Bone structure and strength are enhanced in rats programmed by early overfeeding.

Childhood obesity is growing in prevalence. Obesity and bone dysfunctions may be related disorders, and therefore our aim was to study the impact of the early overfeeding (EO) in offspring bone health since weaning up to adulthood. To induce EO during lactation, litter size was adjusted to 3 male rats per litter (SL). Litter containing 10 pups per mother was the control (NL). Bone tissue was evaluated by dual-energy X-ray absorptiometry, computed tomography, microcomputed tomography, biomechanical tests, and serum analyses. SL offspring presented higher body weight, fat mass, lean mass from 21 up to 180 days, hyperphagia, and higher visceral fat mass. Bone analysis showed that SL offspring presented higher total bone mineral density (BMD) only at 180 days, and higher total bone mineral content and higher bone area from 21 until 180 days. At 180 days, SL offspring presented higher femur BMD and fourth lumbar vertebra (LV4) BMD, higher femoral head radiodensity and LV4 vertebral body radiodensity, lower trabecular pattern factor and trabecular separation, however with higher trabecular number, higher maximal load, resilience, stiffness and break load, and lower break deformation. SL group had, at 180 days, higher osteocalcin and lower C-terminal cross-linked telopeptide of type I collagen (CTX I). We have shown that the excess of fat mass contributed to an increased bone mass, and hypothesized that this increase could be mediated by the hypothyroidism and previous higher thyroid hormone action and hyperleptinemia at weaning. Furthermore, the increased biomechanical loading due to increased body weight probably help us to understand the protective effects obesity exerts upon bone health.

Effects of different doses of soy isoflavones on bone tissue of ovariectomized rats.

AIM: Studies report that hormone replacement prevents osteoporosis, but there are doubts whether isoflavones are really efficient in this process. The aim of this study was to evaluate the effects of different doses of soy isoflavones on bone tissue of ovariectomized rats. METHODS: Forty female rats at the age of 6 months were ovariectomized and, after 3 months, the animals were divided into four groups: GI - Control (treated with drug vehicle); GII - treated with isoflavones (80 mg/kg per day); GIII - treated with isoflavones (200 mg/kg per day) and GIV - treated with isoflavones (350 mg/kg per day). Soy isoflavones were administered by gavage for 90 consecutive days. After treatment, the rats were euthanized and their distal femurs were removed for histological routine, histochemistry and biochemical study. Histological sections were stained with hematoxylin-eosin or subjected to picrosirius red and alcian blue methods. Shafts of femurs were submitted to biochemical assay and tibias were subjected to biophysical and biomechanical tests. RESULTS: In distal femurs, the trabecular bone volume was higher in the groups treated with isoflavones, being higher in GIV, while the cortical bone width and the presence of mature type I collagen fibers were higher in GII. At the trabecular bone region, the percentage of total glycosaminoglycans (GAGs) was higher in GII and the percentage of only sulfated GAGs was higher in GIII, while the higher content of chondroitin sulfate in shafts of femurs was seen in GIV. Biophysical and biomechanical tests in tibias did not differ among the groups. CONCLUSION: Our data indicate that soy isoflavones improve bone quality in femurs of rats by increasing histomorphometric parameters, the content of GAGs and mature type I collagen fibers. These positive effects are dose-dependent and it was different in cortical and trabecular bone.

Effects of soy isoflavones and mechanical vibration on rat bone tissue.

OBJECTIVE: To investigate the effects of soy isoflavones (Iso) and mechanical vibration treatments alone or combined on bone extracellular matrix constituents of ovariectomized rats. METHODS: Forty female Wistar rats at the age of 6 months were ovariectomized (Ovx) and ten were sham-operated (sham). After 3 months, the animals were divided into five groups: GI (sham); GII (Ovx); GIII, ovariectomized and orally treated with isoflavones (200 mg/kg) for 90 consecutive days; GIV, ovariectomized and submitted to vibration for 90 days (5 days/week); GV, ovariectomized and treated with isoflavones plus vibration. After treatments, the rats were euthanized, and their femurs were removed for histological routine and biochemical study. Histological sections were stained with hematoxylin-eosin, picrosirius red and alcian blue. Shaft of femurs were submitted to biochemical assay and tibias were subjected to biophysical and biomechanical tests. RESULTS: Treatments did not have significant effects on the trabecular bone volume, but the combined treatments showed trophic effects on the cortical bone width and area. Bone density and the content of organic material of the tibias were higher in the GIV and GV groups. The GV group showed the highest presence of mature collagen fibers and content of total glycosaminoglycans, while the highest contents of chondroitin sulfate and other sulfated glycosaminoglycans were seen in the GIV group. CONCLUSION: The mechanical vibration treatment is more efficient than soy isoflavones in improving bone quality by increasing the bone density, the content of sulfated glycosaminoglycans and the presence of mature collagen fibers. In addition, the combined interventions have partial trophic and synergistic effects that are bone site-specific in ovariectomized rats.

Effects of resistance training on matrix metalloproteinase-2 activity and biomechanical and physical properties of bone in ovariectomized and intact rats.

The purpose of this study was to investigate the influence of resistance training on the activity of matrix metalloproteinase (MMP)-2 and bone biomechanical properties in ovariectomized and intact rats. Forty-eight female rats were divided into two distinct groups, ovariectomized (OVX) and intact (Int), which were subdivided into three similar subgroups: sedentary, acute exercise and chronic exercise. Rats performed a resistance training for 12 weeks in which animals climbed a vertical ladder of 1.1 m with weights attached to their tails. Sessions were performed with an interval of 3, 4-9 and 8-12 days scaled dynamic movements of climbing. Biomechanical and physical analyses were performed using a universal testing machine, and MMP-2 activity analysis by zymography. Bone density (BD), mineral density (MD), maximum load and fracture load was reduced in sedentary and acute exercise OVX groups compared with the sedentary intact group (P<0.05); in contrast, chronically trained groups (OVX and Int) showed a significant increase in BD, MD and fracture load compared with all the other groups. MMP-2 activity in chronically trained groups also showed a significant increase, while the sedentary OVX group showed a decrease in MMP-2 activity compared with the intact sedentary group (P<0.05). Our results suggest that the resistance training proposed in our work was efficient in reverting the deleterious effects of ovariectomy on bone tissue, and also produced modeling effects in intact rats. On the other hand, ovariectomy reduced the activity of MMP-2 and produced deleterious effects on bone tissue, mimicking menopause intrinsically.

The new ICP minimally invasive method shows that the Monro-Kellie doctrine is not valid.

The Monro-Kellie doctrine states that the interior of the cranium is formed of three main components: blood, fluid and cerebral parenchyma. An increase in the volume of one or more components may increase the intracranial pressure (ICP). This doctrine also affirms that the skull cannot be expanded after the closure of the fontanels. Monro and Kellie's theory has been perfected during the last two centuries. This study leads to a new contribution that proves that even adults' consolidated skulls present volumetric changes as a consequence of ICP variations.

Nocturnal plasma levels of melatonin in quails (Coturnix japonica) injected with l-5-hydroxy-tryptophan.

This study aimed to demonstrate the influence of the systemic administration of l-5-hydroxy-tryptophan (L-HTP) on the plasma levels of melatonin during the dark period in quails. Throughout daylight, the plasma levels of melatonin did not differ significantly, oscillating between 110.2 +/- 15.8 pg.mL(-1) and 157.4 +/- 34.8 pg.mL(-1), from 8 to 16 hours. L-HTP (25 mg.kg(-1), through the intracelomic route) administered at 18 hours lessened significantly the nocturnal increase of the plasma levels of melatonin (controls, 327.3 +/- 20.1 and 315.8 +/- 20.9 pg.mL(-1) vs. 242.1 +/- 24.8 and 217.5 +/- 21 pg.mL(-1), respectively, at 20 and 24 hours, P < 0.05). The results obtained showed that the administration of LHTP reduced the nocturnal melatonin release, possibly by bringing about an increase in serotonin synthesis and synaptic release in the pineal. Therefore, the serotoninergic transmission from the raphe towards the pineal would constitute a mechanism of modulation of the synthesis and melatonin release in quails.

Body adiposity and bone parameters of male rats from mothers fed diet containing flaxseed flour during lactation.

Obesity and osteoporosis may have their origins in early postnatal life. This study was designed to evaluate whether flaxseed flour use during lactation period bears effect on body adiposity and skeletal structure of male rat pups at weaning. At birth, male Wistar rats were randomly assigned to control and experimental (FF) groups, whose dams were treated with control or flaxseed flour diet, respectively, during lactation. At 21 days of age, pups were weaned to assess body mass, length and composition by dual-energy X-ray absorptiometry. The animals were then sacrificed to carry out analysis of serum profile, intra-abdominal adipocyte morphology and femur characteristics. Differences were considered significant when P<0.05. The FF group displayed the following characteristics (P<0.05): higher body mass, length, bone mineral content, bone area and concentrations of osteoprotegerin, osteocalcin and high-density lipoprotein cholesterol; higher levels of stearic, α-linolenic, eicosapentaenoic and docosapentaenoic acids and lower levels of arachidonic acid and cholesterol; smaller adipocyte area; and higher mass, epiphysis distance, diaphysis width, maximal load, break load, resilience and stiffness of femur. Flaxseed flour intake during lactation period promoted adipocyte hypertrophy down-regulation and contributed to pup bone quality at weaning.

Effects of prepubertal manipulation with androgens on the development of sexual differences in the harderian glands of Syrian hamsters.

The onset of sexual differences in the metabolism of porphyrins and melatonin in the Harderian glands of Syrian hamsters was studied. Three weeks after birth, the porphyrin concentrations were already higher in glands of females than in those of males. Castration of 22-day-old male hamsters led to an increase in Harderian porphyrin concentrations, although the levels of intact females were not reached. The administration of testosterone to 22-day-old female hamsters resulted in a marked decrease in porphyrin concentrations. Study of the development of sexual differences in the enzymes involved in melatonin synthesis, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) indicated that not all the sexual differences observed in these glands begin at the same time. Thus, while differences in NAT activity were detected after the age of 3 weeks, male-female differences in HIOMT activity were only observed after 7 weeks. Castration of prepubertal male hamsters lowered NAT but not HIOMT activities. The administration of testosterone to prepubertal female hamsters led to male activity levels in both enzymes. Although circulating androgens seem to have a crucial role in maintaining sexual differences, other hormones including those from the pituitary and thyroid glands are probably also important for generating these sexual differences.

Failure to stimulate melatonin synthesis in the rat pineal gland with an adenosine analog.

Recent studies indicate the presence of adenosine binding sites in rat pinealocytes and an effect of their activation on pineal serotonin metabolism. However, controversial data exist, and reports suggest that the role of adenosine in pineal physiology is complex. On this basis, we evaluated the effects of an adenosine analog (N-ethyl-carboxamido-adenosine: NECA) on in vitro and in vivo melatonin production in the rat pineal gland. In the in vitro protocol, pineals were incubated with NECA (0.5 mM, 1 mM, or 2.5 mM) or isoproterenol (ISO: 10(-6)M) for 4 hr. In the in vivo experiments, animals were given NECA (1 mg/kg IP), ISO (0.5 mg/kg IP) or 1 ml saline diluent and sacrificed 2 hr later. The samples were assayed for pineal N-acetyltransferase (NAT) activity and melatonin concentrations. ISO caused the expected marked rise in NAT activity and melatonin levels in both protocols. NECA was ineffective in causing any modification of the parameters measured. We conclude that the adenosine analog NECA may not be involved in the activation of melatonin production. These data contrast with others in which NECA administration resulted in an increase in melatonin levels. The participation, if any, of the purinergic system in the physiology of the pineal gland is still far from being characterized.

Food restriction retards aging of the pineal gland.

The effects of chronic (40%) food restriction from 6 weeks of age were studied in aging male Fisher 344 rats. When compared with 3-month-old, ad libitum fed rats, pineal N-acetyltransferase (NAT) activity had declined to less than 30% and pineal and serum levels of melatonin to 40% after 28 months when feeding had been ad libitum. Food restriction significantly retarded this development (P less than 0.05) giving NAT and melatonin levels which were twice as high as in the ad libitum fed group. Nighttime levels of pineal serotonin (5-HT) were similar in food-restricted and ad libitum fed old rats but were nearly twice as high (P less than 0.05) as in young rats. There was also a tendency for increased production of 5-hydroxyindoleacetic acid (5-HIAA) in the pineal gland with higher levels of 5-HT. It is concluded that aging in the rat (Fisher 344) is accompanied by a reduction of pineal NAT activity, thereby reducing the production of melatonin and causing a buildup of 5-HT in the pineal gland. It is furthermore proposed that food restriction, which markedly increases the life span and reduces age-related physiological deterioration and diseases in many animals, may mediate some of its effects through a sustained pineal activity in old age.

Melatonin counteracts the 5-aminolevulinic acid-induced rise of rat forebrain tryptophan and serotonin concentrations at night.

When rats were exposed to extended light into the normal dark phase, the administration of 5-aminolevulinic acid (5-ALA) markedly stimulated the accumulation of tryptophan (TRP) and the synthesis of serotonin (5-HT) in the forebrain. When a single injection of melatonin (25 micrograms) was given near the end of the 5-ALA treatment, the rise in forebrain TRP levels was either totally prevented or reversed; likewise, the increased levels of 5-HT were also suppressed. Melatonin by itself also slightly suppressed forebrain TRP levels in non-5-ALA-treated rats. Whether these are pharmacological or physiological effects of melatonin remains to be determined.

In vivo administration of isoproterenol or forskolin during the light phase induces increases in the melatonin content of the Syrian hamster pineal gland without a rise in N-acetyltransferase activity.

In vivo melatonin production was stimulated during the daytime in pineal glands of female Syrian hamsters following the administration of several injections of either isoproterenol, a beta-receptor agonist, or forskolin, an adenylate cyclase stimulator. The large increase in melatonin following either isoproterenol or forskolin administration was not accompanied by significant changes in N-acetyltransferase (NAT) activity. The results suggest that the Syrian hamster pineal gland, as in other species, responds by producing melatonin during the light phase if the stimulus is adjusted to its particular and specific regulatory mechanisms, i.e., if beta-adrenergic stimulation is continued for 4-8 h. The lack of a commensurate increase in NAT activity raises the question of the need of maximal enzymatic activity for a significant rise in melatonin production in the Syrian hamster pineal gland.

Evidence that extremely low frequency Ca(2+)-cyclotron resonance depresses pineal melatonin synthesis in vitro.

The effects of ion cyclotron resonance (ICR)-type magnetic fields on pineal glands were investigated. Both the synthesis and the release of pineal melatonin, the gland's major hormone, were significantly (P less than 0.001 in each case) reduced by Ca(2+)-ICR-exposure, presumably caused by a reduced activity (P less than 0.05) of the enzyme N-acetyltransferase. It is concluded that this type of exposure may help to explain some of the effects of electromagnetic fields on biological systems. An extension to the existing ICR theory is presented which explains that ICR-like conditions may occur under various environmental circumstances.

Involvement of 5-HT3 receptors in the prolactin release induced by immobilization stress in rats.

1. Central serotoninergic participation in prolactin (PRL) secretion regulation is a well-established event. Furthermore, 5-HT may participate in the mediation of stress-induced PRL release. 2. The authors investigated the effect of the blockade of 5-HT3 receptors on the PRL release induced by immobilization stress in male rats. 3. Pretreatment with the 5-HT3 receptor antagonist, ondansetron (0.10-1.0 mg/kg i.p.), inhibited about 50% of the PRL response to immobilization stress. A lower dose of ondansetron (0.05 mg/kg i.p.) had no effect on the PRL response. No dose-response inhibition was observed. 4. It is concluded that serotoninergic activation related to the mediation of the stress-induced PRL response involves the 5-HT3 receptors.

Carbaryl-induced changes in indoleamine synthesis in the pineal gland and its effects on nighttime serum melatonin concentrations.

The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion.

Audiogenic and audiogenic-like seizures: locus of induction and seizure severity determine postictal prolactin patterns.

Audiogenic seizures (AS) are a model of generalized tonic-clonic seizures, evoked by high-intensity (110 dB) acoustic stimulation evaluated by means of behavioral severity indexes (SI). Postictal prolactin (PRL) is a marker of generalized seizures, both in animals and humans. Thus, in the present work we assayed postictal PRL in a) male Wistar AS susceptible (S, n = 5) and AS resistant (R, n = 13) rats made susceptible by specific midbrain lesions. b) In rats electrically stimulated in the central nucleus (CN) of the inferior colliculus (IC) (n = 20), or the cortical IC (CxIC, n = 18). In c) S rats pretreated with either bromocriptine (BRO; 4 mg/kg; SC), a PRL release inhibitor, or vehicle (V), 30 min before the electrical stimulation. Basal PRL was 2-10 ng/ml at time 0. In the S group, only animals with generalized seizures presented a postictal PRL elevation between 5 and 15 min (60-90 ng/ml; p < 0.05). R rats displayed a discrete PRL response lower than that of S animals. CxIC stimulation produced more severe seizures and greater postictal PRL enhancement than CNIC stimulation, always raising at 5-15 min (p < 0.01). BRO blocked the PRL increase even in the presence of higher seizure scores (p < 0.02). The positive correlation between seizure intensity (SI values), site of initiation (central or cortical IC nuclei), and postictal PRL patterns makes this a reliable model for studying the neurochemistry of the postictal phase and the interaction between hormones and epilepsy.

Effect of a selective inhibitor of epinephrine synthesis, SK&F 64 139, on prolactin secretion in the rat.

The participation of central epinephrine in prolactin secretion was investigated in rats by testing the effects of SK&F 64 139, a selective inhibitor of phenylethanolamine-N-methyltransferase. The drug is reported to dramatically reduce brain epinephrine concentration without affecting norepinephrine or dopamine levels. When SK&F 64 139 was administered (40 mg/kg, ip) to male rats or to female rats during diestrus or proestrus, plasma prolactin levels were not different from those of control groups. Lactating and ovariectomized rats, however, responded to the same SK&F 64 139 dose with an increase in plasma prolactin levels: 210.02 +/- 22.72 vs 378.66 +/- 42.57 (mean +/- SEM) for control of lactating rats, and 4.35 +/- 0.52 vs 6.21 +/- 0.60 ng/ml for control ovariectomized rat. These results suggest that epinephrine may play a functional inhibitory role in prolactin secretion in the lactating and ovariectomized rat.

Effects of hemidecortication on the regulation of rat thyroid function.

1. Thyroid function in hemidecorticate and control rats was studied by the measurement of oxygen consumption (under basal conditions and after cold exposure) and radioiodine (131 I) uptake by the thyroid after a low-iodine diet for 30 days. 2. The oxygen uptake of male and female hemidecorticate rats under basal conditions (29 degrees C) did not differ significantly from that of the controls. The values observed for both experimental groups were relatively constant, ranging from 40 to 75 kcal h-1 m-2 for 2 to 50-day-old animals. 3. When hemidecorticate and control rats, age 60-65 days, were exposed to 7-8 degrees C for 5-6 h, the oxygen consumption of the hemidecorticate rats increased (83-84 kcal h-1 m-2) but to a lower extent than that of the controls (105-106 kcal h-1 m-2). 4. The rate of radioiodine (131 I) uptake by the thyroid gland of hemidecorticate rats was also lower than that of controls when the animals were maintained on a low-iodine diet. 5. These results show that, under basal conditions, the hypothalamic-pituitary-thyroid axis of hemidecorticate rats does not differ from that of control rats. However, when exposed to cold the hemidecorticate animals were not able to increase their oxygen consumption to the extent achieved by the control groups.

Effect of copper on the secretion of human growth hormone.

The response of human growth hormone to the oral administration of 5.0 mg copper was studied in plasma from 12 normal adults. Blood samples were collected at 30 min intervals over a period of 240 min after two basal measurements taken at time = -30 and time = 0 min. Six subjects responded to the stimulus with increased growth hormone secretion, revealing a positive correlation with plasma copper levels. The other six subjects presented a similar rise in plasma copper levels but no increase in plasma growth hormone levels. These results suggest that acute, high blood copper levels can increase basal growth hormone secretion in normal individuals, presumably by acting on the hypothalamic center.

Effects of central epinephrine synthesis inhibition on stress-induced prolactin secretion in male rats.

1. The present study was designed to examine the role of central epinephrine pathways in the control of stress-induced prolactin secretion in male adult Wistar rats. 2. Intracerebroventricular administration of two epinephrine synthesis inhibitors, SKF 64139 (5 and 10 micrograms/rat) and LY 134046 (10 and 20 micrograms/rat), 6 h before the onset of immobilization stress blocked prolactin secretion in a dose-dependent manner. Prolactin values before stress were about 4.0 ng/ml and increased to almost 50 ng/ml in the control group. SKF 64139 injection in the higher dose (10 micrograms/rat) induced a complete blockade of the stress-induced prolactin release, whereas partial blockade was observed after the higher dose (20 micrograms/rat) of LY 134046. 3. Salbutamol pretreatment (10 micrograms/rat) completely restored stress-induced prolactin secretion in animals receiving a central injection of both epinephrine synthesis inhibitors under the same conditions as described above. 4. It is suggested that epinephrine pathways in the brain play an important role in the control of prolactin release occurring during immobilization stress.