Effect of Dietary Geranylgeraniol and Green Tea Polyphenols on Glucose Homeostasis, Bone Turnover Biomarkers, and Bone Microstructure in Obese Mice.

Previously, we demonstrated that the administration of either geranylgeraniol (GGOH) or green tea polyphenols (GTP) improved bone health. This study examined the combined effects of GGOH and GTP on glucose homeostasis in addition to bone remodeling in obese mice. We hypothesized that GGOH and GTP would have an additive or synergistic effect on improving glucose homeostasis and bone remodeling possibly in part via suppression of proinflammatory cytokines. Forty-eight male C57BL/6J mice were assigned to a high-fat diet (control), HFD + 400 mg GGOH/kg diet (GG), HFD + 0.5% GTP water (TP), or HFD + GGOH + GTP (GGTP) diet for 14 weeks. Results demonstrated that GTP supplementation improved glucose tolerance in obese mice. Neither GGOH nor GTP affected pancreas insulin or bone formation procollagen type I intact N-terminal, bone volume at the lumbar vertebrae, or bone parameters at the trabecular bone and cortical bone of the femur. There was an interactive effect for serum bone resorption collagen type 1 cross-linked C-telopeptide concentrations, resulting in no-GGOH and no-GTP groups having the highest values. GGOH increased trabecular number and decreased trabecular separation at the lumbar vertebrae. GTP increased trabecular thickness at lumbar vertebrae. The GG group produced the greatest connectivity density and the lowest structure model index. Only GTP, not GGOH, decreased adipokines concentrations (resistin, leptin, monocyte chemoattractant protein-1, and interleukin-6). In an obese male mouse model, individual GGOH and GTP supplementation improved glucose homeostasis, serum CTX, and trabecular microstructure of LV-4. However, the combined GGOH and GTP supplementation compromises such osteoprotective effects on serum CTX and trabecular bone of obese mice.

Osteoprotective effect of green tea polyphenols and annatto-extracted tocotrienol in obese mice is associated with enhanced microbiome vitamin K2 biosynthetic pathways.

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT) × two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using µ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control

Effect of Long-Term Green Tea Polyphenol Supplementation on Bone Architecture, Turnover, and Mechanical Properties in Middle-Aged Ovariectomized Rats.

We investigated the effects of 6-month green tea polyphenols (GTP) supplementation on bone architecture, turnover, and mechanical properties in middle-aged ovariectomized (OVX) rats. Female rats were sham-operated (n = 39, 13/group) or OVX (n = 143, 13/group). Sham-control and OVX-control rats (n = 39) receiving no GTP were assigned for sample collection at baseline, 3, or 6 months. The remaining OVX rats (n = 104) were randomized to 0.15%, 0.5%, 1%, and 1.5% (g/dL) GTP for 3 or 6 months. Blood and bone samples were collected. Relative to the OVX-control group, GTP (1% and 1.5%) lowered serum procollagen type 1 N-terminal propeptide at 3 and 6 months, C-terminal telopeptides of type I collagen at 3 months, and insulin-like growth factor-I at 6 months. GTP did not affect bone mineral content and density. At 6 months, no dose of GTP positively affected trabecular bone volume based on microCT, but a higher cortical thickness and improved biomechanical properties of the femur mid-diaphysis was observed in the 1.5% GTP-treated group. At 3 and 6 months, GTP (0.5%, 1%, and 1.5%) had lower rates of trabecular bone formation and resorption than the OVX-control group, but the inhibitory effects of GTP on periosteal and endocortical bone mineralization and formation at the tibial midshaft were only evident at 3 months. GTP at higher doses suppressed bone turnover in the trabecular and cortical bone of OVX rats and resulted in improved cortical bone structural and biomechanical properties, although it was not effective in preventing the ovariectomy-induced dramatic cancellous bone loss.

Green tea supplementation benefits body composition and improves bone properties in obese female rats fed with high-fat diet and caloric restricted diet.

This study investigated the effects of green tea polyphenols (GTP) supplementation on body composition, bone properties, and serum markers in obese rats fed a high-fat diet (HFD) or a caloric restricted diet (CRD). Forty-eight female rats were fed an HFD ad libitum for 4 months, and then either continued on the HFD or the CRD with or without 0.5% GTP in water. Body composition, bone efficacy, and serum markers were measured. We hypothesized that GTP supplementation would improve body composition, mitigate bone loss, and restore bone microstructure in obese animals fed either HFD or CRD. CRD lowered percent fat mass; bone mass and trabecular number of tibia, femur and lumbar vertebrae; femoral strength; trabecular and cortical thickness of tibia; insulin-like growth factor-I and leptin. CRD also increased percent fat-free mass; trabecular separation of tibia and femur; eroded surface of tibia; bone formation rate and erosion rate at tibia shaft; and adiponectin. GTP supplementation increased femoral mass and strength (P = .026), trabecular thickness (P = .012) and number (P = .019), and cortical thickness of tibia (P < .001), and decreased trabecular separation (P = .021), formation rate (P < .001), and eroded surface (P < .001) at proximal tibia, and insulin-like growth factor-I and leptin. There were significant interactions (diet type × GTP) on osteoblast surface/bone surface, mineral apposition rate at periosteal and endocortical bones, periosteal bone formation rate, and trabecular thickness at femur and lumbar vertebrate (P < .05). This study demonstrates that GTP supplementation for 4 months benefited body composition and improved bone microstructure and strength in obese rats fed with HFD or HFD followed by CRD diet.

Involuntary wheel running improves but does not fully reverse the deterioration of bone structure of obese rats despite decreasing adiposity.

This study investigated whether exercise or antioxidant supplementation with vitamin C and E during exercise affects bone structure and markers of bone metabolism in obese rat. Sprague-Dawley rats, 6-week old, were fed a normal-fat diet (NF, 10 % kcal as fat) and a high-fat diet (HF, 45 % with extra fat from lard) ad libitum for 14 weeks. Then, rats on the high-fat diet were assigned randomly to three treatment groups for additional 12 weeks with forced exercise: HF; HF + exercise (HF + Ex); and HF with vitamin C (0.5 g ascorbate/kg diet) and vitamin E (0.4 g α-tocopherol acetate/kg diet) supplementation + exercise (HF + Ex + VCE). At the end of the study, body weight and fat (%) were similar among NF, HF + Ex, and HF + Ex + VCE, whereas HF had greater body weight and fat (%) than other groups. Compared to NF, HF had elevated serum leptin, tartrate-resistant acid phosphatase (TRAP), and IGF-1; increased trabecular separation and structural model index; and lowered bone mineral density, trabecular connectivity density, and trabecular number in distal femur, while HF + Ex and HF + Ex + VCE had elevated serum TRAP and decreased bone volume/total volume and trabecular number of distal femurs. Compared to HF, HF + Ex and HF + Ex + VCE had decreased serum TRAP and osteocalcin and improved bone structural properties of the distal femur. These findings suggest that exercise, while decreasing body fat, does not fully protect against the negative skeletal effects of existing obesity induced by a high-fat diet. Furthermore, vitamin C and E supplementation has no additional benefits on bone structural properties during exercise.

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.

Soy protein isolates prevent loss of bone quantity associated with obesity in rats through regulation of insulin signaling in osteoblasts.

In both rodents and humans, excessive consumption of a typical Western diet high in saturated fats and cholesterol is known to result in disruption of energy metabolism and development of obesity and insulin resistance. However, how these high-fat, energy-dense diets affect bone development, morphology, and modeling is poorly understood. Here we show that male weanling rats fed a high-fat (HF) diet containing 45% fat and 0.5% cholesterol made with casein (HF-Cas) for 6 wk displayed a significant increase in bone marrow adiposity and insulin resistance. Substitution of casein with soy protein isolate (SPI) in the HF diet (HF-SPI) prevented these effects. Maintenance of bone quantity in the SPI-fed rats was associated with increased undercarboxylated osteocalcin secretion and altered JNK/IRS1/Akt insulin signaling in osteoblasts. The HF-Cas group had significantly greater serum nonesterified free fatty acid (NEFA) concentrations than controls, whereas the HF-SPI prevented this increase. In vitro treatment of osteoblasts or mesenchymal stromal ST2 cells with NEFAs significantly decreased insulin signaling. An isoflavone mixture similar to that found in serum of HF-SPI rats significantly increased in vitro osteoblast proliferation and blocked significantly reduced NEFA-induced insulin resistance. Finally, insulin/IGF1 was able to increase both osteoblast activity and differentiation in a set of in vitro studies. These results suggest that high-fat feeding may disrupt bone development and modeling; high concentrations of NEFAs and insulin resistance occurring with high fat intake are mediators of reduced osteoblast activity and differentiation; diets high in soy protein may help prevent high dietary fat-induced bone impairments; and the molecular mechanisms underlying the SPI-protective effects involve isoflavone-induced normalization of insulin signaling in bone.

Green tea polyphenols improve bone microarchitecture in high-fat-diet-induced obese female rats through suppressing bone formation and erosion.

This study evaluates the effects of green tea polyphenols (GTPs) on bone microarchitecture in high-fat-diet (HFD)-induced obese female rats. Thirty-six 3-month-old female rats were fed either a control diet or a HFD for 4 months. Animals in the control group continued on the control diet for another 4 months. Animals in the HFD group were divided into two groups, with 0.5 g/100 mL GTP (the HFD+GTP group) or without GTP (the HFD group) in drinking water, in addition to the HFD for another 4 months. Compared to the control group, the HFD group increased bone formation and erosion rates at the tibia, decreased trabecular volume and thickness, but had no impact on bone mineral density (BMD), trabecular number (Tb.N), and separation. Compared to the control group, the HFD+GTP group demonstrates a greater Tb.N at the proximal tibia, and a greater trabecular thickness at the femur and the lumbar vertebrae, but a smaller trabecular separation (Tb.Sp) and mineralizing surface at the proximal tibia, and a reduced endocortical mineral apposition rate (MAR) at the tibia shaft. Relative to the HFD group, the HFD+GTP group demonstrates (1) a higher BMD at the femur, a greater trabecular volume, thickness, and number at the proximal tibia, a larger cortical area and thickness at the tibial shaft, and a greater trabecular volume and thickness at the femur and the lumbar vertebrae, (2) a smaller Tb.Sp, MAR, bone formation rate, and eroded surface at the tibia. We concluded that GTP supplementation in drinking water improves bone microarchitecture in the HFD-induced obese female rats, possibly through suppressing bone turnover, resulting in a larger net bone volume.

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.

Green tea polyphenols benefits body composition and improves bone quality in long-term high-fat diet-induced obese rats.

This study investigates the effects of green tea polyphenols (GTPs) on body composition and bone properties along with mechanisms in obese female rats. Thirty-six 3-month-old Sprague Dawley female rats were fed either a low-fat (LF) or a high-fat (HF) diet for 4 months. Animals in the LF diet group continued on an LF diet for additional 4 months, whereas those in the HF diet group were divided into 2 groups: with GTP (0.5%) or without in drinking water, in addition to an HF diet for another 4 months. Body composition, femur bone mass and strength, serum endocrine and proinflammatory cytokines, and liver glutathione peroxidase (GPX) protein expression were determined. We hypothesized that supplementation of GTP in drinking water would benefit body composition, enhance bone quality, and suppress obesity-related endocrines in HF diet-induced obese female rats and that such changes are related to an elevation of antioxidant capacity and a reduction of proinflammatory cytokine production. After 8 months, compared with the LF diet, the HF diet increased percentage of fat mass and serum insulin-like growth factor I and leptin levels; reduced percentage of fat-free mass, bone strength, and GPX protein expression; but had no effect on bone mineral density and serum adiponectin levels in the rats. Green tea polyphenol supplementation increased percentage of fat-free mass, bone mineral density and strength, and GPX protein expression and decreased percentage of fat mass, serum insulin-like growth factor I, leptin, adiponectin, and proinflammatory cytokines in the obese rats. This study shows that GTP supplementation benefited body composition and bone properties in obese rats possibly through enhancing antioxidant capacity and suppressing inflammation.

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.

Green tea and bone health: Evidence from laboratory studies.

Osteoporosis is a major health problem in the elderly. Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly population. Ingestion of green tea and green tea bioactive compounds may be beneficial in mitigating bone loss of this population and decreasing their risk of osteoporotic fractures. This review describes the effect of green tea with its bioactive components on bone health with an emphasis on the following: (i) the etiology of osteoporosis, (ii) evidence of osteo-protective impacts of green tea on bone mass and microarchitecture in various bone loss models in which induced by aging, sex hormone deficiency, and chronic inflammation, (iii) discussion of impacts of green tea on bone mass in two obesity models, (iv) observation of short-term green tea supplementation given to postmenopausal women with low bone mass, (v) possible mechanisms for the osteo-protective effects of green tea bioactive compounds, and (vi) a summary and future research direction of green tea and bone health.

Supplementation with green tea polyphenols improves bone microstructure and quality in aged, orchidectomized rats.

Recent studies show that green tea polyphenols (GTPs) attenuate bone loss and microstructure deterioration in ovariectomized aged female rats, a model of postmenopausal osteoporosis. This study evaluated the efficacy of GTPs at mitigating bone loss and microstructure deterioration along with related mechanisms in androgen-deficient aged rats, a model of male osteoporosis. A 2 (sham vs. orchidectomy) × 2 (no GTP and 0.5% GTP in drinking water) factorial design was studied for 16 weeks using 40 aged male rats. An additional 10 rats (baseline group) were killed at the beginning of study to provide baseline parameters. There was no difference in femoral mineral density between baseline and the sham only group. Orchidectomy suppressed serum testosterone and tartrate-resistant acid phosphatase concentrations, liver glutathione peroxidase activity, bone mineral density, and bone strength. Orchidectomy also decreased trabecular bone volume, number, and thickness in the distal femur and proximal tibia and bone-formation rate in trabecular bone of proximal tibia but increased serum osteocalcin concentrations and bone-formation rates in the endocortical tibial shaft. GTP supplementation resulted in increased serum osteocalcin concentrations, bone mineral density, and trabecular volume, number, and strength of femur; increased trabecular volume and thickness and bone formation in both the proximal tibia and periosteal tibial shaft; decreased eroded surface in the proximal tibia and endocortical tibial shaft; and increased liver glutathione peroxidase activity. We conclude that GTP supplementation attenuates trabecular and cortical bone loss through increasing bone formation while suppressing bone resorption due to its antioxidant capacity.

Green tea polyphenols avert chronic inflammation-induced myocardial fibrosis of female rats.

OBJECTIVE: Green tea proposes anti-inflammatory properties which may attenuate chronic inflammation-induced fibrosis of vessels. This study evaluated whether green tea polyphenols (GTP) can avert fibrosis or vascular disruption along with mechanisms in rats with chronic inflammation. TREATMENTS: Forty 3-month-old female rats were assigned to a 2 (placebo vs. lipopolysaccharide, administration) × 2 (no GTP vs. 0.5% GTP in drinking water) factorial design for 12 weeks. METHODS: Masson's trichrome staining evaluated myocardial fibrosis in coronary vessels and surrounding myocardium. Whole blood specimens were counted for differentials. Spleen tumor necrosis factor-α (TNF-α) mRNA expression was determined by real-time RT-PCR. Data were analyzed by two-way analysis of variance (ANOVA) followed by mean separation procedures. RESULTS: After 12 weeks, lipopolysaccharide administration induced myocardial fibrosis in vessels and surrounding myocardium, spleen TNF-α mRNA expression, and leukocytes, while GTP supplementation in drinking water significantly averted such observation. CONCLUSIONS: GTP attenuates myocardial fibrosis through a suppression of chronic inflammation and innate immune responses.

Protective actions of green tea polyphenols and alfacalcidol on bone microstructure in female rats with chronic inflammation.

This study investigated the effects of green tea polyphenols (GTP) and alfacalcidol on bone microstructure and strength along with possible mechanisms in rats with chronic inflammation. A 12-week study using a 2 (no GTP vs. 0.5%, w/v GTP in drinking water)×2 (no alfacalcidol vs. 0.05 µg/kg alfacalcidol orally, 5×/week) factorial design was employed in lipopolysaccharide (LPS)-administered female rats. A group receiving placebo administration was used to compare with a group receiving LPS administration only to evaluate the effect of LPS. Changes in tibial and femoral microarchitecture and strength of femur were evaluated. Difference in expression of tumor necrosis factor-α (TNF-α) in proximal tibia using immunohistochemistry was examined. Compared to the placebo group, the LPS-administered-only group had significantly lower femoral mass, trabecular volume, thickness and number in proximal tibia and femur, and lower periosteal bone formation rate in tibial shafts but had significantly higher trabecular separation and osteoclast number in proximal tibia and eroded surface in endocortical tibial shafts. Both GTP and alfacalcidol reversed these LPS-induced detrimental changes in femur, proximal tibia and endocortical tibial shaft. Both GTP and alfacalcidol also significantly improved femoral strength, while significantly suppressed TNF-α expression in proximal tibia. There were significant interactions in femoral mass and strength, trabecular separation, osteoclast number and TNF-α expression in proximal tibia. A combination of both showed to sustain bone microarchitecture and strength. We conclude that a protective impact of GTP and alfacalcidol in bone microarchitecture during chronic inflammation may be due to a suppression of TNF-α.

Green tea polyphenols mitigate bone loss of female rats in a chronic inflammation-induced bone loss model.

The purpose of this study was to explore the bioavailability, efficacy and molecular mechanisms of green tea polyphenols (GTP) related to preventing bone loss in rats with chronic inflammation. A 2 [placebo vs. lipopolysaccharide (LPS)]×2 (no GTP vs. 0.5% GTP in drinking water) factorial design enabled the evaluation of effects of LPS administration, GTP levels, and LPS×GTP interaction. Urinary GTP components and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were determined by high-pressure liquid chromatography for bioavailability and molecular mechanism, respectively. Efficacy was evaluated by examining changes in femoral mineral content (BMC) and density (BMD) using dual-energy X-ray absorptiometry, and bone turnover biomarkers [osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP)] using respective ELISA kits. The mRNA expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) in spleen was determined by real-time RT-PCR. Neither LPS administration nor GTP levels affected body weight and femoral bone area throughout the study period. Only GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations. LPS administration led to a decrease in femur BMC and BMD, and serum OC levels, but an increase in serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. GTP supplementation resulted in higher values for femur BMC, BMD and serum OC, but lower values for serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. We conclude that GTP mitigates bone loss in a chronic inflammation-induced bone loss model by reducing oxidative stress-induced damage and inflammation.

Green tea and bone metabolism.

Osteoporosis is a major health problem in both elderly women and men. Epidemiological evidence has shown an association between tea consumption and the prevention of age-related bone loss in elderly women and men. Ingestion of green tea and green tea bioactive compounds may be beneficial in mitigating bone loss of this population and decreasing their risk of osteoporotic fractures. This review describes the effect of green tea or its bioactive components on bone health, with an emphasis on (i) the prevalence and etiology of osteoporosis; (ii) the role of oxidative stress and antioxidants in osteoporosis; (iii) green tea composition and bioavailability; (iv) the effects of green tea and its active components on osteogenesis, osteoblastogenesis, and osteoclastogenesis from human epidemiological, animal, as well as cell culture studies; (v) possible mechanisms explaining the osteoprotective effects of green tea bioactive compounds; (vi) other bioactive components in tea that benefit bone health; and (vii) a summary and future direction of green tea and bone health research and the translational aspects. In general, tea and its bioactive components might decrease the risk of fracture by improving bone mineral density and supporting osteoblastic activities while suppressing osteoclastic activities.