Alterations in the lipids of bone caused by hypervitaminosis A and D

1. The total lipid, phospholipid, total and free fatty acid, free and esterified cholesterol contents of the long bones of normal, hypervitaminotic A, D and A plus D rats were determined. 2. Toxic amounts of vitamin A decreased the total fatty content, whereas toxic amounts of vitamin D increased triglycerides, esterified cholesterol and in particular the phospholipids of bone. 3. An interaction occurred between toxic amounts of vitamins A and D, which prevented, to a large extent, the alterations in bone lipids that occur in hypervitaminosis D. 4. The studies suggest an involvement of vitamin D in lipid metabolism and tend to support the idea that lipids are involved in ossification.     

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver

Summary Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane.To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups.No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.This work was supported by Grants-in-Aid for Co-operative Research (Nos. 437001 and 57370001) from the Ministry of Education, Science and Culture, Japanese Government     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Role of adrenals in the vitamin A-mediated increase in the activities of gluconeogenic enzymes of rat liver.

Oral administration of large doses of vitamin A to rats even for two days was found to cause marked increase in the activities of PEP-carboxykinase, fructose-1, 6-diphosphatase, glucose-6-phosphatase and alanine aminotransferase in liver. However, overdosage of this vitamin failed to enhance the activities of these enzymes in the livers of bilaterally adrenalectomized rats. The adrenalectomy was also found to abolish the vitamin A-mediated increase in the levels of glucose and lactic acid in the blood. Thus, it is concluded that stimulation of gluconeogenesis in hypervitaminosis is, perhaps, caused by the increase in the activities of the key gluconeogenic enzymes of the liver, and that adrenal hormones are directly or indirectly involved in this process.     

Effect of dietary protein on vitamin A levels in plasma and liver of hypervitaminotic-A rats

Level of vitamin A increased in plasma and liver in hypervitaminotic A albino rats fed normal quantity of protein in diet. In low protein fed state vitamin A level in liver increased due to accumulation of vitamin A and lack of carrier protein with an associated decrease of plasma vitamin A. In high protein fed rats the level of vitamin A in plasma increased due to enhanced transport while in liver it decreased. The results indicate that for normal transport of vitamin A adequate plasma protein level is essential.     

Effects of vitamins A and D on the biosynthesis of L-ascorbic acid by rat-liver microsomes.

1. The synthesis of l-ascorbic acid from either d-glucuronolactone or l-gulonolactone by liver microsomes of rats is decreased under conditions of hypervitaminosis A; under hypervitaminosis D the synthesis from d-glucuronolactone is increased and that from l-gulonolactone is not affected. 2. The microsomal conversion of l-gulonolactone into l-ascorbic acid is impaired in liver tissues of rats made deficient with respect to either vitamin A or vitamin D when compared with the controls maintained on stock diet.     

Effect on blood,liver, and kidney variables of age and of dosing rats with lead acetate orally or via the drinking water

Levels of lead in the livers and kidneys of rats increased in proportion to the dose of lead acetate that the rats were given orally or in the drinking water. The activities of delta-aminolevulinic acid dehydratase (DALAD) in blood and liver decreased when the rats were dosed with lead, whereas glutathione levels in the blood increased. The decrease in the activity of blood DALAD was the most sensitive indicator of lead toxicity. Levels of lead in the livers and kidneys decreased after 3, 7, and 14 d of lead withdrawal. The activities of blood DALAD increased after 3 d of lead withdrawal. Groups of rats that initially weighted an average of 140 g were killed at weekly intervals for 6 wk. Blood hematocrits and liver glutathione levels increased, and blood DALAD and activated DALAD from blood decreased with increasing age of the rats. Activated DALAD activities from liver increased after the first week of the study.     

Changes in the proportion of acetyl-CoA carboxylase in the active form in rat liver. Effect of starvation, lactation and weaning

1. The activity of acetyl-CoA carboxylase (EC 6.4.1.2) in extracts of freeze-clamped liver samples from fed or 24 h-starved virgin, pregnant, lactating and weaned rats was measured (i) immediately after preparation of extracts (;I activity'), (ii) after incubation of extracts with partially purified preparations of either rabbit muscle protein phosphatase 1 [Antoniw, Nimmo, Yeaman & Cohen (1977) Biochem. J.162, 423-433] or rabbit liver phosphatase [Brandt, Capulong & Lee (1975) J. Biol. Chem.250, 8038-8044] (;A activity') and (iii) after incubation with 20mm-potassium citrate before or after incubation with phosphatases (;C activity'). 2. Incubation of liver extracts at 30 degrees C without any additions resulted in activation of acetyl-CoA carboxylase that was shown to be due to dephosphorylation of the enzyme by endogenous protein phosphatase activity. This latter activity was not stimulated by Ca(2+) and/or Mg(2+) but was stimulated by 1 mm-Mn(2+). Incubation of extracts with either of the partially purified phosphatases (0.2-0.5 unit) resulted in faster dephosphorylation and activation. The activity achieved after incubation with either of the exogenously added phosphatases was similar. 3. The A and C activities increased during late pregnancy, were lower than in the virgin rat liver during early lactation and increased by 2-fold in liver of mid-lactating rats. Weaning of mid-lactating rats for 24 h resulted in no change in A and C activities but after 48 h weaning they were significantly lower than those in livers from suckled mothers. 4. The I activity followed a similar pattern of changes as the A and C activities during pregnancy and lactation such that, although the I/A and I/C activity ratios tended to be lower during late pregnancy and early lactation, there were no significant changes in I/A and I/C ratios between lactating and virgin animals. However, these ratios were significantly higher in liver from fed 24 h-weaned animals. 5. Starvation (24 h) resulted in a marked decrease in I activity for all animals studied except early-lactating rats. This was due to a combination of a decrease in the concentration of acetyl-CoA carboxylase in liver of starved animals (A and C activities) and a decrease in the fraction of the enzyme in the active form (lower I/C and I/A ratios). The relative importance of the two forms of regulation in mediating the starvation-induced fall in I activity was about equal in livers of virgin, pregnant and lactating animals. However, the decrease in I/A and I/C ratios was of dominating importance in livers of weaned animals. The A/C activity ratios were the same for livers from all animals studied. 6. The maximal activity of fatty acid synthase was also measured in livers and was highly and positively correlated with the A and C activities of acetyl-CoA carboxylase, suggesting that the concentrations of the two enzymes in the liver were controlled coordinately. 7. It is suggested that the lack of correlation between plasma insulin levels and rates of lipogenesis in the transition from the virgin to the lactating state may be explained by different effects of insulin and prolactin on the concentration of acetyl-CoA carboxylase in the liver and on the fraction of the enzyme in the active form.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Inhibition of liver protein degradation in vivo by intensive treatment with cycloheximide.

The AA in experiments performed on male rats treated with 0,5 mCi/kg of cycloheximide have observed an inhibition of liver protein degradation and have suggested two mechanism: 1) cycloheximide prevents the acute proteolytic response induced in fibroblast culture by exposure to serum-deficient media and in rat livers after perfusion probably by inhibiting the lysosomal - autophagic system, without modifications in total activity of lysosome proteinases. 2) It may be that protein degradation rate and level of lysosomal proteinases reinterrelated; the latter probably being a factor controlling the overall cell protein turnover rate.     

The role of vitamin E in metabolism and reception of vitamin D

It was found that calcium exchange disturbances under vitamin E deficiency is due to changes in the metabolism of vitamin D. In vitamin E-deficient rats the serum blood levels of hydroxyvitamin D (25-OHD) showed no significant changes, whereas the concentration of the hormonal form of 1.25-hydroxyvitamin D [1.25(OH)2D], decreased by 40%. In vitro studies showed that the 25-hydroxylase D3 activity in the livers of rats with E-avitaminosis had a tendency to decrease (by 22%), whereas that of 24-hydroxylase dropped drastically (by 52%). The serum blood levels of the parathyroid hormone (PTH) and kidney levels of cAMP under E-avitaminosis were significantly lowered. Preincubation of kidney slices with the adenylate cyclase activator, forskolin, increased the activity of 1-OHase in about the same degree as that in vitamin E-rich rats. The free radical scavenger, BHT, added to kidney slices suppressed the activity of the both enzymes; this finding testifies to the low O2-binding affinity of these monooxygenases. The content of 1.25(OH)2D3 receptors occupied in vivo in the kidneys of vitamin E-deficient rats decreased 2.5-fold; however, the binding of 1.25(OH)2D3-receptor complexes to heterologous DNA was unaffected thereby. The vitamin deficiency in vivo results in the inhibition of vitamin D metabolism in the liver and kidney concomitant with the formation of active metabolites and decreases the concentration of hormone-receptor complexes in target tissues.     

Vitamin A metabolism in rats chronically treated with 3,3',4,4',5,5'-hexabromobiphenyl

Chronic dietary administration of 3,3',4,4',5,5'-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA:retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-3H]retinyl acetate (10 micrograms), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Acid lipase activity in neonatal rat liver cell types. Effect of starvation

The acid lipase activity in the liver of neonatal (1-day-old) rats was studied. It was found that (i) in whole liver, the activity was 50% lower than in adult rats; (ii) in neonatal livers, the activity was 7.7-fold higher in hepatocytes than in hemopoietic cells; (iii) neonatal hepatocytes contained about 25% of the activity detected in adult hepatocytes; (iv) all the differences disappeared when expressed per mg of protein; and (v) starvation did not affect the activity either in adult or in neonatal rat liver.     

Effects of dietary vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of glucocorticoid

The effects of dietary intake of vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of prednisolone were investigated. Rats were divided into five groups. Groups 3, 4, and 5 received a daily supplement in their drinking water of vitamin E, Se, and a combination of vitamin E and Se, respectively, for 30 days. For 3 days subsequently, the control group (group 1) was given a placebo, and the remaining four groups were injected intramuscularly with prednisolone. The tissue samples were collected from each group at 4, 8, 12, 24, and 48 h after the last administration of prednisolone. In the group treated with prednisolone alone, arginase activity in the liver was found to have increased at all the time periods, whereas it had decreased significantly in the heart at 48 h. Arginase activity in the kidneys was not affected by prednisolone. Compared to the control and prednisolone groups, arginase activity in the kidneys and heart of the vitamin E- and Se-supplemented groups was found to be significantly increased at all time periods, however, no difference was seen in the combination group. Arginase activity in the liver of the vitamin E-supplemented group was found to have decreased at all time periods, however, in the Se group compared to the prednisolone group it had reduced at 24 and 48 h only. In the combination group compared to the prednisolone group, liver arginase activity increased constantly up to 12 h returning to normal values at 48 h. Vitamin E and Se in combination may prevent the changes in arginase activity in various tissues caused by prednisolone.     

A functional state of the sphingomyelin cycle and activity of free radical oxidation of rat liver lipids at different phases of starvation

A functional state of the sphingomyeline cycle and its links with processes of free radical lipid oxidation have been investigated in rat liver during starvation of animals without any restriction of access to drinking water at day 1, 2, 3 (phase I) and day 6 (phase II of starvation). The maximal values of the ceramide/sphingomyelin ratio and activities of neutral sphingomyelinase and executive caspase-3 were found in rat livers at day 3 of starvation. Since day 3 of starvation an increase of tumour necrosis factor-α, one of neutral sphingomyelinase activators, was detected in serum. During the major part of phase I of starvation the intensity of liver free radical lipid peroxidation was comparable to that of control due to competent functioning of the antioxidant defense system. Transition of phase I to phase II of starvation (day 6 of the experiment) was accompanied by the development of oxidative stress associated with depletion of the antioxidant defense system. The results obtained in this study suggest that phase I of starvation favors realization of the ceramide-mediated proapoptotic signaling in the liver. In our viewpoint, ceramide-mediated apoptosis is one of mechanisms used optimization of liver cellular population within the frame of metabolic adaptation. In rat liver phase I of starvation was characterized by prevailing of the ceramide-mediated proapoptotic signaling, while in phase II oxidative stress dominated.     

Vitamin K-dependent carboxylase: effect of detergent concentrations, vitamin K status, and added protein precursors on activity

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80-90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.     

Vitamin A metabolism in rats chronically treated with 3,3′,4,4′,5,′-hexabromobiphenyl

Chronic dietary administration of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA: retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-³H]retinyl acetate (10 μg), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.