 18

Pharmacokinetics, CSIR-Central Drug Research Institute, 10 Janakipuram Extn, Sitapur 19

Road, Lucknow 226031, UP, India 20 3Zydus Research Center, Sarkhej-Bavla NH#8A, Moraiya, Ahmedabad 382210, Gujarat, 21

India 22 6Vinayak Cosmetic surgery clinic, I-127, Vipul Khand, Gomatinagar, Lucknow 226010, UP, 23

India 24 7 Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, 25

Lucknow 226014, UP, India 26

27 §

These authors contributed equally 28

29

30

*Address Correspondences to: [email protected] or [email protected] 31

CSIR-Central Drug Research Institute, 10 Janakipuram Extn, Sitapur Road, Lucknow 32

226031, UP, India ; Tel: (+91)-9792366322,9455279614; Fax: (+91)-(522)-2771941 33

34

Word count 5314 35

36

Number of figures 8 37

38

39

40

41

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Diabetes Publish Ahead of Print, published online January 29, 2015

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Abstract 1

2

Type 2 diabetes is associated with increased fracture risk and delayed facture healing; the 3

underlying mechanism however remains poorly understood. Here we made a systematic 4

investigation of skeletal pathology in leptin receptor-deficient diabetic mouse in C57/BLKS 5

background (db). Compared with wild-type (wt), db mice displayed reduced peak bone mass 6

and age-related trabecular and cortical bone loss. Poor skeletal outcome in db was contributed 7

by high glucose and non-esterified fatty acid (NEFA)-induced osteoblast apoptosis that was 8

associated with PPARγ coactivator 1-α (PGC-1α) downregulation and upregulation of 9

skeletal muscle atrogenes in osteoblasts. Osteoblast depletion of the atrogene, muscle ring 10

finger protein-1 (MuRF1) protected against gluco- and lipotoxicity-induced apoptosis. 11

Osteoblast-specific PGC-1α upregulation by 6-C-β-d-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-12

tetrahydroxydihydroflavonol (GTDF), an adiponectin receptor 1 (AdipoR1) agonist as well as 13

metformin in db mice that lacked AdipoR1 expression in muscle but not bone, restored 14

osteopenia to wt levels without improving diabetes. Both GTDF and metformin protected 15

against gluco- and lipotoxicity-induced osteoblast apoptosis and depletion of PGC-1α 16

abolished this protection. While AdipoR1 but not AdipoR2 -depletion abolished protection by 17

GTDF, metformin action was not blocked by AdipoR-depletion. We conclude that PGC-1α 18

upregulation in osteoblasts could reverse type 2 diabetes-associated deterioration in skeletal 19

health. 20

21

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Introduction 1

Type 2 diabetes typically is a mid age-onset (>40 years) metabolic disease that affects 2

multiple organ systems. Increasing evidence indicates that type 2 diabetes is associated with 3

increased fracture risk; especially vertebral and hip fractures in older patients (1-4). Since 4

chronic inflammation participates in diabetes pathogenesis and is also a prerequisite for 5

osteoclast activation; increased bone resorption is considered as the likely cause of increased 6

fracture risk in this disease. Intriguingly however, available studies suggest that in type 2 7

diabetes, there is an increased risk of hip fractures at a higher bone mineral density (BMD) 8

value than non-diabetics (5) and increased incidence of vertebral fractures at BMD values 9

comparable to non-diabetics(6). Thus, the reasons for increased skeletal fragility in type 2 10

diabetes patients remain largely unexplained. 11

Type 2 diabetes patients also display delayed fracture healing resulting in poorer 12

outcomes following hip fracture (7). Reduced fracture healing in type 2 diabetes is attributed 13

to decreased collagen content, defective cross-linking, alterations in collagen sub-type ratios 14

and collagen defects due to accumulation of advanced glycation end-products (8-10), which 15

could impair osteoblast function. These could also lead to compromised bone material 16

strength and increased cortical porosity affecting bone quality as shown in postmenopausal 17

women suffering from type 2 diabetes (11; 12). 18

Several mouse models of type 2 diabetes are available however; they poorly represent 19

human skeletal fragility observed in typical type 2 diabetes patients(13). Unlike typical mid-20

age type 2 diabetes onset in humans, when skeletal maturity has already been attained, in 21

genetically engineered diabetic mice, the onset of diabetes occurs at 4-8 weeks, an age 22

comparable to adolescence in humans at which skeletal maturity has not been attained (14). 23

However, the average onset age is falling in humans and is becoming increasingly common 24

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among those aged less than 30 years including children and adolescents in different ethnic 1

groups (15-18). This early onset diabetes is characterized by increased disease severity and 2

pancreatic β cell failure than typical type 2 diabetes (19-21). Data on the impact of early-3

onset type 2 diabetes on human skeletal health are limited, although a recent report indicates 4

that prediabetic children with impaired glucose tolerance have low mineral content and low 5

bone mass (22). We thus believe that the distinction in skeletal phenotype between early and 6

mid-age-onset type 2 diabetes might be important for the following reason. While, skeleton 7

of children/adolescents predominantly undergoes modeling-directed growth (resulting in net 8

increase in bone mass due to enhanced osteoblastic activity), adult skeletons predominantly 9

experience remodeling (no net bone gain), and thus type 2 diabetes in these two cases may 10

affect skeletal health in different manner. 11

Although monogenic obese and diabetic mice models such as leptin receptor-deficient 12

diabetic mice essentially differ from the polygenic disease origin in human type 2 diabetes, it 13

however, may serve as a suitable model for deciphering the skeletal outcomes in this disease. 14

Here we investigated the skeletal phenotype in leptin receptor-deficient genetically 15

obese diabetic mice (in C57BLKS background; db) that manifest severe diabetes including 16

pancreatic β cell failure. Our study also involved identification of factors crucial for type 2 17

diabetes-induced skeletal effects. Furthermore, modulation of such factors by therapeutic 18

intervention on diabetic skeleton was also assessed. 19

20

21

22

23

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1

2

3

Research Design and Methods 4

Reagents and kits: Cell culture reagents were from Life Technologies. Fine reagents were 5

from Sigma-Aldrich unless indicated otherwise. Globular adiponectin (gAd) was from 6

ATGen Global. 125

I (20 MBq) was from BARC (Mumbai, India). Kits for plasma 7

biochemical parameters were: glucose (Pointe Scientific), lipids and creatinine (Randox 8

Laboratories LTD, Mumbai, India), insulin (Millipore), adiponectin (B-Bridge International 9

Inc), osteocalcin (USCN life Science Inc). ELISA kits: PAI-1, MCP-1, leptin and resistin 10

(Millipore). TUNEL assay kit: Roche Applied Science. GTDF (purity >98%) was purified as 11

reported (23), metformin (Met; purity 97%) and pioglitazone (Pio; purity ≥98%) were from 12

Sigma-Aldrich. 13

Animal Experiments: Wild type (wt; C57BLKS/J) or diabetic (db; BKS.Cg-Dock7m

+/+ 14

Leprdb

/J and B6.db; B6.BKS(D)-Leprdb

/J ) ice were housed at 22±3°C temperature and a 12h 15

light/dark cycle. All animals had access to standard chow diet and water ad libitum. Time 16

course studies were conducted at AALAC accredited animal facility of Zydus Research 17

Centre (ZRC), Ahmedabad following approval from Institutional Animal Ethics Committee 18

(IAEC) of ZRC. The db or wt mice used were originally from Jackson laboratories and the 19

colonies were maintained at ZRC. Drug treatment studies using 10 wk old db mice from 20

Harlan laboratories, Netherlands (BKS.Cg- + Leprdb

/+Leprdb

/OlaHsd) were conducted at 21

Syngene International Ltd (Bangalore, India) in their AALAC accredited animal facility, 22

following ethical approval from IAEC. In both studies, all the animals were randomized into 23

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groups based on blood glucose levels and body weight. Vehicle groups received 0.5% 1

Carboxymethylcellulose and treatment groups received GTDF (10mg/kg), Met (350mg/kg) 2

and Pio (10mg/kg) once a day, by oral gavage for 30 days.The doses of Met in adult human 3

range from 850-2550mg/day which comes to 14.6-42.5mg/kg (considering the average 4

human weight to be 60kg). The adult human dose of Pio is 15-45mg/day which corresponds 5

to 0.25-0.75mg/kg. Formula for dose translation from human to mouse was based on body 6

surface area: human equivalent dose (mg/kg) = animal dose (mg/kg) × (animal Km / human 7

Km)(24). Feed intake and body weight were measured every day and on day 31 the animals 8

were sacrificed. Plasma and tissues were collected and stored at -80oC until further analysis. 9

Micro-computed tomography (µCT): µCT of excised bones was carried out using Sky 10

Scan 1076 CT scanner (Aartselaar) as described earlier for mice bone (25; 26) and following 11

the general guidelines for the assessment of bone microarchitecture in rodents using µCT 12

(27). For scanning, source voltage was set to 50 kV and current, 200 µA. X-ray source 13

rotation step size was 0.84° over a trajectory of 180°. Reconstructions were made using the 14

nRecon V1.6.9.4 software (SkyScan) to create 2D 2000 × 2000 pixel images with a beam 15

hardening correction set to 10% with dynamic range −1,000 to 11,000 Hounsfield units. By 16

drawing ellipsoid contours, trabecular bone was extracted using the CT analyzer software. In 17

the femur epiphysis region, 200 slices were selected leaving 50 slices from the start of the 18

growth plate as a reference point. Cortical parameters were determined at femur mid-19

diaphysis by 2D analysis. From the start of the growth plate as a reference point, 200 slices 20

were selected in the cortical region, leaving 500 slices as offset (to exclude the trabecular 21

region). For BMD calibration, the hydroxyapatite phantom rods of 2mm diameter with 22

known BMD (0.25g/cm3 and 0.75 g/cm

3) were employed. For each analysis, the estimated 23

mineral density of the bone tissue was determined based on the linear correlation between 24

µCT attenuation coefficient and BMD (28). 25

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Bone biomechanical strength: Three-point bending test on femur was performed using a 1

Bone Strength Tester (Muromachi, TK-252C) as previously reported (29). 2

Determination of the bone lining cells: Deparaffinized and hydrated femoral epiphysis 3

sections (5µm) of different groups were stained with H&E and bone lining cells were 4

visualized by light microscopy. 10 sections/mouse (n=6) were used for counting by two 5

independent researchers blinded to experimental design. 6

Cell culture and induction of differentiation: Mouse calvarial osteoblasts (MCO) were 7

obtained from 1-2 day old mouse pups as described earlier (26; 30). Bone marrow cells 8

(BMC) from 10 to 12 wk old male wt and db mice were isolated and were cultured and 9

differentiated into osetoblasts as described earlier (31). 10

Radio iodination of gAd and radioligand binding experiment: 10µg gAd was radio-11

iodinated by iodogen method using precoated iodination tubes (Pierce) according to 12

manufacturer’s instructions. Excess 125

I was removed by using PD 10 desalting column (GE 13

Healthcare). For binding assays, osteoblast and myocytes in 24-well plates were incubated 14

with increasing concentrations of 125

I-gAD in PBS supplemented with 0.1% BSA for 2h (at 15

which binding equilibrium was achieved), following which the cells were washed and lysed. 16

Nonspecific binding for each concentration was determined using 200-fold excess of cold 17

gAD. Specific binding was calculated by subtracting nonspecific binding from total binding. 18

RNAi experiments: siRNAs were from Dharmacon. MCO were transfected with 0.1µM of 19

each siRNA per well using DharmaFECT 1 transfection reagent (Dharmacon) in 6-well 20

plates. 72h after transfection, cells were treated as indicated and were analyzed as required. 21

QPCR, immunoblotting and immunohistochemistry: These studies were performed using 22

standard procedures as previously described (32; 33). Primer sequences for QPCR are listed 23

in supplementary Table 3. Antibodies and dilutions for immunoblotting: anti-PGC-1α 24

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(ST1202; Calbiochem; 1:2000). Anti-AMPK, phospho-AMPK, (Thr 172) and cleaved 1

caspase-3 (Cell Signaling Technology; 1:1000). AdipoR1, AdipoR2, MuRF1 and β-actin 2

antibodies were from Santacruz Biotechnology (1:1000 except β-actin; 1:3000). TUNEL 3

assay was performed as described earlier (33). For immunohistochemistry, femur epiphysis 4

transverse sections (5µm) were deparaffinized, hydrated and after antigen retrieval, incubated 5

with mouse anti-Runx-2 (Abcam, 1:1600) along with anti-PGC-1α (1:2000), pAMPK (1:500) 6

or MuRF1(1:500) at 40C overnight. The sections were then washed and incubated with 7

fluorescent Alexa Fluor-goat anti-mouse and goat anti-rabbit IgG (H + L) (1:1500) (Life 8

Technologies) for 1h at RT. Sections were also stained with DAPI and visualized by 9

fluorescent microscopy. Image-Pro plus 6.1 software (MediaCybernetics) was used for 10

quantification of microscopic data, where five randomly selected fields from six bone 11

sections per group were analyzed. 12

Osteoblast differentiation assay: MCOs at 70-80% confluence were trypsinized and 2 ×103 13

cells/well were seeded in 96-well plates. After 24h, cells were given various treatments for 48 14

h in osteoblast differentiation medium. Alkaline phosphatase (ALP) activity was measured 15

using a fluorometric kit (Biovision) according to the manufacturer’s protocol. 16

Cell viability assay: MCO in 96-well plates (2×103 cells/well) were treated with increasing 17

concentrations of glucose, palimitic acid and dexamethasone (dex) with or without GTDF 18

and metformin for 48h. Osteoblast viability was assessed by MTT assay as reported (33) . 19

Flow cytometry-based determination of apoptosis: Annexin V-FITC Apoptosis Detection 20

kit (Sigma) was used to determine apoptosis. Briefly, the treated cells were trypsinized and 21

washed with PBS. 1×106 cells/mL were resuspended in binding buffer and labeled with 5µL 22

annexinV-FITC and 10µL propidium iodide for 10 minutes in the dark. Cells fluorescence 23

was measured on a FACSCalibur flow cytometer (Becton Dickenson) and analyzed using 24

CellQuest Pro software. 25

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Data analysis and Statistics: Results are expressed as mean ± SE. All data were analyzed 1

using GraphPad Prism 5.0. Statistical analyses were performed using one or two-way 2

ANOVA as appropriate followed by Bonferroni’s post test. 3

4

5

6

7

8

Results 9

Lack of peak bone mass achievement and age-related osteopenia in BKS.Cg-Dock7m

+/+ 10

Leprdb/db/J (db) mice 11

3D µCT-based evaluation of trabecular microarchitecture in 8, 12 and 16 wk old 12

C57BLKS/J (wt) and db revealed that db femur epiphysis at all ages displayed loosely 13

connected trabecular network than wt mice (Fig 1A). wt mice displayed bone gain at 12 wk, 14

characterized by significantly higher BMD, trabecular bone volume (BV/TV), trabecular 15

number (Tb.N) and connectivity density (Conn.D) followed by trabecular loss at 16 wks 16

manifested by the decreases in these parameters (Fig. 1A). db mice did not gain bone at any 17

age and were osteopenic throughout, characterized by significantly lower BMD, BV/TV, 18

Tb.N, trabecular thickness (Tb.Th), Conn.D, and higher trabecular separation (Tb.Sp) than wt 19

(Fig. 1A). Age-based comparison between db mice also revealed progressive osteopenia 20

characterized by significantly higher Tb.sp at 16 wk, and other parameters when compared to 21

8wk old db showed a decreasing trend with age. 22

Femur mid-diaphysis of db mice representing cortical bone showed a thinner cortex 23

than wt mice at all ages (Fig 1B). At 8 wk, BMD, average cortical thickness (Ct.Th), and 24

cortical area (Ct.Ar) were comparable between wt and db, the latter group displayed 25

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significantly lower periosteal perimeter (Ps.Pm) and endocortical perimeter (Ec.Pm). At 12 1

and 16 wk, all cortical parameters in db were significantly lower than corresponding wt 2

groups (Fig. 1B). Like trabecular parameters, cortical parameters including BMD, Ct.Th and 3

Ct.Ar in 12 wk old wt were significantly higher than 8 wk old wt mice, and at 16 wk, these 4

parameters displayed a decreasing trend compared to 8 wk old wt (Fig.1B). db mice showed 5

no cortical gain at any age (Fig. 1B). 6

Osteoblast apoptosis is associated with both primary and secondary osteoporosis (34) 7

and compared to wt femur epiphysis in db mice across all age groups displayed remarkably 8

increased osteoblast apoptosis ( Fig. 1C, Supplementary Figure 1A). 9

Periosteal or bone lining cells serve as a source of osteogenic precursors (35). At 12 10

wk, wt but not db bones displayed significantly higher periosteal cell number compared to the 11

8wk groups (Fig. 1D, Supplementary Figure 1B) and db mice at all ages had significantly 12

lower periosteal cell number compared to wt. (Fig. 1D). The osteogenic surrogate, serum 13

osteocalcin (OCN) level significantly dropped with age in both mice; however as reported 14

earlier (36)db mice had significantly lower OCN level than wt (Fig 1J). 15

Compared to wt non-fasting and fasting glucose was significantly higher in 8wk db 16

mice (>280 and >150 mg/dL respectively) which further increased to >450 and > 200mg/dL 17

at 12 wk and remained steady thereafter (Fig. 1E-F). db mice also displayed significant non-18

fasting and fasting hyperinsulinemia (Fig. 1H-I), however, insulin levels in 16wk db was 19

significantly less than the 8wk old db mice, probably due to β-cell apoptosis which is typical 20

of the db strain. Intriguingly, despite the decrease in insulin, 16wk old db mice had 21

comparable blood glucose to 12wk old db, although we can’t explain it, presumably it was 22

due to elimination of glucose through urine, which again is a trait in db mice. NEFA level in 23

db mice was significantly higher than wt at all ages and no age-dependent change was 24

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observed (Fig. 1G). Consistent with earlier reports (37; 38), db mice at 8 wk displayed a 1

significantly lower adiponectin level than wt which decreased further with age (Fig. 1K). 2

Glucose and palmitate directly induce osteoblast apoptosis 3

We next assessed if NEFA and glucose, the two major mediators of diabetic 4

pathology could directly affect osteoblast viability. Both palmitate and glucose induced loss 5

of mouse calvarial osteoblast (MCO) viability and apoptosis in vitro (Fig 2A-2B). Apoptosis-6

related cysteine protease, caspase-3 activation assay revealed that palmitate and 7

dexamethasone (Dex) but not glucose enhanced cleaved (active) caspase-3 levels (Fig 2C). 8

9

db mice display suppression of PGC-1α and increase in skeletal muscle atrogene 10

expression in bone 11

Consistent with enhanced osteoblast apoptosis, db femur epiphysis displayed 12

significantly higher p53 expression than wt at all ages (Fig. 3A). Among the db group, 16 wk 13

old had significantly higher p53 than the 8wk old (Fig 3A). Consistent with peak bone gain 14

(Fig. 1A-B), wt mice displayed significantly higher Runx2 (key osteogenic factor) expression 15

at 12 wk followed by a decline at 16 wk while Runx2 mRNA in db femur epiphysis was 16

significantly lower than wt at all ages (Fig. 3A). Further, among the db group, Runx2 17

expression significantly declined with age (Fig 3A). 18

Muscular PGC-1α expression is suppressed in diabetes (39; 40). Since in osteoblasts 19

PGC-1α is PTH-responsive (41) and its expression increases during osteoblast differentiation 20

(42), we assessed its skeletal expression. Like Runx2, PGC-1α expression in wt but not db, 21

peaked at 12 wk and then declined and compared to wt, PGC-1α expression in db bones was 22

significantly lower and showed significant decline with age (Fig. 3A). 23

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Diabetes and obesity negatively influence muscular health by increasing atrogenes 1

(43) that are involved in protein catabolism. Increasing PGC-1α expression and activity 2

downregulates these atrogenes and prevents muscle atrophy under diverse stresses (44). Since 3

some of these atrogenes are reported in bone, and genetic ablations of the E3 ubiquitin ligase 4

MuRF1 and lysosomal protease (cathepsin-L) prevent unloading (45) and ovariectomy-5

induced bone loss (46), we assessed their skeletal expression. Compared to wt, db femur had 6

significantly higher MuRF1, atrogin-1 and cathepsin-L transcripts and their levels in db but 7

not wt mice increased significantly with age (Fig. 3A). Consistent with mRNA expression, 8

PGC-1α protein level was drastically lower in db than wt mice of corresponding age groups 9

(Fig. 3B). MuRF1 protein level conversely, was modestly higher in 8 wk old db mice than in 10

wt, but dramatically increased at 12 and 16 wk (Fig. 3B). 11

Consistent with their ability to directly induce osteoblast apoptosis, palmitate or 12

glucose alone robustly enhanced MuRF1 and atrogin-1, and decreased PGC-1α mRNA and 13

protein in MCO (Fig.3C-D; Dex was used as positive control). 14

15

Bones of insulin resistant mice express functional AdipoR1 16

We next asked if modulating PGC-1α expression and activity could ameliorate 17

diabetes-induced osteopenia. Adiponectin signaling via AdipoR1 in particular modulates 18

PGC-1α (47; 48) and therefore we first assessed its expression in wt and diabetic skeleton at 19

different ages. 20

We recently reported that compared to B6.db, db mice at 12 wk displayed severely 21

depleted muscular AdipoR1 protein and impaired response to gAd (32). Interestingly, 22

AdipoR1 was readily detectable in the wt, B6.db and db bones while consistent with our 23

previous report (32) muscular AdipoR1 was markedly reduced in db but not B6.db mice (Fig 24

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4A). In agreement with differential AdipoR1 expression, acute gAd exposure caused AMPK 1

phosphorylation in muscle and bones of wt, but only in bones of db mice (Fig. 4B). 2

To decipher intact AdipoR1 expression in bones while its depletion in db skeletal 3

muscle, we assessed microRNA-221 (miR-221) and RNA binding protein polypyrimidine 4

tract binding protein (PTB) levels as they negatively regulate AdipoR1 expression (49). miR-5

221 level in bone and muscle across all ages was significantly higher in both diabetic strains 6

than wt, although compared to bone, differences in muscle was higher (2-3 fold in bone vs. 3-7

6 fold in muscle; Fig. 4C). PTB expression pattern however was different from miR-221. 8

While 8 wk old bones had similar PTB expression across the groups, 12 and 16 wk old db 9

bones displayed modest but significantly higher PTB expression than both wt and B6.db (Fig. 10

4C). The skeletal muscle however showed a dramatically higher PTB expression in 12 and 16 11

wk old db mice (5-20 fold) than both wt and B6.db. Taken together, PTB and miR-221 12

expression pattern appeared to correlate with the loss of AdipoR1 in skeletal muscle of db 13

mice. 14

GTDF, an AdipoR1 agonist and Met reverses osteopenia in diabetic mice 15

Recently, we showed that the osteoanabolic agent GTDF (23) acting as an AdipoR1 16

agonist ameliorated diabetes in B6.db but not in db mice (32). Since AdipoR1 action in bone 17

induces osteogenic effect (50) and db bones were AdipoR1-intact (Fig. 4A), we reasoned that 18

despite its inability in ameliorating diabetes GTDF may still show osteoanabolic effect in db 19

mice. 20

Testing the effect of GTDF along with standard anti-diabetic drugs necessitated 21

sufficient number of age and sex-matched db mice that was not available in our colony and 22

thus we procured fresh db mice in identical genetic background. This also allowed us to 23

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confirm that the skeletal phenotype observed in db mice (Fig.1) were not due to breeding and 1

maintenance-associated local factors. Trabecular and cortical parameters of both db mice 2

were comparable (Supplementary Figure 2A and 2B) and both displayed AdipoR1 3

expression in bone but not skeletal muscle (Supplementary Figure 2C). These newly 4

acquired db mice were then used for further studies. 5

We treated db mice with GTDF for 4 wk at a dose (10mg/kg) that failed to rescue 6

diabetes in them (32), so that any osteogenic outcome would not be a consequence of 7

improved diabetic phenotype. We compared the skeletal effects of GTDF with clinically used 8

antidiabetic drugs, Met (AMPK/PGC-1α activator) and Pio (PPARγ agonist). 9

Assessment of metabolic parameters revealed while final body weight significantly 10

increased in Pio-treated mice as expected, GTDF or Met did not alter it (Supplementary 11

Table 1). ECHO-MRI data showed that Pio but not GTDF or Met significantly increased fat 12

mass, while none of the treatments altered lean mass or water content (Supplementary Table 13

1). Pio, but not GTDF or Met significantly decreased fasting and non-fasting blood glucose 14

and Pio alone significantly reduced plasma TG and VLDL levels (Supplementary Table 2). 15

In gross observation by µ-CT, deterioration of femoral and tibial trabecular 16

architecture was readily observed in vehicle treated db, while both GTDF and Met caused 17

substantial improvement (Fig 5A). Compared with vehicle-treated db, GTDF-treated db mice 18

femur had significantly higher BV/TV, Tb.N, Tb.Th and Conn.D, and lower Tb.Sp, and all 19

the parameters were comparable to wt (Fig. 5A), suggesting complete trabecular restoration. 20

Although BV/TV, Tb.N and Tb.Th in Met-treated group were restored to the wt level, these 21

mice still had significantly lower Conn.Dn and higher Tb.Sp than wt (Fig. 5A), indicating a 22

partial restoration. Pio modestly but significantly improved BV/TV, Tb.N, Tb.Th and Conn.D 23

in db femur but failed to reduce Tb.Sp, and except Tb.Th and Tb.N, could not restore other 24

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parameters to wt level (Fig. 5A). Tibial trabecular data show that except Tb.Sp, all 1

parameters in db were significantly lower than wt (Fig. 5A). GTDF-treated db mice displayed 2

significant improvement in all tibial parameters and except BV/TV and Tb.Sp, rest were 3

restored to the wt level (Fig. 5A), suggesting substantial tibial restoration. Met caused partial 4

tibial restoration as only Tb.Th and Conn.D were comparable to wt (Fig. 5A). Pio was 5

ineffective in restoring tibial cancellous bone as all parameters were lower than wt (Fig. 5A). 6

Biomechanical strength of femur diaphysis assessed by three-point bending 7

showed db mice had significantly lower strength parameters than wt (data not shown) and 8

GTDF and Met but not Pio-treated db mice displayed significantly higher resistance to 9

bending than vehicle-treated db as evidenced from higher energy, failure load and 10

stiffness (Fig 5B). 11

Further, GTDF and Met but not Pio–treated db had higher periosteal cell number 12

(Fig. 5C and Supplementary Figure 3). Osteogenic effect of GTDF and Met was further 13

evident from significantly higher Runx2 and OCN mRNAs in femur epiphysis of db mice 14

treated with them whilst Pio had no effect (Fig 5D). Further, GTDF and Met but not Pio 15

significantly lowered expression of adipogenic markers PPARγ and C/EBPα in db (Fig 16

5D). 17

In support of direct effects of GTDF and Met on osteoblasts, the cytotoxic and 18

apoptosis-inducing effects of glucose and palmitate on osteoblasts were mitigated by GTDF 19

and Met but not Pio (Fig 5E and F). 20

AMPK is a key downstream mediator of AdipoR1 signaling, which phosphorylates 21

and thereby activates PGC-1α (47). Osteoblasts in vehicle-treated db femur epiphysis had 22

severely depleted pAMPK compared to wt (Fig. 6A). Consistent with AdipoR1-intact status 23

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of diabetic bone; GTDF-treated db epiphysis had significantly higher pAMPK than vehicle-1

treated db (Fig. 6A). Met activates AMPK and thereby stimulates osteoblast differentiation 2

(51; 52) and consistent with this finding Met-treated db mice displayed significantly higher 3

pAMPK than vehicle control (Fig. 6A). Pio-treated db also had modest but significantly 4

higher pAMPK than vehicle-treated db (Fig. 6A). Similar to AMPK, PGC-1α expression in 5

osteoblasts was severely lower in vehicle-treated db mice and GTDF and Met but not Pio 6

restored it (Fig. 6A and Supplementary Fig. 4). MuRF1 displays an inverse correlation with 7

PGC-1α [(Fig. 3 and (44)] and here too MuRF1 was strongly expressed in vehicle-treated db 8

osteoblasts and GTDF and Met but not Pio–treated db mice its expression was significantly 9

lowered (Fig. 6A and Supplementary Fig. 4). These results were confirmed by 10

immunoblotting using whole marrow-free femur and tibia, where, while PGC-1α was 11

undetectable in vehicle and Pio -treated db, it was strongly expressed in GTDF or Met-treated 12

db mice (Fig 6B). Conversely, MuRF-1 level was high in vehicle and pio -treated db bones 13

while in GTDF and Met –treated bones it was drastically lower (Fig 6B). 14

AdipoR1 and PGC-1αααα mediate the effects of GTDF in osteoblasts 15

We next determined the roles of AdipoR1, AdipoR2, PGC-1α and MuRF1 in 16

osteoblasts by individually silencing them. Fig 7A shows confirmation of silencing of these 17

proteins in osteoblasts. 18

MuRF1 depletion reduced both palmitate and glucose-induced apoptosis in MCOs by 19

>50% (Fig 7B), suggesting the mediatory role of this atrogene in osteoblast apoptosis. 20

We asked if the protection conferred by GTDF against palmitate- and glucose-21

mediated MCO apoptosis (Figure 5G) was AdipoR1-dependent. Indeed siAdipoR1 but not 22

siAdipoR2 or siC (non-silencing control siRNA) abolished anti-apoptotic effects of GTDF 23

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(Fig. 7C and Supplementary Fig. 5). siPGC-1α also abrogated GTDF effect (Fig 7C). As 1

expected, the anti-apoptotic effect of Met was attenuated by siPGC-1α but not siAdipoR1 2

(Fig 7C). 3

Since both GTDF and Met restored trabecular and cortical parameters in db mice, 4

which essentially indicated osteoanabolic actions of these compounds, we assessed GTDF 5

and Met-mediated osteoblast differentiation. In control MCO (siC transfected) ALP activity 6

was significantly stimulated by GTDF and Met (Fig 7D, gAd and BMP-2 was used as 7

positive controls for AdipoR1 and differentiation respectively). Silencing PGC-1α abolished 8

the induction of ALP activity stimulated by all agents (Fig 7D). Upon AdipoR1 silencing, the 9

ALP stimulatory effect of gAd or GTDF but not BMP-2 was abolished (Fig 7D). Silencing 10

AdipoR2 failed to impact the stimulatory effect of gAd or GTDF on ALP activity (Fig 7D). 11

These data suggest the specific role of AdipoR1 and PGC-1α in GTDF-mediated osteoblast 12

differentiation. Interestingly, basal ALP activity in MCOs was also significantly depleted in 13

presence of siPGC-1α and siAdipoR1 but not siAdipoR2 (Fig. 7D) indicating that the 14

autonomous activities of two proteins might also be required for osteoblast differentiation. 15

16

Discussion 17

Although there are numerous rodent models of type 2 diabetes, few have undergone 18

thorough assessment of bone structure in relation to peak bone mass achievement and age-19

related bone loss. Amongst various mouse models that represent human obesity-associated 20

with type 2 diabetes, leptin receptor-deficient mice in a C57-BLKS background represent 21

severe phenotypes (53). In our hands, wt mice in C57-BLKS background displayed peak 22

bone mass achievement at both trabecular and cortical sites at 12 wk whilst db mice did 23

not. Several reports have shown osteopenia in leptin receptor-deficient mice (6; 13) with 24

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the exception of one (54) which showed high bone mass compared to wt littermates. Our 1

data corroborates the finding of Williams et al., showing trabecular osteopenia in 2

approximately 11 week old male db (36). In addition, db mice showed severe age-related 3

bone loss. From these observations, it appears that db mice had an early termination of 4

peak bone gain and/or early onset of age-related bone loss. Bone accrual is critically 5

dependent on osteoblast number and function and since db mice displayed robust apoptosis 6

in osteoblasts at all ages, it appears that lack of viable osteoblasts and resultant deficiency 7

in osteoblast function in the db bones was responsible for their failure in peak bone mass 8

achievement and accelerated the development of age-related osteopenia. Bone formation is 9

consistently lower in type 2 diabetes patients compared to non-diabetics, as evidenced by 10

lower serum osteocalcin (55) and as reported before(36), we have observed that db mice 11

too had markedly lower osteocalcin levels at all ages than wt. 12

Glucose and palmitate induced mouse primary osteoblast apoptosis at pathologically 13

relevant concentrations. The extent of cytotoxicity induced by palmitate and glucose was 14

comparable to dex, a potent inducer of osteopenia (33). It thus appears that the combined 15

effect of glucotoxicity and lipotoxicity causes osteopenia that is equivalent in severity with 16

Dex and diabetic bones indeed shared common features of Dex-induced osteoporosis as bone 17

was lost at both trabecular and cortical sites. 18

Tumor suppressor p53 is a negative regulator of osteoblast differentiation as it 19

suppresses Runx2 expression and hypermorphic p53 mutation in mice causes osteopenia (56). 20

In db bones, p53 was increased and Runx2 suppressed, suggesting reduced osteoblast number 21

and differentiation. Skeletal muscle atrogenes were also elevated in the db bones and showed 22

age-related increase, which could also negatively impact osteoblast survival and 23

differentiation. In fact silencing MuRF1 did confer robust protection against palmitate and 24

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glucose-mediated MCO apoptosis. It is possible that other atrogenes (atrogin-1 and cathepsin-1

L) may too play key roles in osteopenia induced by various stresses, as these factors were 2

also induced in db bones and glucose and palmitate-treated MCO. Recent reports support 3

such notion as mice lacking these atrogenes are protected from osteopenia under diverse 4

stresses (45; 46). By contrast, the muscle anabolic factor, PGC-1α exhibited an age-related 5

decline in db mice. Therefore it appears that like skeletal muscle, a reciprocal relationship 6

between MuRF1 and PGC-1α exists in osteoblasts. Thus loss of osteoblast population and 7

function in db bones may occur due to suppression of PGC-1α and induction of atrogenes. 8

The regulation and possible interaction of p53 and Runx2 with MuRF1 and PGC-1α would 9

be interesting future topic of investigation. 10

AdipoR1 increases insulin sensitivity and promotes cellular energy expenditure by 11

activating AMPK and PGC-1α and AdipoR1 activation represents an attractive therapeutic 12

approach for the treatments of obesity and type 2 diabetes (48). Previously, we showed that 13

db skeletal muscle is deficient in AdipoR1 expression (32). By contrast, here we showed that 14

diabetic bones are AdipoR1-intact. The explanation behind this appears to involve two 15

negative AdipoR1 regulators, PTB and miR-221 (49), as both, especially PTB, were 16

expressed at much higher levels in skeletal muscle than bones of diabetic mice. 17

Adiponectin and its receptors are expressed in bone marrow stromal cells that suggest 18

their potential role in bone metabolism (57). C57BL6/J mice treated with adenoviral-derived 19

adiponectin had increased trabecular bone mass and enhanced mineralization activity of 20

osteoblasts (58). Mice harboring porcine AdipoR1 transgene had higher bone volume and 21

trabecular number than the age- and sex matched controls (50). Cultures of bone marrow 22

stromal cells from adiponectin knockout mice show significantly less osteogenesis than 23

cultures from adiponectin-intact mice (57). Together, these reports suggest that adiponectin 24

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may regulate bone formation in autocrine/paracrine manner in addition to endocrine mode. 1

Although AdipoR1 expression in db bones was comparable to wt, osteopenia in db could be 2

attributed to their observed hypo-adiponectinemia. 3

To investigate whether AdipoR1 in bone and more specifically osteoblasts of diabetic 4

mice could be pharmacologically targeted to mitigate osteopenia through an osteoanabolic 5

mechanism, we made use of GTDF. We have shown that GTDF improved diabetic 6

phenotypes in B6.db mice with intact AdipoR1 in skeletal muscle and liver but not db that 7

lacked functional AdipoR1 in these tissues (32). Thus db mice with skeletal (but not 8

muscular) expression of functional AdipoR1 allowed us to selectively activate AdipoR1 in 9

bone without correcting the diabetic phenotype. The most common clinically used drugs Met 10

and Pio were used at their pharmacologically relevant doses for comparison of skeletal 11

effects between the drugs. 12

Neither GTDF nor Met improved diabetic phenotypes in db but Pio was modestly 13

effective, where although it failed to reduce NEFA yet it reduced fasting and non-fasting 14

serum glucose, which however were still significantly higher than in wt. GTDF and Met 15

improved trabecular microarchitecture, increased bone strength and bone lining cells. Pio was 16

mostly ineffective as some improvement in femur epiphysis was seen but tibia metaphysis, 17

where bone is actually accrued less than femur, showed no improvement. Also, strength 18

parameters were not improved by Pio and neither did it increase periosteal cell number. The 19

modest skeletal improvement by Pio could be due to modest mitigation of diabetic 20

phenotypes as Pio failed to confer protection against gluco- and lipotoxicity on isolated 21

osteoblasts. The skeletal improvement by GTDF and Met without altering diabetic phenotype 22

as well as the protection imparted by them on isolated osteoblasts against glucose and 23

palmitate assault clearly indicates that GTDF and Met had direct osteoblastic effect. Our data 24

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also gains support from previous reports where Met has been shown to directly induce 1

osteoblast differentiation (51; 59)) and protect against high glucose-mediated inhibition of 2

osteoblast growth (60). 3

Although, both GTDF and Met restored bones of db mice by likely preservation of 4

osteoblasts against gluco- and lipotoxicity, the molecular mechanism of action of the two 5

were different. Silencing AdipoR1 but not AdipoR2 abrogated the pro-survival and 6

differentiation-promoting effects of GTDF thus suggesting its AdipoR1-specific effect in 7

osteoblasts as observed earlier in myocytes (32). Skeletal effects of Met however was 8

blocked by silencing PGC-1α but not AdipoR1, suggesting that the osteoanabolic effect of 9

the drug was mediated by PGC-1α and not AdipoR1, which was supported by the fact that 10

Met failed to replace 125

I-gAd in a radioligand competition assay (data not shown). Salient 11

findings of our study are summarized in Fig 8. 12

13

In conclusion, this study showed that diabetic mice in BLKS background have poor 14

achievement of peak bone mass and age-related development of severe osteopenia and, 15

inducing and activating PGC-1α in osteoblasts by GTDF or Met completely reverses 16

osteopenia in these mice without correcting diabetic phenotypes. 17

Author Contributions: SS conceived the study, MPK, AKS, AAJ, MY, SoS, HK, AG, JSM, 18

MCT, SK, GKN and MMG performed experiments, MPK, AKS, JRG, NC and SS designed 19

experiments, AKT, RR, MMG, MRJ, RM, JRG, NC and SS supervised experiments, all 20

authors analyzed data and contributed to discussion. NC and SS wrote the paper. NC is the 21

guarantor of this work and, as such, had full access to all the data in the study and takes 22

responsibility for the integrity of the data and the accuracy of the data analysis. 23

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Acknowledgements: SS and NC acknowledge funding from CSIR network project ASTHI. 1

Authors acknowledge sophisticated analytical instrument facility of CDRI for helps with 2

confocal microscopy and flowcytometry. AKS and AG were supported by fellowships from 3

CSIR. MPK and MY were supported by ICMR. HK, JSM, SoS and SK were supported by 4

UGC. 5

Conflict of interest statement: AAJ and MRJ are employees of Zydus Research Center, the 6

research and development arm of Cadila Healthcare Ltd. All authors have no conflict of 7

interest. 8

9

10

11

12

13

References 14

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41. Nervina JM, Magyar CE, Pirih FQ, Tetradis S: PGC-1alpha is induced by parathyroid 1 hormone and coactivates Nurr1-mediated promoter activity in osteoblasts. Bone 2 2006;39:1018-1025 3 42. Chen CT, Shih YR, Kuo TK, Lee OK, Wei YH: Coordinated changes of mitochondrial 4 biogenesis and antioxidant enzymes during osteogenic differentiation of human 5 mesenchymal stem cells. Stem Cells 2008;26:960-968 6 43. Akhmedov D, Berdeaux R: The effects of obesity on skeletal muscle regeneration. 7 Front Physiol 2013;4:371 8 44. Sandri M, Lin J, Handschin C, Yang W, Arany ZP, Lecker SH, Goldberg AL, Spiegelman 9 BM: PGC-1alpha protects skeletal muscle from atrophy by suppressing FoxO3 action and 10 atrophy-specific gene transcription. Proc Natl Acad Sci U S A 2006;103:16260-16265 11 45. Kondo H, Ezura Y, Nakamoto T, Hayata T, Notomi T, Sorimachi H, Takeda S, Noda M: 12 MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to 13 lead to bone loss. J Cell Biochem 2011;112:3525-3530 14 46. Potts W, Bowyer J, Jones H, Tucker D, Freemont AJ, Millest A, Martin C, Vernon W, 15 Neerunjun D, Slynn G, Harper F, Maciewicz R: Cathepsin L-deficient mice exhibit abnormal 16 skin and bone development and show increased resistance to osteoporosis following 17 ovariectomy. Int J Exp Pathol 2004;85:85-96 18 47. Iwabu M, Yamauchi T, Okada-Iwabu M, Sato K, Nakagawa T, Funata M, Yamaguchi M, 19 Namiki S, Nakayama R, Tabata M, Ogata H, Kubota N, Takamoto I, Hayashi YK, Yamauchi 20 N, Waki H, Fukayama M, Nishino I, Tokuyama K, Ueki K, Oike Y, Ishii S, Hirose K, Shimizu 21 T, Touhara K, Kadowaki T: Adiponectin and AdipoR1 regulate PGC-1alpha and 22 mitochondria by Ca(2+) and AMPK/SIRT1. Nature 2010;464:1313-1319 23 48. Yamauchi T, Kadowaki T: Physiological and pathophysiological roles of adiponectin and 24 adiponectin receptors in the integrated regulation of metabolic and cardiovascular 25 diseases. Int J Obes (Lond) 2008;32 Suppl 7:S13-18 26 49. Lustig Y, Barhod E, Ashwal-Fluss R, Gordin R, Shomron N, Baruch-Umansky K, Hemi R, 27 Karasik A, Kanety H: RNA-binding protein PTB and microRNA-221 coregulate AdipoR1 28 translation and adiponectin signaling. Diabetes 2014;63:433-445 29 50. Lin YY, Chen CY, Chuang TY, Lin Y, Liu HY, Mersmann HJ, Wu SC, Ding ST: Adiponectin 30 receptor 1 regulates bone formation and osteoblast differentiation by GSK-3beta/beta-31 catenin signaling in mice. Bone 2014;64:147-154 32 51. Molinuevo MS, Schurman L, McCarthy AD, Cortizo AM, Tolosa MJ, Gangoiti MV, Arnol V, 33 Sedlinsky C: Effect of metformin on bone marrow progenitor cell differentiation: in vivo 34 and in vitro studies. Journal of bone and mineral research : the official journal of the 35 American Society for Bone and Mineral Research 2010;25:211-221 36 52. Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T: Metformin enhances the 37 differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation 38 as well as eNOS and BMP-2 expression. Biochem Biophys Res Commun 2008;375:414-419 39 53. Davis RC, Castellani LW, Hosseini M, Ben-Zeev O, Mao HZ, Weinstein MM, Jung DY, Jun 40 JY, Kim JK, Lusis AJ, Peterfy M: Early hepatic insulin resistance precedes the onset of 41 diabetes in obese C57BLKS-db/db mice. Diabetes 2010;59:1616-1625 42 54. Ducy P, Amling M, Takeda S, Priemel M, Schilling AF, Beil FT, Shen J, Vinson C, Rueger 43 JM, Karsenty G: Leptin inhibits bone formation through a hypothalamic relay: a central 44 control of bone mass. Cell 2000;100:197-207 45 55. Diaz-Lopez A, Bullo M, Juanola-Falgarona M, Martinez-Gonzalez MA, Estruch R, Covas 46 MI, Aros F, Salas-Salvado J: Reduced serum concentrations of carboxylated and 47 undercarboxylated osteocalcin are associated with risk of developing type 2 diabetes 48 mellitus in a high cardiovascular risk population: a nested case-control study. J Clin 49 Endocrinol Metab 2013;98:4524-4531 50 56. Wang X, Kua HY, Hu Y, Guo K, Zeng Q, Wu Q, Ng HH, Karsenty G, de Crombrugghe B, 51 Yeh J, Li B: p53 functions as a negative regulator of osteoblastogenesis, osteoblast-52 dependent osteoclastogenesis, and bone remodeling. J Cell Biol 2006;172:115-125 53

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57. Shinoda Y, Yamaguchi M, Ogata N, Akune T, Kubota N, Yamauchi T, Terauchi Y, 1 Kadowaki T, Takeuchi Y, Fukumoto S, Ikeda T, Hoshi K, Chung UI, Nakamura K, Kawaguchi 2 H: Regulation of bone formation by adiponectin through autocrine/paracrine and 3 endocrine pathways. J Cell Biochem 2006;99:196-208 4 58. Oshima K, Nampei A, Matsuda M, Iwaki M, Fukuhara A, Hashimoto J, Yoshikawa H, 5 Shimomura I: Adiponectin increases bone mass by suppressing osteoclast and activating 6 osteoblast. Biochem Biophys Res Commun 2005;331:520-526 7 59. Jang WG, Kim EJ, Bae IH, Lee KN, Kim YD, Kim DK, Kim SH, Lee CH, Franceschi RT, 8 Choi HS, Koh JT: Metformin induces osteoblast differentiation via orphan nuclear receptor 9 SHP-mediated transactivation of Runx2. Bone 2011;48:885-893 10 60. Shao X, Cao X, Song G, Zhao Y, Shi B: Metformin rescues the MG63 osteoblasts against 11 the effect of high glucose on proliferation. J Diabetes Res 2014;2014:453940 12 61. Kim JE, Ahn MW, Baek SH, Lee IK, Kim YW, Kim JY, Dan JM, Park SY: AMPK activator, 13 AICAR, inhibits palmitate-induced apoptosis in osteoblast. Bone 2008;43:394-404 14

15

16

17

18

Figure Legends 19

Figure 1. Evaluation of bone phenotypes in 8, 12 and 16 wk old wt and db mice. A. Age 20

and genotype-dependent changes in trabecular microarchitecture. µCT analysis was carried 21

out using Sky Scan 1076 CT scanner (Aartselaar). Left panel shows representative images 22

from 3D-µCT analysis of femur epiphyses (trabecular bone) from 8, 12 and 16 wk old wt and 23

db mice. Bar graphs on the right show quantification. B. Age and genotype-dependent 24

changes in cortical parameters. Femur mid-diaphysis representing cortical bone was analyzed 25

by µCT. Left: 2D-µCT; representative images. Right: Quantification. A and B; n=6 26

bones/group. C. db mice display enhanced osteoblast apoptosis. Quantification of apoptotic 27

osteoblasts was performed by dual TUNEL (DNA fragmentation marker) and Runx2 28

(osteoblast marker) staining of femur epiphysis sections (6 bones/group; 5 fields/bone) 29

followed by confocal microscopy (Carl Zeiss LSM 510 Meta). Runx2+ve, TUNEL+ve cells 30

were plotted as percent of total Runx2+ve cells, representative images in Supplementary 31

Figure 1A. D. db mice display reduced bone lining cells. H&E stained femur epiphyses 32

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sections (6 bones/group; 5 fields/bone) were used for counting by light microscopy by two 1

independent researchers blinded to experimental design; Representative microscopic images 2

in Supplementary Figure 1B. E-J. Serum biochemical parameters in wt and db mice, 3

N=6/group. All graphs in the figure are mean ± SE. *8 wk vs. 12 or 16 wk age groups, #wt vs. 4

db (corresponding age groups). *,#

p<0.05, **,##

p<0.01, ***,###

p<0.001 as determined by two 5

way ANOVA followed by Bonferroni’s post test. Tb.N; average trabecular number per unit 6

length, Conn.Dn; trabecular connectivity normalized by tissue volume, Tb.Sp; mean distance 7

between trabeculae. 8

9

10

Figure 2. Glucose and free fatty acid (palmitate) are sufficient for inducing osteoblast 11

apoptosis. A. Glucose and palmitate induce loss of osteoblast viability. MCO were treated 12

for 24h as indicated and cell viability was determined by an MTT assay and plotted as % 13

viable cells compared to vehicle treated controls. Dexamethasone (Dex) was used as positive 14

control. The glucose and long chain free fatty acid (palmitate) concentrations used here were 15

based on available references (12; 24; 54; 61). Data are mean ± SE of 3 independent 16

experiments performed in triplicates. *P<0.05, ***P<0.001 compared to vehicle treated 17

control as determined by one way ANOVA followed by Bonferroni’s post test. B. Glucose 18

and palmitate induce osteoblast apoptosis in a concentration-dependent manner. Mouse 19

calvarial osteoblasts were treated for 24h as indicated and apoptosis was analyzed by 20

Annexin-V-FITC and PI staining followed by analysis in a FACScalibur flowcytometer 21

(Beckton and Dickinson). Representative dot plots are shown. Right panel shows 22

quantification from 2 independent experiments. C. Palmitate but not glucose induces caspase-23

3 activation. MCOs were treated for 24h as indicated and cleaved caspase-3 levels was 24

determined by immunoblotting. Data are representative of 3 independent experiments. V; 25

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Vehicle; V for Dex treatment was 0.1% DMSO. V for glucose treated group was medium 1

containing 5.5mM glucose and V for palmitate group was medium supplemented with 4% 2

BSA. 3

Figure 3. Diabetic bones exhibit enhanced skeletal muscle atrogenes and suppressed 4

PGC-1α expression. A. Quantitative real time PCR (QPCR)-based gene expression analysis. 5

Total RNA from femur epiphyses of indicated mice (N= 3/group) were isolated, reverse 6

transcribed and were assessed for expression of indicated genes in triplicates. β-actin was 7

used for normalization. Data are mean ± SE. *8 wk vs. 12 or 16 wk age groups, #wt vs. db 8

*,#

p<0.05, **,##

p<0.01, ***,###

p<0.001 as determined by two way ANOVA followed by 9

Bonferroni’s post test. B. Immunoblot-based evaluation of PGC-1α and MuRF1 protein 10

levels in femur epiphyses. β-actin was used as loading control. N=3. C. QPCR-based gene 11

expression analyses in MCO. MCO were treated as indicated for 24h and mRNA expressions 12

were analyzed. β-actin was used for normalization. Data are mean ± SE of 3 independent 13

experiments performed in triplicates. *p<0.05, **p<0.01, ***p<0.001 as determined by one 14

way ANOVA followed by Bonferroni’s post test. D. Evaluation of PGC-1α and MuRF1 15

expression by immunoblotting. β-actin was used as loading control. Data are representative 16

of 3 independent experiments with similar results. 17

Figure 4. db bone but not skeletal muscle express AdipoR1, bind to gAd and induce 18

AMPK phosphorylation. A. Evaluation of AdipoR1 protein level in wt or diabetic mice. 19

Total protein was isolated from femur epiphysis or extensor digitorum longus (EDL) muscles 20

from wt, B6.db or db mice and AdipoR1 expression was determined by immunoblotting. 21

N=3. B. Radioligand saturation assays in isolated osteoblasts or myocytes. Osteoblasts 22

derived from bone marrow cultures or myoblasts isolated from 12 wk old wt or db mice were 23

differentiated in their respective differentiation mediums. Cells were incubated with 125

I-gAd 24

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(specific activity 407.43 Ci/mmole) for 2h (at this time point binding equilibrium was 1

reached) and following washes the cells were lysed and radioactivity was determined in a 2

gamma counter. Data are mean ± SE of 3 independent experiments performed in duplicates. 3

Photomicrographs above the line graphs show representative phase-contrast images of the 4

cells before they were used for 125

IgAd binding assay (objective 10×, magnification 100×). C. 5

db bones but not skeletal muscle display gAd-sensitivity. wt or db mice were 6

intraperitoneally injected with V (PBS) or gAd. 30 min following injection, mice were 7

sacrificed and total protein from EDL muscles and marrow free femurs were used for 8

determination of pAMPK and AMPK levels by immunoblotting. N=3/group. D. 9

Determination of age and strain-dependent bone and skeletal muscle expression of PTB and 10

miR-221. For miR-221, total miRNA from bone and gastrocnemius muscle were isolated 11

using a micro RNA isolation kit. miRNA-221 values were normalized with U6 expression 12

and plotted. For PTB, total RNA isolated from the same tissues was used. Data are mean ± 13

SEM of 3 independent experiments performed in triplicates. *8 wk vs. 12 or 16 wk age 14

groups, #wt vs. B6.db or db, @

B6.db vs. db. *,#,@

p<0.05, **,##

p<0.01, ***,###,@@@

p<0.001 as 15

determined by two way ANOVA followed by Bonferroni’s post test. 16

Figure 5. AdipoR1 agonist GTDF and AMPK/PGC-1α activator metformin (Met) but 17

not PPARγγγγ agonist pioglitazone (Pio) improved bone phenotype in diabetic mice. 12 wk 18

old db mice were treated with GTDF or Pio (10mg/kg b.w.) or Met (350mg/kg b.w.) for 4 wk 19

(comparison of skeletal parameters with db and AdipoR1 expression are in Supplementary 20

Figure 2A-C. Metabolic parameters and the plasma biochemistry are in Supplementary 21

Tables 1 and 2). All graphs are mean ± SE. *wt vs db (all treatments), #vehicle-treated db vs 22

other treatment groups. A. Evaluation of trabecular restoration by GTDF, Met and Pio. Femur 23

and tibia epiphyses (white and black bars respectively) from indicated animals (n=10/group) 24

were evaluated by µCT. Representative images are shown along with quantification data 25

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below. B. Evaluation of bone strength. Bone strength parameters of the same animals were 1

determined by 3-point bending test. N=10/group. C. GTDF and Met but not Pio increase the 2

number of trabecular lining cells. H&E stained femur epiphyses sections (10 bones/group) 3

were used for counting by two independent researchers blinded to experimental design. 4

Representative images are in Supplementary Figure 3. D. GTDF and Met enhanced 5

osteoblast formation markers and suppressed adipogenic marker expressions in femur 6

epiphysis. Femur epiphysis from db mice (n=3; performed in triplicates) treated as indicated 7

were assessed for the expression of indicated mRNAs by QPCR. E. GTDF and Met but not 8

Pio ameliorate palmitate and glucose-induced loss of osteoblast viability. MCOs were 9

pretreated with GTDF (0.1µM), Met (100µM) or Pio (1µM) for 24h followed by treatment 10

with indicated concentrations of glucose or palmitate for a further 24h. Cell viability then was 11

assessed by MTT assay. Data are mean ± SEM of three independent experiments performed 12

in triplicates. F. GTDF and Met protect against palmitate and glucose-induced apoptosis. 13

MCO were pretreated with GTDF (0.1µM) or Met (100µM) for 24h followed by 24h 14

incubation in medium containing indicated concentration of glucose or palmitate. Apoptosis 15

was assessed by Annexin-V-FITC and PI staining followed by flowcytometry. Representative 16

dot plots from 2 independent experiments with similar results are shown. *V vs. treatment 17

groups.*,#

p<0.05, **,##

p<0.01, ***,###

p<0.001 as determined by one way ANOVA followed 18

by Bonferroni’s post test. 19

Figure 6. GTDF and Met but not Pio induce pAMPK, PGC-1α and suppress MuRF1 20

expression in db mice. A. Evaluation of osteoblast expressions of pAMPK, PGC-1α and 21

MuRF1. Femur epiphysis sections obtained from 6 mice/group were probed with indicated 22

antibodies and visualized by fluorescent microscopy (Carl zeiss, AX10-Imager. M2). 23

Representative photomicrographs are shown (objective 20×, magnification 200×). 24

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Microscopic quantifications are given as percent cell count. B. PGC-1α and MuRF1 1

expression in femur or tibia epiphyses from mice treated as indicated. N=3. *Comparison of 2

db with wt mice. #Comparison between vehicle-treated db mice with GTDF, Met or Pio-3

treated groups. *,#

p<0.05, **, ##

p<0.01, ***, ###

p<0.001 as determined by one way ANOVA 4

followed by Bonferroni’s post test. 5

Figure 7. MuRF1 is necessary for palmitate or glucose-induced osteoblast apoptosis and 6

AdipoR1 and PGC-1α are required for GTDF-induced protection against palmitate and 7

glucose-mediated osteoblast apoptosis. A. siRNA-mediated silencing of PGC-1α, MuRF1, 8

AdipoR1 and AdipoR2. MCO were transfected with 0.1µM of each siRNA and expression of 9

indicated proteins was assessed by immunoblotting. Data are representative of 3 independent 10

experiments with similar results. B. MuRF1 depletion protects against glucose and palmiate-11

induced osteoblast apoptosis. 48h after transfection with indicated siRNAs, MCO were 12

incubated in medium containing indicated concentrations of palmitate or glucose for further 13

24h. Apoptosis was then assessed by flowcytometry. One representative set of dot plots from 14

2 independent experiments is shown. Bar graph is quantification from two independent 15

experiments (mean±SE). C. GTDF protection against glucose and palmitate-induced 16

osteoblast apoptosis was compromised by AdipoR1 and PGC-1α silencing. 48h after 17

transfection with indicated siRNAs, MCO were incubated in medium containing glucose 18

(22mM) or palmitate (500µM) supplemented with vehicle, GTDF (0.1µM) or Met (100µM) 19

for further 24h. Apoptosis was then assessed by flowcytometry. One representative set of dot 20

plots from 2 independent experiments is shown. Bar graph is quantification from two 21

independent experiments. AdipoR2 silencing failed to influence GTDF or Met effects; 22

supplementary Figure 5. D. GTDF and gAd-mediated osteoblast differentiation is 23

dependent on PGC-1α and AdipoR1 but not AdipoR2. 48h after transfection with indicated 24

siRNAs, MCO were incubated in differentiation medium supplemented with vehicle, GTDF 25

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(0.1µM), gAd (1µg/ml), BMP2 (0.1µg/ml) or Met (100µM). 24h after treatment, osteoblast 1

differentiation was determined by fluorometric quantification of the differentiation marker 2

alkaline phosphatase (ALP). Data are mean ± SE of 3 independent experiments performed in 3

duplicates. *Vehicle vs. treatment groups, #siC vs. other siRNA transfected groups. 4

***p<0.001, #p<0.05,

###p<0.001 as determined by two way ANOVA followed by 5

Bonferroni’s post test. 6

Figure 8. Schematic diagram summarizing the pathophysiological mechanisms of bone 7

loss in db mice and a potential osteoanabolic role of AdipoR1. In osteoblasts gluco-8

/lipotoxicity induces downregulation (-) of pAMPK and PGC-1α (a key regulator of cellular 9

energy metabolism and whose expression and activity are modulated by AdipoR1) and 10

upregulation (+) of several skeletal muscle atrogenes (atrophy related genes that are involved 11

in protein catabolism) resulting in impaired survival and differentiation of these cells. 12

Osteopenia in db mice is associated with reduced expression of osteogenic genes likely due to 13

decreased PGC-1α and increased atrogenes. Atrogenes could in turn activate p53 leading to 14

inhibition of osteoblast survival and function. Osteopenia in db mice could be reversed by 15

GTDF (an AdipoR1 agonist) or Met (acting independent of AdipoR1 activation) but both 16

converge to PGC-1α to promote osteogenic genes and suppress atrogenes that ultimately 17

improve skeletal health by a likely osteoanabolic mechanism. (Stimulation) and 18

(inhibition). 19

Supplementary information includes Supplementary Figures 1-5 and and Supplementary 20

tables 1-3. 21

22

23

24

25

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2

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Supplementary Figure 1. Age and diabetes-dependent changes in osteoblast viability and

periosteal cell number. A. Bones from db mice display apoptotic osteoblasts. 5µm thick femur

sections from indicated mice were analyzed by TUNEL and Runx2 staining followed by

confocal microscopy (bars 50µm). DAPI was used as a nuclear stain. Representative images are

shown. N=6 bones/group; 5 fields/bone section. B. db bones display severe depletion of

perisoteal cells. Femur epiphysis sections were stained with H&E and visualized by light

microscopy. Arrows indicate perisoetal cells (flattened nuclei). Representative images are shown

(objective 40X, magnification 400X). N=6 bones/group; 5 fields/bone section.

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Supplementary Figure 2. db mice from ZRC and Harlan (New db) display comparative

skeletal features. A. µCT derived parameters for trabecular microarchitecture between 14 wk

old wt, db and new db (n= 6 bones/group), were compared. B. µCT derived cortical parameters

between 14 wk old wt, db and new db (n= 6 bones/group), were compared.*p,0.05, **p<0.01,

***p<0.001 as determined by one way ANOVA followed by Bonferroni’s post test. C. Both db

and New db retain skeletal but not muscle AdipoR1 expression. AdipoR1 expression was

determined in femur epiphyses or EDL muscles by immunoblotting. N=3.

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Supplementary Figure 3. GTDF and Met but not Pio enhance surface lining cells in

diabetic mice. Femur epiphysis sections were stained with H&E and visualized by light

microscopy (objective 40X, magnification 400X). Arrows indicate perisoetal cells (flattened

nuclei). Representative images are shown. N=6 bones/group; 5 fields/bone section.

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Supplementary Figure 4. GTDF and Met but not Pio enhance PGC-1α and suppresses

MuRF1 mRNA expression. Total RNA from femur epiphyses of vehicle (V) or GTDF, Met or

Pio –treated db mice were analyzed for PGC-1a and MuRF1 expression. N=3. **p<0.001 as

determined by one way ANOVA followed by Bonferroni’s post test.

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Supplementary Figure 5. Silencing AdipoR2 fails to affect GTDF action of gluocose and

palmitate -induced osteoblast apoptosis. 48h after transfection with indicated siRNAs, MCO

were incubated in medium containing glucose (22mM) or palmitate (500µM) supplemented with

vehicle, GTDF (0.1µM) or Met (100µM) for further 24h. Apoptosis was then assessed by

flowcytometry. One representative set of dot plots from 2 independent experiments is shown.

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Supplementary Table 1. Pioglitazone but not GTDF and Met alter metabolic parameters in

diabetic mice. Mice were subjected to Echo MRI (Model no. 700) on indicated days. Fed

glucose was measured as described in methods section. *p<0.05, ***p<0.001 as determined by

one way ANOVA between different treatment groups (corresponding ages) followed by

Bonferroni’s post test.

ECHO MRI V GTDF

(10mg/kg)

Metformin

(350 mg/kg)

Pioglitazone

(10mg/kg)

Day 0 26 0 26 0 26 0 26

Fat mass (g) 25.79±0.

66

27.03±0.

76

25.63±0.

42

28.16±0.

66

25.06±0.47 28.50±1.

92

25.35±0.

51

34.31±0.

51*

Lean mass (g) 18.32±0.

50

18.03±0.

30

17.98±0.

45

17.67±0.

18

19.01±0.34 19.94±0.

67

18.81±0.

41

20.14±0.

42

Free water (g) 0.36±0.1

0

0.41±0.0

8

0.90±0.6

0

0.96±0.2

6

1.75±1.17 0.84±0.4

2

0.32±0.1

4

0.05±0.0

3

Total water

(g)

12.94±0.

55

12.84±0.

26

13.18±0.

40

12.80±0.

16

13.98±

0.44

14.12±0.

48

13.95±0.

34

14.43±0.

28

Other Parameters

Body weight 46.8±

0.9

49.5±

1.2

47.5±

0.4

49.9±

0.9

46.7±

0.4

50.8±

2.2

46.9±

0.8

57±

0.8***

Fed Glucose

(mg/dl)

337±11 531±

22

334±11

611±

34

357±26

529±

50

355±24 368±

41***

Number of

animals

16 16 16 16 6 6 6 6

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Supplementary Table 2. Comparison of fasting serum biochemical parameters in db mice

treated as indicated for 28 days. ND; not done. *p<0.05, ***p<0.001 as determined by one

way ANOVA followed by Bonferroni’s post test.

V GTDF

(10mg/kg)

Metformin

(350 mg/kg)

Pioglitazone

(10mg/kg)

Glucose

(mg/dL)

590±

48

516±

44

479±

55

340±

56***

Insulin

(ng/mL)

1.59±

0.34

1.81±

0.25

ND ND

NEFA

(mM)

0.8±0.09 0.8±0.06 1.3±0.2 1.1±0.1

TC

(mg/dL)

140±10 159±26 154±18 156±7

TG

(mg/dL)

83±6 96±16 87±8 52±5*

LDL

(mg/dL)

81±6 92±15 79±10 80±4

VLDL

(mg/dL)

17±1 19±3 17±2 10±1*

HDL

(mg/dL)

74±6 79±11 73±6 66±4

PAI-1

(pg/mL)

20117±

3544

10933±

4491

ND ND

MCP-1

(pg/mL)

13±4 25±9 ND ND

Leptin

(pg/mL)

108316±

23410

95392±

33863

ND ND

Resistin

(pg/ml)

1904±

442

2168±

486

ND ND

Creatinine

(mg/dL)

0.6±0.12 0.7±0.06 0.5±0.1 0.8±0.02

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Supplementary Table 3. Oligonucleotide sequences used for qPCR. Orientation (5’����3’)

Gene

name Forward Reverse

p53 CCGTGTTGGTTCATCCCTGTA

TTTTGGATTTTTAAGACAGAGTCTTTG

TA

Runx2 AAGTGCGGTGCAAACTTTCT TCTCGGTGGCTGCTAGTGA

Cathapsin-

L

CAAATAAGAATAAATATTGGCTTGT

CA TGTAGCCTTCCATACCCCATT

Atrogin-1 AGTGAGGACCGGCTACTGTG GATCAAACGCTTGCGAATCT

MuRF1 CCTGCAGAGTGACCAAGGA GGCGTAGAGGGTGTCAAACT

GLU-L AGTCTGAAGGCTCCAACAGC AAGGGGTCTCGAAACATGG

PTB TCTACCCAGTGACCCTGGAC GAGCTTGGAGAAGTCGATGC

PGC-1α AGCCGTGACCACTGACAACGAG CTGCATGGTTCTGAGTGCTAAG

PPARγ CAGGCCGAGAAGGAGAAGCT GGCTCGCAGATCAGCAGACT

C/EBPα AAACAACGCAACGTGGAGAC TGTCCAGTTCACGGCTCAG

OCN CTGACAAAGCCTTCATGTCCAA GCGGGCGAGTCTGTTCACTA

β-actin CCTCACCCTCCCAAAAGC GTGGACTCAGGGCATGGA

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