Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery

An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons.     

Length of cell cycle in vitro and sister-chromatid exchange (SCE) frequency in bone marrow cells from severely malnourished rats

Cell proliferation and SCE frequency were evaluated through differential staining of sister chromatids in cultured bone marrow cells from rats malnourished during the lactation period. Cell proliferation was studied in vitro in sequential analysis every 5 h in cultures from 20 to 40 h of incubation. Results show a longer generation cycle in malnourished rat cells, revealing a delay in cell proliferation. Cells of this group of animals showed a higher percentage of first-cycle metaphases and lacked third-cycle metaphases even after 40 h of culture. This shows that the damage caused to cells of undernourished organisms used in this experiment persists even when they are placed in a nutrient-rich medium. The SCE frequency did not show differences between malnourished rats and their controls.     

Frequency of sister-chromatid exchange (SCE) in bone-marrow cells of severely malnourished animals during early life

The frequency of sister-chromatid exchange (SCE) was examined in bone-marrow cells of 21-day-old Wistar rats malnourished during lactation and well-nourished controls of the same age. Malnutrition was obtained by increasing the litter size to 15 pups per mother. SCE were scored in 25 consecutive second-division metaphases in the femoral bone marrow cells from each animal. The average SCE in the malnourished animals was significantly higher than in the control group (p less than 0.01). The distribution of SCE per mitosis was also significantly higher in the malnourished animals (p less than 0.001). These results indicate that malnutrition per se during early life can increase SCE in the bone marrow of experimental animals.     

The effect of different anamnesis during ontogeny on higher nervous activity in rats.

The consequences of malnutrition, experimental experience and sensory deprivation during early postnatal life were investigated in adult rats. The higher nervous activity of experimental animals was evaluated by means of a battery of tests (exploratory activity, avoidance reaction, preference of a small chamber and a water maze). Rats were divided into 5 groups, i. e. control, trained (with previous experimental experience), light deprived, malnourished naive (12-14 animals in the litter) and malnourished trained. From experimental data obtained it may be concluded that the level of nutrition as well as sensory deprivation or additional stimulation during development are important factors the influence of which persists and modifies the somatic development and the higher nervous activity in adult animals. Light deprived and malnourished rats showed retarded somatic development and worse parameters of higher nervous activity. The difference between groups could be altered by augmented stimulation. Previous experimental experience and intensive handling seems to be an important factor which influences the higher nervous activity of experimental animals.     

Post-natal diet determines insulin resistance in fetally malnourished, low birthweight rats (F1) but diet does not modify the insulin resistance of their offspring (F2)

We examined the effects of prenatal and postnatal nutrition on birthweight and insulin sensitivity, indicated by the glucose/insulin (G/I) ratio, in adult rats (F1 generation) and in their adult offspring (F2 generation). Rat pups (F1) whose dams consumed low-protein diets during gestation (malnourished) consumed either nutritionally adequate (control) or high-fat diets ad libitum post-weaning. The offspring of these rats (F2) were maintained on the same diets as their respective dams. Separate pups (F1) whose dams consumed high-fat diets during gestation (over-nourished) were maintained on high-fat diets post-weaning, as were their offspring (F2). Birthweights were significantly reduced in all fetally malnourished F1 animals. At approximately 70 d of age, fasting insulin sensitivity in over-nourished F1 rats was significantly reduced compared to controls regardless of whether they were malnourished or over-nourished in utero; however, fetally malnourished F1 rats consuming control diets post-natally had significantly greater fasting insulin sensitivity than control animals. At 30 and 120 min post-glucose load, insulin sensitivity was reduced 12-65% in both groups of over-nourished F1 rats as compared to the fetally malnourished F1 rats consuming the control diet. Birthweights were significantly lower in F2 animals whose dams (F1) were fetally malnourished and weaned to high fat diets. Insulin sensitivity was significantly reduced in all F2 animals versus control animals, regardless of dietary treatment. Thus, post-natal diets alter insulin sensitivity in fetally malnourished, adult rats; and maternal malnutrition during gestation results in insulin resistance in offspring, irrespective of offsprings' birthweight or diet.     

Baroreflex dysfunction in rats submitted to protein restriction

Earlier studies from the authors' laboratory showed that malnourishment induces alterations in the cardiovascular homeostasis increasing the basal mean arterial pressure and heart rate. In this study, the authors evaluated whether the sympathetic and parasympathetic efferent activities contribute to changes in the cardiovascular homeostasis through altered modulation of the arterial baroreflex of malnourished rats. After weaning, male Fischer rats were given 15% (Normal Protein--NP) or 6% (Low Protein--LP) protein diet for 35 d. The baroreflex gain and latency were evaluated before and after selective autonomic blockades in control and malnourished rats. It was observed that malnourishment affected the baroreflex gain in response to activation and deactivation of the arterial baroreflex. Moreover, malnourished rats showed increased baroreflex latency as compared to that of control rats. Regarding the autonomic efferent activity directed to the heart, the data showed increased sympathetic and decreased parasympathetic efferent activities in malnourished rats, and such alterations could be related to the observed changes in the arterial baroreflex gain as well as in the basal mean arterial pressure and heart rate.     

Stingless bee honey (Tetragonula laeviceps): Chemical composition and their potential roles as an immunomodulator in malnourished rats

Honey is rich in bioactive compounds, phenolic acids, and flavonoids and is an antioxidant and an immunomodulator. The objectives of this study were to determine the honey chemical composition of Indonesian stingless bees and their potential roles as an immunomodulator in the malnourished rats. Tetragonula laeviceps honey was used to analyses of chemical composition was obtained from three different geographical origins were Depok Sleman, Bayan Lombok, and Nglipar Gunungkidul. Thirty-two rats were divided into four groups of 8 rats and placed in individual cages. The experimental designed was as follows: T1 = normal rats + without honey (0–7 weeks), T2 = normal rats + with honey of 1.8 g/kg BW/day (0–7 weeks), T3 = malnourished honey of 1.8 g/kg BW/day started from 2 weeks after the malnourished condition (2–7 weeks). The results showed that the chemical composition of Tetragonula laeviceps honey from three different geographical origins were vitamin C content (6.49–13.58 mg/100 g), total phenolic content (0.65–2.30% GAE/100 g), total flavonoid content (0.28–1.00 mg QE/g), and antioxidant activity DPPH (61.43–90.28%). The application of fresh honey from stingless bee that was offered to either normal or malnourished rats were increased lymphocytes proliferation and decreased the production of both proinflammatory markers, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) from tissue culture supernatant of lymphocytes (p < 0.01). Data from this study clearly indicates the potential role of honey from stingless bee as an immunomodulator in malnourished rats.     

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.     

Age- and training-related changes in the collagen metabolism of rat skeletal muscle

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.     

Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450

Cytochrome P450-dependent oxidation of arachidonic acid was studied in liver microsomes from normal fed, protein-energy malnourished, and refed rats. The overall rate of arachidonic acid oxidation was very similar in microsomes from the three groups, but microsomes from malnourished rats showed a higher turnover rate than microsomes from normal fed and refed rats. The regiospecificity of cytochrome P450 oxidation of arachidonic acid was drastically altered by the animal nutritional status. Thus, protein-energy malnutrition results in a clear stimulation of total omega and omega-1 hydroxylation, concomitant with a marked decrease in olefin epoxidation and allyllic oxidations. These changes, as well as the documented biological activity of some of the cytochrome P450 arachidonate metabolites, suggest that protein-energy deficiency might help to select P450 isozymes which are probably involved in key monooxygenation reactions of physiological substrates.     

Quantitative ultrastructural study of somatotropic cells in malnourished weanling rats

A quantitative ultrastructural study of somatotropic cells was carried out in 21-day-old malnourished rats after maternal food restriction during the nursing period. Changes represented mainly by a reduced cytoplasmic mass and diminished secretory material were found. A decrease in growth hormone secretion of malnourished weanling rats is suggested.     

Protein synthesis, myosin ATPase activity and myofibrillar protein composition in hearts from tumour-bearing rats and mice.

Growing rats and adult weight-stable mice bearing a transplantable methylcholanthrene-induced sarcoma were compared with animals with various states of malnutrition. Heart protein synthesis was measured in vivo. Myocardial RNA, myofibrillar protein composition and the Ca2+-activated ATPase activity in heavy chains of native myosin were measured. 'Fingerprints' were made from myosin by trypsin treatment to evaluate possible structural changes in the protein. Cardiac protein-synthesis rate was decreased by 20% in growing tumour-bearing rats, by 35% in protein-malnourished (rats) and by 47% in starved rats, compared with freely fed controls (P less than 0.05). Adult tumour-bearing mice showed no significant decrease in myocardial protein synthesis. Pair-weighed control mice had significantly depressed heart protein synthesis. Protein translational efficiency was maintained in both tumour-bearing rats and mice, but was decreased in several groups of malnourished control animals. The Ca2+-activated myosin ATPase activity was decreased in all groups of malnourished animals, including tumour-bearing mice and rats, without any evidence of a change in cardiac isomyosin composition. We conclude that loss of cardiac muscle mass in tumour disease is communicated by both depressed synthesis and increased degradation largely owing to anorexia and host malnutrition. Increased adrenergic sensitivity in hearts from tumour-bearing and malnourished animals is not communicated by increased Ca2+-activated ATPase activity. This may be down-regulated in all groups with malnutrition, without any observable alterations in the isomyosin profile.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.     

Effects of chlordecone and malnutrition on immune response in rats

Effect of Chlordecone (Cd) and malnutrition on total body and spleen weights, and plaque forming cells (PFC) were studied. Rats were fed on normal, calcium (CaD), protein (PD) or Ca+P-deficient diets containing 0, 10 or 100 ppm of Cd for 2 or 4 weeks. High (95−100%) mortality was observed in malnourished rats treated with 100 ppm of Cd for 4 weeks. A slight decrease in body weight and an increase in spleen weight was observed in normal but not malnourished rats treated with 10 ppm of Cd for 4 weeks. PFC were significantly increased in both malnourished and Cd-treated rats. Similar increase in PFC was observed in rats fed on CaD but not PD diet containing 10 or 100 ppm of Cd. Whereas, rats fed on Ca+P-D diet containing 100 ppm of Cd exhibited a significant decrease in PFC.     

Increased L-CPT-1 activity and altered gene expression in pancreatic islets of malnourished adult rats: a possible relationship between elevated free fatty acid levels and impaired insulin secretion

Intrauterine growth restriction is associated with chronically elevated levels of serum fatty acids and reduced glucose-stimulated insulin secretion. Lipid metabolism in pancreatic beta cells is critical for the regulation of insulin secretion, and the chronic exposure to fatty acids results in higher palmitate oxidation rates and an altered insulin response to glucose. Using a rat model of isocaloric protein restriction, we examined whether pre- and postnatal protein malnutrition influences the properties of pancreatic islet carnitine palmitoyltransferase-1 (liver isoform, L-CPT-1), a rate-limiting enzyme that regulates fatty acid oxidation in mitochondria. The activity of L-CPT-1 in pancreatic islets increased in the low protein (LP), although the L-CPT-1 mRNA levels were unaffected by malnutrition. The susceptibility of enzyme to inhibition by malonyl-CoA was unaltered and the content of malonyl-CoA was reduced in LP cells. Because the mitochondrial oxidation of fatty acids is related to the altered expression of a number of genes encoding proteins involved in insulin secretion, the levels of expression of insulin and GLUT-2 mRNA were assessed. A reduced expression of both genes was observed in malnourished rats. These results provide further evidence that increased L-CPT-1 activity and changes in gene expression in pancreatic islets may be involved in the reduced insulin secretion seen in malnourished rats.     

Utilization of tyrosine and tryptophan for protein synthesis by undernourished developing rat brain

Incorporation of tracer doses of radiolabeled tryptophan and tyrosine into brain proteins was investigated in rats malnourished during gestation and lactation. Age and time dependent increases in the radioactivity was observed in the whole homogenate and in the TCA insoluble fraction. Protein malnourished rats showed increased incorporation of tryptophan and tyrosine. However the diet restricted (Pair-fed) animals showed increased incorporation of tyrosine only. The increased incorporation may probably be due to changes in the pool size of the amino acids and effective recycling of the amino acids. The enhanced utilization in protein synthesis may also probably offer a mechanism for conservation of these amino acids.     

Diurnal Behavioral and Metabolic Patterns in Chronic Malnourished Rats

The temporal organization of behavioral patterns and metabolic activity was assessed in chronic low protein malnourished rats and their nutritional controls at 60 days of age. Spontaneous activity was video-recorded during 3 days for 10 min every hour and the frequency of exploration, grooming and ingestive behaviors was obtained, their basal metabolic rate was measured during 5 min every hour for one 24 h cycle. Rats of both nutritional groups showed a clear diurnal rhythmicity in all behavioral patterns, characterized by an increase of activity during the dark phase and a decrease during the light phase. Effects of malnutrition were selective and produced mainly a general decrease of the mean value and amplitude of the rhythm of ingestive behavior, as well as a bimodal pattern and rhythmic components of 12, 24 and 30 h according to autocorrelation and cosinor analysis. In both groups, the metabolic rate showed a bimodal diurnal rhythm, which fitted significantly to a 12 h period model and was not affected in malnourished rats. These data provide evidence that the circadian system is highly resistant to malnutrition. In malnourished rats rhythmicity of ingestive behavior was specifically affected, probably because the relation between metabolic processes and ingestive behaviors may be enhanced as a compensatory mechanism aimed to counteract the deficient diet composition.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.