Véronique Coxam

Oxidative stress in bone remodelling and disease

Oxidative stress is characterised by an increased level of reactive oxygen species (ROS) that disrupts the intracellular reduction-oxidation (redox) balance. Although initially shown to be involved in aging, physiological roles for ROS in regulating cell functions and mediating intracellular signals have emerged. In bone tissues, recent studies have demonstrated that ROS generation is a key modulator of bone cell function and that oxidative status influences the pathophysiology of mineralised tissues. Here, we review the crucial role of oxidative stress in bone pathophysiology, and discuss the possibility that ROS production might be a relevant therapeutic target under certain conditions. Further studies will be needed to investigate whether manipulation of the redox balance in bone cells represents a useful approach in the design of future therapies for bone diseases.

Rutin Inhibits Ovariectomy-Induced Osteopenia in Rats

Several studies suggest that polyphenols might exert a protective effect against osteopenia. The present experiment was conducted to observe the effects of rutin (quercetin‐3‐O‐glucose rhamnose) on bone metabolism in ovariectomized (OVX) rats. Thirty 3‐month‐old Wistar rats were used. Twenty were OVX while the 10 controls were sham‐operated (SH). Among the 20 OVX, for 90 days after surgery 10 were fed the same synthetic diet as the SH or OVX ones, but 0. 25% rutin (OVX + R) was added. At necropsy, the decrease in uterine weight was not different in OVX and OVX + R rats. Ovariectomy also induced a significant decrease in both total and distal metaphyseal femoral mineral density, which was prevented by rutin consumption. Moreover, femoral failure load, which was not different in OVX and SH rats, was even higher in OVX + R rats than in OVX or SH rats. In the same way, on day 90, both urinary deoxypyridinoline (DPD) excretion (a marker for bone resorption) and calciuria were higher in OVX rats than in OVX + R or SH rats. Simultaneously, plasma osteocalcin (OC) concentration (a marker for osteoblastic activity) was higher in OVX + R rats than in SH rats. High‐performance liquid chromatography (HPLC) profiles of plasma samples from OVX + R rats revealed that mean plasma concentration of active metabolites (quercetin and isorhamnetin) from rutin was 9.46 + 1 μM, whereas it was undetectable in SH and OVX rats. These results indicate that rutin (and/or its metabolites), which appeared devoid of any uterotrophic activity, inhibits ovariectomy‐induced trabecular bone loss in rats, both by slowing down resorption and increasing osteoblastic activity.

Amylin and Bone Metabolism in Streptozotocin-Induced Diabetic Rats

Amylin (AMY) is a 37 amino acid peptide cosecreted with insulin (INS) by pancreatic β‐cells and absent in type 1 diabetes, a condition frequently associated with osteopenia. AMY binds to calcitonin receptors, lowers plasma calcium concentration, inhibits osteoclast activity, and stimulates osteoblasts. In the present study, we examined the effects of AMY replacement on bone loss in a streptozotocin (STZ)‐induced rodent model type 1 diabetes. Of 50 male Wistar rats studied, 40 were made diabetic with intraperitoneal STZ (50 mg/kg; plasma glucose concentrations >11 mM within 5 days). Ten nondiabetic control (CONT) rats received citrate buffer without STZ. Diabetic rats were divided into four groups (n = 10/group) and injected subcutaneously with rat AMY (45 mg/kg), INS (12 U/kg), both (same doses), or saline (STZ; diabetic controls) once per day. After 40 days of treatment and five 24‐h periods of urine collection for deoxypyridinoline (DPD), the animals were killed, blood was sampled, and femurs were removed. The left femur was tested for mechanical resistance (three‐point bending). The right femur was tested for total, diaphyseal (cortical bone), and metaphyseal (trabecular bone) bone densities using dual‐energy X‐ray absorptiometry (DXA). Bone was ashed to determine total bone mineral (calcium) content. None of the treatments had any significant effect on femoral length and diameter. Untreated diabetic rats (STZ; 145 ± 7N) had lower bone strength than did nondiabetic CONT (164 ± 38; p < 0.05). Total bone mineral density (BMD; g/cm2) was significantly lower in STZ (0. 2523 ± 0. 0076) than in CONT (0.2826 ± 0.0055), as were metaphyseal and diaphyseal densities. Diabetic rats treated with AMY, INS, or both had bone strengths and bone densities that were indistinguishable from those in nondiabetic CONT. Changes in bone mineral content paralleled those for total BMD (T‐BMD). Plasma osteocalcin (OC) concentration, a marker for osteoblastic activity, was markedly lower in untreated diabetic rats (7. 6 ± 0. 9 ng/ml); p < 0. 05) than in nondiabetic CONT (29. 8 ± 1. 7; p < 0. 05) or than in AMY (20. 1 ± 0. 7; p < 0. 05). Urinary DPD excretion, a marker for bone resorption, was similar in untreated and AMY‐treated diabetic rats (35.0 ± 3.1 vs. 35.1 ± 4.4 nmol/mmol creatinine), intermediate in rats treated with INS (49.9 ± 2.7), and normalized in diabetic rats treated with both agents (58.8 ± 8.9 vs. 63.2 ± 4.5 in CONT). Thus, in our STZ rat model of diabetic osteopenia, addition of AMY improved bone indices apparently by both inhibiting resorption and stimulating bone formation.

Olive oil and its main phenolic micronutrient (oleuropein) prevent inflammation-induced bone loss in the ovariectomised rat

The present study was designed to evaluate the effect of olive oil and its main polyphenol (oleuropein) in ovariectomised rats with or without inflammation. Rats (6 months old) were ovariectomised or sham-operated as control. Ovariectomised rats were separated into three groups receiving different diets for 3 months: a control diet with 25 g peanut oil and 25 g rapeseed oil/kg (OVX), the control diet with 50 g olive oil/kg or the control diet with 0.15 g oleuropein/kg. The sham-operated group was given the same control diet as OVX. Inflammation was induced 3 weeks before the end of the experiment by subcutaneous injections of talc (magnesium silicate) in one-half of each group. The success of ovariectomy was verified at necropsy by the atrophy of uterine horns. Inflammation, oleuropein or olive oil intakes did not have any uterotrophic activity, as they had had no effect on uterus weight. The plasma concentration of alpha-1-acid glycoprotein (an indicator of inflammation) was increased in OVX rats with inflammation. With regard to bone variables, osteopenia in OVX was exacerbated by inflammation, as shown by a decrease in metaphyseal and total femoral mineral density. Both oleuropein and olive oil prevented this bone loss in OVX rats with inflammation. At necropsy, oleuropein and olive oil consumption had had no effect on plasma osteocalcin concentrations (marker of bone formation) or on urinary deoxypyridinoline excretion (marker of bone resorption). In conclusion, oleuropein and olive-oil feeding can prevent inflammation-induced osteopenia in OVX rats.

Hesperidin inhibits ovariectomized-induced osteopenia and shows differential effects on bone mass and strength in young and adult intact rats

The main aim of this study was to investigate the bone-sparing effect of hesperidin, one of the main flavonoid present in oranges, in two age groups of ovariectomized female rats, compared with their intact controls. Young (3 mo) and adult (6 mo) female Wistar rats were sham operated (SH) or ovariectomized (OVX) and then pair-fed for 90 days a casein-based diet supplemented or not with 0.5% hesperidin (Hp; n = 10/group). In older rats, Hp intake led to a partial inhibition of OVX-induced bone loss, whereas a complete inhibition was obtained in younger animals. At both ages, while plasma osteocalcin concentrations were unchanged, urinary excretion of deoxypyridinoline was reduced by Hp intake, suggesting that Hp was able to slow down bone resorption. Unexpectedly, in intact young rats, Hp consumption resulted in a significant increase in bone mineral density (BMD). Indeed, 6-mo-old HpSH rats had a similar BMD to 9-mo-old nontreated SH adult rats, suggesting an accelerated bone mass gain in the young rats. In contrast, in intact adult rats, Hp did not further increase BMD but did improve their bone strength. The results of this study show a protective effect of Hp on bone loss in OVX rats of both ages without uterine stimulation and accompanied by a lipid-lowering effect. The unexpected and intriguing findings obtained in intact rats showing improved BMD in young rats and improved femoral load in adult rats merit further investigation. The bone and lipid benefits of hesperidin make it an attractive dietary agent for the management of the health of postmenopausal women.

Bone Mass in Obese Diabetic Zucker Rats: Influence of Treadmill Running

Some controversy exists in the literature concerning bone mineral densitry (BMD) in obese, diabetic, leptin-resistant Zucker rats. To investigate this question further, we measured body composition and femoral bone mineral density (BMD) (by dual energy X-ray absorptiometry) in 10 male and 10 female 6 month-old Zucker rats and their homozygous lean controls. Fat mass (percent from body weight) was about 3 times higher in fatty rats than in lean controls. Total, diaphyseal, and distal metaphyseal BMD, total femoral Ca content, and femoral failure load were lower in Zucker rats than in controls. Moderate treadmill running (35% - 40% VO2 max, 20-50 minutes day, 6 days/ week, for 89 days) increased BMD in these animals, possibly by inhibiting bone resorption, as evidenced by no change in plasma osteocalcin concentration but decreased urinary deoxypyridinoline excretion in fatty runners.

Prevention of Bone Loss by Phloridzin, an Apple Polyphenol, in Ovariectomized Rats under Inflammation Conditions

Aging and sex hormones related changes lead to inflammatory and oxidant conditions, which are involved in the pathogenesis of osteoporosis. Recent studies have suggested that polyphenols may exert a protective effect in such conditions. We assessed the effect of phloridzin (Phlo), a flavonoid exclusively found in apple, on bone metabolism in ovariectomized (OVX) or sham-operated (SH) rats with and without inflammation. Six-month-old Wistar rats were allocated to two equal groups that received either a control diet or a diet supplemented with 0.25% Phlo for 80 days. Three weeks before necropsy, inflammation was induced by subcutaneous injection of talc in 10 animals of each group. At necropsy, ovariectomy decreased both total (T-BMD) and metaphyseal (M-BMD) femoral bone mineral density (P < 0.01). Inflammation conditions, checked by an increase in the spleen weight and α1-acid glycoprotein concentration in OVX rats, exacerbated the decrease in T-BMD (g/cm2) (as well as M-BMD) observed in castrated animals (P < 0.05). Daily Phlo intake prevented ovariectomy-induced bone loss in conditions of inflammation as shown by T-BMD and M-BMD (P < 0.05). At the diaphyseal site, BMD was improved by Phlo in OVX rats with or without inflammation (P < 0.05). These results could be explained by changes in bone remodeling as the increased urinary deoxypyridinoline excretion in OVX and OVXinf animals was prevented by the polyphenol-rich diet (P < 0.001), while plasma osteocalcin concentration was similar in all experimental groups. In conclusion, Phlo consumption may provide protection against ovariectomy-induced osteopenia under inflammation conditions by improving inflammation markers and bone resorption.

Muscle and bone, two interconnected tissues

As bones are levers for skeletal muscle to exert forces, both are complementary and essential for locomotion and individual autonomy. In the past decades, the idea of a bone-muscle unit has emerged. Numerous studies have confirmed this hypothesis from in utero to aging works. Space flight, bed rest as well as osteoporosis and sarcopenia experimentations have allowed to accumulate considerable evidence. Mechanical loading is a key mechanism linking both tissues with a central promoting role of physical activity. Moreover, the skeletal muscle secretome accounts various molecules that affect bone including insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-2), interleukin-6 (IL-6), IL-15, myostatin, osteoglycin (OGN), FAM5C, Tmem119 and osteoactivin. Even though studies on the potential effects of bone on muscle metabolism are sparse, few osteokines have been identified. Prostaglandin E2 (PGE2) and Wnt3a, which are secreted by osteocytes, osteocalcin (OCN) and IGF-1, which are produced by osteoblasts and sclerostin which is secreted by both cell types, might impact skeletal muscle cells. Cartilage and adipose tissue are also likely to participate to this control loop and should not be set aside. Indeed, chondrocytes are known to secrete Dickkopf-1 (DKK-1) and Indian hedgehog (Ihh) and adipocytes produce leptin, adiponectin and IL-6, which potentially modulate bone and muscle metabolisms. The understanding of this system will enable to define new levers to prevent/treat sarcopenia and osteoporosis at the same time. These strategies might include nutritional interventions and physical exercise.

Phyto-oestrogens and bone health.

As oestrogen deficiency is the main cause in the pathogenesis of osteoporosis hormone-replacement therapy remains the mainstay for prevention. However, prophylaxis by hormone-replacement therapy is limited. Phyto-oestrogens, which are weakly-oestrogenic compounds present in plants, deserve particular mention because emerging data support the suggestion that they may prevent bone loss associated with the menopause. In the past few years extensive research using animal models has provided convincing data to indicate a significant improvement in bone mass or other end points following feeding with soyabean. Moreover, observational studies relate the lower incidence of osteoporosis among women in the Eastern world to a diet rich in phyto-oestrogens. However, it is not valid to extrapolate to the Western situation. The varied clinical trials that have been published suggest that isoflavones reduce bone loss in women in the early period post menopause, but a definitive result requires more investigations of the effect of phyto-oestrogens on bone health that have substantial sample size and are of long duration. In addition, the clinical efficacy of soya foods in preventing osteopenia depends on their intestinal metabolism. Thus, phyto-oestrogens are a source for putative innovative dietary health intervention for post-menopausal women. However, more data are necessary, particularly in relation to their effect on the risk of fracture.

One-month exposure to soy isoflavones did not induce the ability to produce equol in postmenopausal women.

Objective:As more and more postmenopausal women are taking soy isoflavone supplementation for relieving menopausal symptoms, we investigated the impact of chronic exposure on their bioavailability, with focus on achievable plasma concentrations and potential stimulation of the capacity to produce equol.Subjects:A total of 12 Caucasian postmenopausal women.Intervention:Volunteers ingested 100 mg isoflavones/day (aglycone equivalents, in cereal bars and yoghurts) for 1 month. Plasma concentrations of metabolites at 2, 4, 6, 8, 10, 12 and 24 h postdose, as well as urinary excretion in fractions over 36 h were compared between days 1 and 30.Results:Similar plasma kinetic curves were obtained at day 1 and day 30 for genistein and daidzein. Maximum plasma concentrations were 1.68±0.68 μmol/l on day 1 compared to 2.27±0.76 μmol/l on day 30 for daidzein (P=0.056), and 3.88±1.50 μmol/l on day 1 compared to 5.30±2.38 μmol/l on day 30 for genistein (P=0.091). Urinary excretion of daidzein and genistein did not differ significantly between days 1 and 30. Maximum plasma concentration of equol increased significantly from 0.31±0.27 to 0.99±0.51 μmol/l for equol-producer volunteers (P=0.046). However, the seven volunteers who were classified as non-equol producers on day 1 did not acquire the ability to produce equol after 1-month exposure.Conclusions:Chronic exposure to isoflavones in postmenopausal women resulted in plasma concentrations as high as 2.5–5 μmol/l of each isoflavone, but did not induce the ability to produce equol.Sponsorship:French Ministry of Research (AQS program A01347) and Beghin-Meiji (doctoral fellowship).