Effect of rhythmic alternation of protein depletion and repletion on serum protein synthesis.

The authors attempted to determine whether repeated protein depletion would produce changes in the organism's response to this unfavourable situation indicative of the preservation of information on past depletion and of its inclusion in the formation of the organism's defences against repetition of this unphysiological state. It can be concluded from the results that the anticipated trend was demonstrated. The question of the site of origin, storage and action of this information was not resolved.     

Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome

Zinc (Zn) deficiency is a problem world-wide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224–1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immunoaffinity column. An unnamed protein that was related to immunoglobulins was observed in the immunodepleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future.     

Kinetics of taurine depletion and repletion in plasma, serum, whole blood and skeletal muscle in cats

The relationship between taurine concentrations of plasma, whole blood, serum and skeletal muscle during taurine depletion and repletion was investigated in cats, to identify the most useful indicators of taurine status. Sixteen cats were fed a purified diet containing either 0 or 0.15 g/kg taurine for 5 months. Treatments were then reversed and the taurine concentration was measured during repletion and depletion phases. Plasma taurine exhibited the fastest rate (slow component) of depletion (t 1/2 = 4.8 wk), followed by serum (5.3 wk), whole blood (6.2 wk), and skeletal muscle (11.2 wk). Whole blood taurine was the first to replete at a rate of 0.74 wk to 1/2 maximal repletion, followed by serum (2.1 wk), skeletal muscle (3.5 wk), and plasma (3.5 wk). Whole blood more closely reflected skeletal muscle taurine concentrations than plasma during depletion, while plasma taurine concentrations appear to be the most valuable predictor of skeletal muscle taurine concentrations during repletion. This study suggests that the best clinical method to evaluate the taurine status of the cat is the determination and interpretation of both plasma and whole blood taurine concentrations.     

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca²⁺ depletion caused by EGTA treatment and Ca²⁺ repletion after Ca²⁺ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca²⁺ depletion and repletion. In the early stage after Ca²⁺ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca²⁺ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca²⁺ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.     

Induction of hepatocytes in the pancreas of copper-depleted rats following copper repletion

Pancreatic hepatocytes are induced in rats maintained on copper-deficient diet containing 0.6% D-penicillamine for 8-10 weeks, followed by copper repletion. These induced hepatocytes are morphologically and functionally very similar to parenchymal cells of the liver. Immunofluorescence stains demonstrated the presence of albumin and catalase in these cells. Stains for pancreatic enzymes and hormones were negative. As expected, the hypolipidemic compound, ciprofibrate, induced peroxisome proliferation in these cells. These results indicate that a simple depletion and repletion of copper can trigger transdifferentiation in the pancreas of adult rats.     

Effect of calcium depletion and subsequent repletion on parathyroids,parafollicular (C) cells and bone in the growing pig

Summary The ultrastructure of the chief cells of the parathyroid gland and thyroid parafollicular (C) cells and the morphology of bone in calcium depletion and subsequent repletion were examined in young growing pigs. A low calcium diet resulted in osteopenia, increased removal of the cartilaginous core, osteoclasia and osteocytic osteolysis. Subsequent repletion quickly returned bone to normal. In pigs fed the low calcium diet, there was a marked depletion of secretory granules but a striking increase in the number of microtubules in chief cells. Increasing the calcium content of the diet to normal quickly returned the ultrastructural appearance of chief cells to apparent normal. In the initial response to calcium repletion, chief cells exhibited large number of lysosomes and occasionally prominent paracrystalloid bodies. Electron microscopic examination of parafollicular (C) cells of the thyroid gland failed to reveal differences in ultrastructure between test and control pigs. These findings support the view that bone resorption following calcium deficiency may be the result of a secondary hyperparathyroidism rather than of calcium deficiency per se.Supported by U.S.P.H.S. Grant A.M. 12957 from the Division of Arthritis and Metabolic Diseases     

Sodium depletion of rats induces a mitochondrial protein in the zona glomerulosa of the adrenal cortex

Sodium restriction of rats resulted in a marked increase in the concentration of a mitochondrial protein with a mol. wt of 49000 in the zona glomerulosa of the adrenal cortex. The same protein had been previously induced in potassium deficient rats by potassium repletion. Since both sodium depletion and potassium repletion are known to stimulate the activity of the mitochondrial mixed function oxidase involved in the conversion of corticosterone to aldosterone, the present observation supports the hypothesis that the potassium-induced protein might represent cytochrome P-450CMO (corticosterone methyl oxidase).     

Methylenetetrahydrofolate reductase 677C-->T polymorphism affects DNA methylation in response to controlled folate intake in young women

DNA methylation is critical for normal genomic structure and function and is dependent on adequate folate status. A polymorphism (677C-->T) in a key folate enzyme, methylenetetrahydrofolate reductase (MTHFR), may impair DNA methylation when folate intake is inadequate and may increase the risk of reproductive abnormalities. The present study was designed to evaluate the effect of the MTHFR 677C-->T polymorphism on changes in global DNA methylation in young women consuming a low folate diet followed by repletion with the current Recommended Dietary Allowance (RDA). Women (age 20-30 years) with the TT (variant; n = 19) or CC (n = 22) genotype for the MTHFR 677C-->T polymorphism participated in a folate depletion-repletion study (7 weeks, 115 microg DFE/day; 7 weeks, 400 microg DFE/day). DNA methylation was measured at baseline, week 7, and week 14 using a [3H]methyl acceptance assay and a novel liquid chromatography tandem mass spectrometry assay of the DNA bases methylcytosine and cytosine. [3H]Methyl group acceptance tended to increase (P = 0.08) during depletion in all subjects, indicative of a decrease in global DNA methylation. During repletion, the raw change and the percent change in the methylcytosine/total cytosine ratio increased (P = 0.03 and P = 0.04, respectively) only in the subjects with the TT genotype. Moderate folate depletion in young women may cause a decrease in overall DNA methylation. The response to folate repletion suggests that following folate depletion women with the MTHFR 677 TT genotype have a greater increase in DNA methylation with folate repletion than women with the CC genotype.     

Altered metabolic response of iron-deficient women during graded, maximal exercise.

Metabolic responses during a standardized, progressive, maximal work capacity test on a cycle ergometer were studied in 11 women, mean age 28 (SEM 2) years, at admission to the study, after their body iron stores were depleted by diet, phlebotomy and menstruation for about 80 days and after iron repletion by diet for about 100 days, including daily iron supplementation (0.9 mmol iron as ferrous sulfate) for the last 14 days of repletion. Iron depletion was characterized by a decline (P less than 0.05) in hemoglobin, ferritin and body iron balance. Iron repletion, including supplementation, increased (P less than 0.05) hemoglobin, ferritin and iron balance. No changes were observed in cardiovascular and ventilatory responses or peak oxygen uptake. Iron depletion was associated with a reduced (P less than 0.05) rate of oxygen utilization, total oxygen uptake and aerobic energy expenditure, and elevated (P less than 0.05) peak respiratory exchange ratio and post-exercise concentration of lactate. Reduction of body iron stores without overt anemia affects exercise metabolism by reducing total aerobic energy production and increasing glycolytic metabolism.     

Osteocyte-Derived Insulin-Like Growth Factor I Is Not Essential for the Bone Repletion Response in Mice

The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both Igf1 osteocyte conditional knockout (cKO) mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by the reduction in osteoclast number, indicating that deficient Igf1 expression in osteocytes also did not have deleterious effects on the bone repletion response. The lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected since previous studies show that these Igf1 osteocyte cKO mice exhibited impaired developmental growth and displayed complete resistance to bone anabolic effects of loading. These studies suggest that there is a dichotomy between the mechanisms necessary for anabolic responses to mechanical loading and the regulatory hormonal and anabolic skeletal repletion following low dietary calcium challenge. In conclusion, to our knowledge this study has demonstrated for the first time that osteocyte-derived IGF-I, which is essential for anabolic bone response to mechanical loading, is not a key regulatory factor for bone repletion after a low calcium challenge.     

Effects of Iron Deficiency on Serotonin Uptake In Vitro by Rat Brain Synaptic Vesicles

Abstract: The effects of moderate and severe degrees of iron deficiency on brain and liver nonhaem iron levels and 5-hydroxytryptamine (serotonin; 5-HT) uptake by synaptic vesicles in vitro were investigated in experimental rats. Data obtained suggested that in both moderate and severe forms of iron deficiency, 5-HT uptake by brain synaptic vesicles is decreased and is accompanied by a reduction in brain and liver nonhaem iron levels. On repletion with iron for 4 weeks, the deficient group of rats showed a normalisation of 5-HT uptake by synaptic vesicles and liver nonhaem iron content, whereas the brain nonhaem iron concentration still showed a significant deficit. The data thus suggest that changes in the uptake of 5-HT by brain synaptic vesicles that accompany iron depletion and repletion are more rapid than changes in the total nonhaem iron concentration in the brain. The observation that 5-HT uptake by brain synaptic vesicles is decreased in iron deficiency suggests a probable role for iron in 5-HT storage in rat brain.     

Time course of changes in plasma concentrations of the growth related hormones during protein restriction in the domestic fowl (Gallus domesticus)

Experiments were conducted to evaluate the possible role of circulating growth hormones triiodothyronine (T3), thyroxine (T4), and insulin-like growth factor I (somatomedin-C; IGF-I) in the elevation of plasma growth hormone (GH) which occurs in protein-restricted chickens. Plasma hormone changes were determined over a 2-week period of protein depletion by feeding a 5% protein diet as well as a similar period of protein repletion with a 20% protein diet. The rise in plasma GH was observed in two separate studies. Plasma concentrations of T4, T3, and IGF-I were all depressed in protein-restricted chicks prior to or concurrent with the GH elevation. In the protein repletion time course study, T4 and T3 concentrations were normalized prior to or concurrent with plasma GH normalization. However, IGF-I concentrations in repleted chicks did not return to control levels until after normal levels of GH were observed. These data suggest that thyroid hormones may play a greater role in the regulation of GH secretion during periods of malnourishment than IGF-I; the latter being currently thought to be a peripherally circulating inhibitor of GH release in animals.     

Development of bone mineralization and body composition of replacement gilts fed a calcium and phosphorus depletion and repletion strategy

This study investigated the ability of replacement gilts to adapt their calcium and phosphorus utilization and their kinetics in bone mineralization to compensate for modified intake of these nutrients by applying a novel Ca and P depletion and repletion strategy. A total of 24 gilts were fed according to a two-phase feeding program. In the first phase, gilts (60–95 kg BW) were fed ad libitum a depletion diet providing either 60% (D60; 1.2 g digestible P/kg) or 100% (D100; 2.1 g digestible P/kg) of the estimated P requirement. In the second phase, gilts (95–140 kg BW) were fed restrictively (aim: 700–750 g/d BW gain) a repletion diet. Half of the gilts from each depletion diet were randomly assigned to either a control diet or a high-P diet (R100 and R160; with 2.1 and 3.5 g digestible P/kg, respectively) according to a 2 × 2 factorial design, resulting in four treatments: D60-R100, D60-R160, D100-R100 and D100-R160. Dual-energy X-ray absorptiometry was used to measure whole-body bone mineral content (BMC), bone mineral density (BMD) and lean and fat tissue mass on each gilt at 2-week intervals. The depletion and repletion diets, fed for 5 and 8 weeks, respectively, did not influence growth performance. The D60 gilts had a reduced BMC and BMD from the second week onwards and ended (95 kg BW) with 9% lower values than D100 gilts (P < 0.001). During repletion, D60 gilts completely recovered the deficit in bone mineralization from the second and fourth week onwards, when fed R160 (D60-R160 vs D100-R160) or R100 (D60-R100 vs D100-R100) diets, respectively (treatment × time interaction, P < 0.001); thus, the depletion diets did not affect these values at 140 kg BW. These results illustrate the rapid homeostatic counter-regulation capacity of dietary Ca and P, and they show the high potential to limit dietary digestible P concentration by completely excluding the use of mineral phosphates during the depletion phase, representative of the fattening period, without causing any detrimental effects to gilts at mating. The gilts were able to recover their BMC deficit between their selection at 95 kg BW and first mating at 140 kg BW by increasing their dietary Ca and P efficiency. Finally, excess dietary digestible P, requiring increased amounts of mineral phosphates, further increased the gilts’ BMC.     

Relative effects of glycogen depletion and previous exercise on muscle force and endurance capacity

Endurance capacity of human vastus lateralis muscles was observed 24 h after hard exercise followed by either a carbohydrate-restricted or a carbohydrate-loaded diet (depletion and repletion conditions). In a control condition the subjects did no previous exercise and ate their normal diet. Each of these conditions was followed by an experimental protocol in which the five male subjects made a series of alternating 25-s static contractions of each leg at 50% maximal voluntary contraction until one leg failed to achieve the required force (Tlim). Glycogen concentration before the experimental protocol in both legs was significantly lower in the depletion than in the repletion condition. Muscle lactate and creatine phosphate concentrations were within normal limits before the static contractions. The number of contractions the repleted (12.7 +/- 2.2) and depleted (10.3 +/- 1.5) legs could sustain before Tlim were not different from each other, but both were 35% (P less than 0.05) fewer than the control (17.6 +/- 3.0). Surface electromyogram (EMG) amplitude was higher in depleted than in repleted or control muscles. At Tlim, EMG amplitude was maximal, creatine phosphate was 50-70% depleted, and lactate increased fourfold. Average glycogen utilization per contraction in both the repletion and depletion conditions was 5.8 mmol/kg dry wt, but postexercise lactate concentrations were lower in depleted (14.4 +/- 3.6 mmol/kg dry wt) than in repleted (43.2 +/- 7.4) muscles. The EMG frequency distribution shifted downward in all conditions during the experimental protocol and was independent of muscle lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of blood serum constituents to production of and recovery from a kwashiorkor-like syndrome in the young pig.

Twenty-six 3-week-old genetically obese pigs were fed in two experiments to determine the serum chemistry profile during severe protein malnutrition and repletion. Severe protein deficiency was produced in pigs fed the high-fat, low-protein diet (growth failure, rough hair, low serum total protein and albumin). In Experiment 1, blood was sampled from the anterior vena cava of each pig five times during depletion and three times during repletion to determine serum total cholesterol, high density lipoprotein (HDL)-cholesterol, triglycerides, total protein, albumin, glucose, Ca, inorganic P, Mg, Na, K, Cl, total bilirubin, urea N, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase. In Experiment 2, blood was sampled weekly for 8 weeks for serum total cholesterol, HDL-cholesterol, triglycerides, albumin, glucose, Ca, P, Mg and alkaline phosphatase. HDL-cholesterol was increased (P less than 0.01) and albumin was decreased (P less than 0.01) in protein-deficient pigs in both experiments. Creatinine, total bilirubin, gamma-glutamyltransferase, alanine aminotransferase, and aspartate aminotransferase were elevated in protein-deficient pigs compared with controls after 7 weeks of depletion. Inorganic P (P less than 0.01), Ca (P less than 0.01), and Mg (P less than 0.05) concentrations were depressed in protein-depleted pigs compared with controls in both experiments. After 8 weeks of repletion in Experiment 1, all elements except inorganic P were similar in the two groups. Short-term, severe, protein malnutrition affected lipid, electrolyte, and structural mineral metabolism and indices of liver function in the absence of parasites, diarrhea, and infection. The effects were reversed after 8 weeks of repletion. We conclude that the elevated serum cholesterol in protein deficiency is related primarily to an increase in the HDL fraction.     

Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion

Disruption of theactin cytoskeleton in proximal tubule cells is a key pathophysiologicalfactor in acute renal failure. To investigate dynamic alterations ofthe actin cytoskeleton in live proximal tubule cells,LLC-PK₁₀ cells were transfected with an enhanced yellowfluorescence protein (EYFP)-actin construct, and a clone with stableEYFP-actin expression was established. Confluent live cells werestudied by confocal microscopy under physiological conditions or duringATP depletion of up to 60 min. Immunoblots of stabletransfected LLC-PK₁₀ cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicatedcolocalization with G-actin as well. Beyond previous findings, weobserved differential subcellular disassembly of F-actin structures:stress fibers tagged with EYFP-actin underwent rapid and completedisruption, whereas cortical and microvillar actin disassembled atslower rates. In parallel, ATP depletion induced the formation ofperinuclear EYFP-actin aggregates that colocalized with F-actin. DuringATP depletion the G-actin fraction of EYFP-actin substantiallydecreased while endogenous and EYFP-F-actin increased. Duringintracellular ATP repletion, after 30 min of ATP depletion, there was ahigh degree of agreement between F-actin formation from EYFP-actin andendogenous actin. Our data indicate that EYFP-actin did not alter thecharacteristics of the endogenous actin cytoskeleton or the morphologyof LLC-PK₁₀ cells. Furthermore, EYFP-actin is a suitableprobe to study the spatial and temporal dynamics of actin cytoskeletonalterations in live proximal tubule cells during ATP depletion and ATP repletion.

     

Renal and hepatic glutathione pool modifications in response to depletion treatments

In this study we examined the response of the renal and hepatic glutathione (GSH) pool in rats to drastic GSH depletion treatments. For this purpose, we used a protein-free diet, starvation, and the injection of varying doses of diethyl maleate as depleting agents. We analysed GSH levels in both kidney and liver tissue homogenates after rats were fed a protein-free diet for 2 or 7 days or starved for 1, 2, or 3 days, as well as after diethyl maleate administration in a single maximal dose or in varying doses. The results indicated that the liver GSH pool was always more labile than the kidney GSH pool. Moreover, kidney GSH levels were almost unchanged after 7 days on a protein-free diet or after 2 days of starvation, while liver showed significant changes in GSH levels. When we analysed the repletion rate, kidney had higher kinetic parameters (k = 0.148 h-1) than liver (0.097 h-1). We conclude that efficient mechanisms of maintaining GSH levels exist in the kidney and these may serve to avoid GSH diminution and hence preserve renal function during states of GSH depletion.     

Phospholipid hydroperoxide glutathione peroxidase in various mouse organs during selenium deficiency and repletion

An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of 10 or 500 micrograms/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ more sensitive to oxidative stress.