Bone tissue changes in rats during prolonged restriction of motor activity

The objective of this investigation was to measure the effect of prolonged restriction of motor activity (hypokinesia) of rats on the mass, density, mineral composition, reconstruction parameters and elemental composition of their bone tissue. The studies were done during 90 days of hypokinesia (HK) on 90 male Wistar rats equally divided into two groups: (1) vivarium control rats (VCR) and (2) hypokinetic rats (HKR). For the simulation of the hypokinetic effect the HKR group was kept for 90 days in small individual cages made of wood that restricted the movements of rats in all directions without hindering food and water intakes. During the prehypokinetic period of 15 days and during the hypokinetic period of 90 days bone mass, bone density, bone calcium and phosphorus concentrations, bone reconstruction parameters and elemental composition of bones were determined. During the same periods food intake and body weight losses were also measured. In the HKR group signs of osteoporosis in the spongy structures of the tubular bones were observed; they also showed significant decrease in rat femur weight, and in cross section of the rat femur, and in mineral concentrations of the femoral head when compared with the VCR group. The HKR group also show a significant decrease in food intake and body weight when compared with the VCR group. The corresponding parameters did not change significantly in the VCR group when compared with the baseline control values. It was concluded that prolonged exposure to HK induced osteoporosis and structural changes in bones. This apparently occurred due to inhibition of bone tissue formation in the HKR group.     

Daily zinc supplementation effect on zinc deficiency in rats during prolonged restriction of motor activity

The objective of this investigation was to evaluate the effect of 47 mg zinc supplementation on deficiency of zinc in rats during 98 d of restriction of motor activity (hypokinesia), which appeared by higher plasma zinc concentration. One Hundred 13-week-old Sprague-Dawley male rats weighing 360–390 g were used to perform the studies: They were equally divided into four groups: 1. Unsupplemented control animals (UCA); 2. Unsupplemented hypokinetic animals (UHA); 3. Supplemented control animals (SCA); and 4. Supplemented hypokinetic animals (SHA). For the simulation of the effect of hypokinesia (HK), the UHA and SHA were kept in small individual cages made of wood, which restricted their movements in all directions without hindering food and water intake. The SCA and SHA received daily with their food an additional amount of zinc. Before and during the experimental period of 98 d, plasma, urinary and fecal zinc, balance of zinc, food intake, and body weight were determined at different intervals. In the SHA and UHA, the concentration of zinc in plasma, and the elimination of zinc in urine and feces increased significantly when compared with the SCA and UCA, whereas the balance of zinc was negative. The body weight and food intake decreased significantly in the SHA and UHA when compared with the SCA and UCA. The increased plasma concentration of zinc in both the SHA and UHA groups was in contrast to the observed hypozincnemia during prolonged immobilization as during prolonged hospitalization. This reaction suggests that there may be some other mechanisms that are affecting the process of control and regulation of zinc metabolism during prolonged HK. It was concluded that exposure to prolonged restriction of motor activity of rats induces significant increases in plasma concentration, fecal and urinary elimination of zinc in the presence of negative zinc balance and regardless the daily intake of large amounts of zinc with their food, leading to zinc deficiency.     

Water metabolic parameter changes in rhesus monkeys during exposure to prolonged restriction of motor activity

The objective of this investigation was to determine the effect of prolonged restriction of motor activity (hypokinesia [HK]) on several parameters of water metabolism in primates. The studies were performed on 12 rhesus monkeys aged 4–5 yr (5.10–6.85 kg) during the hypokinetic period of 90 d and during the prehypokinetic period of 30 d. They were divided into two equal groups: the first group was placed under ordinary vivarium conditions (vivarium control animals) and the second group was subjected to 90 d of HK (hypokinetic animals). For the simulation of the hypokinetic effect, the primates were immobilized on their abdomens in special tables. The legs of the monkeys were immobilized with hip and knee joints extended. The primates retained freedom of movement at elbow, wrist, and ankle. During the preexperimental period of 30 d and during the experimental period of 90 d, the following variables were determined: body weight, total body fluid content, specific total body fluid, mean fluid consumed and eliminated in urine, specific plasma resistance, hematocrit level, and plasma concentrations of sodium (Na) and potassium (K). In the hypokinetic primates, body weight decreased significantly when compared to the controls. Mean fluid intake, total body fluid, and specific total body fluid decreased, whereas mean daily fluid loss and specific mean daily fluid elimination increased significantly. Specific plasma resistance, hematocrit level, and plasma electrolyte concentrations increased significantly when compared to the control primates. It was concluded that prolonged restriction of motor activity induces significant changes in water metabolic parameters of primates leading in decreased total water content of the body.     

Daily magnesium supplementation on serum and urinary magnesium changes in rats during prolonged restriction of motor activity

The objective of this investigation was to determine whether a plentiful magnesium (Mg²⁺) supplementation might be used to normalize or prevent Mg deficiency. This is manifested by increased rather than decreased serum Mg²⁺ concentration as is observed during prolonged hospitalization, which is developed during prolonged hypokinesia (HK) (decreased motor activity). Eighty male Wistar rats with an initial body weight of 370–390 g were used to perform the studies: They were equally divided into four groups:

1. Unsupplemented control animals (UCA);
2. Supplemented control animals (SCA);
3. Unsupplemented hypokinetic animals (UHA); and
4. Supplemented hypokinetic animals (SHA).

For the simulation of the hypokinetic effect, the hypokinetic animals were kept in small individual cages made of wood, which restricted their movements in all directions without hindering food and water intake. The control and hypokinetic supplemental animals receive 0.9 mg/mL Mg sulfate daily with their drinking water. Prior to and during the experimental period, urinary excretions of Mg, calcium, and phosphate along with their concentrations in serum, water intake, and urine excretion, and body weight were determined in the control and hypokinetic animals. In the supplemental and unsupplemental hypokinetic rats, urinary excretions and serum concentrations of electrolytes increased significantly, whereas serum concentration and urinary excretion thereof remained unchanged in the supplemented and unsupplemented control animals. It was concluded that a daily intake of large amounts of Mg supplementation cannot be used to prevent or normalize Mg deficiency in rats during prolonged exposure to HK.     

Adenylate cyclase and guanylate cyclase activity in the isolated kidney glomerulus of the rat]

Isolated rat renal glomeruli contain an adenylate cyclase system and guanylate cyclase system. Adenylate cyclase was strikingly activated by purified parathyroid hormone, epinephrine, prostaglandin I2 and histamine. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role of this hormone in regulation of glomerular filtration rate. Guanylate cyclase was strikingly activated by CA2+, nitrate derivatives such as sodium nitroprusside. Its role remained still unknown.     

Contralateral neuro-motor apparatus under unilateral restriction of motor activity in rat

Electromyographic recordings of contralateral m. gastrocnemius of the rat after unilateral section of Achilles' tendon (tenotomy) were studied. Motor reflex, Hoffman reflex and firing of motor units of contralateral m. gastrocnemius were recorded in control and in 10 days after tendotomy. Motor units changed their firing in the background and in reaction to sciatic nerve stimulation. The results of study showed that unilateral section of Achilles' tendon increased the excitability of single motoneurons of contralateral spinal senters.     

Mitochondrial activity of rat kidney during ontogeny

The development of oxidative metabolism was studied from the late fetal to adult stages in mitochondria isolated from rat kidney. We used the oxygen consumption rate, as an index of inner membrane activity and citrate synthase and fumarase activities as an index of matrix activity and cytochrome c oxidase activity as an index of the number of mitochondria. Fumarase and citrate synthase activities displayed different developmental patterns, suggesting that these Krebs cycle enzymes did not mature synchronously. In fetal mitochondria, net oxygen consumption measured in the presence of succinate or glutamate as substrate, was low; it increased during the day after birth and reached adult level between days 10 and 15. During this period, the levels of citrate synthase and cytochrome c oxidase activity did not change significantly in the isolated mitochondrial fraction. However, in fetal and adult kidney homogenates, these levels increased four-fold, suggesting a corresponding increase in the number of mitochondria. Most of these increases occurred during the 15 days after birth. These results suggest that in rat kidney, mitochondrial maturation precedes the maturation of reabsorptive ion transport and does not limit its development.     

Scanning electron microscopy of the kidney glomerulus during development

Consecutive stages of renal glomerulogenesis were studied in white rats using scanning electron microscopy of microvessel corrosion casts and specimens dried through critical point. The splitting of ingrowing capillaries was shown to be the leading mechanism of glomerulogenesis. The differentiation of endothelial and epithelial components of glomerular filter involves two main processes: progressive spreading and arborization of cells. A suggestion is put forward that the growth and spatial reorganization of cytoskeletal elements in the endothelial and epithelial cells are the basic mechanisms of their differentiation during glomerulogenesis.     

Zinc metabolism in endurance-trained volunteers during prolonged restriction of muscular activity and chronic hyperhydration

The objective of this study was to evaluate the effects of hypokinesia (HK) and fluid- and salt supplementation (FSS) on zinc metabolism in endurance-trained volunteers (ETV) for a period of 364 d. Thirty long-distance runners aged 22–25 yr with a peak V_O2 of 67 mL/min/kg with an average 13.8 km/d running distance were chosen as subjects. They were equally divided into three groups:

Potassium loading effect on potassium balance in athletes during prolonged restriction of muscular activity

Negative potassium balance during hypokinesia (decreased number of kilometers taken/day) is not based on the potassium shortage in the diet, but on the impossibility of the body to retain potassium. To assess this hypothesis, we study the effect of potassium loading on athletes during prolonged hypokinesia (HK). Studies were done during 30 d of a pre-HK period and during 364 d of an HK period. Forty male athletes aged 23–26 yr were chosen as subjects. They were divided equally into four groups: unloaded ambulatory control subjects (UACS), unloaded hypokinetic subjects (UHKS), loaded hypokinetic subjects (LHKS), and loaded ambulatory control subjects (LACS). For the simulation of the hypokinetic effect, the LHKS and UHKS groups were kept under an average running distance of 1.7 km/d. In the LACS and LHKS groups, potassium loading tests were done by administering 95.35 mg KC1 per kg body weight. During the pre-HK and HK periods and after KC1 loading tests, fecal and urinary potassium excretion, sodium and chloride excretion, plasma potassium, sodium and chloride concentration, and potassium balance were measured. Plasma renin activity (PRA) and plasma aldosterone concentration was also measured. Negative potassium balance increased significantly (p < -0.01) in the UHKS and LHKS groups when compared with the UACS and LACS groups. Plasma electrolyte concentration, urinary electrolyte excretion, fecal potassium excretion, PRA, and PA concentration increased significantly (p ≤ 0.01) in the LHKS and UHKS groups when compared with LACS and UACS groups. Urinary and fecal potassium excretion increased much more and much faster in the LHKS group than in the UHKS group. By contrast, the corresponding parameters change insignificantly in the UACS and LACS groups when compared with the base line control values. It was concluded that urinary and fecal potassium excretion increased significantly despite the presence of negative potassium balance; thus, negative potassium balance may not be based on potassium shortage in the diet because of the impossibility of the body to retain potassium during HK.     

The GOAT-ghrelin system is not essential for hypoglycemia prevention during prolonged calorie restriction

Objective

Ghrelin acylation by ghrelin O-acyltransferase (GOAT) has recently been reported to be essential for the prevention of hypoglycemia during prolonged negative energy balance. Using a unique set of four different genetic loss-of-function models for the GOAT/ghrelin/growth hormone secretagogue receptor (GHSR) system, we thoroughly tested the hypothesis that lack-of-ghrelin activation or signaling would lead to hypoglycemia during caloric deprivation.

Methodology

Male and female knockout (KO) mice for GOAT, ghrelin, GHSR, or both ghrelin and GHSR (dKO) were subjected to prolonged calorie restriction (40% of ad libitum chow intake). Body weight, fat mass, and glucose levels were recorded daily and compared to wildtype (WT) controls. Forty-eight hour blood glucose profiles were generated for each individual mouse when 2% or less body fat mass was reached. Blood samples were obtained for analysis of circulating levels of acyl- and desacyl-ghrelin, IGF-1, and insulin.

Principal Findings

Chronic calorie restriction progressively decreased body weight and body fat mass in all mice regardless of genotype. When fat mass was depleted to 2% or less of body weight for 2 consecutive days, random hypoglycemic events occurred in some mice across all genotypes. There was no increase in the incidence of hypoglycemia in any of the four loss-of-function models for ghrelin signaling including GOAT KO mice. Furthermore, no differences in insulin or IGF-1 levels were observed between genotypes.

Conclusion

The endogenous GOAT-ghrelin-GHSR system is not essential for the maintenance of euglycemia during prolonged calorie restriction.     

Effect of prolonged restriction of overall motor activity on the hemomicrocirculatory bed of the retina of the rabbit

In 56 rabbits kept in tight cages for 4, 8, 12, 16 and 20 weeks, effect of hypokinesia on structure of hemomicrocirculatory bed of the retina has been studied. The retina is digested in tripsin and then it is exfoliated, and the vessels are stained after Shiff. Diameters of arterioles, capillaries and venules are measured, their relations to each other and number of vessels per square unit are taken into consideration. Restriction of the motor activity for 4 weeks results in narrowing of arterioles, capillaries and venules, in increasing venuloarteriolar coefficient, in decreasing number of vessels per square unit, as compared to the norm. In 8 weeks of hypokinesia the diameter of all links of the hemomicrocirculatory bed is evenly increasing, but it does not reach the normal value; the number of the vessels is keeping to decrease; the venuloarteriolar coefficient is at the same level. In 12, 16 and 20 weeks changes in the diameter and the number of the vessels are poorly pronounced, have a wavy character and diversily directed. The value of the venuloarteriolar coefficient is kept constant. As a whole, the data obtained demonstrate that at a long hypokinesia the animals adapt to the new conditions of existence, and the state of the reticular hemomicrocirculatory bed stabilizes.     

Magnesium loading effect on magnesium deficiency in endurance-trained subjects during prolonged restriction of muscular activity

The aim of this study was to evaluate the effect of magnesium (Mg) loading (10.0 mg Mg/kg body wt) and daily Mg supplements (5.0 mg Mg/kg body wt) on Mg deficiency shown by increased and not by decreased serum Mg concentration during hypokinesia (decreased km number/d). The studies were done during 30 d of prehypokinesia and 364 d of hypokinesia (HK) periods. Forty endurance-trained volunteers aged 22–26 yr with a peak VO2 max of 66.3 mL·kg⁻¹ min⁻¹ and with an average 15.0 km/d running distance were chose as subjects. They were equally divided into four groups:

Podocytes in glomerulus of rat kidney express a characteristic 44 KD protein

We describe a new monoclonal antibody (MAb) directed against glomerular visceral epithelial cells (podocytes), generated by immunization with isolated rat kidney glomeruli. In immunoblotting experiments this MAb (IgG1 subclass) reacted with a 44 KD protein. In cryostat sections of normal rat kidney the MAb stained glomerular podocytes; therefore, we called the antigen pp44 (podocyte protein 44 KD). On 0.5-micron cryostat sections the signal could be more precisely ascribed to the podocyte foot processes, whereas the cell bodies appeared virtually unreactive. On ultra-thin frozen sections pp44 was found within the cytoplasm of podocyte foot processes at their origin from their parent processes. The podocyte cell membrane was not labeled. All other parts of the nephron were unreactive. An additional but weaker immunoreaction was found in the arterial endothelium; the endothelia of other vessels (peritubular capillaries, veins) were negative. In human kidney anti-pp44 revealed the same staining pattern as in rat kidney. The expression of pp44 was also studied in newborn rat kidney. The early stages of glomerular development (renal vesicle, S-shaped body) were negative. pp44 first appeared during the capillary loop stage, i.e., when formation of podocyte foot processes commences. In comparing the present results with published data, pp44 is clearly different from other antigens thus far described in podocytes. From the results of this investigation we conclude that pp44 represents a novel cytoplasmic protein of podocytes. Our data suggest a cytoskeletal role for pp44 in preserving the complex architecture of podocytes. This idea is confirmed by the simultaneous appearance of foot processes and anti-pp44 immunoreactivity during glomerular development.     

Effect of daily hyperhydration on fluid-electrolyte changes in endurance-trained volunteers during prolonged restriction of muscular activity

The aim of this investigation was to evaluate the effect of a daily intake of fluid and salt supplementation on fluid and electrolyte losses in endurance-trained volunteers during prolonged restriction of muscular activity (hypokinesia). The studies were performed on 30 long-distance runners aged 23–26 who had a peak oxygen uptake of 65.5 mL/kg/min and had taken 13.8 km/d on average prior to their participation in the study. The volunteers were divided into three groups: The volunteers in the first group were placed under normal ambulatory conditions (control subjects), the second group of volunteers subjected to hypokinesia alone (hypokinetic subjects), and the third group of volunteers was submitted to HK and consumed daily 0.1 g sodium chloride (NaCl)/kg body wt and 26 mL water/kg body wt (hyperhydrated subjects). The second and third group of volunteers were kept under an average of 2.7 km/d for 364 d. During the pre-experimental period of 60 d and during the experimental period of 364 d sodium, potassium, calcium, and magnesium in urine and plasma were determined. Blood was also assayed for osmolality, hemoglobin, hematocrit, plasma volume, plasma renin activity and plasma aldosterone. Mean arterial blood pressure was also determined. In the hyperhydrated volunteers plasma volume and arterial blood pressure increased, whereas plasma osmolality, plasma renin activity, plasma aldosterone, hematocrit, hemoglobin concentration, and urinary excretion and concentrations of electrolytes in plasma decreased. In the hypokinetic volunteers, plasma volume and arterial blood pressure decreased significantly, whereas hematocrit values, hemoglobin concenfration, plasma osmolality, plasma renin activity, plasma aldosterone, and electrolytes in urine and plasma increased significantly during the experimental period. It was concluded that chronic hyperhydration may be used in minimizing fluid and electrolyte losses in endurance-trained volunteers during prolonged restriction of muscular activity.     

Daily copper supplement effects on copper balance in trained subjects during prolonged restriction of muscular activity

The aim of this study was to assess the effect of a daily intake of copper supplements on negative copper balance during prolonged exposure to hypokinesia (decreased number of kilometers per day). During hypokinesia (HK), negative copper balance is shown by increased, not by decreased, serum copper concentration, as it happens in other situations. Studies were done during a 30-d prehypokinetic period and a 364-d hypokinetic period. Forty male trained volunteers aged 22–26 yr with a peak oxygen uptake of 66.4 mL/min/kg and with an average of 13.7 km/d running distance were chosen as subjects. They were equally divided into four groups: unsupplemented ambulatory control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented hypokinetic subjects (SHKS), and supplemented ambulatory control subjects (SACS). The SACS and SHKS groups took 0.09 mg copper carbonate/kg body weight daily. The SHKS and UHKS groups were maintained under an average running distance of 1.7 km/d, whereas the SACS and UACS groups did not experience any modifications in their normal training routines. During the 30-d prehypokinetic period and the 346-d hypokinetic period, urinary excretion of copper, calcium, and magnesium and serum concentrations of copper, calcium, and magnesium were measured. Copper loss in feces and copper balance was also determined. In both UHKS and SHKS groups, urinary excretion of copper, calcium, and magnesium and concentrations of copper, magnesium, and calcium in serum increased significantly when compared with the SACS and UACS groups. Loss of copper in feces was also increased significantly in the SHKS and UHKS groups when compared with the UACS and SACS groups. Throughout the study, the copper balance was negative in the SHKS and UHKS groups, whereas in the SACS and UACS groups, the copper balance was positive. It was concluded that a daily intake of copper supplements cannot be used to prevent copper deficiency shown by increased copper concentration. Copper supplements also failed to prevent negative copper balance and copper losses in feces and urine in endurancetrained subjects during prolonged exposure to HK.