Gestational and lactational exposure to BPA, but not BPS, negatively impacts trabecular microarchitecture and cortical geometry in adult male offspring.

Bisphenol-A (BPA) and bisphenol-S (BPS) are endocrine disrupting chemicals (EDCs) found primarily in plastics. Estrogen is a primary hormonal regulator of skeletal growth and development; however, the impact of gestational BPA or BPS exposure on skeletal health of offspring remains relatively unknown. Here, adult female mice were randomized into three treatment groups: 200 µg BPA/kg BW (BPA), 200 µg BPS/kg BW (BPS) or control (CON). Animals were then further randomized to exercising (EX) or sedentary (SED) groups. Treatment continued through mating, gestation, and lactation. One male offspring from each dam (n = 6-8/group) was assessed at 16 weeks of age to evaluate effects of EDC exposure on the adult skeleton. Cortical geometry of the mid-diaphysis and trabecular microarchitecture of the distal femur were assessed via micro-CT. Biomechanical strength and mineral apposition rate of the femoral diaphysis were assessed via three-point bending and dynamic histomorphometry, respectively. Two-factor ANOVA or ANCOVA were used to determine the effects of maternal EX and BPA or BPS on trabecular and cortical bone outcomes. Maternal EX led to a significant decrease in body fat percentage and bone stiffness, independent of EDC exposure. Offspring exposed to BPA had significantly lower trabecular bone volume, trabecular number, connectivity density, cortical thickness, and greater trabecular spacing compared to BPS or CON animals. In conclusion, gestational BPA, but not BPS, exposure negatively impacted trabecular microarchitecture and cortical geometry in adult male offspring. If these findings translate to humans, this could have significant public health impacts on expecting women or those seeking to become pregnant.

Gestational and lactational exposure to BPA or BPS has minimal effects on skeletal outcomes in adult female mice.

Bisphenol-A (BPA) and bisphenol-S (BPS) are estrogen disrupting chemicals (EDCs) found in the environment and common household items. Estrogen is a primary hormonal regulator of bone growth and development; however, the impact of gestational BPA or BPS exposure on skeletal health of offspring remains relatively unknown. In this longitudinal study, adult female mice were randomized into three groups: 200 µg BPA/kg BW (BPA), 200 µg BPS/kg BW (BPS) or control (CON). Animals in each group were further randomized to exercise treatment (EX) or sedentary (SED) control, resulting in six overall groups. BPA/BPS/CON and EX/SED treatment were initiated prior to mating and continued through mating, gestation, and lactation. One female offspring from each dam (n = 6/group) was assessed at 17 weeks of age to evaluate effects of EDC exposure on the adult skeleton. Cortical geometry of the mid-diaphysis and trabecular microarchitecture of the distal femur were assessed via micro-computed tomography. Biomechanical strength and mineral apposition rate of the femoral diaphysis were assessed via three-point bending and dynamic histomorphometry, respectively. Sclerostin expression was measured using immunohistochemistry. Two-factor ANOVA or ANCOVA were used to determine the effects of maternal exercise and BPA or BPS exposure on trabecular and cortical bone outcomes, respectively. Consistent with prior studies, there were no significant differences in body weight, femoral length, cortical geometry, trabecular microarchitecture, or biomechanical strength between groups in female offspring. In conclusion, gestational BPA exposure and maternal exercise have minimal impact on skeletal outcomes in female adult offspring.

Voluntary Wheel Running Partially Compensates for the Effects of Global Estrogen Receptor-α Knockout on Cortical Bone in Young Male Mice.

Estrogen receptor-α knockout (ERKO) in female, but not male, mice results in an impaired osteogenic response to exercise, but the mechanisms behind this ability in males are unknown. We explored the main and interactive effects of ERKO and exercise on cortical geometry, trabecular microarchitecture, biomechanical strength, and sclerostin expression in male mice. At 12 weeks of age, male C57BL/6J ERKO and WT animals were randomized into two groups: exercise treatment (EX) and sedentary (SED) controls, until 22 weeks of age. Cortical geometry and trabecular microarchitecture were measured via µCT; biomechanical strength was assessed via three-point bending; sclerostin expression was measured via immunohistochemistry. Two-way ANOVA was used to assess sclerostin expression and trabecular microarchitecture; two-way ANCOVA with body weight was used to assess cortical geometry and biomechanical strength. ERKO positively impacted trabecular microarchitecture, and exercise had little effect on these outcomes. ERKO significantly impaired cortical geometry, but exercise was able to partially reverse these negative alterations. EX increased cortical thickness regardless of genotype. There were no effects of genotype or exercise on sclerostin expression. In conclusion, male ERKO mice retain the ability to build bone in response to exercise, but altering sclerostin expression is not one of the mechanisms involved.

Soy Protein Isolate Suppresses Bone Resorption and Improves Trabecular Microarchitecture in Spontaneously Hyperphagic, Rapidly Growing Male OLETF Rats.

BACKGROUND: Traditionally, milk proteins have been recommended for skeletal health; recently, soy proteins have emerged as popular alternatives. Excess adiposity appears detrimental to skeletal health, as obese adolescents have increased fracture rates compared with healthy controls. However, soy protein effects on skeletal health during excess adiposity remain unknown. OBJECTIVE: The study objective was to examine the effects of isocaloric diets containing milk protein isolate (MPI), soy protein isolate (SPI), or a 50/50 combination (MIX) as the sole protein source on metabolic health indicators and bone outcomes in rapidly growing, hyperphagic, male Otsuka Long Evans Tokushima Fatty (OLETF) rats. METHODS: OLETF rats, aged 4 wk, were randomly assigned to 3 treatment groups (MPI, SPI, or MIX, n = 20 per group) and provided with access to experimental diets ad libitum for 16 wk. RESULTS: Body mass did not differ between the groups, but SPI had lower percentage body fat than MPI (P = 0.026). Insulin was lower in MPI than in MIX (P = 0.033) or SPI (P = 0.044), but fasting blood glucose was not different between the groups. SPI significantly reduced serum cholesterol compared with MPI (P = 0.001) and MIX (P = 0.002). N-terminal propeptide of type I collagen (P1NP) was higher in MIX than MPI (P = 0.05); C-terminal telopeptide of type 1 collagen (CTx) was higher in MPI than SPI (P < 0.001) and MIX (P < 0.001); the P1NP to CTx ratio was significantly higher in SPI and MIX than in MPI (P < 0.001). Trabecular separation was reduced in SPI compared with MPI (P = 0.030) and MIX (P = 0.008); trabecular number was increased in SPI compared with MIX (P = 0.038). No differences were seen in cortical geometry and biomechanical properties. CONCLUSIONS: In the context of excess adiposity, soy- and milk-based proteins have comparable effects on cortical bone geometry and biomechanical properties, whereas soy-based proteins favorably affect the trabecular microarchitecture, and the combination of both proteins may offer additional benefits to bone remodeling in rapidly growing male OLETF rats.

Soy protein improves tibial whole-bone and tissue-level biomechanical properties in ovariectomized and ovary-intact, low-fit female rats.

BACKGROUND: Osteoporosis and related fractures, decreased physical activity, and metabolic dysfunction are serious health concerns for postmenopausal women. Soy protein might counter the negative effects of menopause on bone and metabolic health due to the additive or synergistic effects of its bioactive components. OBJECTIVE: To evaluate the effects of ovariectomy (OVX) and a soy-protein diet (SOY) on bone outcomes in female, low-capacity running (LCR) rats selectively bred for low aerobic fitness as a model of menopause. METHODS: At 27 weeks of age, LCR rats (N = 40) underwent OVX or sham (SHAM) surgery and were randomized to one of two isocaloric and isonitrogenous plant-protein-based dietary treatments: 1) soy-protein (SOY; soybean meal); or, 2) control (CON, corn-gluten meal), resulting in four treatment groups. During the 30-week dietary intervention, animals were provided ad libitum access to food and water; body weight and food intake were measured weekly. At completion of the 30-week intervention, body composition was measured using EchoMRI; animals were fasted overnight, euthanized, and blood and hindlimbs collected. Plasma markers of bone formation (osteocalcin, OC; N-terminal propeptide of type I procollagen, P1NP) and resorption (tartrate-resistant acid phosphatase, TRAP5b; C-terminal telopeptide of type I collagen, CTx) were measured using ELISA. Tibial trabecular microarchitecture and cortical geometry were evaluated using µCT; and torsional loading to failure was used to assess cortical biomechanical properties. Advanced glycation end-product (AGE) content of the femur was measured using a fluorimetric assay, and was expressed relative to collagen content measured by a colorimetric OH-proline assay. Two-factor ANOVA or ANOVCA was used to test for significant main and interactive effects of ovarian status (OV STAT: OVX vs. SHAM) and DIET (SOY vs. CON); final body weight was included as a covariate for body-weight-dependent cortical geometry and biomechanical properties. RESULTS: OVX had significantly greater CTx than SHAM; SOY did not affect bone turnover markers. OVX adversely affected trabecular microarchitecture as evidenced by reduced BV/TV, trabecular thickness (Tb.Th), trabecular number (Tb.N), and connectivity density (Conn.D), and by increased trabecular separation (Tb.Sp) and structural model index (SMI). SOY increased BV/TV only in ovary-intact animals. There was no effect of OVX or SOY on tibial cortical geometry. In SHAM and OVX rats, SOY significantly improved whole-bone strength and stiffness; SOY also increased tissue-level stiffness and tended to increase tissue-level strength (p = 0.067). There was no effect of OVX or SOY on AGE content. CONCLUSION: Soy protein improved cortical bone biomechanical properties in female low-fit rats, regardless of ovarian hormone status.

Exercise initiated after the onset of insulin resistance improves trabecular microarchitecture and cortical bone biomechanics of the tibia in hyperphagic Otsuka Long Evans Tokushima Fatty rats.

The present study extends our previous findings that exercise, which prevents the onset of insulin resistance and type 2 diabetes (T2D), also prevents the detrimental effects of T2D on whole-bone and tissue-level strength. Our objective was to determine whether exercise improves bone's structural and material properties if insulin resistance is already present in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is hyperphagic due to a loss-of-function mutation in cholecystokinin-1 receptor (CCK-1 receptor), which leads to progressive obesity, insulin resistance and T2D after the majority of skeletal growth is complete. Because exercise reduces body mass, which is a significant determinant of bone strength, we used a body-mass-matched caloric-restricted control to isolate body-mass-independent effects of exercise on bone. Eight-wk old, male OLETF rats were fed ad libitum until onset of hyperglycemia (20weeks of age), at which time they were randomly assigned to three groups: ad libitum fed, sedentary (O-SED); ad libitum fed, treadmill running (O-EX); or, sedentary, mild caloric restriction to match body mass of O-EX (O-CR). Long-Evans Tokushima Otsuka rats served as the normophagic, normoglycemic controls (L-SED). At 32weeks of age, O-SED rats had T2D as evidenced by hyperglycemia and a significant reduction in fasting insulin compared to OLETFs at 20weeks of age. O-SED rats also had reduced total body bone mineral content (BMC), increased C-terminal telopeptide of type I collagen (CTx)/tartrate resistant acid phosphatase isoform 5b (TRAP5b), decreased N-terminal propeptide of type I procollagen (P1NP), reduced percent cancellous bone volume (BV/TV), trabecular number (Tb.N) and increased trabecular separation (Tb.Sp) and structural model index (SMI) of the proximal tibia compared to L-SED. T2D also adversely affected biomechanical properties of the tibial diaphysis, and serum sclerostin was increased and ß-catenin, runt-related transcription factor 2 (Runx2) and insulin-like growth factor-I (IGF-I) protein expression in bone were reduced in O-SED vs. L-SED. O-EX or O-CR had greater total body bone mineral density (BMD) and BMC, and BV/TV, Tb.N, Tb.Sp, and SMI compared to O-SED. O-EX had lower CTx and CR greater P1NP relative to O-SED. O-EX, not O-CR, had greater cortical thickness and area, and improved whole-bone and tissue-level biomechanical properties associated with a 4-fold increase in cortical bone ß-catenin protein expression vs. O-SED. In summary, EX or CR initiated after the onset of insulin resistance preserved cancellous bone volume and structure, and EX elicited additional benefits in cortical bone.

Insulin-Stimulated Bone Blood Flow and Bone Biomechanical Properties Are Compromised in Obese, Type 2 Diabetic OLETF Rats.

Type 2 diabetes (T2D) increases skeletal fragility and fracture risk; however, the underlying mechanisms remain to be identified. Impaired bone vascular function, in particular insulin-stimulated vasodilation and blood flow is a potential, yet unexplored mechanism. The purpose of this study was to determine the effects of T2D on femoral biomechanical properties, trabecular microarchitecture, and insulin-stimulated bone vasodilation by comparison of hyperphagic Otsuka Long-Evans Tokushima Fatty (OLETF) rats with normoglycemic control OLETF rats. Four-week old, male OLETF rats were randomized to two groups: type 2 diabetes (O-T2D) or normoglycemic control (O-CON). O-T2D were allowed ad libitum access to a rodent chow diet and O-CON underwent moderate caloric restriction (30% restriction relative to intake of O-T2D) to maintain normal body weight (BW) and glycemia until 40 weeks of age. Hyperphagic O-T2D rats had significantly greater BW, body fat, and blood glucose than O-CON. Total cross-sectional area (Tt.Ar), cortical area (Ct.Ar), Ct.Ar/Tt.Ar, and polar moment of inertia of the mid-diaphyseal femur adjusted for BW were greater in O-T2D rats versus O-CON. Whole-bone biomechanical properties of the femur assessed by torsional loading to failure did not differ between O-T2D and O-CON, but tissue-level strength and stiffness adjusted for BW were reduced in O-T2D relative to O-CON. Micro-computed tomography (µCT) of the distal epiphysis showed that O-T2D rats had reduced percent bone volume, trabecular number, and connectivity density, and greater trabecular spacing compared with O-CON. Basal tibial blood flow assessed by microsphere infusion was similar in O-T2D and O-CON, but the blood flow response to insulin stimulation in both the proximal epiphysis and diaphyseal marrow was lesser in O-T2D compared to O-CON. In summary, impaired insulin-stimulated bone blood flow is associated with deleterious changes in bone trabecular microarchitecture and cortical biomechanical properties in T2D, suggesting that vascular dysfunction might play a causal role in diabetic bone fragility. © 2017 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.