Abstract
Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a and less sympathetic innervation compared to wildtype (+/+)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hr), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2 and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wildtype. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wildtype and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular BV/TV loss in the distal femur of Misty mice without affecting wildtype. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.
Keywords: bone, brown adipose tissue, DOCK7, Misty, thermogenesis
Introduction
Control of energy balance is a highly integrated part of metabolism and involves a number of tissues including the hypothalamus, white adipose tissue (WAT) and brown adipose tissue (BAT). In addition, emerging evidence indicates that skeletal metabolism is regulated by systemic energy balance (1–3). For example, leptin is released from WAT when there is excess energy and signals to the hypothalamus to increase energy expenditure and reduce appetite (4, 5). Evidence suggests that leptin also decreases skeletal mass through a hypothalamic-sympathetic relay by uncoupling bone remodeling units leading to suppressed bone formation and enhanced bone resorption (6–8). Consistent with this, sympathetic signaling through the β2-adrenergic receptor (β2AR) results in bone loss (9). Interestingly, the skeleton in turn regulates the expression of insulin and adiponectin in pancreatic β-cells and adipocytes, respectively, by modulating the ratio of un- and under-carboxylated osteocalcin (OCN) relative to total osteocalcin (10). These lines of evidence suggest that energy and skeletal metabolism are mutually interactive and that the sympathetic nervous system (SNS) is critical in this network.
BAT is essential for energy homeostasis in rodents, hibernating animals and neonates. Accumulating evidence from clinical studies suggests the presence of functional BAT in adult humans, although its role in modulating energy metabolism is not clear (11–14). In neonates and small animals such as rodents, BAT contributes to the maintenance of core body temperature and adaptive thermogenesis in response to external stimuli such as food intake and cold exposure. In rodents, a defect in BAT function often results in low body temperature and intolerance to cold exposure due to impaired adaptive thermogenesis. However, the SNS compensates for the lack of BAT in an attempt to maintain body temperature. For example, in adult Ucp1−/− mice, there is no functional BAT, but SNS tone is enhanced, WAT appears ‘brown-like’, and body temperature is maintained at thermo-neutrality (15). Similarly, stimulation of the SNS by treatment with a β3-adrenergic receptor agonist increases metabolic rate in peripheral tissues including WAT (16, 17), and an elevated metabolic rate in WAT causes a morphological change of white adipocytes into brown-like adipocytes accompanied by an increase in mitochondrial content. Thus, elevated sympathetic tone induced by BAT dysfunction causes increased energy expenditure in the peripheral WAT leading to a lean phenotype and a greater metabolic rate. However, increased energy expenditure cannot fully compensate for low body temperature because the thermogenic capacity of peripheral tissues is not as efficient as that in BAT. Thus, BAT plays an important role in energy metabolism in collaboration with the hypothalamic-sympathetic network and affects the systemic alteration of body composition.
The relationship of BAT function to skeletal metabolism in rodents has not previously been studied. Interestingly, in a recent study of younger women, Bredella and colleagues demonstrated a strong positive correlation between BAT volume (by PET) and bone mineral density (18). Similar findings in adolescents were noted by Ponrartana et al, although the correlation became non-significant when muscle mass was included in a multiple regression analysis (19). Notwithstanding, because the sympathetic nervous system regulates skeletal metabolism in a negative manner, we hypothesized that BAT dysfunction drives the SNS and leads to bone loss due to the disruption of the bone remodeling unit (20). To shed light on this issue, we took advantage of Misty mice, which have reduced BAT function, and analyzed their skeletal phenotype (21). The diluted coat color and white belly spot of Misty mice were originally used as a phenotypic marker for the prediction of the Lepr (db) genotype because Lepr (db) locus co-segregates with the locus affected by the Misty mutation. Recently, DOCK7, a Rho family guanine exchange factor (GEF) belonging to the DOCK180 protein family, which has been implicated in axon formation and Schwann cell migration (22, 23), was reported to be responsible for the phenotype of Misty mice (24). DOCK7 is a 2130 amino acid protein and is involved in the function of Rho family of small GTPase such as Rac1, cdc42 and RhoA (25). DOCK7 contains the evolutionarily conserved Dock homology region (DHR)-1 and DHR-2 domains (25–27). The DHR-2 domain has been shown to be necessary for the exchange of GDP to GTP on the GTPases, whereas the DHR-1 domain has been implicated in the interaction with phosphatidylinositol (3,5)-bisphosphate (25–27). Misty mice possess a 43-bp insertion in Exon18, which generates a premature stop codon (24). The truncation occurs in the middle of the DHR-1 domain and if translated, the truncated Misty protein would completely lack the DHR-2 domain (24). Therefore, the Misty mutation in DOCK7 is likely a loss of function mutation although this awaits confirmatory studies.
In this study we demonstrate that the Misty mice have accelerated age-dependent trabecular bone loss due to impaired bone formation and increased bone resorption in both a cell and non-cell autonomous manner. In respect to the latter, trabecular bone loss in Misty mice was slowed by treatment with a β-adrenergic receptor antagonist. These lines of evidence demonstrate that BAT function is involved in skeletal metabolism in part through modulating the SNS.
Materials and Methods
Mice
B6.D2(BKS)-Dock7m/m mice, which we refer to as Misty (m/m) mice, were purchased from Jackson Laboratory (Bar Harbor, Maine). Misty mice were backcrossed to C57BL/6J (Jackson Laboratory) and bred as heterozygous matings to produce Misty mice and wildtype littermate controls. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Maine Medical Center Research Institute.
Dual-energy X-ray absorptiometry (DXA)
Dual-energy X-ray absorptiometry (DXA) for whole body and femoral areal bone mineral density (aBMD, g/cm2) and body composition exclusive of the head were performed using the PIXImus (GE-Lunar) as previously described (28). The PIXImus was calibrated daily with a phantom provided by the manufacturer.
MicroCT
Microarchitecture of distal trabecular bone and midshaft cortical bone were analyzed in femora and vertebrae (L5) by high resolution micro-computed tomography (resolution 10 μm, VivaCT-40, Scanco Medical AG, Bassersdorf, Switzerland). Bones were scanned at energy level of 55 kVp, and intensity of 145 μA. The VivaCT-40 is calibrated weekly using a phantom provided by Scanco. Trabecular bone volume fraction and micro-architecture were evaluated in the secondary spongiosa, starting proximately at 0.6 mm proximal to the distal femoral growth plate, and extending proximally 1.5 mm. Approximately 230 consecutive slices were made at 10.5 μm interval at the distal end of the growth plate and extending in a proximal direction, and 180 contiguous slices were selected for analysis. A fixed threshold of 220 was used to separate bone from soft tissue in all samples. Measurements included trabecular bone volume/total volume (Tb.BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp.) and connectivity density. Scans for the cortical region were measured at the mid-point of each femur, with an isotropic pixel size of 21 μm and slice thickness of 21 μm, and used to calculate the average bone area (BA), total cross-sectional area (TA), bone area/total area (BA/TA), and cortical thickness (Ct.Th.). For mid-shaft analysis, the cortical shell was contoured by user-defined threshold of 260 and iterated through all 50 slices. All scans were analyzed using manufacturer software (Scanco, version 4.05). Acquisition and analysis of microCT data were performed in accordance with recently published guidelines (29).
Bone Histomorphometry
Static and dynamic histomorphometry measures were analyzed between Misty and control mice at 16 weeks of age. Mice were injected with 20 mg/kg calcein and demeclocycline intraperitoneally 7 days and 2 days, respectively, before sample collection. Femurs were analyzed as described previously (28) and standard nomenclature was used (30).
Adipose Tissue Histology and Immunohistochemistry
Brown adipose tissue was fixed in 10% neutral buffered formalin and then transferred to 70% ethanol after 24 hours. Samples were paraffin embedded, sectioned and stained with hematoxylin and eosin. Anti-UCP1 (ab23841) and anti-tyrosine hydroxylase (TH, ab112) antibodies were purchased from Abcam (Cambridge, MA). TH stain: unstained paraffin embedded sections were incubated for 1 hr at room temperature with a 1:700 dilution of anti-TH primary antibody or overnight at 4°C with a 1:250 dilution of anti-UCP-1 primary antibody and developed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
Analysis of Body Temperature
Thermal Signature Analysis ImagIR (Seahorse Bioscience, North Billerica, MA, performed by JAX Phenotyping Services, The Jackson Laboratory, Bar Harbor, ME) was used for the analysis of interscapular thermal change in response to intraperitoneal administration of β3 adrenergic receptor agonist (1.0 mg/kg BW, BRL 37344) according to the manufacturer’s protocol. Body temperature was monitored for 90 minutes after the injection of BRL 37344. Core (rectal) temperature was measured with a ThermoWorks Microtherma 2 (Alpine, UT).
Cold Exposure
Wildtype and Misty mice were subjected to 4°C temperatures (in previously cooled cages containing standard bedding, food and water) for six hours. Mice were observed every 15 minutes for signs of distress. Rectal temperature was monitored every hour.
Brown Adipocyte Culture
Primary brown adipocytes from wildtype and Misty mice were isolated from the interscapular preformed brown adipose tissue (BAT) of neonates (P3) and cultured with modifications according to previously published protocols (31–33). Briefly, BAT was excised and minced under sterile conditions and incubated with isolation buffer (0.123 M NaCl, 1.3 mm CaCl2, 5 mM glucose, 100 mM Hepes, 4% BSA and 0.1% collagenase P) for 40 min at 37°C. Cells were washed and resuspended in primary culture medium (high glucose DMEM, 20% FBS and 1% Penn Strep) and plated at 20,000 cells/cm2. Cells were maintained in primary culture medium (with daily media changes) until confluence, at which time they were trypsinized and plated at 4,000 cells/cm2 in differentiation media (high glucose DMEM, 10% FBS, 20 nM insulin and 1 nM triiodo-L-thyronine (T3)), changed every other day. At confluence, media was changed to induction media (differentiation media, 0.125 mM indomethacin, 0.5 mM IBMX and 5 μM dexamethasone) for two days. Cells were then changed back to differentiation media and maintained for 4 days at which time they were fixed and stained with Oil Red O.
Calvarial Osteoblast Culture
Calvarial osteoblasts (COB) were isolated from wildtype and Misty neonates (P3) as previously described (34). Briefly, calvariae were digested with collagenase P and trypsin and plated in DMEM supplemented with 10% FBS, and nonessential amino acids. Osteoblastogenesis of primary COB was induced with 8 mM β-glycerophosphate and 50 μg/mL of ascorbic acid in αMEM at confluence (day 7) and maintained until day 21.
Real-time PCR
Total RNA was prepared using an RNeasy Mini Kit (Qiagen, Valencia, CA) for cell culture samples or using standard TRIZOL (Sigma, St. Louis, MO) method for tissues. cDNA was generated using a random hexamer and reverse transcriptase (Superscript III, Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. mRNA expression analysis was carried out using an iQ SYBR Green Supermix with an iQ5 thermal cycler and detection system (Bio-Rad, Hercules, CA). Hprt was used as an internal standard control gene for all quantification (35). Primers were designed and tested to be 95–100% efficient by PrimerDesign (Southampton, UK) unless otherwise noted. All primer sequences are listed in Supplementary Table S1.
Propranolol Treatment
Propranolol (Roxane Laboratories, Columbus, OH) was administered at a concentration of 0.5 mg/ml in the drinking water of female Misty and control mice for 8 weeks, from 12–20 weeks of age. Propranolol-containing water was replaced three times per week. Untreated Misty and control mice had normal drinking water.
Serum Parameters
Serum concentrations of P1NP and CTx were measured with the Rat/Mouse P1NP EIA and the RatLaps EIA, respectively (Immunodiagnostic Systems, Scottsdale, AZ). The assay sensitivities were 0.7 and 2 ng/ml for P1NP and CTx, respectively. The intraassay variations were 6.3% and 6.9% and the interassay variations were 8.5% and 12%, respectively for both assays. All measurements were performed in duplicate.
Indirect Calorimetry
Indirect calorimetry measurements were performed using the Promethion® Metabolic Cage System (Sable Systems, Las Vegas, NV) located in the Physiology Core Department of Maine Medical Research Institute. Mice were subjected to a standard 12 hr light/dark cycle during the study which consisted of a 12 hr acclimation period followed by a 72 hour sampling duration. Data shown are representative of the 24 hr average of this period. Each cage in the 8 cage system consists of a cage with standard bedding, a food hopper, water bottle and “house- like enrichment tube” for body mass measurements connected to load cells for continuous monitoring as well as a 11.5 cm running wheel connected to a magnetic reed switch to record revolutions. Ambulatory activity and position were monitored using the XYZ beam arrays with a beam spacing of 0.25 cm. Respiratory gases were measured using the GA-3 gas analyzer (Sable Systems, Las Vegas, NV) utilizing a pull-mode, negative pressure system. Air flow is measured and controlled by the FR-8 (Sable Systems, Las Vegas, NV), with a set flow rate of 2000 mL/min. Oxygen consumption and carbon dioxide production were reported in mL/min. Water vapor is continuously measured and its dilution effect on O2 and CO2 are mathematically compensated for in the analysis stream (36). Energy expenditure was calculated using the Weir equation: Kcal/hr = 60*(0.003941*VO2+0.001106*VCO2) and respiratory quotient (RQ) was calculated as the ratio of VCO2/VO2. Ambulatory activity and wheel running were determined simultaneously with the collection of the calorimetry data. Data acquisition and instrument control were performed using the MetaScreen software v. 1.7.2.3 and the obtained raw data was processed using ExpeData v. 1.5.4 (Sable Systems, Las Vegas, NV) using an analysis script detailing all aspects of data transformation. Correlations were performed between energy expenditure and lean, fat and total body mass to determine if these variables could explain changes in energy expenditure (37–39).
Western Blot
Tibiae were isolated from female Misty and wildtype mice at 8 wks of age, flash frozen, and crushed under liquid nitrogen conditions. Particulates were incubated for 5 minutes at 95°C in Laemmli buffer and protein concentration of the supernatant was determined by BCA assay. Protein (31μg) of wildtype and Misty bone lysates was electrophoresed on 4–15% Tris-glycine gel and transferred to an Immun-Blot PVDF membrane for western blot analysis. Blots were probed with antibodies to DOCK7 (1:1000) (22), β-actin (1:10,000), ECL™-peroxidase labeled anti-rabbit HRP (1:2000), and ECL™-peroxidase labeled anti-mouse HRP (1:10,000).
Statistical analysis
All data are expressed as the mean ± standard error of the mean (SEM) unless otherwise noted. Results were analyzed for statistically significant differences using Student’s t-test or ANOVA followed by Bonferroni’s multiple comparison posthoc test where appropriate. All statistics, including regression analysis, were performed with Prism 6 statistical software (GraphPad Software, Inc. La Jolla, CA). Statistical significance was set at p<0.05.
Results
Misty mice are small with less fat-free mass
To initially examine body composition of Misty mice, we performed DEXA measurements for lean, fat and bone mass (Figure 1). Female Misty mice had approximately 10% lower body mass than wildtype at 4, 8, 12 and 16 weeks. This difference could be largely accounted for by reduced fat-free mass. Interestingly, fat mass in Misty mice varied depending on age, with significantly higher fat mass at 8 weeks and lower fat mass at 16 weeks. Both total and femoral areal bone mineral density (aBMD) and bone mineral content (aBMC) were significantly lower in Misty compared to wildtype at all time points examined (femoral aBMD data not shown). Male Misty mice had similar defects in body composition as the female mice (not shown).
Figure 1. The small size of Misty mice can be attributed to reduced fat-free mass and low bone mass.
Female Misty (open squares) and wildtype (closed circles) littermates were scanned at 4, 8, 12 and 16 weeks of age with a Lunar PIXImus Densitometer for body composition, including total body and femur areal bone mineral density (aBMD) and bone mineral content (aBMC). Points represent mean ± standard error of n=9–15 per group. *p<0.05, **p<0.01 compared to age-matched wildtype.
Trabecular bone loss is accelerated in Misty from 8 to 16 weeks of age
Female Misty mice have significantly lower distal femur trabecular BV/TV (Tb.BV/TV) compared to wildtype at both 8 and 16 weeks of age (Table I and Figure 2), with lower connectivity density (Conn.D) and trabecular number (Tb.N), as well as increased trabecular separation. Trabecular thickness (Tb.Th) was not different between wildtype and Misty at either age. Two-way analysis of variance (ANOVA) demonstrated a significant genotype-age interaction in both the BV/TV and connectivity density parameters. Thus, Tb.BV/TV declined 63% between 8 and 16 weeks of age in Misty, whereas it declined only by 49% in wildtype mice over the same period. Similar to females, male Misty mice also had low distal femur Tb.BV/TV at 8 and 16 weeks of age (Table S2). However, the acceleration in bone loss with age was not present in male mice.
Table I.
μCT of female wildtype and Misty femur and vertebrae at 8 and 16 weeks of age.
| 8 weeks | 16 weeks | 2-way ANOVA p-value | |||||
|---|---|---|---|---|---|---|---|
| +/+ (n=10) | m/m (n=10) | +/+ (n=10) | m/m (n=15) | Interaction | Age | Genotype | |
| Femur Midshaft | |||||||
| Ct.Th (mm) | 0.149 ± 0.003 | 0.126 ± 0.003** | 0.181 ± 0.004 | 0.165 ± 0.003** | 0.390 | < 0.001 | < 0.001 |
| Tt.Ar (mm2) | 1.10 ± 0.02 | 1.24 ± 0.02** | 1.78 ± 0.03 | 1.78 ± 0.03 | 0.011 | < 0.001 | 0.008 |
| Ct.Ar (mm2) | 0.65± 0.02 | 0.56 ± 0.01** | 0.77 ± 0.02 | 0.70 ± 0.02* | 0.555 | < 0.001 | < 0.001 |
| Ct.Ar/Tt.Ar (%) | 37.3 ± 0.5 | 31.2 ± 0.6** | 43.5 ± 0.7 | 39.7 ± 0.6** | 0.122 | < 0.001 | < 0.001 |
| Distal Femur | |||||||
| BV/TV (%) | 13.2 ± 0.6 | 7.3 ± 0.5** | 6.7 ± 0.4 | 2.7 ± 0.3** | 0.031 | < 0.001 | < 0.001 |
| Conn.D (1/mm3) | 132 ± 9 | 54 ± 7** | 52 ± 6 | 9 ± 2** | 0.006 | < 0.001 | < 0.001 |
| Tb.N (1/mm) | 4.64 ± 0.10 | 3.03 ± 0.07** | 3.35 ± 0.13 | 2.08 ± 0.13** | 0.123 | < 0.001 | < 0.001 |
| Tb.Th (mm) | 0.048 ± 0.000 | 0.049 ± 0.001 | 0.049 ± 0.001 | 0.049 ± 0.001 | 0.636 | 0.965 | 0.559 |
| Tb.Sp (mm) | 0.210 ± 0.005 | 0.335 ± 0.009** | 0.301 ± 0.016 | 0.500 ± 0.032** | 0.066 | < 0.001 | < 0.001 |
| Vertebrae (L5) | |||||||
| BV/TV (%) | 26.2 ± 0.6 | 21.9 ± 0.5** | 24.4 ± 0.6 | 18.2 ± 1.2** | 0.230 | 0.001 | < 0.001 |
| Conn.D (1/mm3) | 199 ± 8 | 157 ± 7** | 127 ± 7 | 87 ± 9** | 0.871 | < 0.001 | < 0.001 |
| Tb.N (1/mm) | 5.22 ± 0.09 | 4.60 ± 0.10** | 4.23 ± 0.10 | 3.72 ± 0.17* | 0.646 | < 0.001 | < 0.001 |
| Tb.Th (mm) | 0.0503 ± 0.0004 | 0.0505 ± 0.0003 | 0.0573 ± 0.0015 | 0.0557 ± 0.0021* | 0.068 | < 0.001 | 0.148 |
| Tb.Sp (mm) | 0.185 ± 0.004 | 0.209 ± 0.004** | 0.230 ± 0.007 | 0.264 ± 0.012* | 0.541 | < 0.001 | 0.001 |
Values are expressed as the mean ± SEM. Abbreviations: Ct, cortical; Th, thickness; Tt, total; Ar, area; BV, bone volume; TV, total volume; Conn.D, connectivity density; Tb, trabecular; N, number; Sp, separation.
p<0.01 vs age matched wildtype.
p<0.05 vs age matched wildtype.
Figure 2. Trabecular bone loss in Misty was accelerated with age.
(A) Representative images of distal femur microarchitecture, examined in female wildtype (+/+) and Misty (m/m) mice at 8 and 16 weeks of age. (B) Trabecular (Tb.) bone volume fraction (BV/TV), thickness (Th), number (N), separation (Sp) were measured. White (8-week) and gray (16-week) bars represent mean ± standard error. ap<0.05 compared to genotype-matched 8-week group. bp<0.05 compared to age matched wildtype group.
In female Misty L5 vertebrae, Tb.BV/TV was significantly lower than wildtype at both 8 and 16 weeks (Table I). Remarkably, at 20 weeks of age vertebral BV/TV was more than 50% lower than age-matched wildtype controls (Table S4). Similar to the femur, connectivity density and trabecular number were significantly reduced while trabecular spacing was elevated in Misty vertebrae. Additionally, trabecular thickness was significantly reduced at the 16-week time point only. Two-way ANOVA did not demonstrate a significant interaction between genotype and age in any of the vertebral parameters.
Cortical thickness was significantly lower in female Misty femur midshaft than in wildtype at both ages (Table I). Although mid-femoral cross-sectional area was higher in Misty at 8 weeks of age compared to wildtype, mid-femoral cross-sectional area was not different between wildtype and Misty at 16 weeks of age, suggesting impaired periosteal expansion. Similar to trabecular bone mass, changes in cortical bone from the Misty mutation were not as profound, albeit still significant, in males compared to females (Table S2).
Trabecular bone changes are attributable to both reduced bone formation and increased resorption
To determine which components of the basic multicellular unit were affected by the Misty mutation, we performed static and dynamic histomorphometry on the proximal tibia of both female and male mice at 16 weeks of age. Changes in trabecular microarchitecture by histomorphometry (not shown) were consistent with those from μCT (Table I). Percent mineralized surface (MS/BS), bone formation rate (BFR/BS), osteoblast number (N.Ob/B.Pm) and osteoid thickness were markedly reduced in Misty compared to wildtype, although the mineral apposition rate did not differ by genotype. Additionally, osteoclast surface (Oc.S), osteoclast number (N.Oc/B.Pm) and percent eroded surface (ES/BS) were all significantly elevated. Misty bone marrow also had 63% more adipocytes than wildtype marrow. Thus, a marked reduction in the number of osteoblasts recruited to the bone surface, as well as an increase in osteoclast number and activity accounts for the trabecular bone changes in Misty female mice. Misty male mice had increased osteoclast surface and osteoclast number, but did not have altered osteoblast parameters compared to wildtype at 16 weeks of age, which could account for the less profound trabecular bone loss phenotype in males (Table S2). To fully understand the mechanism of low bone mass in Misty, the remaining studies were performed primarily in female mice.
Interscapular brown adipose tissue is present, but less functional, in Misty mice
To examine whether the absence of brown fat previously described in Misty could alter bone metabolism, we first set out to confirm the absence of pre-formed brown fat (21). To our surprise, Misty mice did indeed have measureable interscapular brown adipose tissue (BAT), which was not different in size, but more variable in Misty compared to wildtype (Figure 3A). Misty BAT tended to have more lipid droplets and less UCP-1 staining (Figure 3B,C). To determine whether the BAT present in Misty was functional, we used infrared imaging to examine the temperature of the interscapular region. Despite a normal core temperature, the interscapular surface temperature was significantly lower in Misty compared to wildtype at 12 weeks of age (Figure 3D,E). Interestingly, stimulation of BAT function with the β3 adrenergic receptor agonist BRL37344 resulted in increased interscapular temperature and increased Pgc1a in both genotypes, indicating Misty BAT is responsive to direct activation (Figure 3F,G). Although it appears that the BRL37344-induced temperature and Pgc1a increases are slightly higher in Misty compared to wildtype, there are no significant genotype X treatment interactions by two-way ANOVA (p=0.61 and p=0.35, respectively). We hypothesized that the reduced BAT temperature at baseline could be the result of impaired sympathetic signaling to BAT, so we quantified sympathetic nerve fibers in wildtype and Misty BAT. The number of tyrosine hydroxylase (TH) positive fibers was significantly reduced in Misty BAT compared to wild type (Figure 3 H,I). To determine whether DOCK7 could play a cell autonomous role in BAT, we differentiated primary brown adipocytes from the stromal vascular fraction of the interscapular BAT of wildtype and Misty mice but found no difference in Oil Red O staining between the two groups (Figure 3J).
Figure 3. Brown adipose tissue (BAT) is present in Misty mice.
(A) Interscapular brown adipose tissue was removed from wildtype (+/+) and Misty (m/m) mice at P2 and weighed (n=5–6). (B) Hematoxylin and eosin stain and (C) UCP-1 immunohistochemistry at P2 (n=5–6). Brown stain is positive for UCP-1 and nuclei are blue. (D) Interscapular temperature measured by infrared imaging at 12 weeks of age (n=8) and (E) core temperature measured with a rectal probe at 8 weeks of age (n=10). (F) Interscapular temperature measured by infrared imaging (n=8) and (G) Pgc1a expression in interscapular BAT (n= 6–8), 3 hours after injection of vehicle or the β3AR agonist BRL37344. (H) Representative images and (I) quantification of tyrosine hydroxylase (TH) positive sympathetic nerve fibers in BAT from 16 week old +/+ and m/m mice. Brown stain (indicated by black arrows) is positive for tyrosine hydroxylase and nuclei are blue. Three random images from n=5 mice per genotype were quantified. (J) Oil red O stain of primary brown adipocyte culture. Image representative of two experiments performed in triplicate with BAT from 4–6 pups per genotype. Bars represent mean ± standard error. Scale bars = 50 μm.
Misty white adipose tissue has an elevated response to cold exposure
To determine whether the observed reduction in BAT temperature and sympathetic innervation had physiologic consequences we exposed wildtype and Misty mice to cold (4°C) for 6 hours. After an initial drop in core temperature, both genotypes maintained core temperature at approximately 37°C throughout the experiment (Figure 4A). BAT expression of Pgc1a was increased in both genotypes compared to ambient temperature controls, however the level of expression in cold-exposed Misty mice did not reach that of cold-exposed wild type, likely due to a significant reduction in baseline Pgc1a expression (Figure 4B). In order to maintain core temperature in the presence of significantly lower BAT temperature, sympathetic innervation and Pgc1a expression, we hypothesized that Misty mice must compensate through another mechanism of heat generation. Indeed, inguinal white adipose tissue (WAT) from cold-exposed Misty mice had increased expression of genes associated with “browning” of white adipose tissue (Pdk4, Foxc2, Pgc1a and Acadl) compared to ambient temperature controls (Figure 4C). Inguinal WAT from wildtype mice did not show the same increases noted in Misty. Interestingly, the elevation of Pgc1a and Acadl in cold-exposed Misty occurred despite significantly lower baseline (room temperature) expression of these genes in Misty compared to wildtype.
Figure 4. Cold-induced thermogenesis in WAT of Misty mice is accompanied by altered gene expression in bone.
Eight week old wildtype and Misty mice were subjected to 4°C temperature for six hours or maintained at ambient temperature. (A) Rectal temperature of cold-treated mice was measured every hour. (B) Pgc1a expression in interscapular BAT. (C–G) Ucp1, Pdk4, Foxc2, Pgc1a and Acadl expression in inguinal WAT. (H–I) Runx2 and Rankl expression in tibia. N=5–10. *p<0.05.
Bone responds to cold temperature
Browning of WAT is under the control of the sympathetic nervous system, therefore, we hypothesized that Misty mice have elevated sympathetic tone, which could partially account for their low trabecular bone volume and accelerated trabecular bone loss after 8 weeks of age. We tested this hypothesis with two strategies. First, to examine whether bone metabolism responds to cold, we measured whole tibia gene expression of bone markers after cold exposure. Runx2 was significantly suppressed by cold in both wildtype and Misty mice (Figure 4D). Furthermore, expression of the osteoclast recruitment factor Rankl was elevated by cold exposure in Misty, but not wildtype tibiae.
Increased sympathetic tone accounts for accelerated trabecular bone loss in Misty femur
To test whether elevated sympathetic activity could cause bone loss under ambient conditions as Misty mice age, we treated wildtype and Misty mice with the non-selective β2 adrenergic receptor (β2AR) antagonist propranolol, which would block sympathetic signaling at the level of the osteoblast. Although propranolol did not alter bone mass in wildtype mice, it increased total bone mineral content (BMC) accrual and slowed distal femur trabecular bone loss in Misty mice from 12 to 20 weeks of age (Figure 5A–C). Increased Tb.Th and Tb.N accompanied the high trabecular BV/TV in Misty mice treated with propranolol (Figure 5D,E). Interestingly, trabecular BV/TV was significantly increased in vertebrae from both wildtype and Misty mice treated with propranolol (Table S4). Additionally, propranolol suppressed the marker of bone resorption, serum Ctx, in Misty mice but not wildtype (Figure 5F). Propranolol did not alter serum P1NP, the marker of bone formation, in either wildtype or Misty mice (Figure 5G).
Figure 5. β-adrenergic receptor blockade slows age-related trabecular bone loss in Misty mice.
Wildtype and Misty mice were administered propranolol from 12 to 20 weeks of age. (A) Representative μCT images of the distal femur trabecular bone. (B) aBMC was measured using DEXA. (C–E) Trabecular BV/TV, Tb.Th and Tb.N were measured by μCT. (F–G) Serum Ctx and P1NP were measured by EIA. N=7–9. *p<0.05.
Low energy expenditure in Misty mice is due to decreased muscle mass
We hypothesized that high sympathetic tone in Misty would be accompanied by increased energy expenditure, so we measured energy expenditure of Misty and wildtype female mice at 8 and 16 weeks of age. Contrary to our initial hypothesis, Misty mice had significantly lower energy expenditure (EE) at 8 weeks (Table III) and 16 weeks (data not shown). On the other hand, energy expenditure was significantly and directly related to fat-free mass (r2=0.971, Table IV and Figure S1). Hence, we suspected that the low EE was related to reduced lean/muscle mass in Misty mice. Consistent with this, when EE was divided by fat-free mass, the difference between the two genotypes disappeared (29.3 ± 0.3 kcal/kg*hr in wildtype vs. 28.8 ± 0.2 kcal/kg*hr in Misty, p=0.40). To confirm altered muscle mass in Misty mice, we weighed soleus, gastrocnemius and thigh muscle in 8-week-old wildtype and Misty mice. Gastrocnemius and thigh muscles from Misty mice weighed significantly less than wildtype, consistent with the reduced fat-free mass and energy expenditure noted previously (Table III).
Table III.
Body composition and metabolic variables in 8-week female Misty and wildtype mice.
| +/+ (n=4–8) | m/m (n=4–8) | p-value | |
|---|---|---|---|
|
|
|||
| Body Mass (g) | 21.1 ± 0.8 | 18.3 ± 0.4 | 0.042 |
| FFM (g) | 17.7 ± 0.5 | 15.2 ± 0.5 | 0.025 |
| Fat (g) | 2.9 ± 0.3 | 3.1 ± 0.2 | 0.696 |
| Activity (meters/day) | 7241 ± 922 | 4795 ± 476 | 0.057 |
| RER | 0.847 ± 0.009 | 0.782 ± 0.039 | 0.155 |
| EE (Kcal/hr) | 0.516 ± 0.02 | 0.411 ± 0.03 | 0.020 |
| Thigh (mg) | 225 ± 7 | 201 ± 6 | 0.017 |
| Gastrocnemius (mg) | 91 ± 2 | 74 ± 2 | <0.001 |
| Soleus (mg) | 6.2 ± 0.3 | 6.0 ± 0.9 | 0.843 |
Abbreviations: FFM, fat-free mass; RER, respiratory exchange ratio; EE, energy expenditure.
Table IV.
Linear regression analysis of total EE vs. body composition in the Misty mouse model.
| r2 | p-value | |
|---|---|---|
|
|
||
| Total Body Mass | 0.87 | 0.0023 |
| Fat-Free Mass | 0.97 | <.0001 |
| Fat Mass | 0.04 | 0.68 |
DOCK7 has a significant role in osteoblast function
Although sympathetic tone was higher in Misty mice and this could be linked to lower bone mass, it appeared unlikely that the increase in sympathetic activity could account for the marked reduction in the recruitment of osteoblasts, nor the fact that bone mass in Misty mice was reduced as early as 4 weeks of age (Figure 1). Therefore, we hypothesized that the protein mutated in Misty mice, DOCK7, could have a cell autonomous role that had not been previously described in osteoblasts. First we found that the DOCK7 protein is detectable in whole femur lysates and Dock7 mRNA is detectable in calvarial osteoblasts (Figure 6) as well as in MC3T3-E1 cells at all stages of differentiation (not shown). Second, although mRNA for Dock7 is detectable in Misty mice (Figure 6A), protein expression (using an N-terminal antibody to an epitope of DOCK7 upstream of the truncation) is absent in whole bone lysates from Misty mice (Figure 6B). Additionally, we did not detect a protein product at or near the predicted 76 kDa molecular weight that we would expect if indeed a truncated DOCK7 protein was being produced. Consistent with reduced osteoblast number in vivo (Table II), bone marrow stromal cell cultures derived from Misty mice had significantly lower colony forming units (CFU-F) compared to those derived from wildtype (data not shown). Similarly, primary calvarial osteoblasts from Misty neonates had reduced alkaline phosphatase staining at day 7 and 21 and reduced Von Kossa stained mineral at day 21 compared to wildtype (Figure 6C). Although the early osteoblast marker Runx2 was unchanged, expression levels of alkaline phosphatase and osteocalcin were both reduced at day 21 (Figure 6D). Taken together, these data support the tenet that DOCK7 plays an important role in osteoblast recruitment and differentiation and that in the Misty mice there are both cell autonomous and non-cell autonomous effects which contribute to low bone mass.
Figure 6. Loss of function of DOCK7 reduces calvarial osteoblastogenesis in vitro.
Calvarial osteoblasts were isolated from Misty and wildtype neonates and differentiated into osteoblasts. (A) Dock7 expression at day 21. (B) DOCK7 protein expression from whole femur lysates from wildtype and Misty mice. (C) Calvarial osteoblast alkaline phosphatase stain at day 7 and alkaline phosphatase and Von Kossa stain at day 21. (D) Expression of osteoblast differentiation markers in calvarial osteoblast cultures at day 21. Gene expression and images representative of two experiments, each performed in triplicate. *p<0.05.
Table II.
Proximal tibia histomorphometry of female wildtype and Misty mice at 16 weeks of age.
| +/+ (n=4–7) | m/m (n=7) | |
|---|---|---|
|
|
||
| MS/BS (%) | 39.3 ± 1.9 | 24.8 ± 3.5* |
| MAR (μm/day) | 1.99 ± 0.21 | 1.34 ± 0.35 |
| BFR/BS(μm3/μm2/year) | 286 ± 38 | 138 ± 43* |
| Ob.S/BS (%) | 9.92 ± 3.27 | 3.08 ± 1.86 |
| N.Ob/B.Pm (/mm) | 8.43 ± 2.67 | 2.03 ± 1.13* |
| OS/BS (%) | 5.65 ± 2.27 | 1.32 ± 1.32 |
| O.Th (μm) | 2.34 ± 0.75 | 0.33 ± 0.33* |
| Oc.S/BS (%) | 1.43 ± 0.26 | 3.32 ± 0.73* |
| N.Oc/B.Pm (/mm) | 0.56 ± 0.11 | 1.50 ± 0.33* |
| ES/BS (%) | 0.48 ± 0.20 | 1.87 ± 0.52* |
| N.Ad/T.Ar (/mm2) | 32.0 ± 4.5 | 52.3 ± 6.2* |
Abbreviations: MS/BS: mineralized surface/bone surface, MAR: mineral apposition rate, BFR/BS: bone formation rate/bone surface, ObS/BS: osteoblast surface/bone surface, N.Ob/B.Pm: number of osteoblast/bone perimeter, OS/BS: osteoid surface/bone surface, O.Th: osteoid thickness, Oc.S/BS: osteoclast surface/bone surface, N.Oc/B.Pm: number of osteoclasts/bone perimeter, ES/BS: erosion surface/bone surface, N.Ad/T.Ar: number of adipocyte/total area.
N/A: not available
p<0.05 compared to control
p<0.01 compared to control
Discussion
Although accumulating evidence demonstrates that skeletal metabolism is under the control of systemic energy balance and that sympathetic tone plays an integral role in this regulation (1, 40, 41), the role of BAT in bone remodeling is not well understood. Misty mice were originally described as having no brown adipose tissue (21); hence our rationale for testing the effect of BAT on bone mass. However, we determined that BAT is indeed present, albeit partially functional (Figure 3). Notwithstanding, we hypothesized that BAT dysfunction could increase sympathetic signaling to bone and cause uncoupled skeletal remodeling. Indeed, Misty mice do have low trabecular and cortical bone mass that is likely due to a combination of cell-autonomous and indirect (sympathetically mediated) effects. Additionally, we demonstrate for the first time that bone responds, albeit adversely, to cold temperature, even in wildtype B6 mice.
BAT, which is present in neonates, was thought to become non-functional by adulthood; however, accumulating evidence from PET scanning suggests the existence of functional BAT in many adults (11–14). Moreover, stimulation of BAT activity has become a potential target for new drugs to combat obesity (42). Nevertheless, it remains to be elucidated whether BAT dysfunction is involved in the pathogenesis of diseases involving altered energy metabolism such as obesity. UCP-1 is critical for brown fat function, serving as the uncoupling protein in mitochondria necessary for proton transfer and heat generation. Ucp1−/− mice gained weight and fat mass when placed at thermoneutrality on a high fat diet, but became resistant to diet-induced obesity when the ambient temperature was decreased (43, 44). This is best explained by the activation of an alternative thermogenic program induced to maintain body temperature and through sympathetic stimulation that increases the metabolic rate in non-BAT tissues. However, the thermogenic capacity of non-BAT tissues is not as efficient as BAT, and requires more calories to maintain body temperature, resulting in the increased energy expenditure and decreased adiposity of some animal models. Similar to Ucp1−/− mice, Misty mice had reduced body temperature and increased markers of thermogenesis in WAT in response to cold (Figure 4), suggesting this alternative thermogenic network is likely activated (45, 46).
It is now well established that skeletal metabolism is under the regulation of the central nervous system, which is mediated through the modulation of sympathetic tone (1). Pharmacological activation of the β2 adrenergic receptor has been shown to result in low bone mass, whereas pharmacological suppression can increase bone mass in certain individuals, suggesting that activation of sympathetic nervous system is a negative regulator for bone mass. In line with this, both global β2-adrenergic receptor (Adrb2) knockout mice, and osteoblast conditionally deleted Adrb2 mice have high bone mineral density (9, 47). Activation of the sympathetic nervous system stimulates bone resorption in part by increasing the expression of Rankl in osteoblasts, whereas it suppresses bone formation, thus uncoupling the bone remodeling unit and causing bone loss (9). In our study, the low bone mass phenotype of Misty mice was accompanied by uncoupling of bone remodeling; i.e. impaired bone formation and increased bone resorption, which resembles the skeletal characteristics of the mice treated with β-adrenergic agonists. Importantly, blockade of β-adrenergic sympathetic tone slowed, although did not totally reverse trabecular bone loss in Misty mice (Figure 5). These lines of evidence demonstrate that elevated sympathetic tone is in part responsible for the reduced bone mass phenotype in Misty mice.
Although Misty mice showed a remarkable age-dependent bone loss phenotype, areal BMD in Misty mice was impaired as early as 4 weeks of age (Figure 1) and trabecular bone mass was not completely rescued by propranolol treatment (Figure 5), suggesting that the Misty mutation in DOCK7 contributes to the skeletal phenotype independent of sympathetic tone. Indeed, calvarial osteoblast differentiation was impaired in Misty mice compared with controls (Figure 6), which is consistent with reduced osteoblast numbers and percent mineralizing surface in vivo (Table II). A function for DOCK7 in osteoblasts has not been previously described. However, others have demonstrated that DOCK7 is important for axon specification and ErbB2-dependent Schwann cell migration (22, 23). DOCK7 knockdown in radial glial progenitor cells suppresses differentiation while overexpression promotes differentiation to neurons (48). We expect that a loss of function of DOCK7 in osteoblasts precursors could affect migration, adhesion and differentiation and a better understanding of these processes could have important physiologic and pathophysiolgic consequences. Thus, a cell-autonomous effect of a presumed loss of function of Dock7, together with an increase in sympathetic tone contribute to the remarkable skeletal phenotype in Misty mice.
Although activation of thermogenesis in WAT is under the control of the SNS and increases energy expenditure in WAT itself, total body energy expenditure was lower in Misty mice compared to wildtype due to a dramatic reduction in fat-free mass, specifically skeletal muscle (Tables III-IV, Figure S1). Whether the Dock7 mutation in Misty mice has a direct effect on muscle development remains to be clarified, and we therefore cannot exclude that some of the low bone mass in Misty could be explained by reduced muscle mass and/or strength.
In sum, we have proposed a novel concept that skeletal metabolism is influenced by BAT function, through modulation of the sympathetic nervous system. Several lines of evidence suggest that sympathetic tone increases with age and in one large cohort study, higher heart rate was a strong and independent predictor of hip fracture risk (49). Moreover, PET imaging suggests that BAT function decreases in older individuals (50). Given the possibility that BAT dysfunction could affect age-related bone loss, beta blockade to enhance bone mass may be a plausible therapeutic target for some individuals with increased SNS tone. Moreover, recent attention has been given to the induction of thermogenesis in WAT to fight obesity. Our findings suggest that targeting thermogenesis may have negative bone consequences, which should be further investigated when developing therapeutics.
Supplementary Material
Figure 7. Model of SNS-mediated bone changes in response to cold.
Cold temperature induces heat generation in brown adipose tissue (BAT) and inducible BAT (iBAT) if necessary (such as with BAT dysfunction in Misty mice). These sympathetically mediated events can also lead to uncoupled skeletal remodeling and accelerated trabecular bone loss with age. Additionally, the Misty mutation in Dock7 has a cell-autonomous role in osteoblasts, through suppression of differentiation.
Acknowledgments
This work was supported by NIH grants AR061932 to KJM, AG040217 to CJR, AR045433 to CJR, DK084970 to CJR. This work was also supported by NIH grants P20 RR18789 and P20 RR15555 to Don M. Wojchowski and P30 RR030927 to Robert Friesel, and by institutional support from Maine Medical Center. The authors thank Leslie Kozak for experimental input and Linda Van Aelst for the DOCK7 antibody. The authors thank Terry Henderson, David Maridas, Casey Doucette and the Investigative Histopathology Laboratory at Michigan State University for technical assistance and Anyonya Guntur for critical reading of the manuscript.
Funding sources: AR061932 to KJM, AG040217 to CJR, AR045433 to CJR, DK084970 to CJR.
Footnotes
Disclosures: All authors state that they have no conflicts of interest.
KJM, KAB, VED, MK, and MLB contributed to design, data acquisition, analysis and interpretation. SAB, PL, SL contributed to data acquisition, analysis and interpretation. MCH, RB and CJR contributed to design and interpretation. KJM and MK drafted the initial manuscript and the remaining authors critically revised the manuscript. All authors approved the final version of the manuscript.
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