SEARCH

SEARCH BY CITATION

Keywords:

  • BONE;
  • BROWN ADIPOSE TISSUE;
  • DOCK7;
  • MISTY;
  • THERMOGENESIS

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

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 wild-type (+/+)). 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 hours), 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 wild-type. 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 wild-type and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild-type. 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.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

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 uncarboxylated and undercarboxylated 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 thermoneutrality.[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[18] demonstrated a strong positive correlation between BAT volume (by positron emission tomography [PET]) and bone mineral density. Similar findings in adolescents were noted by Ponrartana and colleagues,[19] although the correlation became nonsignificant when muscle mass was included in a multiple regression analysis. 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 the Lepr (db) locus cosegregates 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 guanosine triphosphatase (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 guanosine diphosphate (GDP) to guanosine triphosphate (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 exon 18, 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-autonomous and noncell-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.

Subjects and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Mice

B6.D2(BKS)-Dock7m/m mice, which we refer to as Misty (m/m) mice, were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Misty mice were backcrossed to C57BL/6J (Jackson Laboratory) and bred as heterozygous matings to produce Misty mice and wild-type 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

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, Fairfield, CT, USA), as described.[28] The PIXImus was calibrated daily with a phantom provided by the manufacturer.

Micro–computed tomography

Microarchitecture of distal trabecular bone and midshaft cortical bone were analyzed in femora and vertebrae (L5) by high-resolution micro–computed tomography (µCT) (VivaCT-40, 10-µm resolution; 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 microarchitecture 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 intervals 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 µCT 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[28] and standard nomenclature was used.[30]

Adipose tissue histology and immunohistochemistry

BAT 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–uncoupling protein 1 (UCP1, ab23841) and anti–tyrosine hydroxylase (TH, ab112) antibodies were purchased from Abcam (Cambridge, MA, USA). For TH stain, unstained paraffin-embedded sections were incubated for 1 hour 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, USA).

Analysis of body temperature

Thermal Signature Analysis ImagIR (Seahorse Bioscience, North Billerica, MA, USA; performed by JAX Phenotyping Services, The Jackson Laboratory, Bar Harbor, ME, USA) was used for the analysis of interscapular thermal change in response to intraperitoneal administration of β3 adrenergic receptor agonist (1.0 mg/kg body weight [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, USA).

Cold exposure

Wild-type and Misty mice were subjected to 4°C temperatures (in previously cooled cages containing standard bedding, food, and water) for 6 hours. Mice were observed every 15 minutes for signs of distress. Rectal temperature was monitored every hour.

Brown adipocyte culture

Primary brown adipocytes from wild-type and Misty mice were isolated from the interscapular preformed BAT of neonates (P3) and cultured with modifications according to 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% bovine serum albumin [BSA], and 0.1% collagenase P) for 40 minutes at 37°C. Cells were washed and resuspended in primary culture medium (high glucose DMEM, 20% fetal bovine serum [FBS], and 1% Penicillin-Streptomycin [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 4000 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 3-isobutyl-1-methylxanthine [IBMX], and 5 µM dexamethasone) for 2 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 wild-type and Misty neonates (P3) as 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 α modified essential medium (α-MEM) at confluence (day 7) and maintained until day 21.

Real-time PCR

Total RNA was prepared using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) for cell culture samples or using a standard TRIzol (Sigma, St. Louis, MO, USA) method for tissues. cDNA was generated using a random hexamer and reverse transcriptase (Superscript III; Invitrogen, Carlsbad, CA, USA) 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, USA). Hprt was used as an internal standard control gene for all quantification.[35] Primers were designed and tested to be 95% to 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, USA) 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 to 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 amino-terminal propeptide of type I procollagen (P1NP) and cross-linked C-telopeptide (CTX) were measured with the Rat/Mouse P1NP enzyme immunoassay (EIA) and the RatLaps EIA, respectively (Immunodiagnostic Systems, Scottsdale, AZ, USA). 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, USA) located in the Physiology Core Department of Maine Medical Research Institute. Mice were subjected to a standard 12-hour light/dark cycle during the study, which consisted of a 12-hour acclimation period followed by a 72-hour sampling duration. Data shown are representative of the 24-hour average of this period. Each cage in the eight-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) using a pull-mode, negative-pressure system. Air flow is measured and controlled by the FR-8 (Sable Systems), with a set flow rate of 2000 mL/min. Oxygen consumption and carbon dioxide production were reported in milliliters per minute (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/h = 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) 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

Tibias were isolated from female Misty and wild-type mice at 8 weeks of age, flash-frozen, and crushed under liquid nitrogen conditions. Particulates were incubated for 5 minutes at 95°C in Laemmli buffer and the protein concentration of the supernatant was determined by bicinchoninic acid (BCA) assay. Protein (31 µg) of wild-type and Misty bone lysates was electrophoresed on 4% to 15% Tris-glycine gel and transferred to an Immun-Blot polyvinylidene fluoride (PVDF) membrane for Western blot analysis. Blots were probed with antibodies to DOCK7 (1:1000),[22] β-actin (1:10,000), enhanced chemiluminescence (ECL)-peroxidase–labeled anti-rabbit horseradish peroxidase (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 post hoc test where appropriate. All statistics, including regression analysis, were performed with Prism 6 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at p < 0.05.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Misty mice are small with less fat-free mass

To initially examine body composition of Misty mice, we performed DXA measurements for lean, fat, and bone mass (Fig. 1). Female Misty mice had approximately 10% lower body mass than wild-type 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 aBMD and bone mineral content (aBMC) were significantly lower in Misty compared to wild-type 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).

image

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 wild-type (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 ± SEM of n = 9–15 per group. *p < 0.05, **p < 0.01 compared to age-matched wild-type.

Download figure to PowerPoint

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 wild-type at both 8 and 16 weeks of age (Table 1, Fig. 2A, B), 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 wild-type and Misty at either age. Two-way 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 wild-type 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 (Supplementary Table S2). However, the acceleration in bone loss with age was not present in male mice.

Table 1. µCT of Female Wild-Type and Misty Femur and Vertebrae at 8 and 16 Weeks of Age
 8 weeks16 weeksTwo-way ANOVA p
+/+ (n = 10)m/m (n = 10)+/+ (n = 10)m/m (n = 15)InteractionAgeGenotype
  • Values are expressed as mean ± SEM.

  • µCT = micro–computed tomography; 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 <0.05 versus age-matched wild-type.

  • **

    p <0.01 versus age-matched wild-type.

Femur midshaft
Ct.Th (mm)0.149 ± 0.0030.126 ± 0.003**0.181 ± 0.0040.165 ± 0.003**0.390<0.001<0.001
Tt.Ar (mm2)1.10 ± 0.021.24 ± 0.02**1.78 ± 0.031.78 ± 0.030.011<0.0010.008
Ct.Ar (mm2)0.65 ± 0.020.56 ± 0.01**0.77 ± 0.020.70 ± 0.02*0.555<0.001<0.001
Ct.Ar/Tt.Ar (%)37.3 ± 0.531.2 ± 0.6**43.5 ± 0.739.7 ± 0.6**0.122<0.001<0.001
Distal femur
BV/TV (%)13.2 ± 0.67.3 ± 0.5**6.7 ± 0.42.7 ± 0.3**0.031<0.001<0.001
Conn.D (1/mm3)132 ± 954 ± 7**52 ± 69 ± 2**0.006<0.001<0.001
Tb.N (1/mm)4.64 ± 0.103.03 ± 0.07**3.35 ± 0.132.08 ± 0.13**0.123<0.001<0.001
Tb.Th (mm)0.048 ± 0.0000.049 ± 0.0010.049 ± 0.0010.049 ± 0.0010.6360.9650.559
Tb.Sp (mm)0.210 ± 0.0050.335 ± 0.009**0.301 ± 0.0160.500 ± 0.032**0.066<0.001<0.001
Vertebrae (L5)
BV/TV (%)26.2 ± 0.621.9 ± 0.5**24.4 ± 0.618.2 ± 1.2**0.2300.001<0.001
Conn.D (1/mm3)199 ± 8157 ± 7**127 ± 787 ± 9**0.871<0.001<0.001
Tb.N (1/mm)5.22 ± 0.094.60 ± 0.10**4.23 ± 0.103.72 ± 0.17*0.646<0.001<0.001
Tb.Th (mm)0.0503 ± 0.00040.0505 ± 0.00030.0573 ± 0.00150.0557 ± 0.0021*0.068<0.0010.148
Tb.Sp (mm)0.185 ± 0.0040.209 ± 0.004**0.230 ± 0.0070.264 ± 0.012*0.541<0.0010.001
image

Figure 2. Trabecular bone loss in Misty was accelerated with age. (A) Representative images of distal femur microarchitecture, examined in female wild-type (+/+) and Misty (m/m) mice at 8 and 16 weeks of age. (B) Trabecular (Tb) bone volume fraction (BV/TV), thickness (Th), number (N), and separation (Sp) were measured. White (8-week) and gray (16-week) bars represent mean ± SEM. ap < 0.05 compared to genotype-matched 8-week group; bp < 0.05 compared to age matched wild-type group.

Download figure to PowerPoint

In female Misty L5 vertebrae, Tb.BV/TV was significantly lower than wild-type at both 8 and 16 weeks (Table 1). Remarkably, at 20 weeks of age vertebral BV/TV was more than 50% lower than age-matched wild-type controls (Supplementary Table S4). Similar to the femur, connectivity density and trabecular number were significantly reduced, whereas 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 wild-type at both ages (Table 1). Although mid-femoral cross-sectional area was higher in Misty at 8 weeks of age compared to wild-type, mid-femoral cross-sectional area was not different between wild-type 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 (Supplementary 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 1). 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 wild-type, although the mineral apposition rate did not differ by genotype (Table 2). 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 wild-type 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 wild-type at 16 weeks of age, which could account for the less profound trabecular bone loss phenotype in males (Supplementary Table S3). To fully understand the mechanism of low bone mass in Misty, the remaining studies were performed primarily in female mice.

Table 2. Proximal Tibia Histomorphometry of Female Wild-Type and Misty Mice at 16 Weeks of Age
 +/+ (n = 4–7)m/m (n = 7)
  • 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 adipocytes/total area.

  • *

    p < 0.05 compared to control.

  • **p < 0.01 compared to control.

MS/BS (%)39.3 ± 1.924.8 ± 3.5*
MAR (μm/d)1.99 ± 0.211.34 ± 0.35
BFR/BS(μm3/μm2/year)286 ± 38138 ± 43*
Ob.S/BS (%)9.92 ± 3.273.08 ± 1.86
N.Ob/B.Pm (1/mm)8.43 ± 2.672.03 ± 1.13*
OS/BS (%)5.65 ± 2.271.32 ± 1.32
O.Th (μm)2.34 ± 0.750.33 ± 0.33*
Oc.S/BS (%)1.43 ± 0.263.32 ± 0.73*
N.Oc/B.Pm (1/mm)0.56 ± 0.111.50 ± 0.33*
ES/BS (%)0.48 ± 0.201.87 ± 0.52*
N.Ad/T.Ar (1/mm2)32.0 ± 4.552.3 ± 6.2*

Interscapular BAT 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 preformed brown fat.[21] To our surprise, Misty mice did indeed have measureable interscapular BAT, which was not different in size, but more variable in Misty compared to wild-type (Fig. 3A). Misty BAT tended to have more lipid droplets and less UCP-1 staining (Fig. 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 wild-type at 12 weeks of age (Fig. 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 (Fig. 3F, G). Although it appears that the BRL37344-induced temperature and Pgc1a increases are slightly higher in Misty compared to wild-type, 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 wild-type and Misty BAT. The number of tyrosine hydroxylase (TH) positive fibers was significantly reduced in Misty BAT compared to wild-type (Fig. 3H, 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 wild-type and Misty mice but found no difference in Oil Red O staining between the two groups (Fig. 3J).

image

Figure 3. Brown adipose tissue (BAT) is present in Misty mice. (A) Interscapular BAT 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 to 6 pups per genotype. Bars represent mean ± SEM. Scale bars = 50 µm.

Download figure to PowerPoint

Misty WAT has an elevated response to cold exposure

To determine whether the observed reduction in BAT temperature and sympathetic innervation had physiologic consequences, we exposed wild-type 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 (Fig. 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 (Fig. 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 WAT from cold-exposed Misty mice had increased expression of genes associated with “browning” of WAT (Pdk4, Foxc2, Pgc1a, and Acadl) compared to ambient temperature controls (Fig. 4C–G). 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 wild-type.

image

Figure 4. Cold-induced thermogenesis in WAT of Misty mice is accompanied by altered gene expression in bone. Eight-week-old wild-type and Misty mice were subjected to 4°C temperature for 6 hours or maintained at ambient temperature. (A) Rectal temperature of cold-treated mice was measured every hour. (B) Pgc1a expression in interscapular BAT. (CG) Ucp1, Pdk4, Foxc2, Pgc1a, and Acadl expression in inguinal WAT. (H, I) Runx2 and Rankl expression in tibia. n = 5–10. *p < 0.05.

Download figure to PowerPoint

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 wild-type and Misty mice (Fig. 4H). Furthermore, expression of the osteoclast recruitment factor Rankl was elevated by cold exposure in Misty, but not wild-type tibias (Fig. 4I).

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 wild-type and Misty mice with the nonselective 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 wild-type mice, it increased total BMC accrual and slowed distal femur trabecular bone loss in Misty mice from 12 to 20 weeks of age (Fig. 5AC). Increased Tb.Th and Tb.N accompanied the high trabecular BV/TV in Misty mice treated with propranolol (Fig. 5D, E). Interestingly, trabecular BV/TV was significantly increased in vertebrae from both wild-type and Misty mice treated with propranolol (Supplementary Table S4). Additionally, propranolol suppressed the marker of bone resorption, serum CTX, in Misty mice but not wild-type (Fig. 5F). Propranolol did not alter serum P1NP, the marker of bone formation, in either wild-type or Misty mice (Fig. 5G).

image

Figure 5. β-adrenergic receptor blockade slows age-related trabecular bone loss in Misty mice. Wild-type 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 DXA. (CE) Trabecular BV/TV, Tb.Th, and Tb.N were measured by µCT. (FG) Serum CTX and P1NP were measured by EIA. n = 7–9. *p < 0.05.

Download figure to PowerPoint

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 wild-type 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 3) 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 4, Supplementary Fig. 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*h in wild-type versus 28.8 ± 0.2 kcal/kg*h in Misty, p = 0.40). To confirm altered muscle mass in Misty mice, we weighed soleus, gastrocnemius, and thigh muscle in 8-week-old wild-type and Misty mice. Gastrocnemius and thigh muscles from Misty mice weighed significantly less than wild-type, consistent with the reduced fat-free mass and energy expenditure noted previously (Table 3).

Table 3. Body Composition and Metabolic Variables in 8-Week-Old Female Misty and Wild-Type Mice
 +/+ (n = 4–8)m/m (n = 4–8)p
  1. Values are mean ± SEM.

  2. FFM = fat-free mass; RER = respiratory exchange ratio; EE = energy expenditure.

Body mass (g)21.1 ± 0.818.3 ± 0.40.042
FFM (g)17.7 ± 0.515.2 ± 0.50.025
Fat (g)2.9 ± 0.33.1 ± 0.20.696
Activity (m/d)7241 ± 9224795 ± 4760.057
RER0.847 ± 0.0090.782 ± 0.0390.155
EE (kcal/h)0.516 ± 0.020.411 ± 0.030.020
Thigh (mg)225 ± 7201 ± 60.017
Gastrocnemius (mg)91 ± 274 ± 2<0.001
Soleus (mg)6.2 ± 0.36.0 ± 0.90.843
Table 4. Linear Regression Analysis of Total EE Versus Body Composition in the Misty Mouse Model
 r2p
  1. EE = energy expenditure.

Total body mass0.870.0023
Fat-free mass0.97<0.0001
Fat mass0.040.68

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 (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 6B).

image

Figure 6. Loss of function of DOCK7 reduces calvarial osteoblastogenesis in vitro. Calvarial osteoblasts were isolated from Misty and wild-type neonates and differentiated into osteoblasts. (A) Dock7 expression at day 21. (B) DOCK7 protein expression from whole-femur lysates from wild-type 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.

Download figure to PowerPoint

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 2), bone marrow stromal cell cultures derived from Misty mice had significantly lower fibroblast colony-forming units (CFU-F) compared to those derived from wild-type (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 wild-type (Fig. 6C). Although the early osteoblast marker Runx2 was unchanged, expression levels of alkaline phosphatase and osteocalcin were both reduced at day 21 (Fig. 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 noncell autonomous effects which contribute to low bone mass (Fig. 7).

image

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.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

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 BAT[21]; hence our rationale for testing the effect of BAT on bone mass. However, we determined that BAT is indeed present, albeit partially functional (Fig. 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 wild-type B6 mice.

BAT, which is present in neonates, was thought to become nonfunctional 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 (Fig. 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 SNS 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 BMD.[9, 47] Activation of the SNS 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; ie, 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 (Fig. 5). These lines of evidence demonstrate that elevated sympathetic tone is in part responsible for the reduced bone mass phenotype in Misty mice (Fig. 7).

Although Misty mice showed a remarkable age-dependent bone loss phenotype, aBMD in Misty mice was impaired as early as 4 weeks of age (Fig. 1) and trabecular bone mass was not completely rescued by propranolol treatment (Fig. 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 (Fig. 6), which is consistent with reduced osteoblast numbers and percent mineralizing surface in vivo (Table 2). 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, whereas 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 (Fig. 7).

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 wild-type due to a dramatic reduction in fat-free mass, specifically skeletal muscle (Tables 3 and 4, Supplementary Fig. 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 SNS. 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.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

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 GM103465 to Don M. Wojchowski and P30 GM103392 to Robert Friesel, and by institutional support from Maine Medical Center. We thank Leslie Kozak for experimental input and Linda Van Aelst for the DOCK7 antibody. We 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.

Authors' roles: KJM, KAB, VED, MK, and MLB contributed to design, data acquisition, analysis, and interpretation. SAB, PL, and 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.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information
  • 1
    Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, Armstrong D, Ducy P, Karsenty G. Leptin regulates bone formation via the sympathetic nervous system. Cell. 2002 Nov 1; 111(3):30517.
  • 2
    Fulzele K, Riddle RC, DiGirolamo DJ, Cao X, Wan C, Chen D, Faugere MC, Aja S, Hussain MA, Bruning JC, Clemens TL. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell. 2010 Jul 23; 142(2):30919.
  • 3
    Ferron M, Wei J, Yoshizawa T, Del Fattore A, DePinho RA, Teti A, Ducy P, Karsenty G. Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell. 2010 Jul 23; 142(2):296308.
  • 4
    Friedman JM. Leptin at 14 y of age: an ongoing story. Am J Clin Nutr. 2009 Mar; 89(3):973S9S.
  • 5
    Yadav VK, Oury F, Suda N, Liu ZW, Gao XB, Confavreux C, Klemenhagen KC, Tanaka KF, Gingrich JA, Guo XE, Tecott LH, Mann JJ, Hen R, Horvath TL, Karsenty G. A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure. Cell. 2009 Sep 4; 138(5):97689.
  • 6
    Yadav VK, Karsenty G. Leptin-dependent co-regulation of bone and energy metabolism. Aging (Albany NY). 2009 Nov; 1(11):9546.
  • 7
    Shi Y, Yadav VK, Suda N, Liu XS, Guo XE, Myers MG Jr, Karsenty G. Dissociation of the neuronal regulation of bone mass and energy metabolism by leptin in vivo. Proc Natl Acad Sci U S A. 2008 Dec 23; 105(51):2052933.
  • 8
    Ducy P, Amling M, Takeda S, Priemel M, Schilling AF, Beil FT, Shen J, Vinson C, Rueger JM, Karsenty G. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell. 2000 Jan 21; 100(2):197207.
  • 9
    Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, Kondo H, Richards WG, Bannon TW, Noda M, Clement K, Vaisse C, Karsenty G. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature. 2005 Mar 24; 434(7032):51420.
  • 10
    Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F, Ducy P, Karsenty G. Endocrine regulation of energy metabolism by the skeleton. Cell. 2007 Aug 10; 130(3):45669.
  • 11
    Nedergaard J, Bengtsson T, Cannon B. Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab. 2007 Aug; 293(2):E44452.
  • 12
    van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ. Cold-activated brown adipose tissue in healthy men. N Engl J Med. 2009 Apr 9; 360(15):15008.
  • 13
    Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, Taittonen M, Laine J, Savisto NJ, Enerback S, Nuutila P. Functional brown adipose tissue in healthy adults. N Engl J Med. 2009 Apr 9; 360(15):151825.
  • 14
    Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB, Kuo FC, Palmer EL, Tseng YH, Doria A, Kolodny GM, Kahn CR. Identification and importance of brown adipose tissue in adult humans. N Engl J Med. 2009 Apr 9; 360(15):150917.
  • 15
    Ukropec J, Anunciado RP, Ravussin Y, Hulver MW, Kozak LP. UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice. J Biol Chem. 2006 Oct 20; 281(42):3189408.
  • 16
    Granneman JG, Burnazi M, Zhu Z, Schwamb LA. White adipose tissue contributes to UCP1-independent thermogenesis. Am J Physiol Endocrinol Metab. 2003 Dec; 285(6):E12306.
  • 17
    Grujic D, Susulic VS, Harper ME, Himms-Hagen J, Cunningham BA, Corkey BE, Lowell BB. Beta3-adrenergic receptors on white and brown adipocytes mediate beta3-selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice. J Biol Chem. 1997 Jul 11; 272(28):1768693.
  • 18
    Bredella MA, Fazeli PK, Freedman LM, Calder G, Lee H, Rosen CJ, Klibanski A. Young women with cold-activated brown adipose tissue have higher bone mineral density and lower Pref-1 than women without brown adipose tissue: a study in women with anorexia nervosa, women recovered from anorexia nervosa, and normal-weight women. J Clin Endocrinol Metab. 2012 Apr; 97(4):E58490.
  • 19
    Ponrartana S, Aggabao PC, Hu HH, Aldrovandi GM, Wren TA, Gilsanz V. Brown adipose tissue and its relationship to bone structure in pediatric patients. J Clin Endocrinol Metab. 2012 Aug; 97(8):26938.
  • 20
    Motyl KJ, Rosen CJ. Temperatures rising: brown fat and bone. Discov Med. 2011 Mar; 11(58):17985.
  • 21
    Sviderskaya EV, Novak EK, Swank RT, Bennett DC. The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat. Genetics. 1998 Jan; 148(1):38190.
  • 22
    Watabe-Uchida M, John KA, Janas JA, Newey SE, Van Aelst L. The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18. Neuron. 2006 Sep 21; 51(6):72739.
  • 23
    Yamauchi J, Miyamoto Y, Chan JR, Tanoue A. ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration. J Cell Biol. 2008 Apr 21; 181(2):35165.
  • 24
    Blasius AL, Brandl K, Crozat K, Xia Y, Khovananth K, Krebs P, Smart NG, Zampolli A, Ruggeri ZM, Beutler BA. Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function. Proc Natl Acad Sci U S A. 2009 Feb 24; 106(8):270611.
  • 25
    Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005 Feb; 6(2):16780.
  • 26
    Cote JF, Vuori K. GEF what? Dock180 and related proteins help Rac to polarize cells in new ways. Trends Cell Biol. 2007 Aug; 17(8):38393.
  • 27
    Miyamoto Y, Yamauchi J. Cellular signaling of Dock family proteins in neural function. Cell Signal. 2010 Feb; 22(2):17582.
  • 28
    DeMambro VE, Clemmons DR, Horton LG, Bouxsein ML, Wood TL, Beamer WG, Canalis E, Rosen CJ. Gender-specific changes in bone turnover and skeletal architecture in igfbp-2-null mice. Endocrinology. 2008 May; 149(5):205161.
  • 29
    Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 2010 Jul; 25(7):146886.
  • 30
    Dempster DW, Compston JE, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR, Parfitt AM. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 2013 Jan; 28(1):217.
  • 31
    Kawai M, Green CB, Lecka-Czernik B, Douris N, Gilbert MR, Kojima S, Ackert-Bicknell C, Garg N, Horowitz MC, Adamo ML, Clemmons DR, Rosen CJ. A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-gamma nuclear translocation. Proc Natl Acad Sci U S A. 2010 Jun 8; 107(23):1050813.
  • 32
    Fasshauer M, Klein J, Kriauciunas KM, Ueki K, Benito M, Kahn CR. Essential role of insulin receptor substrate 1 in differentiation of brown adipocytes. Mol Cell Biol. 2001 Jan; 21(1):31929.
  • 33
    Fasshauer M, Klein J, Ueki K, Kriauciunas KM, Benito M, White MF, Kahn CR. Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes. J Biol Chem. 2000 Aug 18; 275(33):2549401.
  • 34
    Rosen CJ, Ackert-Bicknell CL, Adamo ML, Shultz KL, Rubin J, Donahue LR, Horton LG, Delahunty KM, Beamer WG, Sipos J, Clemmons D, Nelson T, Bouxsein ML, Horowitz M. Congenic mice with low serum IGF-I have increased body fat, reduced bone mineral density, and an altered osteoblast differentiation program. Bone. 2004 Nov; 35(5):104658.
  • 35
    Vengellur A, LaPres JJ. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts. Toxicol Sci. 2004 Dec; 82(2):63846.
  • 36
    Lighton JR, Turner RJ. The hygric hypothesis does not hold water: abolition of discontinuous gas exchange cycles does not affect water loss in the ant Camponotus vicinus. J Exp Biol. 2008 Feb; 211(Pt 4):5637.
  • 37
    Kaiyala KJ, Schwartz MW. Toward a more complete (and less controversial) understanding of energy expenditure and its role in obesity pathogenesis. Diabetes. 2011 Jan; 60(1):1723.
  • 38
    Arch JR, Hislop D, Wang SJ, Speakman JR. Some mathematical and technical issues in the measurement and interpretation of open-circuit indirect calorimetry in small animals. Int J Obes (Lond). 2006 Sep; 30(9):132231.
  • 39
    Kaiyala KJ, Morton GJ, Leroux BG, Ogimoto K, Wisse B, Schwartz MW. Identification of body fat mass as a major determinant of metabolic rate in mice. Diabetes. 2010 Jul; 59(7):165766.
  • 40
    Farr JN, Charkoudian N, Barnes JN, Monroe DG, McCready LK, Atkinson EJ, Amin S, Melton LJ 3rd, Joyner MJ, Khosla S. Relationship of sympathetic activity to bone microstructure, turnover, and plasma osteopontin levels in women. J Clin Endocrinol Metab. 2012 Nov; 97(11):421927.
  • 41
    Veldhuis-Vlug AG, El Mahdiui M, Endert E, Heijboer AC, Fliers E, Bisschop PH. Bone resorption is increased in pheochromocytoma patients and normalizes following adrenalectomy. J Clin Endocrinol Metab. 2012 Nov; 97(11):E20937.
  • 42
    Tseng YH, Cypess AM, Kahn CR. Cellular bioenergetics as a target for obesity therapy. Nat Rev Drug Discov. 2010 Jun; 9(6):46582.
  • 43
    Liu X, Rossmeisl M, McClaine J, Riachi M, Harper ME, Kozak LP. Paradoxical resistance to diet-induced obesity in UCP1-deficient mice. J Clin Invest. 2003 Feb; 111(3):399407.
  • 44
    Feldmann HM, Golozoubova V, Cannon B, Nedergaard J. UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality. Cell Metab. 2009 Feb; 9(2):2039.
  • 45
    Seale P, Bjork B, Yang W, Kajimura S, Chin S, Kuang S, Scime A, Devarakonda S, Conroe HM, Erdjument-Bromage H, Tempst P, Rudnicki MA, Beier DR, Spiegelman BM. PRDM16 controls a brown fat/skeletal muscle switch. Nature. 2008 Aug 21; 454(7207):9617.
  • 46
    Petrovic N, Walden TB, Shabalina IG, Timmons JA, Cannon B, Nedergaard J. Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes. J Biol Chem. 2010 Mar 5; 285(10):715364.
  • 47
    Bouxsein ML, Devlin MJ, Glatt V, Dhillon H, Pierroz DD, Ferrari SL. Mice lacking beta-adrenergic receptors have increased bone mass but are not protected from deleterious skeletal effects of ovariectomy. Endocrinology. 2009 Jan; 150(1):14452.
  • 48
    Yang YT, Wang CL, Van Aelst L. DOCK7 interacts with TACC3 to regulate interkinetic nuclear migration and cortical neurogenesis. Nat Neurosci. 2012 Sep; 15(9):120110.
  • 49
    Kado DM, Lui LY, Cummings SR. Rapid resting heart rate: a simple and powerful predictor of osteoporotic fractures and mortality in older women. J Am Geriatr Soc. 2002 Mar; 50(3):45560.
  • 50
    Nedergaard J, Bengtsson T, Cannon B. Three years with adult human brown adipose tissue. Ann N Y Acad Sci. 2010 Nov;1212;E2036.

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr1943-sm-0001-SupplFigS1.tif321KSupplementary Figure S1
jbmr1943-sm-0002-SupplTabs.doc63KSupplementary Tables
jbmr1943-sm-0003-SupplFigLegend.doc19KSupplementary Figure Legend

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.