Ppargamma Expression Adipose Tissue High Fat Diet

Deficiency of PPARγ in Bone Marrow Stromal Cells Does not Prevent High-Fat Diet-Induced Bone Deterioration in Mice

Jay J Cao,

Grand Forks Human Nutrition Research Center, Agricultural Research Service

, USDA, Grand Forks, ND,

USA

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Brian R Gregoire,

Grand Forks Human Nutrition Research Center, Agricultural Research Service

, USDA, Grand Forks, ND,

USA

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Kim G Michelsen,

Grand Forks Human Nutrition Research Center, Agricultural Research Service

, USDA, Grand Forks, ND,

USA

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Xingming Shi

Department of Neuroscience and Regenerative Medicine, Augusta University

, Augusta, GA,

USA

Department of Orthopaedic Surgery, Augusta University

, Augusta, GA,

USA

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Revision received:

08 April 2021

ABSTRACT

Background

Bone marrow osteoblasts and adipocytes are derived from a common mesenchymal stem cell and have a reciprocal relationship. Peroxisome proliferator-activated receptor gamma (PPARγ), a regulator for adipocyte differentiation, may be a potential target for reducing obesity and increasing bone mass.

Objectives

This study tested the hypothesis that bone-specific Pparg conditional knockout (cKO), via deletion of Pparg from bone marrow stromal cells (BMSC) using Osterix 1 (Osx1)-Cre, would prevent high-fat (HF) diet–induced bone deterioration in mice.

Methods

PPARγ cKO (PPARγ^fl/fl: Osx1-Cre) and floxed littermate control (PPARγ^fl/fl Osx1-Cre^– ) mice that were 6 weeks old were randomly assigned to 4 groups (n = 12/group, 6 male and 6 female) and fed ad libitum with either a normal-fat (NF) purified diet (3.85 kcal/g; 10% energy as fat) or an HF diet (4.73 kcal/g; 45% energy as fat) for 6 mo. Bone structure, body composition, and serum bone-related cytokines were measured. Data were analyzed by 2-way ANOVA with Tukey post hoc comparison.

Results

The HF diet decreased the tibial and lumbar vertebrae trabecular bone volume/total volume (BV/TV) by 28% and 18%, respectively, compared to the NF diet (P < 0.01). PPARγ cKO mice had 23% lower body fat mass and 9% lower lean mass than control mice. PPARγ cKO mice had 41% greater tibial trabecular BV/TV compared to control mice. None of trabecular bone parameters at the second lumbar vertebra were affected by genotype. PPARγ cKO mice had decreased cortical thickness compared to control mice. PPARγ cKO mice had a 14% lower (P < 0.01) serum concentration of leptin and a 35% higher (P < 0.05) concentration of osteocalcin compared with control mice.

Conclusions

These data indicate that PPARγ has site-specific impacts on bone structures in mice and that knockout PPARγ in BMSC increased bone mass (BV/TV) in the tibia but not the lumbar vertebrae. PPARγ disruption in BMSC did not prevent HF diet–induced bone deterioration in mice.

Introduction

Obesity is a major public health problem affecting 36% of the population in the United States (1), and is associated with many chronic disorders (2–4). Osteoporosis, a disease characterized by low bone mass and microarchitectural deterioration, affects about 10 million Americans over the age of 50 (5, 6). Although obesity is generally associated with increased bone mineral density and content in humans (7, 8), studies have also documented detrimental effects of obesity on bone in humans (9) and mice (10, 11).

However, the pathophysiological effects of obesity on bone are complex: the mechanisms through which obesity affects bone metabolism are not well defined, and multiple mechanisms have been proposed (9, 11, 12). One possible mechanism is that because both marrow osteoblasts (bone-forming cells) and adipocytes (energy-storing cells) are derived from a common mesenchymal stem cell (13), obesity would increase bone marrow adipocyte differentiation and fat accumulation at the expense of decreasing osteoblast differentiation and bone formation (12). In supporting this mechanism, studies have demonstrated that the decreased bone marrow osteoblastogenesis with aging or estrogen deficiency is accompanied by increased marrow adipogenesis (14, 15). Experimentally, agents that inhibit adipogenesis stimulate osteoblast differentiation, and vice versa (16–19).

Obesity affects bone metabolism through systemic or local cytokines secreted by adipocytes, especially proinflammatory cytokines. Obesity is associated with low-grade elevated chronic inflammation because adipose tissue is infiltrated with the increased amount of macrophages, which are an important source of proinflammatory cytokines (20–22). Proinflammatory cytokines, such as TNFα, IL-1β, and IL-6, promote osteoclast activity and bone resorption (10, 23–25).

The specific mechanism controlling the fate of a mesenchymal stem cell, to either adipocytes or osteoblasts, is the area of interest for our research. Peroxisome proliferator-activated receptor gamma (PPARγ), as a ligand-activated nuclear receptor and a master regulator of adipocyte differentiation (26), has been considered a primary target for potential anti-osteoporosis and anti-obesity therapy (27–29).

Homozygous global PPARγ deficient (PPARγ^–/–) mice are embryonically lethal due to placental dysfunction, and heterozygous global PPARγ deficient (PPARγ^+/–) mice are resistant to high-fat (HF) diet–induced obesity and insulin resistance (30) and show a high bone mass phenotype (31). To overcome embryonic lethality and further investigate PPARγ and adipocyte functions in specific tissue, mice with various tissue-specific PPARγ deletions have been generated. For example, deletion of PPARγ in the adipose tissue of mice protects against HF diet–induced obesity and insulin resistance (32) and increases bone formation (33).

Several mouse models with bone-specific PPARγ deletion have been generated (29, 34, 35). We previously demonstrated that adult mice with bone-specific deletion of the PPARγ gene from osteoprogenitor cells (PPARγ^fl/fl: Col3.6Cre) have a higher bone mass [bone volume/total volume (BV/TV)] during development than control mice (29) and that deletion of the PPARγ gene from bone marrow mesenchymal stromal cells (BMSC; PPARγ^fl/fl: Dermo1-Cre) protects against cortical bone loss in aging mice (34).

However, the extent to which bone marrow adipocytes affect bone in diet-induced obesity has not been investigated. In this study, we hypothesized that bone marrow adipocyte deficiency would prevent bone deterioration in HF diet–induced obesity. To test this hypothesis, we generated mice in which the PPARγ gene was deleted from BMSC/progenitor cells using Osterix 1 (Osx1)-Cre, which is expressed in the earliest stages of commitment to the osteoblast lineage (36).

Methods

Animals, breeding, and genotyping

Double-floxed PPARγ (PPARγ^fl/fl) mice were kindly donated by Dr. Frank J Gonzales at the National Cancer Institute (37) and rederived at The Jackson Laboratory. In addition, a breeding pair of Osx1-GFP::Cre [B6 .Cg-Tg(Sp7-tTA, tetO-EGFP/cre)1Amc/J] mice were purchased from The Jackson Laboratory. The same Osx1 Cre line has been used successfully in bone-related research (35).

Mice were housed in the Tecniplast HEPA-ventilated micro-isolator caging system in an isolated barrier room with a 12-hour light/dark cycle. The animal protocol for the study was approved by the USDA Agricultural Research Service Grand Forks Human Nutrition Research Center Institutional Animal Care and Use Committee. Animals were maintained and processed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Mice were fed Purina Rat Chow #5012 (Ralston-Purina) during the breeding period before the experiment started.

PPARγ conditional knockout (cKO, PPARγ^fl/fl: Osx1-Cre) mice were created by breeding Osx1-Cre transgenic mice with PPARγ^fl/fl mice using standard breeding procedures as previously described (29, 34, 35, 37). Genotyping was performed around weaning and analyzed using primers and PCR conditions previously described (29, 38). In brief, ear genomic DNA was extracted and amplified with 1 forward primer (F1: 5'-CTCCAATGTTCTCAAACTTAC-3') and 2 reverse primers (R1: 5'-GATGAGTCATGTAAGTTGACC-3'; R2: 5'-GTATTCTATGGCTTCCAGTG C-3'). The expected sizes of the PCR products are ∼250 bp for a wild-type allele, ∼285 bp for a floxed allele, and ∼450 bp for a null allele. PCR analysis of Cre was performed using primers 5'-GCATTTCTGGGGATTGCTTA-3' (forward) and 5'-GTCATCCTTAGCGCCGTAAA-3' (reverse). The expected size of a PCR product for Cre is ∼350 bp.

At 6 weeks of age, floxed littermate control (PPARγ^fl/fl Osx1-Cre^–) and cKO mice were randomly assigned to 4 groups (n = 12/group, 6 male and 6 female) and fed ad libitum with either a normal-fat (NF) purified diet (3.85 kcal/g; 10% energy as fat; D12450B) or an HF diet (4.73 kcal/g; 45% energy as fat with extra fat from lard; D12451), which were formulated based on AIN-93G (39) and purchased from Research Diet for 6 mo (Supplemental Table 1). The diets were formulated based on energy, with concentrations by weight of essential nutrients adjusted to be greater in the HF diet than in the NF diet. Mice had free access to diets and water throughout the study. Body mass was recorded every 2 wk and food consumption was recorded for 2 consecutive days every 2 wk.

Body composition

Body composition (fat compared with lean mass) was measured 1 d before starting the experimental diets and at the end of the study by an EchoMRI-100 whole-body composition analyzer (Echo Medical Systems, LLC) according to the manufacturer's instruction and as previously described in detail (25).

Bone structure evaluation with micro–computed tomography

The tibia and the second lumbar vertebra (LV2) of each mouse were scanned at an isotopic voxel size of 12 μm and were analyzed for bone structure using a Scanco micro–computed tomography (μCT) scanner 40 (Scanco Medical AG) with the setting as previously described (10, 40). For the tibial trabecular bone, 100 slices starting from about 0.1 mm distal to the growth plate were chosen for analyses. For the LV2 analysis, the entire secondary spongiosa between the cranial and the caudal area was scanned and analyzed. For assessment of cortical indices, a scan of 100 slices at the tibial mid-diaphysis was performed. The recommended guidelines for μCT scanning (41) and bone histomorphometry nomenclature were followed (42).

Measurements of serum biochemical markers

Serum concentrations of bone biochemical markers were determined using commercial anti-mouse ELISA kits according to the manufacturers' instructions, with kits for adiponectin and IL-6 (R&D Systems), bone-specific alkaline phosphatase (BALP, Cusabio Technology), osteocalcin (Biomedical Technologies Inc.), leptin (ALPCO Diagnostics), and tartrate-resistant acid phosphatase 5b (TRAP, Immunodiagnostic System). The same cytokine was measured for all samples with the same batch kits on the same day to minimize variation. Quality control of each bone biomarker was performed using 2 control samples in duplicate for each plate. The control samples with known concentrations (low and high values) were supplied by the respective kit manufacturers. The concentrations of the low- and high-value control samples must fall within the recommended range for the assay to be valid. The calculated CVs for biomarkers were 2.3% for adiponectin, 4.2% for IL-6, 3.2% for BALP, 3.7% for leptin, 0.6% for osteocalcin, and 1.5% for TRAP.

Measurement of mRNA in bone

Total RNA was obtained from pulverized bone samples that included bone marrow using Trizol (Invitrogen), reverse transcribed, and amplified and quantified using a Sequence Detection System (SDS 7300) as previously described (10). Relative mRNA expression was normalized to the expression of Gapdh mRNA in the same sample. The sequence information of oligonucleotide primers is presented in Supplemental Table 2. All oligonucleotide primers for PCR amplification were designed using PrimerQuest software, synthesized, and purified by HPLC (Integrated DNA Technologies).

Statistical analyses

Data are expressed as means ± SDs. The main effects of dietary fat, genotype, sex, and their interactions were analyzed using 3-factor ANOVA with JMP (version 15.0.0, SAS Institute, Inc.). Data from both sexes were then pooled and analyzed using 2-factor ANOVA (or repeated 2-factor ANOVA for body weight changes in Supplemental Figure 1) because no interactions were detected between sex and fat level or genotype. Tukey contrasts were performed to compare group means if the fat-by-genotype interaction was significant. In all analyses, P values ≤ 0.05 and > 0.05 were considered statistically significant and nonsignificant, respectively. A power analysis indicated that detecting a significant difference in the primary outcome variable of tibial BV/TV (3 percentage points) among the treatments with 0.90 power and an alpha of 0.05 (10) would require 12 mice/group.

Results

There were no differences in initial body weight between the NF and HF groups, but PPARγ cKO mice had 11% lower body weight than control mice (Figure 1A). All animals gained weight (the body weight changes are presented in Supplemental Figure 1). Body masses of mice fed the HF diet were significantly greater than the masses of those fed the NF diet (P < 0.01). Compared to those in the floxed littermate control group, PPARγ cKO mice had a 15% lower body mass at the end of study (P < 0.05). We further examined the extent to which the HF diet or genotype affected body composition. The HF diet increased the fat mass for both floxed littermate control and cKO mice (P < 0.05) but did not affect the lean body mass (P > 0.05; Figure 1B andC). Floxed littermate control mice had more fat mass than PPARγ cKO mice before the study (P < 0.01) and at the end of the study (P = 0.05). Floxed littermate control mice also had more lean mass at the beginning (P < 0.05) and the end of the study (P < 0.05) than PPARγ cKO mice.

FIGURE 1

(A) Body mass, (B) fat mass, (C) lean mass, and (D) food and energy intake of floxed littermate control (PPARγ^fl/fl Osx1-Cre^–) or PPARγ cKO (PPARγ^fl/fl: Osx1-Cre) mice fed either a purified normal-fat (10% energy as fat) or a high-fat (45% energy as fat) diet for 6 mo. Values are means ± SDs (n = 12). The main effects of dietary fat and genotype and their interactions were analyzed using 2-factor ANOVA. Abbreviations: C-HF, control mice fed a high-fat diet; C-NF, control mice fed a normal-fat diet; cKO, conditional knockout; cKO-NF, conditional knockout mice fed a NF diet; cKO-HF, conditional knockout mice fed an HF diet.

FIGURE 1

The mean food intake (Figure 1D) by animals fed the HF diets was lower than the intake by those fed the NF diet (P < 0.01) but the total energy intake was similar between groups (P = 0.80) due to the higher energy content of the HF diet (3.85 kcal/g compared with 4.73 kcal/g for the NF and HF diets, respectively). Since the diet was formulated based on energy, with the concentrations by weight of essential nutrients adjusted to be greater in the HF diet than that in the NF diet, while food intake was lower for the mice fed the HF diet compared to those fed the NF diet (2.85 g compared with 3.53 g for the HF and NF diets, respectively), the intake of micronutrients, including Ca, was the same for both groups (Supplemental Table 3). PPARγ cKO mice had reduced food intake and energy intake compared with floxed littermate control mice (P < 0.05).

The effects of the HF diet and genotype on bone structures in the proximal tibia, mid-diaphysis of the tibia, and LV2 were evaluated with μCT (Table 1;Figure 2). As expected, the HF diet adversely affected the 3-dimensional bone microstructure. Compared with the NF diet, the HF diet decreased the tibial and lumbar vertebrae trabecular BV/TV (P < 0.01) by 28% and 18%, respectively. Mice fed the HF diet also had lower bone mineral density (BMD) at the tibia (P < 0.01) and LV2 (P = 0.01). The HF diet did not affect any tibial mid-diaphysis parameters. PPARγ cKO mice had greater tibial BV/TV, a higher trabecular number (Tb.N), greater connectivity density (Conn.Dn), a higher BMD, and a lower structure model index (SMI) compared to control mice. None of the trabecular bone parameters at LV2 were affected by genotype. PPARγ cKO mice had a lower bone area, total area, medullary area (Me.Ar), bone perimeter, cortical thickness, and BMD compared to control mice. There were no significant interactions between diet and genotype on other bone parameters (P > 0.05), except the tibial structure mode index (SMI; P < 0.05). PPARγ cKO mice fed the HF diet had a higher SMI than those fed the NF diet, indicating the adverse effect of the HF diet on the bones of PPARγ cKO mice.

FIGURE 2

Representative images of (A) proximal tibia trabecular bone, (B) tibial mid-diaphysis cortical bone, and (C) the second lumbar vertebra trabecular bone (data are presented in Table 1) of floxed littermate control (PPARγ^fl/fl Osx1-Cre^–) or PPARγ cKO (PPARγ^fl/fl: Osx1-Cre) mice fed either a purified normal-fat (10% energy as fat) or a high-fat (45% energy as fat) diet for 6 mo. For the tibial trabecular bone, 100 slices starting from about 0.1 mm distal to the growth plate were chosen for analyses. For the second lumbar vertebra analysis, the entire secondary spongiosa between the cranial and the caudal area was scanned and analyzed. For assessment of cortical indices, a scan of 100 slices at the tibial mid-diaphysis was performed. Abbreviations: C-HF, control mice fed a high-fat diet; C-NF, control mice fed a normal-fat diet; cKO, conditional knockout; cKO-NF, conditional knockout mice fed a NF diet; cKO-HF, conditional knockout mice fed an HF diet.

FIGURE 2

TABLE 1

Bone structural properties of proximal tibia and lumbar vertebrae from floxed littermate control or PPARγ cKO mice fed either a purified normal-fat or a high-fat diet for 6 mo ¹

					ANOVA (P value)
Indices	C-NF	cKO-NF	C-HF	cKO-HF	Fat	Genotype	Fat × Genotype
Proximal tibia (trabecular bone)
BV/TV, %	7.47 ± 1.88	9.07 ± 3.75	4.34 ± 1.38	7.60 ± 3.26	<0.01	<0.01	0.30
Tb.N, mm^–1	2.72 ± 0.56	3.52 ± 1.20	2.44 ± 0.72	2.99 ± 1.51	0.19	0.03	0.70
Tb.Th, mm	0.06 ± 0.01	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.01	0.99	0.47	0.65
Tb.Sp, mm	0.38 ± 0.08	0.32 ± 0.13	0.44 ± 0.12	0.41 ± 0.17	0.05	0.16	0.69
Conn.Dn, mm^–3	18.3 ± 17.9	44.0 ± 35.9	5.3 ± 4.2	40.7 ± 53.4	0.40	<0.01	0.61
SMI	2.64 ± 0.48^ab	2.46 ± 0.31^b	3.06 ± 0.43^a	2.32 ± 0.39^b	0.24	<0.01	<0.05
BMD, mg hydroxyapatite/ccm	115 ± 23	140 ± 44	74 ± 32	115 ± 45	<0.01	<0.01	0.43
Tibial mid-diaphysis (cortical bone)
B.Ar, mm²	0.73 ± 0.06	0.65 ± 0.05	0.71 ± 0.06	0.63 ± 0.06	0.24	<0.01	0.90
T.Ar, mm²	1.08 ± 0.10	0.97 ± 0.08	1.05 ± 0.08	0.94 ± 0.09	0.29	<0.01	0.97
Me.Ar, mm²	0.35 ± 0.07	0.31 ± 0.04	0.34 ± 0.04	0.30 ± 0.04	0.51	0.01	0.95
B.Pm, mm	6.3 ± 0.4	6.0 ± 0.4	6.2 ± 0.3	5.8 ± 0.4	0.43	<0.01	0.71
Ct.Th, mm	0.26 ± 0.03	0.23 ± 0.01	0.24 ± 0.01	0.23 ± 0.01	0.19	<0.01	0.30
BMD, mg hydroxyapatite/ccm	787 ± 70	748 ± 38	772 ± 41	750 ± 33	0.64	0.03	0.51
Second lumbar vertebra (trabecular bone)
BV/TV, %	22.3 ± 6.6	21.3 ± 3.0	18.0 ± 5.5	17.5 ± 3.3	<0.01	0.62	0.85
Tb.N, mm^–1	4.3 ± 0.7	4.5 ± 0.7	4.3 ± 0.7	4.1 ± 0.9	0.33	0.87	0.36
Tb.Th, mm	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.45	0.50	0.22
Tb.Sp, mm	0.23 ± 0.04	0.22 ± 0.04	0.23 ± 0.04	0.25 ± 0.05	0.24	0.79	0.38
Conn.Dn, mm^–3	116 ± 36	152 ± 44	120 ± 46	119 ± 72	0.32	0.25	0.24
SMI	0.63 ± 0.77	0.70 ± 0.45	1.16 ± 0.75	1.16 ± 0.42	<0.01	0.84	0.86
BMD, mg hydroxyapatite/ccm	240 ± 59	232 ± 28	203 ± 50	200 ± 32	0.01	0.68	0.87

					ANOVA (P value)
Indices	C-NF	cKO-NF	C-HF	cKO-HF	Fat	Genotype	Fat × Genotype
Proximal tibia (trabecular bone)
BV/TV, %	7.47 ± 1.88	9.07 ± 3.75	4.34 ± 1.38	7.60 ± 3.26	<0.01	<0.01	0.30
Tb.N, mm^–1	2.72 ± 0.56	3.52 ± 1.20	2.44 ± 0.72	2.99 ± 1.51	0.19	0.03	0.70
Tb.Th, mm	0.06 ± 0.01	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.01	0.99	0.47	0.65
Tb.Sp, mm	0.38 ± 0.08	0.32 ± 0.13	0.44 ± 0.12	0.41 ± 0.17	0.05	0.16	0.69
Conn.Dn, mm^–3	18.3 ± 17.9	44.0 ± 35.9	5.3 ± 4.2	40.7 ± 53.4	0.40	<0.01	0.61
SMI	2.64 ± 0.48^ab	2.46 ± 0.31^b	3.06 ± 0.43^a	2.32 ± 0.39^b	0.24	<0.01	<0.05
BMD, mg hydroxyapatite/ccm	115 ± 23	140 ± 44	74 ± 32	115 ± 45	<0.01	<0.01	0.43
Tibial mid-diaphysis (cortical bone)
B.Ar, mm²	0.73 ± 0.06	0.65 ± 0.05	0.71 ± 0.06	0.63 ± 0.06	0.24	<0.01	0.90
T.Ar, mm²	1.08 ± 0.10	0.97 ± 0.08	1.05 ± 0.08	0.94 ± 0.09	0.29	<0.01	0.97
Me.Ar, mm²	0.35 ± 0.07	0.31 ± 0.04	0.34 ± 0.04	0.30 ± 0.04	0.51	0.01	0.95
B.Pm, mm	6.3 ± 0.4	6.0 ± 0.4	6.2 ± 0.3	5.8 ± 0.4	0.43	<0.01	0.71
Ct.Th, mm	0.26 ± 0.03	0.23 ± 0.01	0.24 ± 0.01	0.23 ± 0.01	0.19	<0.01	0.30
BMD, mg hydroxyapatite/ccm	787 ± 70	748 ± 38	772 ± 41	750 ± 33	0.64	0.03	0.51
Second lumbar vertebra (trabecular bone)
BV/TV, %	22.3 ± 6.6	21.3 ± 3.0	18.0 ± 5.5	17.5 ± 3.3	<0.01	0.62	0.85
Tb.N, mm^–1	4.3 ± 0.7	4.5 ± 0.7	4.3 ± 0.7	4.1 ± 0.9	0.33	0.87	0.36
Tb.Th, mm	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.45	0.50	0.22
Tb.Sp, mm	0.23 ± 0.04	0.22 ± 0.04	0.23 ± 0.04	0.25 ± 0.05	0.24	0.79	0.38
Conn.Dn, mm^–3	116 ± 36	152 ± 44	120 ± 46	119 ± 72	0.32	0.25	0.24
SMI	0.63 ± 0.77	0.70 ± 0.45	1.16 ± 0.75	1.16 ± 0.42	<0.01	0.84	0.86
BMD, mg hydroxyapatite/ccm	240 ± 59	232 ± 28	203 ± 50	200 ± 32	0.01	0.68	0.87

Abbreviations: B.Ar, cross-sectional bone area; BMD, bone mineral density; B.Pm, bone perimeter; BV, bone volume; C-HF, control mice fed a high-fat diet; C-NF, control mice fed a normal-fat diet; cKO, conditional knockout; cKO-HF, conditional knockout mice fed a high-fat diet; cKO-NF, conditional knockout mice fed a normal-fat diet; Conn.Dn, connectivity density; Ct.Th, cortical thickness; Me.Ar, cross-sectional medullary area; SMI, structure model index; T.Ar, cross-sectional total area; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; TV, total volume.

Values are means ± SDs (n = 12). Labeled means in a row without a common letter differ at a P value ≤ 0.05. The main effects of dietary fat and genotype and their interactions were analyzed using a 2-factor ANOVA. Control mice were PPARγ^fl/fl Osx1-Cre⁻ mice and cKO mice were PPARγ^fl/fl: Osx1-Cre mice.

TABLE 1

Bone structural properties of proximal tibia and lumbar vertebrae from floxed littermate control or PPARγ cKO mice fed either a purified normal-fat or a high-fat diet for 6 mo ¹

					ANOVA (P value)
Indices	C-NF	cKO-NF	C-HF	cKO-HF	Fat	Genotype	Fat × Genotype
Proximal tibia (trabecular bone)
BV/TV, %	7.47 ± 1.88	9.07 ± 3.75	4.34 ± 1.38	7.60 ± 3.26	<0.01	<0.01	0.30
Tb.N, mm^–1	2.72 ± 0.56	3.52 ± 1.20	2.44 ± 0.72	2.99 ± 1.51	0.19	0.03	0.70
Tb.Th, mm	0.06 ± 0.01	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.01	0.99	0.47	0.65
Tb.Sp, mm	0.38 ± 0.08	0.32 ± 0.13	0.44 ± 0.12	0.41 ± 0.17	0.05	0.16	0.69
Conn.Dn, mm^–3	18.3 ± 17.9	44.0 ± 35.9	5.3 ± 4.2	40.7 ± 53.4	0.40	<0.01	0.61
SMI	2.64 ± 0.48^ab	2.46 ± 0.31^b	3.06 ± 0.43^a	2.32 ± 0.39^b	0.24	<0.01	<0.05
BMD, mg hydroxyapatite/ccm	115 ± 23	140 ± 44	74 ± 32	115 ± 45	<0.01	<0.01	0.43
Tibial mid-diaphysis (cortical bone)
B.Ar, mm²	0.73 ± 0.06	0.65 ± 0.05	0.71 ± 0.06	0.63 ± 0.06	0.24	<0.01	0.90
T.Ar, mm²	1.08 ± 0.10	0.97 ± 0.08	1.05 ± 0.08	0.94 ± 0.09	0.29	<0.01	0.97
Me.Ar, mm²	0.35 ± 0.07	0.31 ± 0.04	0.34 ± 0.04	0.30 ± 0.04	0.51	0.01	0.95
B.Pm, mm	6.3 ± 0.4	6.0 ± 0.4	6.2 ± 0.3	5.8 ± 0.4	0.43	<0.01	0.71
Ct.Th, mm	0.26 ± 0.03	0.23 ± 0.01	0.24 ± 0.01	0.23 ± 0.01	0.19	<0.01	0.30
BMD, mg hydroxyapatite/ccm	787 ± 70	748 ± 38	772 ± 41	750 ± 33	0.64	0.03	0.51
Second lumbar vertebra (trabecular bone)
BV/TV, %	22.3 ± 6.6	21.3 ± 3.0	18.0 ± 5.5	17.5 ± 3.3	<0.01	0.62	0.85
Tb.N, mm^–1	4.3 ± 0.7	4.5 ± 0.7	4.3 ± 0.7	4.1 ± 0.9	0.33	0.87	0.36
Tb.Th, mm	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.45	0.50	0.22
Tb.Sp, mm	0.23 ± 0.04	0.22 ± 0.04	0.23 ± 0.04	0.25 ± 0.05	0.24	0.79	0.38
Conn.Dn, mm^–3	116 ± 36	152 ± 44	120 ± 46	119 ± 72	0.32	0.25	0.24
SMI	0.63 ± 0.77	0.70 ± 0.45	1.16 ± 0.75	1.16 ± 0.42	<0.01	0.84	0.86
BMD, mg hydroxyapatite/ccm	240 ± 59	232 ± 28	203 ± 50	200 ± 32	0.01	0.68	0.87

					ANOVA (P value)
Indices	C-NF	cKO-NF	C-HF	cKO-HF	Fat	Genotype	Fat × Genotype
Proximal tibia (trabecular bone)
BV/TV, %	7.47 ± 1.88	9.07 ± 3.75	4.34 ± 1.38	7.60 ± 3.26	<0.01	<0.01	0.30
Tb.N, mm^–1	2.72 ± 0.56	3.52 ± 1.20	2.44 ± 0.72	2.99 ± 1.51	0.19	0.03	0.70
Tb.Th, mm	0.06 ± 0.01	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.01	0.99	0.47	0.65
Tb.Sp, mm	0.38 ± 0.08	0.32 ± 0.13	0.44 ± 0.12	0.41 ± 0.17	0.05	0.16	0.69
Conn.Dn, mm^–3	18.3 ± 17.9	44.0 ± 35.9	5.3 ± 4.2	40.7 ± 53.4	0.40	<0.01	0.61
SMI	2.64 ± 0.48^ab	2.46 ± 0.31^b	3.06 ± 0.43^a	2.32 ± 0.39^b	0.24	<0.01	<0.05
BMD, mg hydroxyapatite/ccm	115 ± 23	140 ± 44	74 ± 32	115 ± 45	<0.01	<0.01	0.43
Tibial mid-diaphysis (cortical bone)
B.Ar, mm²	0.73 ± 0.06	0.65 ± 0.05	0.71 ± 0.06	0.63 ± 0.06	0.24	<0.01	0.90
T.Ar, mm²	1.08 ± 0.10	0.97 ± 0.08	1.05 ± 0.08	0.94 ± 0.09	0.29	<0.01	0.97
Me.Ar, mm²	0.35 ± 0.07	0.31 ± 0.04	0.34 ± 0.04	0.30 ± 0.04	0.51	0.01	0.95
B.Pm, mm	6.3 ± 0.4	6.0 ± 0.4	6.2 ± 0.3	5.8 ± 0.4	0.43	<0.01	0.71
Ct.Th, mm	0.26 ± 0.03	0.23 ± 0.01	0.24 ± 0.01	0.23 ± 0.01	0.19	<0.01	0.30
BMD, mg hydroxyapatite/ccm	787 ± 70	748 ± 38	772 ± 41	750 ± 33	0.64	0.03	0.51
Second lumbar vertebra (trabecular bone)
BV/TV, %	22.3 ± 6.6	21.3 ± 3.0	18.0 ± 5.5	17.5 ± 3.3	<0.01	0.62	0.85
Tb.N, mm^–1	4.3 ± 0.7	4.5 ± 0.7	4.3 ± 0.7	4.1 ± 0.9	0.33	0.87	0.36
Tb.Th, mm	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.45	0.50	0.22
Tb.Sp, mm	0.23 ± 0.04	0.22 ± 0.04	0.23 ± 0.04	0.25 ± 0.05	0.24	0.79	0.38
Conn.Dn, mm^–3	116 ± 36	152 ± 44	120 ± 46	119 ± 72	0.32	0.25	0.24
SMI	0.63 ± 0.77	0.70 ± 0.45	1.16 ± 0.75	1.16 ± 0.42	<0.01	0.84	0.86
BMD, mg hydroxyapatite/ccm	240 ± 59	232 ± 28	203 ± 50	200 ± 32	0.01	0.68	0.87

Mice fed the HF diet had higher (P < 0.01) serum concentrations of leptin and the bone resorption marker TRAP and a lower (P < 0.05) concentration of osteocalcin than those fed the NF diet (Figure 3). Compared with control mice, PPARγ cKO mice had a 14% lower (P < 0.01) serum concentration of leptin and a 35% higher (P < 0.05) concentration of osteocalcin. There were no differences in serum concentrations of adiponectin, BALP, and IL-6 between mice fed the HF and NF diets or between control and PPARγ cKO mice.

FIGURE 3

Serum concentrations of (A) leptin, (B) adiponectin, (C) BALP, (D) IL-6, (E) osteocalcin, and (F) TRAP of floxed littermate control (PPARγ^fl/fl Osx1-Cre^–) or PPARγ cKO (PPARγ^fl/fl: Osx1-Cre) mice fed either a purified normal-fat (10% energy as fat) or a high-fat (45% energy as fat) diet for 6 mo. Values are means ± SDs (n = 12). The main effects of dietary fat and genotype and their interactions were analyzed using 2-factor ANOVA. Abbreviations: BALP, bone-specific alkaline phosphatase; C-HF, control mice fed a high-fat diet; C-NF, control mice fed a normal-fat diet; cKO, conditional knockout; cKO-NF, conditional knockout mice fed a normal-fat diet; cKO-HF, conditional knockout mice fed a high-fat diet; TRAP, tartrate-resistant acid phosphatase.

FIGURE 3

The HF diet increased Pparg (P < 0.05) and leptin (P < 0.01) expression in whole bone with marrow (P < 0.01;Figure 4), and there were no differences in their expressions between control and PPARγ cKO mice (P > 0.05).

FIGURE 4

Expression of Pparg and Lep in femurs of floxed littermate control (PPARγ^fl/fl Osx1-Cre^–) or PPARγ cKO (PPARγ^fl/fl: Osx1-Cre) mice fed either a purified normal-fat (10% energy as fat) or a high-fat (45% energy as fat) diet for 6 mo. Values are means ± SDs (n = 12). The main effects of dietary fat and genotype and their interactions were analyzed using 2-factor ANOVA. Abbreviations: C-HF, control mice fed a high-fat diet; C-NF, control mice fed a normal-fat diet; cKO, conditional knockout; cKO-NF, conditional knockout mice fed a normal-fat diet; cKO-HF, conditional knockout mice fed a high-fat diet; Lep, leptin.

FIGURE 4

Discussion

In this study, we examined the role of marrow adipocytes in bone-related changes in an HF diet–induced obese mouse model using animals carrying a deletion of the PPARγ gene in Osx1-expressing bone marrow stromal cells and using their floxed littermate as controls. Our data showed that Pparg cKO mice (PPARγ^fl/fl: Osx1-Cre) have an increased trabecular bone mass (BV/TV) in the proximal tibia and an increased serum concentration of osteocalcin, a bone formation marker, but with decreased fat mass and a lower serum leptin concentration, compared to their littermate controls. We found that Pparg cKO mice were not protected from the detrimental impact of an HF diet on bone structure, a finding different from our original hypothesis.

As expected, we found that the HF diet increased the body mass but negatively affected the bone structure at the tibia and LV2, which has been repeatedly demonstrated (43–46). Similarly, leptin-deficient (ob/ob) mice are obese and weigh twice as much as wild-type animals but have a lower bone mass at the weight-bearing bone femur (47). Apparently, the mechanical loading and stimulation of bone formation conferred by body weight does not counter the detrimental effects of excessive adiposity on bone (47). The mechanisms through which obesity or adiposity negatively affect bone health have been extensively discussed and reviewed (9, 11, 12). Obesity in humans is multifaceted and the micronutrient intake is not always at the recommended level; the use of rodent models of obesity allows us to control the intake of nutrients and to delineate the mechanisms underlying the negative impact of adiposity upon bone health.

The noteworthy finding of this study is that, contrary to our hypothesis, our data show that the knockout of Pparg in BMSC improved bone outcome measures but did not protect against bone deterioration in HF diet–induced obesity. Bone is a dynamic organ that undergoes significant turnover via a process involving bone resorption by osteoclasts and bone formation by osteoblasts (48). HF diet–induced obesity is known to increase the production of proinflammatory cytokines and increase osteoclast-mediated bone resorption (24, 25, 49), despite the increase in body weight or osteoblast number or activity (10, 24). Considering osteoclast differentiation and function are not affected by a global Pparg deficiency (31) or bone-specific Pparg deficiency (29), it is possible that the HF diet could induce osteoclastogenesis that is not regulated by PPARγ in the mesenchymal lineage. Osteoclasts are derived from hematopoietic precursors of the monocyte-macrophage lineage (48), and mice lacking Pparg in osteoclast lineage cells (Tie2-Cre: PPARγ^f/f) developed osteopetrosis, characterized by an increased bone mass (50). Therefore, it is possible that PPARγ also plays an important role in obesity-induced osteoclastogenesis and bone resorption, in addition to osteoblastogenesis and bone formation. In addition, the possibility of systemic effects of PPARγ needs to be explored.

One of the proposed mechanisms relating to the detrimental effects of obesity on bone is that both adipocytes and osteoblasts are derived from a common mesenchymal stem cell (13), and the increase in 1 population in the bone marrow would decrease the other. Existing evidence does support a reciprocal relationship between bone marrow adipocytes and osteoblasts (14–19). Our data in part support this mechanism, in that mice carrying deletion of Pparg in Osx1-expressing cells (PPARγ^fl/fl: Osx1-Cre) had increased tibial Tb.N, BMD, and Conn.Dn and decreased cortical thickness. Decreased cortical thickness and/or Me.Ar in Pparg cKO mice could indicate modeling for growth was limited when compared to the control mice. In addition, the serum concentration of osteocalcin was increased compared to the concentration in floxed littermate control mice, indicating an increase in the osteoblast number and/or activity and bone formation. Similar to our findings, Sun et al. (35), using the same Pparg cKO model, also reported that the femoral Tb.N was significantly increased and trabecular separation was decreased in Pparg cKO mice. However, it is possible that this is due to a limitation of bone modeling for growth rather than to an increase in bone mass (BV/TV) in the proximal tibia per se, since Pparg cKO mice were smaller than the controls.

Interestingly, we found that deletion of the Pparg gene did not affect the bone structural parameters at lumbar vertebrae. Contrary to the findings of this study, we found in an earlier study that mice deficient in Pparg from cells expressing a 3.6-kb type I collagen promoter fragment (PPARγ^fl/fl: Col3.6-Cre) exhibited moderate reductions in BMD and bone mineral content (BMC) in the fourth LV but not in the femur (29). Mice with Pparg deletion of Dermo1-expressing mesenchymal lineage cells (PPARγ^fl/fl: Dermo1-Cre) had increased BMD and BMC in both the femur and LV at 6 mo but not at 21 mo of age (34). We do not have a good explanation for the discrepancies in these site-specific bone structural changes among the different studies. Contrasting phenotypes in the femur and spine have also been reported in leptin-deficient ob/ob mice (47). Different cKO models in these studies could contribute to these different results. Other mechanisms or pathways might be involved. For example, mechanical loading conferred by body weight may have differential effects on long bones, such as the tibia in our study, compared to the lumbar spine. David et al. (17) found that mechanical loading downregulates PPARγ in BMSC and adipogenesis while increasing osteoblastogenesis.

The finding that Pparg cKO mice had a lower fat mass than floxed littermate controls was unexpected, since adipocyte deletion should have been restricted to bone marrow. Similar to this observation, we also found that mice with Pparg deletion in Dermol-expressing cells had a reduction in whole-body adiposity (34). While Osx1 was identified as being expressed in developing bones, which is required for osteoblast differentiation and bone formation (51), whether or how much it is expressed or its function in other tissues besides bone has not been investigated. It is also possible that bone-specific ablation of adipocytes would affect peripheral adipogenesis and/or adipocyte functions through certain feedback loops, such as bone-secreted cytokines. For example, osteocalcin, an osteoblast-secreted protein used as a bone formation marker, has been demonstrated to behave as a hormone that regulates glucose and energy metabolism (52). Osteocalcin-deficient (osteocalcin ^–/–) mice have abnormal visceral fat (53), and long-term treatment of wild-type mice with nanomolar amounts of osteocalcin can significantly decrease the body weight and fat mass of mice fed an HF diet (52). Whether the decrease in fat mass in Pparg cKO mice is the consequence of the increase in the serum concentration of osteocalcin, as found in this study, or of other bone-secreted cytokines remains to be investigated.

Obviously, the relationship between obesity and bone is multifaceted, and PPARγ is 1 of the important factors regulating both obesity and bone development. Bone-secreted cytokines can affect energy, the glucose metabolism, and adiposity (as discussed above), and bone itself is also modulated by obesity or paracrine effects of adipokines, such as leptin and adiponectin (11, 54, 55).

In summary, this study demonstrated that mice deficient of Pparg in BMSC had a decreased fat mass and an increased bone mass (BV/TV) in the proximal tibia but not in lumbar vertebrae when compared with the PPARγ-floxed littermate controls. Deletion of PPARγ did not prevent HF diet–induced bone deterioration in mice. Further studies are needed to understand the complex relationship between adipocytes or adiposity and bone metabolism, which may help develop therapeutic agents to prevent or treat both obesity and osteoporosis.

Acknowledgments

We thank Dr. Frank J. Gonzalez, National Cancer Institute, National Institute of Health, for providing floxed PPARγ mice. We thank James Lindlauf for help in animal breeding and care and food intake measurements, and Matthew J. Picklo for critical review of the manuscript.

The authors' responsibilities were as follows—JJC: contributed to the study design, implementation, data analysis and interpretation, and manuscript preparation; XS: contributed to the study design, data interpretation, and manuscript preparation: BRG and KGM: contributed to the study design, the experiment execution, data collection, and manuscript revision; and all authors: read and approved the final manuscript.

Notes

This work was supported by the Agricultural Research Service of the USDA (#3062-51000-053-00D). Funding support for XS was from the National Institute on Aging of the National Institutes of Health under Award Number R01AG046248.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer. The findings and conclusions in this manuscript are those of the authors and should not be construed to represent any official USDA or US Government determination or policy.

Author disclosures: Funding support for XS was from the National Institute on Aging of the National Institutes of Health under Award Number R01AG046248. JJC is a member of the Journal's Editorial Board. All other authors report no conflicts of interest.

Supplemental Tables 1–3 and Supplemental Figure 1 are available from the "Supplementary data" link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn.

Abbreviations used: B.Ar, bone area; BALP, bone-specific alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; BMSC, bone marrow stromal cells; B.Pm, bone perimeter; BV, bone volume; cKO, conditional knockout; Conn.D, connectivity density; Ct.Th, cortical thickness; HF, high-fat; LV2, second lumbar vertebra; Me.Ar, medullary area; NF, normal-fat; Osx1, Osterix 1; PPARγ, peroxisome proliferator-activated receptor gamma; SMI, structure model index; T.Ar, total area; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; TRAP, tartrate-resistant acid phosphatase; TV, total volume; μCT, micro–computed tomography.

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This work is written by (a) US Government employee(s) and is in the public domain in the US.

Supplementary data

Ppargamma Expression Adipose Tissue High Fat Diet

Source: https://academic.oup.com/jn/article/151/9/2697/6296112

Ord Giatead76

Ppargamma Expression Adipose Tissue High Fat Diet

Deficiency of PPARγ in Bone Marrow Stromal Cells Does not Prevent High-Fat Diet-Induced Bone Deterioration in Mice

ABSTRACT

Introduction

Methods

Animals, breeding, and genotyping

Body composition

Bone structure evaluation with micro–computed tomography

Measurements of serum biochemical markers

Measurement of mRNA in bone

Statistical analyses

Results

Discussion

Acknowledgments

Notes

References

Supplementary data

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