Jay J. Cao

High-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice

Body mass has a positive effect on bone health. Whether mass derived from an obesity condition or excessive fat accumulation is beneficial to bone has not been established; neither have the mechanisms by which obesity affects bone metabolism. The aim of this study was to examine the effects of obesity on bone structure and osteoblastic expression of key markers involved in bone formation and resorption in a diet-induced obesity mouse model. Six-wk-old male C57BL/6 mice (n=21) were assigned to two groups and fed either a control (10 kcal% energy as fat) or high-fat diet (HFD, 45 kcal% energy as fat) for 14 weeks. Bone marrow stromal/osteoblastic cells (BMSC) were cultured. Osteoprogenitor activity [alkaline phosphatase (ALP) positive colonies] and mineralization (calcium nodule formation) were determined. Gene expression was measured using quantitative real-time PCR. Bone structure of proximal and midshaft tibia was evaluated by micro-computed tomography. Mice fed the HFD were 31% heavier (P<0.01) than those fed the control diet. There were more ALP positive colony forming units at d 14 and calcium nodules at d 28 of culture by BMSC from HFD mice than from control mice (P<0.01). Receptor activator of NF-kappaB ligand (RANKL) mRNA levels and the ratio of RANKL to osteoprotegerin expression in HFD animals was higher (P<0.01) than in control diet animals. Serum tartrate-resistant acid phosphatase levels were higher in HFD fed mice when compared to control diet fed mice (P<0.05). There were no significant differences in tibial fat-free weight, length, and cortical parameters of midshaft between the two groups. Compared with control mice, tibial trabecular bone volume was reduced, and trabecular separation was increased in HFD mice. Trabecular number was lower (P<0.05) and connectivity density tended to be less (P=0.07) in HFD mice than in control mice. In conclusion, our data indicate that obesity induced by a high-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice.

Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial

The purpose of this work was to determine the effects of varying levels of dietary protein on body composition and muscle protein synthesis during energy deficit (ED). A randomized controlled trial of 39 adults assigned the subjects diets providing protein at 0.8 (recommended dietary allowance; RDA), 1.6 (2X‐RDA), and 2.4 (3X‐RDA) g kg–1 d–1 for 31 d. A 10‐d weight‐maintenance (WM) period was followed by a 21 d, 40% ED. Body composition and postabsorptive and postprandial muscle protein synthesis were assessed during WM (d 9‐10) and ED (d 30‐31). Volunteers lost (P<0.05) 3.2 ± 0.2 kg body weight during ED regardless of dietary protein. The proportion of weight loss due to reductions in fat‐free mass was lower (P<0.05) and the loss of fat mass was higher (P<0.05) in those receiving 2X‐RDA and 3X‐RDA compared to RDA. The anabolic muscle response to a protein‐rich meal during ED was not different (P>0.05) from WM for 2X‐RDA and 3X‐RDA, but was lower during ED than WM for those consuming RDA levels of protein (energy × protein interaction, P<0.05). To assess muscle protein metabolic responses to varied protein intakes during ED, RDA served as the study control. In summary, we determined that consuming dietary protein at levels exceeding the RDA may protect fat‐free mass during short‐term weight loss.—Pasiakos, S. M., Cao, J. J., Margolis, L. M., Sauter, E. R., Whigham, L. D., McClung, J. P., Rood, J. C., Carbone, J. W., Combs, G. F., Jr., Young, A. J. Effects of high‐protein diets on fat‐free mass and muscle protein synthesis following weight loss: a randomized controlled trial. FASEB J. 27, 3837–3847 (2013). www.fasebj.org

Diet-induced obesity alters bone remodeling leading to decreased femoral trabecular bone mass in mice

Obesity‐derived body mass may be detrimental to bone health through not well‐defined mechanisms. In this study we determined changes in bone structure and serum cytokines related to bone metabolism in diet‐induced obese mice. Mice fed a high‐fat diet (HFD) had higher serum tartrate‐resistant acid phosphatase (TRAP) and leptin but lower osteocalcin concentrations than those fed the normal‐fat diet. The HFD increased multinucleated TRAP‐positive osteoclasts in bone marrow compared to the control diet. Despite being much heavier, mice fed the HFD had lower femoral bone volume, trabecular number, and connectivity density and higher trabecular separation than mice on the control diet. These findings suggest that obesity induced by a HFD increases bone resorption that may blunt any positive effects of increased body weight on bone.

Selenium in Bone Health: Roles in Antioxidant Protection and Cell Proliferation

Selenium (Se) is an essential trace element for humans and animals, and several findings suggest that dietary Se intake may be necessary for bone health. Such findings may relate to roles of Se in antioxidant protection, enhanced immune surveillance and modulation of cell proliferation. Elucidation of the mechanisms by which Se supports these cellular processes can lead to a better understanding of the role of this nutrient in normal bone metabolism. This article reviews the current knowledge concerning the molecular functions of Se relevant to bone health.

Selenium Deficiency Decreases Antioxidative Capacity and Is Detrimental to Bone Microarchitecture in Mice

Selenium (Se), an essential mineral, plays a major role in cellular redox status and may have beneficial effects on bone health. The objective of this study was to determine whether Se deficiency affects redox status and bone microarchitecture in a mouse model. Thirty-three male C57BL/6J mice, 18 wk old, were randomly assigned to 3 groups. Mice were fed either a purified, Se-deficient diet (SeDef) containing ∼0.9 μg Se/kg diet, or Se-adequate diets containing ∼100 μg Se/kg diet from either selenomethionine (SeMet) or pinto beans (SeBean) for 4 mo. The Se concentration, glutathione peroxidase (GPx1) activity, and GPx1 mRNA in liver were lower in the SeDef group than in the SeMet or SeBean group. The femoral trabecular bone volume/total volume and trabecular number were less, whereas trabecular separation was greater, in the SeDef group than in either the SeMet or SeBean group (P < 0.05). Bone structural parameters between the SeMet and SeBean groups did not differ. Furthermore, Serum concentrations of C-reactive protein, tartrate-resistant acid phosphatase, and intact parathyroid hormone were higher in the SeDef group than in the other 2 groups. These findings demonstrate that Se deficiency is detrimental to bone microarchitecture by increasing bone resorption, possibly through decreasing antioxidative potential.

Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis

This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×‐RDA), or 2.4 (3×‐RDA) g · kg–1·· d–1 for 31 d. A 10‐d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)‐mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence‐based assays, immunoblot analysis, and real‐time qRT‐PCR In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up‐regulation of UPS‐associated gene expression, as mRNA expression of the atrogenes muscle RING finger‐1 (MuRF1) and atrogin‐1 were 1.2‐ and 1.3‐fold higher (P<0.05) for ED than for WM. However, mixed‐meal consumption attenuated UPS‐mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits β1, β2, and β5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin‐1 mRNA expression during ED, consuming a protein‐containing mixed meal attenuates Ub‐mediated proteolysis.—Carbone, J. W., Margolis, L. M., McClung, J. P., Cao, J. J., Murphy, N. E., Sauter, E. R., Combs, G. F., Jr., Young, A. J., Pasiakos, S. M., Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis. FASEB J. 27, 5104–5111 (2013). www.fasebj.org

Calcium homeostasis and bone metabolic responses to high-protein diets during energy deficit in healthy young adults: a randomized controlled trial

BACKGROUND Although consuming dietary protein above current recommendations during energy deficit (ED) preserves lean body mass, concerns have been raised regarding the effects of high-protein diets on bone health. OBJECTIVE The objective was to determine whether calcium homeostasis and bone turnover are affected by high-protein diets during weight maintenance (WM) and ED. DESIGN In a randomized, parallel-design, controlled trial of 32 men and 7 women, volunteers were assigned diets providing protein at 0.8 [Recommended Dietary Allowance (RDA)], 1.6 (2 × RDA), or 2.4 (3 × RDA) g · kg(-1) · d(-1) for 31 d. Ten days of WM preceded 21 d of ED, during which total daily ED was 40%, achieved by reduced dietary energy intake (∼30%) and increased physical activity (∼10%). The macronutrient composition (protein g · kg(-1) · d(-1) and % fat) was held constant from WM to ED. Calcium absorption (ratio of (44)Ca to (42)Ca) and circulating indexes of bone turnover were determined at day 8 (WM) and day 29 (ED). RESULTS Regardless of energy state, mean (±SEM) urinary pH was lower (P < 0.05) at 2 × RDA (6.28 ± 0.05) and 3 × RDA (6.23 ± 0.06) than at the RDA (6.54 ± 0.06). However, protein had no effect on either urinary calcium excretion (P > 0.05) or the amount of calcium retained (P > 0.05). ED decreased serum insulin-like growth factor I concentrations and increased serum tartrate-resistant acid phosphatase and 25-hydroxyvitamin D concentrations (P < 0.01). Remaining markers of bone turnover and whole-body bone mineral density and content were not affected by either the protein level or ED (P > 0.05). CONCLUSION These data demonstrate that short-term consumption of high-protein diets does not disrupt calcium homeostasis and is not detrimental to skeletal integrity. This trial was registered at www.clinicaltrials.gov as NCT01292395.

Dietary protein level and source differentially affect bone metabolism, strength, and intestinal calcium transporter expression during ad libitum and food-restricted conditions in male rats.

High-protein (HP) diets may attenuate bone loss during energy restriction. The objective of the current study was to determine whether HP diets suppress bone turnover and improve bone quality in male rats during food restriction and whether dietary protein source affects this relation. Eighty 12-wk-old male Sprague Dawley rats were randomly assigned to consume 1 of 4 study diets under ad libitum (AL) control or restricted conditions [40% food restriction (FR)]: 1) 10% [normal-protein (NP)] milk protein; 2) 32% (HP) milk protein; 3) 10% (NP) soy protein; or 4) 32% (HP) soy protein. After 16 wk, markers of bone turnover, volumetric bone mineral density (vBMD), microarchitecture, strength, and expression of duodenal calcium channels were assessed. FR increased bone turnover and resulted in lower femoral trabecular bone volume (P < 0.05), higher cortical bone surface (P < 0.001), and reduced femur length (P < 0.01), bending moment (P < 0.05), and moment of inertia (P = 0.001) compared with AL. HP intake reduced bone turnover and tended to suppress parathyroid hormone (PTH) (P = 0.06) and increase trabecular vBMD (P < 0.05) compared with NP but did not affect bone strength. Compared with milk, soy suppressed PTH (P < 0.05) and increased cortical vBMD (P < 0.05) and calcium content of the femur (P < 0.01) but did not affect strength variables. During AL conditions, transient receptor potential cation channel, subfamily V, member 6 was higher for soy than milk (P < 0.05) and HP compared with NP (P < 0.05). These data demonstrate that both HP and soy diets suppress PTH, and HP attenuates bone turnover and increases vBMD regardless of FR, although these differences do not affect bone strength. The effects of HP and soy may be due in part to enhanced intestinal calcium transporter expression.

N-Acetylcysteine Supplementation Decreases Osteoclast Differentiation and Increases Bone Mass in Mice Fed a High-Fat Diet

Obesity induced by high-fat (HF) diets increases bone resorption, decreases trabecular bone mass, and reduces bone strength in various animal models. This study investigated whether N-acetylcysteine (NAC), an antioxidant and a glutathione precursor, alters glutathione status and mitigates bone microstructure deterioration in mice fed an HF diet. Forty-eight 6-wk-old male C57BL/6 mice were randomly assigned to 4 treatment groups (n = 12 per group) and fed either a normal-fat [NF (10% energy as fat)] or an HF (45% energy as fat) diet ad libitum with or without NAC supplementation at 1 g/kg diet for 17 wk. Compared with the NF groups, mice in the HF groups had higher body weight, greater serum leptin concentrations and osteoclast differentiation, and lower trabecular bone volume, trabecular number, and connectivity density (P < 0.05). NAC supplementation increased the serum-reduced glutathione concentration and bone volume and decreased osteoclast differentiation in HF-fed mice (P < 0.05). We further demonstrated that osteoclast differentiation was directly regulated by glutathione status. NAC treatment of murine macrophage RAW 264.7 cells in vitro increased glutathione status and decreased osteoclast formation. These results show that NAC supplementation increases the bone mass of obese mice induced by an HF diet through elevating glutathione status and decreasing bone resorption.

Alpha-1 antitrypsin reduces ovariectomy-induced bone loss in mice

Proinflammatory cytokines are primary mediators of bone loss in estrogen deficiency. This study determined whether alpha‐1 antitrypsin (AAT), a multifunctional protein with proteinase inhibitor and anti‐inflammatory activities, mitigates bone loss induced by estrogen deficiency. Mice were either sham‐operated or ovariectomized and injected with either AAT or phosphate buffered saline (PBS). Ovariectomy resulted in decreased wet uterus weight, significant bone loss, increased serum leptin concentrations, and higher body weight compared to sham. AAT injection increased tibial trabecular bone volume/total volume and trabecular thickness compared to PBS injection in ovariectomized mice. Ovariectomized mice with AAT treatment had higher uterus weight, lower serum osteocalcin levels, fewer bone marrow tartrate‐resistant acid phosphatase–positive osteoclasts, and less expression of calcitonin receptor in bone than that in PBS‐injected mice. These data demonstrate that AAT mitigates ovariectomy‐induced bone loss in mice possibly through inhibiting osteoclast activity and bone resorption.