Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis

The objective of the present study was to explore the bone protective role of blueberry in an ovariectomized rat model. Thirty 6-month-old female Sprague-Dawley rats were either sham-operated (Sham) or ovariectomized (Ovx) and divided into three groups: Sham, Ovx (control), Ovx+blueberry (5% blueberry w/w). After 100 days of treatment, rats were euthanized, and blood and tissues were collected. Bone mineral density (BMD) and content of whole body, right tibia, right femur and fourth lumbar vertebra were assessed via dual-energy X-ray absorptiometry. As expected, Ovx resulted in loss of whole-body, tibial, femoral, and 4th lumbar BMD by approximately 6%. Blueberry treatment was able to prevent the loss of whole-body BMD and had an intermediary effect on prevention of tibial and femoral BMD when compared to either Sham or Ovx controls. The bone-protective effects of blueberry may be due to suppression of Ovx-induced increase in bone turnover, as evident by lowered femoral mRNA levels of alkaline phosphatase, collagen type I and tartrate-resistant acid phosphatase to the Sham levels. Similarly, serum osteocalcein levels were also lower in the blueberry group when compared to the Ovx control group, albeit not significantly. In summary, our findings indicate that blueberry can prevent bone loss as seen by the increases in BMD and favorable changes in biomarkers of bone metabolism.     

Modified activity-stress paradigm in an animal model of the female athlete triad.

The exercising woman with nutritional deficits and related menstrual irregularities is at risk of compromising long-term bone health, i.e., the female athlete triad. There is no animal model of the female athlete triad. The purpose of this study was to examine long-term energy restriction in voluntary wheel-running female rats on estrous cycling, bone mineral content, and leptin levels. Twelve female Sprague-Dawley rats (age 34 days) were fed ad libitum and given access to running wheels during an initial 14-wk period, providing baseline and age-related data. Daily collection included dietary intake, body weight, estrous cycling, and voluntary running distance. At 4 mo, rats were randomized into two groups, six restrict-fed rats (70% of ad libitum intake) and six rats continuing as ad libitum-fed controls. Energy intake, energy expenditure, and energy availability (energy intake - energy expenditure) were calculated for each animal. Serum estradiol and leptin concentrations were measured by RIA. Femoral and tibial bone mineral density and bone mineral content (BMC) were determined by dual-energy X-ray absorptiometry. Restrict-fed rats exhibited a decrease in energy availability during Weight Loss and Anestrous phases (P = 0.002). Compared with controls after 12 wk, restrict-fed rats showed reduced concentrations of serum estradiol (P = 0.002) and leptin (P = 0.002), lower ovarian weight (P = 0.002), and decreased femoral (P = 0.041) and tibial (P = 0.05) BMC. Decreased energy availability resulted in anestrus and significant decreases in BMC, estrogen and leptin levels, and body weight. Finally, there is a critical level of energy availability to maintain estrous cycling.     

High-impact exercise strengthens bone in osteopenic ovariectomized rats with the same outcome as Sham rats.

The effect of jump exercise on middle-aged osteopenic rats was investigated. Forty-two 9-mo-old female rats were either sham-operated (Sham) or ovariectomized (OVX). Three months after surgery, the rats were divided into the following groups: Sham sedentary, Sham exercised, OVX sedentary, and OVX exercised. Rats in the exercise groups jumped 10 times/day, 5 days/wk, for 8 wk, with a jumping height of 40 cm. Less than 1 min was required for the jump training. After the experiment, the right tibia and femur were dissected, and blood was obtained from each rat. OVX rats were observed to have increased body weights and decreased bone mass in their tibiae and femurs. Jump-exercised rats, on the other hand, had significantly increased tibial bone mass, strength, and cortical areas. The bone mass and strength of OVX exercised rats increased to approximately the same extent as Sham exercised rats, despite estrogen deficiency or osteopenia. Our data suggest that jump exercise has beneficial effects on lower limb bone mass, strength, bone mineral density, and morphometry in middle-aged osteopenic rats, as well as in Sham rats.     

Effects of ovariectomy and hindlimb unloading on skeletal muscle

Female rats(7-8 mo old, n = 40) wererandomly placed into the intact control (Int) and ovariectomizedcontrol (Ovx) groups. Two weeks after ovariectomy, animals were furtherdivided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomizedhindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situcontractile tests were performed on soleus (Sol), plantaris (Plan),peroneus longus (Per), and extensor digitorum longus (EDL) muscles.Body weight and Sol mass were ~22% larger in Ovx than in Int groupand ~18% smaller in both HU groups than in Int rats (Ovx × HUinteraction, P < 0.05), and therewas a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading(atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles wasunaffected by either ovariectomy or hindlimb unloading. Time to peaktwitch tension for EDL and one-half relaxation times for Sol, Plan,Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that1) ovariectomy led to similarincreases of ~20% in body weight and plantar flexor mass;2) hindlimb unloading may haveprevented ovariectomy-related muscle growth;3) greater atrophy may have occurredin Sol and Plan of Ovx animals compared with controls; and4) removal of ovarian hormonalinfluence decreased skeletal muscle contraction times.

     

Ovariectomy prevents the recovery of atrophied gastrocnemius skeletal muscle mass.

The recovery of atrophied muscle mass in animals is thought to be dependent on a number of factors including hormones, cytokines, and/or growth factor expression. The Akt/mammalian target of rapamycin (mTOR) signaling pathway is believed to be activated by these various factors, resulting in skeletal muscle growth through the initiation of protein synthesis. It was hypothesized that surgical removal of the ovaries (Ovx) may alter activation of the Akt/mTOR signaling pathway, a mechanism necessary for muscle regrowth. To test this, 36 Sprague-Dawley rats underwent Ovx or sham surgeries. A portion of the animals were then subjected to hindlimb unloading (HLU) for 28 days. After HLU, one group of Sham and Ovx rats underwent a 14-day recovery period in which the animals were allowed free cage ambulation. The HLU animals demonstrated approximately 21-27% reduction in medial gastrocnemius muscle mass irrespective of whether the ovaries were intact or not. The Sham animals that were reloaded recovered their atrophied muscle mass; however, the Ovx group failed to recover any of the atrophied muscle mass with reloading. The failure to recover muscle mass in the Ovx group was associated with reduced phosphorylation levels of both Akt and p70s6k, whereas in the Sham recovery animals no reductions were found in Akt phosphorylation and significant increases in p70s6k activation were detected. Finally, no differences were detected in mTOR phosphorylation in any of Sham or Ovx groups. These results suggest that ovariectomy surgeries could be detrimental to the recovery of atrophied muscle mass.     

Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.     

Prepubertal OVX increases IGF-I expression and bone accretion in C57BL/6J mice

It is generally well accepted that the pubertal surge in estrogen is responsible for the rapid bone accretion that occurs during puberty and that this effect is mediated by an estrogen-induced increase in growth hormone (GH)/insulin-like growth factor (IGF) action. To test the cause and effect relationship between estrogen and GH/IGF, we evaluated the consequence of ovariectomy (OVX) in prepubertal mice (C57BL/6J mice at 3 wk of age) on skeletal changes and the GH/IGF axis during puberty. Contrary to our expectations, OVX increased body weight (12-18%), bone mineral content (11%), bone length (4%), bone size (3%), and serum, liver, and bone IGF-I (30-50%) and decreased total body fat (18%) at 3 wk postsurgery. To determine whether estrogen is the key ovarian factor responsible for these changes, we performed a second experiment in which OVX mice were treated with placebo or estrogen implants. In addition to observing similar results compared with our first experiment, estrogen treatment partially rescued the increased body weight and bone size and completely rescued body fat and IGF-I levels. The increased bone accretion in OVX mice was due to increased bone formation rate (as determined by bone histomorphometry) and increased serum procollagen peptide. In conclusion, contrary to the known estrogen effect as an initiator of GH/IGF surge and thereby pubertal growth spurt, our findings demonstrate that loss of estrogen and/or other hormones during the prepubertal growth period effect leads to an increase in IGF-I production and bone accretion in mice.     

葛根异黄酮对去卵巢大鼠骨质疏松症的影响

通过对3月龄Wister大鼠,手术切除双侧卵巢后7天,每天灌胃TIP40mg/kg和10mg/kg,并设去卵巢组(OVX)、假手术组(Sham)和尼尔雌醇阳性对照组(OVX-E2),在给药3个月时,测定大鼠股骨骨矿密度(BMD)、骨钙及血清钙水平等,研究葛根异黄酮(TIP)对由雌激素缺乏引起的骨质疏松症的防治作用。结果TIP40mg/kg的BMD比去卵巢组显著提高了18.1%;使胫骨和血清钙含量显著增加;使去卵巢大鼠的脾脏重量系数和胸腺重量系数明显恢复;并可明显控制大鼠的体重。葛根异黄酮可能具有雌激素样活性,并有改善骨质疏松症的生物学活性。     

Estrogen deficiency decreases ischemic tolerance in the aged rat heart: Roles of PKCdelta, PKCepsilon, Akt, and GSK3beta

The mechanisms underlying the age-dependent reversal of female cardioprotection are poorly understood and complicated by findings that estrogen replacement is ineffective at reducing cardiovascular mortality in postmenopausal women. Although several protective signals have been identified in young animals, including PKC and Akt, how these signals are affected by age, estrogen deficiency, and ischemia-reperfusion (I/R) remains unknown. To determine the independent and combined effects of age and estrogen deficiency on I/R injury and downstream PKC-Akt signaling, adult and aged female F344 rats (n = 12/age) with ovaries intact or ovariectomy (Ovx) were subjected to I/R using Langendorff perfusion (31-min global-ischemia). Changes in cytosolic (s), nuclear (n), mitochondrial (m) PKC (delta, epsilon) levels, and changes in total Akt and mGSK-3beta phosphorylation after I/R were assessed by Western blot analysis. Senescence increased infarct size 50% in ovary-intact females (P < 0.05), whereas no differences in LV functional recovery or estradiol levels were observed. Ovx reduced functional recovery to a greater extent in aged compared with adult rats (P < 0.05). In aged (vs. adult), levels of m- and nPKC(-delta, -epsilon) were markedly decreased, whereas mGSK3beta levels were increased (P < 0.05). Ovx led to greater levels of sPKC(-delta, -epsilon) independent of age (P < 0.05). I/R reduced p-Akt(Ser473) levels by 57% and increased mGSK-3beta accumulation 1.77-fold (P < 0.05) in aged, ovary-intact females. These data suggest, for the first time, that estrogen alone cannot protect the aged female myocardium from I/R damage and that age- and estrogen-dependent alterations in PKC, Akt, and GSK-3beta signaling may contribute to loss of ischemic tolerance.     

Evaluation of the Preventive Effect of Isoflavone Extract on Bone Loss in Ovariectomized Rats

To examine a potential role for soybean phytoestrogens in postmenopausal bone loss, twenty-four 12-week-old Sprague-Dawley rats were divided randomly into 4 groups and given controlled diets for 16 weeks. The treatment groups were as followed: sham operated, ovariectomized (OVX) control, OVX + isoflavone extract (6.25 g/kg), and OVX + 17β-estradiol (4 mg/kg). OVX treatments reduced femoral and fourth lumbar vertebral bone density and mineral content (p<0.01), decreased uterine weight (p<0.01), accelerated body weight increases (p<0.05), and increased the activities (p<0.01) of both serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Supplementation with isoflavone prevented the losses of bone density and mineral content caused by OVX (p<0.01). Although both isoflavone and 17β-estradiol exhibited similar bone-sparing ability on the OVX-induced bone loss, the effect of isoflavone was not the same as that of 17β-estradiol on the serum ALP and TRAP, body weight increase, and uterine weight change. We concluded that dietary supplementation with soybean isoflavone can prevent postmenopausal bone loss via a different mechanism of estrogen in OVX rats.     

Effect of calcium stress on the skeleton mass of intact and ovariectomized rats

Female rats were ovariectomized (Ovx) or sham-operated (control) at 18 weeks and the entire skeleton obtained at 24 weeks (baseline) or after an additional 31 day (28 week) interval on a normal (1.0%) or deficient (0.02%) calcium diet. Ovx rats showed a 42% increase in whole body bone resorption (3H-tetracycline loss) in the absence of calcium stress (1.0% calcium diet) and a 70% increase in resorption with morphological evidence of dramatic loss of cancellous bone mass when placed on calcium-deficient (0.02%) diets. Ovx rats kept on the 1.0% calcium diet showed a significant increase in both their body weight (30.2%) and total bone mass (11.6%) compared to baseline sham-operated controls. However, the total skeleton mass of these animals was significantly reduced (-20%) from that predicted by calculations based on body weight. Maintaining animals on calcium-deficient diets had no significant effect on the total skeleton mass of either control or Ovx rats in comparison with age-matched controls on 1.0% diets. It was further determined that an increase in bone mass between 24 and 28 weeks in rats receiving 1.0% dietary calcium occurred in both the axial and appendicular skeleton and was proportionately similar between control and Ovx groups. However, in animals subjected to dietary calcium stress during this interval, the decreased skeletal growth noted was confined primarily to the axial skeleton. The data indicate that ovariectomy or ovariectomy plus calcium stress does not result in loss of total bone mass during the interval of dramatically increased resorption and rapid loss of cancellous bone. The results suggest that the deterioration in individual bone structural and mechanical integrity due to ovariectomy or dietary calcium deficiency may not be attributed to overt loss in total bone mass but may involve a redistribution of bone mass.     

Effects of different exercise modes on mineralization, structure, and biomechanical properties of growing bone.

Weight bearing during exercise plays an important role in improving the mechanical properties of bone. The effect on bone of non-weight-bearing exercise such as swimming remains controversial. To investigate the effects of exercise mode on growing bone, 29 male Wistar rats (7 wk old) were randomly assigned to a running exercise group (Run, n = 9), a swimming exercise group (Swim, n = 10), or a nonexercise control group (Con, n = 10). During an 8-wk training session (20-60 min/day, 5 days/wk), the Run rats were trained at progressively increasing running speeds (12-22 m/min), and weights attached to the tail of the Swim rats were progressively increased from 0 to 2% of their body weight. The bone mineral density of the proximal tibiae of the Run rats was significantly higher than in the Swim (P < 0.05). Femoral wet weights of the two exercise groups were significantly higher than in the control group (P < 0.05). Interestingly, the percent difference between the tissue wet weight and dry weight (water content ratio), which is related to bone mechanical properties, was significantly higher in the tibiae of the Swim rats and the femora of both exercise groups compared with controls (P < 0.05). Extrinsic as well as intrinsic biomechanical material properties were measured in a three-point bending test. Bone mechanical properties of the tibiae and femora of rats in the Swim and Run groups were significantly greater than those in the control group (P < 0.05). In summary, different modes of exercise may benefit bone mechanical properties in different ways. The specific effects of swimming exercise (non-weight-bearing exercise) on bone require further study.     

Evaluation of the preventive effect of isoflavone extract on bone loss in ovariectomized rats

To examine a potential role for soybean phytoestrogens in postmenopausal bone loss, twenty-four 12-week-old Sprague-Dawley rats were divided randomly into 4 groups and given controlled diets for 16 weeks. The treatment groups were as followed: sham operated, ovariectomized (OVX) control, OVX + isoflavone extract (6.25 g/kg), and OVX + 17beta-estradiol (4 mg/kg). OVX treatments reduced femoral and fourth lumbar vertebral bone density and mineral content (p<0.01), decreased uterine weight (p<0.01), accelerated body weight increases (p<0.05), and increased the activities (p<0.01) of both serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Supplementation with isoflavone prevented the losses of bone density and mineral content caused by OVX (p<0.01). Although both isoflavone and 17beta-estradiol exhibited similar bone-sparing ability on the OVX-induced bone loss, the effect of isoflavone was not the same as that of 17beta-estradiol on the serum ALP and TRAP, body weight increase, and uterine weight change. We concluded that dietary supplementation with soybean isoflavone can prevent postmenopausal bone loss via a different mechanism of estrogen in OVX rats.     

The thyroid C cells of ovariectomized rats treated with estradiol

The structure and function of thyroid C cells were studied in ovariectomized (Ovx) adult female rats without and after chronic treatment with estradiol dipropionate (EDP). A peroxidase–antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume, nuclei, and relative volume density were evaluated in comparison with sham-operated control rats using a stereological method. The number of C cells was calculated. CT content in the sera was determined by radioimmunoassay. Ovariectomy (Ovx) led to a 21% increase in body weight (P<0.005), while treatment of Ovx rats with EDP decreased body weight by 25% (P<0.01). The immunoreactivity for CT in C cells of the Ovx rats was markedly increased. Significant decreases in the volume of C cells (by 13%; P<0.05) and serum CT (by 45%) were recorded, while the C cell number increased by 59% (P<0.05) in relation to the corresponding controls. The treatment of Ovx rats with EDP caused conspicuous degranulation of the C cells. The cellular volume was increased by 11% and serum CT by 36% in comparison with Ovx animals. At the same time a decrease in C cell number by 29% (P<0.05) was evident. It may be concluded that estradiol deficiency after Ovx reduced the synthesis and release of CT, while chronic treatment of these animals with EDP had a positive effect on the secretory activity of thyroid C cells.     

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Chronic upper airway resistive loading induces growth retardation via the GH/IGF-I axis in prepubescent rats.

The effect of upper airway loading on longitudinal bone growth and various components of the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis has not been fully elucidated. In the present study, the effect of chronic resistive airway loading (CAL) in a prepubescent rat model on linear bone growth and weight gain was investigated. We hypothesize that CAL induced in prepubescent rats will lead to impaired longitudinal growth due to impairment in circulating and liver GH/IGF-I parameters. The tracheae of 22-day-old rats were obstructed by tracheal banding to increase inspiratory esophageal pressure. The GH/IGF-I markers were analyzed using ELISA, RT-PCR, and Western immunoblot analysis 14 days after surgery. Animals exhibited impaired longitudinal growth as demonstrated by reduction of tibia and tail length gains by 40% (P < 0.0001) and body weight gain by 24% (P < 0.0001). No differences were seen in total body energy balance, i.e., oxygen consumption, daily food intake, or arterial blood gases. Circulating GH, IGF-I, and IGF binding protein-3 (IGFBP-3) levels were reduced by 40% (P = 0.037), 30% (P < 0.006), and 27% (P = 0.02), respectively, in the CAL group. Liver IGF-I mRNA level decreased by 20% (P < 0.0002), whereas GH receptor mRNA and protein expression were unchanged. We conclude that impaired longitudinal growth in prepubescent CAL rats is related to a decrease in GH, IGF-I, and IGFBP-3 levels.     

How cancellous and cortical bones adapt to loading and growth hormone

There is great interest in the relationships between growth hormone (GH), muscle loading and bone, in part, because GH increases muscle mass which provides the largest signals that control bone modeling and remodeling. This study was designed to examine the effects of GH and muscle loading by exercise (EX) independently and in combination on bone and skeletal muscle. Thirteen-month-old female F344 rats were divided into 6 groups: Group 1, baseline controls (B); Group 2, agematched controls (C); Group 3, GH treated (2.5 mg rhGH/kg b. wt/day, 5 days per week); Group 4, voluntary wheel running exercise (EX); Group 5, GH+EX, and rats in Group 6 were food restricted (FR) to lower their body weight and examine the effects of decreased muscle load on bone. All animals, except the baseline controls, were sacrificed after 4.5 months. Growth hormone increased the body weight and tibial muscle mass of the rats markedly, while EX caused a slight decrease in body weight and partially inhibited the increase caused by GH in the GH+EX group. Food restriction greatly decreased body weight below that of age-matched controls but neither FR nor EX had a significant effect on the mass of the muscles around the tibia. Growth hormone and EX independently increased tibial diaphyseal cortical bone area (p<0.0001), cortical thickness (p<0.0001), cortical bone mineral content (p<0.0001), periosteal perimeter (p<0.0001) and bone strength-strain index (SSI) (p<0.0001). The effects of GH were more marked, and the combination of GH and EX produced additive effects on many of the tibial diaphyseal parameters including bone SSI. GH+EX, but not GH or EX alone caused a significant increase in endocortical perimeter (p<0.0001). In the FR rats, cortical bone area and cortical mineral content increased above the baseline level (p<0.0001) but were below the levels for age-matched controls (p<0.0001). In addition, marrow area, endocortical perimeter and endocortical bone formation rate increased significantly in the FR rats (p<0.01, p<0.0001, p<0.0001). Three-point bending test of right tibial diaphysis resulted in maximum force (Fmax) values that reflected the group differences in indices of tibial diaphyseal bone mass except that GH+EX did not produce additive effect on Fmax. The latter showed good correlation with left tibial diaphyseal SSI (r=0.857, p<0.0001) and both indices of bone strength correlated well with tibial muscle mass (r=0.771, Fmax; r=0.700, SSI; p<0.0001). We conclude that the bone anabolic effects of GH with or without EX may relate, in part, to increased load on bone from tibial muscles and body weight, which were increased by the hormone. The osteogenic effects of EX with or without GH may relate, in part, to increased frequency of muscle load on bone as EX decreased body weight (p<0.05) but had no significant effect on tibial muscle mass. The enhanced loss of endocortical bone by FR may relate, in part, to decreased load on bone due to low body weight (p<0.0001) as FR did not cause a significant decrease in tibial muscle mass (p=0.357). The roles of humoral and local factors in the bone changes observed remain to be established.     

Lipid peroxidation in liver tissue of ovariectomized and pinealectomized rats: effect of estradiol and progesterone supplementation

The present study aimed to determine the effect of estradiol-progesterone supplementation and pinealectomy on lipid peroxidation of liver tissue in ovariectomized rats. The study was carried out on 36 adult Sprague-Dawley female rats, which weighed 200-250 g. The rats were divided into 6 groups: Group 1: Sham Ovariectomy (Sham-Ovx), Group 2: Ovariectomy (Ovx), Group 3: Ovx + Estradiol-Progesterone supplementation (Ovx + H), Group 4: Sham Pinealectomy and Ovx (Sham Pnx -Ovx), Group 5: Ovx -Pnx, Group 6: Ovx -Pnx + H. Malondialdehyde (MDA), reduced form of glutathione (GSH) and glutathione peroxidase (GSH-Px) levels were determined in liver tissue of rats. The highest MDA levels and the lowest GSH-Px levels were determined in the ovariectomized-pinealectomized group, whereas the lowest MDA was in the Sham-Ovx group, and the highest GSH-Px levels were found in the Sham-Ovx and Ovx + Hormone supplemented group. Furthermore, the highest GSH levels were in group 1 and lowest levels were in group 5. The findings of this study demonstrate that ovariectomy led to lipid peroxidation in liver tissues of rats. Pinealectomy in addition to ovariectomy, increases lipid peroxidation, but, estradiol and progesterone supplementations to the ovariectomized-pinealectomized rats protect against lipid peroxidation to a significant extent.     

Effects of chronic NH4Cl dosage and swimming exercise on bone metabolic turnover in rats

To determine the effects of ammonium chloride (NH4Cl) dosage and swimming exercise training during 4 weeks on bone metabolic turnover in rats, seven-week-old female 24 Wister-Kyoto (WKY) rats were investigated by bone status including bone mineral density (BMD) and biomechanical markers from blood and urine. Twenty-four rats (initial weight: 191.2+/-7.6 g) were randomly divided into four groups: baseline (8 weeks old) control group (n=6, BC), 4-week control group (n=6, Con), 4-week swimming exercise loading group (n=6, Swim) and 4-week chronic NH4Cl dosage group (n=6, Acid). All rats were fed an AIN93M diet (Ca: 0.5%, P: 0.3%), and both Con and Swim groups were pair-fed by feeding volume of the NH4Cl dosage group. The acid group only received 0.25 M NH4Cl distilled water ad libitum. At the end of the experimental period, rats were sacrificed with blood drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the Swim group, 24-hour urinary deoxypiridinoline (Dpd) excretion, reflecting bone resorption, was significantly increased (p<0.05) with a tendency towards decrease of BMD (N.S.), and body weight and abdominal fat weight were decreased in approximately 7% (p<0.05) and 58% (p<0.001), as compared with age matched Con rats. In the Acid group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased approximately 2.1-fold (p<0.05) and 2.0-fold (p<0.01), respectively, with increase of kidney weight as much as in the Con groups. Serum Ca and P concentration, as well as urinary Dpd excretion were, however, not significantly changed. These results suggest that blood Ca and P concentrations in the chronic acidosis condition during the 4-weeks might be maintained by hypercalciuria and hyperphosphaturia with kidney disorder, and swimming exercise training leads to decrease in BMD with stimulation of bone resorption and reduction of body fat.     

Regulation of UCP1, UCP2, and UCP3 mRNA expression in brown adipose tissue, white adipose tissue, and skeletal muscle in rats by estrogen

The effects of ovariectomy (OVX) and estrogen substitution on body weight, body composition, food intake, weight gain, and expression of uncoupling proteins (UCPs) in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle were studied in four groups of rats: (1) Sham-operated rats (N = 8), (2) ovariectomized rats (OVX - E) (N = 8), (3) estrogen-treated OVX rats (OVX + E) (N = 8), and (4) OVX rats on energy restriction (OVX - E + D) (N = 8). OVX was associated with an increase in food intake and body weight gain during a 5-week study period compared to sham-operated rats. The estrogen-substituted rats had a significantly lower food intake and weight gain during the 5 weeks compared to the sham-operated group. However, we also included a nontreated OVX group that was allowed to eat only enough chow to match the weight gain of the sham-operated group. To match the weight gain in the two groups, the OVX group had to consume 16% less chow than the sham-operated group. In BAT, the UCP1 expression was significantly lower in estrogen-deficient rats compared to either intact rats or estrogen-substituted rats, whereas UCP2 and UCP3 mRNA expression was similar in BAT from all four groups. In WAT, both estrogen-deficient groups had significantly lower UCP2 mRNA expression compared to the control rats and estrogen-treated rats; In contrast, the UCP3 mRNA expression in WAT was similar in all four groups. Finally, in skeletal muscle the OVX group on mild energy restriction had reduced UCP3 mRNA expression compared to control, OVX, and estrogen-treated rats. In contrast, the UCP2 mRNA expression in skeletal muscle was similar in all four groups. Thus, the findings that estrogen deficiency is followed by reduced UCP1 expression in BAT and reduced UCP2 expression in WAT in association with weight gain probably caused by a decrease in energy expenditure might indicate that UCPs play a role for the estrogen-mediated changes in body weight and energy expenditure.