Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.     

Colchicine increases hepatic alkaline phosphatase activity

In vivo administration of colchicine increases the activity of alkaline phosphatase significantly in the livers of rats. Prior treatment with cycloheximide prevented the induction of the enzyme by colchicine suggesting that de novo protein synthesis was essential for the effect of colchicine on alkaline phosphatase activity. Bilateral adrenalectomy did not affect the response of alkaline phosphatase following the administration of colchicine. This indicates that the rise in the level of alkaline phosphatase in liver caused by colchicine is not secondary to the release of glucocorticoids.     

Nystatin increases hepatic alkaline phosphatase activity

The activity of alkaline phosphatase is increased significantly in the livers of rats after the intraperitoneal administration of nystatin. Studies with cycloheximide suggested that de novo protein synthesis was essential for the effect of nystatin on alkaline phosphatase activity. The induction of hepatic alkaline phosphatase by nystatin is not secondary to the release of glucocorticoids because the response of the enzyme to the administration of nystatin was not affected by bilateral adrenalectomy.     

Effects of adrenalectomy on urea synthesis in rats

The effect of depletion of glucocorticoids on the dynamics of hepatic amino-N conversion was examined 2 and 7 days after adrenalectomy in a total of 22 rats substituted by adrenaline. The capacity of urea synthesis was studied by infusion of alanine under steady state conditions with arterial concentrations of alanine between 7.3 and 11.6 mmol/l. The animals were nephrectomized and the capacity was calculated as accumulation of urea in total body water corrected for intestinal hydrolysis. Adrenalectomy reduced the capacity of urea synthesis to 55% of the capacity for control rats and reduced the alanine metabolic rate to 60%. In control rats the urea synthesis exceeded the alanine infusion by indicating an extrahepatic tissue release of amino acids. This difference disappeared after adrenalectomy. The body weight and food intake did not change during the study period. Thus lack of glucocorticoids influences the in vivo nitrogen economy both by decreasing the liver function as to conversion of amino-nitrogen and by decreasing release of tissue amino-nitrogen.     

Rapid oxidative stress induced by N-nitrosamines

We have investigated the generation of prooxidant state shortly after administration of N-nitrosamines (NA) to rats. N-Nitrosodimethylamine (NDMA) was found to increase ethane exhalation (EE) rapidly in a dose-related manner. EE remained elevated for several days after single doses of NDMA. Similarly, lipid peroxidation (LP) in the liver (measured by four methods) increased rapidly showing a peak 20 min after NDMA dose. The increase of LP was preceded by a decrease in retinol concentration in the liver. N-Nitrosodiethanolamine, too, increased EE and LP in the liver, whereas N-nitrosomethylbenzylamine had no effect. Thus, hepatocarcinogenic NA induced LP in their target tissue, and the LP enhancing effects of NA were not related to their acute toxic effects.     

Induction of cytochrome P-450 by glucocorticoids in rat liver. II. Evidence that glucocorticoids regulate induction of cytochrome P-450 by a nonclassical receptor mechanism

In the companion report we used primary cultures of adult rat hepatocytes to demonstrate that glucocorticoids comprise a "class" of compounds that stimulate de novo synthesis of a form of cytochrome P-450 (P450PCN) indistinguishable from that induced by the nonhormonal steroid pregnenolone 16 alpha-carbonitrile (PCN). Because induction of P450PCN is stereospecific for glucocorticoids and is dependent on the concentration of and the length of exposure to steroids it seemed possible that P450PCN represented another of the many genes whose expression is coordinately regulated by glucocorticoids bound to their specific cytoplasmic receptor and translocated into the nucleus. However, in cultured hepatocytes treated with glucocorticoids, synthesis of P450PCN failed to parallel synthesis of a typical glucocorticoid-responsive liver function, tyrosine aminotransferase, in the time course of induction, in the concentrations of glucocorticoids required for half-maximal induction, and in the order of effective steroids ranked by potency. Indeed, two moderately potent inducers of P450PCN either failed to induce tyrosine aminotransferase (spironolactone) or actually antagonized induction of tyrosine aminotransferase synthesis by glucocorticoids (PCN). Moreover, in the same cultures in which glucocorticoid induction of tyrosine aminotransferase was blocked by the presence of PCN or other previously identified antiglucocorticoids, synthesis of P450PCN was actually enhanced. We conclude that synthesis of P450PCN is a specific glucocorticoid-responsive liver function evoked by a novel mechanism readily distinguishable from the classic glucocorticoid receptor pathway.     

Inhibition of Ca-induced calcium release from mitochondria by antiischemic phenol-based antioxidants

Effects of different inhibitors of lipid peroxidation (LP), such as sulphur-containing oligoquinone hypoxen, natural flavonoid dihydroquercetin (DHQ), and β-ionol, on Ca²⁺-induced calcium release from rat liver mitochondria (RLM) were investigated during oxidation of various substrates. The hypothesis about interrelation between antioxidant properties and influence of selected substances on spontaneous calcium release from mitochondria was verified. Degree of antioxidant activity of the selected substances was estimated by the inhibition of LP induced by Fe²⁺/ATP complex in phospholipid emulsion or in rat liver mitochondria (RLM). According to the inhibition efficacy the investigated substances were ordered as follows: β-ionol ≫ hypoxen > DHQ. 50% inhibition of oxygen consumption during LP of phospholipid emulsion was reached in presence of 3.2 ± 0.6 μM of β-ionol, 15.0 ± 1.1 μM of hypoxen, or 19.8 ± 1.7 μM of DHQ. Among the investigated antioxidants hypoxen only decreased spontaneous release of calcium from RLM after calcium accumulation by RLM. The impact of the antioxidants onto calcium current depended on the oxidized substrate. Hypoxen effect was most expressed during the oxidation of NAD-dependent substrate. The direct relationship between the antioxidant activity of the selected antioxidants and their influence on calcium transport in RLM was not revealed. The results indicate that the choice of antiischemic preparations should not only rely on their antioxidant activities.     

Adrenalectomy-Induced Increase of Brain Protein Synthesis Is Antagonized by Corticosterone Replacements in Free-Moving Rats

Abstract: The autoradiographic method with l [³⁵S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of me-thionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.     

Effect of gamma irradiation on liver metallothionein synthesis and lipid peroxidation in rats.

Several studies have recently shown that metallothionein (MT), a protein characterized by a high thiol content and that binds Zn2+ and Cu+, might be involved in the protection against oxidative stress and can act as a free radical scavenger. Oxidative stresses, such as irradiation, increase lipid peroxidation (LP) and subsequent tissue damage through free radical production. The induction of hepatic MT synthesis by gamma-irradiation (20 Gy) at 8, 24, 30 and 48 hrs. post-irradiation in two different age groups of Sprague-Dawley rats (39-40 and 48-49 days old) was studied. LP measured by the thiobarbituric acid reactive substances (TBARS) assay and Cu and Zn levels in liver have also been determined. In the younger group, the gamma-irradiation induced hepatic MT synthesis and increased LP that peaked 24 hrs. after irradiation. During the first 30 hrs. post-irradiation, a positive and statistically significant correlation between hepatic MT content and LP level in liver was found. In the older group, liver MT synthesis was only increased 1.7-fold and LP levels were not altered at 24 hrs. post-irradiation compared with sham-irradiated rats.Therefore it appears that LP is not necessary for induction of MT synthesis by gamma-irradiation.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates of intact organs

A number of growth phenomena observed in vitro have shown that cells, at high densities, produce and release substances which, when they have reached a given concentration, arrest further growth. In vivo, these possibilities can be studied on the model of rapid regeneration of the rat liver after 65-70% partial hepatectomy (PH). We evaluated the course of liver regeneration after PH in animals treated with dialysates (DIA) of intact rat tissues. In addition to kidney and lymph node DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on male rats weighing 210-240 g. The relevant DIA was administered 24 h prior to PH; the controls were given physiological saline. The animals were killed just before PH and 24, 48, 30 and 72 h and 14 days after. DIA obtained from intact liver tissue inhibited the regeneration process induced by PH and its effect persisted 48 h after PH. Compared with the controls and with the rats given kidney DIA, DNA synthesis in the liver 24 h after PH was reduced to 77%. After spleen DIA, several (still hypothetical) factors probably acted together synergically (factors belonging to the immune system--RES--and spleen-produced factors capable of promoting proliferation of the hepatocytes--the "portal blood factor"). We arrived at this conclusion from an evaluation of liver DNA synthesis 24 h after 24h after PH, when synthesis was altogether markedly raised, but attained far higher values after the administration of spleen DIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein synthesis in liver,skeletal muscle,and brown adipose tissue of rats fed a protein-deficient diet

Feeding protein-deficient diets to rats is known to stimulate diet-induced thermogenesis and activate brown adipose tissue (BAT). The fact that BAT protein content, unlike that of other tissues, is unnaffected by protein deficiency prompted us to measure tissue protein synthesis in vivo in animals maintained on normal- (18.8%) and low- (7.6%) protein (LP) diets. Protein synthesis was depressed in the liver of the LP rats due to a fall in RNA activity, with no change in RNA content, and synthesis was also reduced in skeletal muscle from the LP group, but this was due to decreased RNA content with no change in RNA activity. Conversely, protein synthesis, RNA, DNA, and protein content of interscapular BAT were all unaltered in protein-restricted animals. These data indicate that, unlike liver, skeletal muscle, and whole carcass, BAT protein synthesis is not reduced in protein-restricted rats, and this may be related to activation of thermogenesis in the tissue.     

Oleoyl-estrone increases adrenal corticosteroid synthesis gene expression in overweight male rats

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10 nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5α-reductase was 3× larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands’ corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

Effect of cycloheximide on the induction of tryptophan oxygenase mRNA by hydrocortisone invivo

Hydrocortisone increases rat liver tryptophan oxygenase mRNA activity as measured by a translational assay. Pretreatment of rats with cycloheximide thirty minutes before hydrocortisone administration largely prevents the hormonal induction of tryptophan oxygenase mRNA. Tryptophan oxygenase mRNA activity begins to increase after a lag of at least 30 to 60 minutes after hydrocortisone injection. These results suggest that the synthesis of intermediary protein(s) is required for the induction of tryptophan oxygenase mRNA by glucocorticoids.     

Rapid inhibitory effect of corticosterone on histamine release from rat peritoneal mast cells.

Glucocorticoids are steroids endowed with powerful anti-inflammatory properties, which are routinely believed to require several hours to take effect through modulation of gene expression. Our recent report has shown that glucocorticoids could inhibit allergic reaction within 10 minutes, which the classical genomic mechanism could not explain. Histamine is thought to be one of major mediators in the allergic reaction, and IgE-mediated histamine release from mast cells plays a pivotal role in allergic diseases. Here, we have determined a rapid effect of corticosterone on histamine release from rat peritoneal mast cells, using fluorometric assay. The results showed that corticosterone could inhibit antigen-induced histamine release from rat peritoneal cells within 15 minutes (p<0.05), which could be mimicked by membrane-impermeable BSA conjugated corticosterone (p<0.05). Neither glucocorticoid nuclear receptor antagonist nor protein synthesis inhibitor could block the rapid action (p<0.05). The study provided evidence that nongenomic mechanism might be involved in rapid effect of glucocorticoids on mast cells in allergic disease.     

Lymphocytolytic activity of glucocorticoids in vitro:51Cr release under equilibrium conditons

Summary Cytolytic activity of glucocorticoids in vitro is assessed by measuring radiochromium release from steroid-treated thymic lymphocytes under the equilibrium conditions provided by a continuous-labeling technique. Isotope release is a glucocorticoid-specific effect produced at physiological concentrations and is virtually abolished by inhibitors of RNA and protein synthesis. The relative lytic potencies of the steroids tested are comparable to those reported for glucocorticoids as measured by other methods. This procedure not only possesses the advantages typical of isotopic techniques in general, but, in addition, circumvents the problem of “spontaneous” label release associated with the pulse-labeling method. It is a useful alternative to the morphologic examination of cells or the estimation of cell viability for determination of glucocorticoid cytolytic activity. This work was supported by the Medical Research Council of Canada (MA-2996).     

Taurine supplementation restores glucose and carbachol-induced insulin secretion in islets from low-protein diet rats: involvement of Ach-M3R, Synt 1 and SNAP-25 proteins

Isolated islets from low-protein (LP) diet rats showed decreased insulin secretion in response to glucose and carbachol (Cch). Taurine (TAU) increases insulin secretion in rodent islets with a positive effect upon the cholinergic pathway. Here, we investigated the effect of TAU administration upon glucose tolerance and insulin release in rats fed on a normal protein diet (17%) without (NP) or with 2.5% of TAU in their drinking water (NPT), and LP diet fed rats (6%) without (LP) or with TAU (LPT). Glucose tolerance was found to be higher in LP, compared to NP rats. However, plasma glucose levels, during ipGTT, in LPT rats were similar to those of controls. Isolated islets from LP rats secreted less insulin in response to increasing glucose concentrations (2.8-22.2 mmol/L) and to 100 μmol/L Cch. This lower secretion was accompanied by a reduction in Cch-induced internal Ca(2+) mobilization. TAU supplementation prevents these alterations, as judged by the higher secretion induced by glucose or Cch in LPT islets. In addition, Ach-M3R, syntaxin 1 and synaptosomal associated protein of 25 kDa protein expressions in LP were lower than in NP islets. The expressions of these proteins in LPT were normalized. Finally, the sarcoendoplasmatic reticulum Ca(2+)-ATPase 3 protein expression was higher in LPT and NPT, compared with controls. In conclusion, TAU supplementation to LP rats prevented alterations in glucose tolerance as well as in insulin secretion from isolated islets. The latter effect involves the normalization of the cholinergic pathway, associated with the preservation of exocytotic proteins.     

Calcium administration increases calcium-binding protein regucalcin concentration in the liver of rats

The alteration of regucalcin concentrations in the liver and serum of rats administered orally calcium is investigated. Rats received a single oral administration of calcium chloride solution (25, 50 and 75 mg Ca/100 g body weight). The administration of calcium (50 mg/100 g) produced a significant increase in liver regucalcin concentration between 30 and 180 min after the administration, while serum regucalcin concentration was not altered appreciably. The effect of calcium administration increasing liver regucalcin concentration was also seen with the dose of 25 mg/100 g. When liver cytosol prepared from normal rats was incubated for 6 h in the presence of 10 M Ca²⁺, the cytosolic regucalcin concentration at 3 and 6 h of incubation was decreased about 20% (p<0.05) as compared with the value at zero time point, indicating that the presence of Ca²⁺ does not inhibit the decomposition of liver cytosolic regucalcin. Moreover, serum regucalcin concentration was not significantly altered by the incubation for 6 h at 37°C, indicating a stability of regucalcin in rat serum. This suggests that the calcium administration-induced in liver regucalcin concentration is not based on the inhibition of regucalcin release from liver to serum. The present study demonstrates that regucalcin in the liver is clearly increased by calcium administration, presumably due to stimulating the protein synthesis.     

The role of glycogen synthase phosphatase in the glucocorticoid-induced deposition of glycogen in foetal rat liver.

1. The mechanism that underlies the induction of glycogen synthesis in the foetal rat liver by glucocorticoids was reinvestigated in conditions where the accumulation of glycogen is either precociously induced with dexamethasone or inhibited by steroid deprivation. It appears that glucocorticoids act as the physiological trigger for glycogen synthesis by inducing both glycogen synthase (a known effect) and its activating enzyme, glycogen synthase phosphatase. 2. The activity of glycogen synthase phosphatase in adult liver stems from the interaction of two protein components [Doperé, Vanstapel & Stalmans (1980) Eur. J. Biochem. 104, 137--146]. Two independent experimental approaches indicate that the cytosolic 'S-component' is already well developed in the foetal liver before the onset of glycogen synthesis. The manifold glucocorticoid-dependent increase in synthase phosphatase activity during late gestation must be attributed to the specific development of the glycogen-bound 'G-component'.