Dehydroepiandrosterone (DHEA) facilitates liver regeneration after partial hepatectomy in rats

In this study we investigated whether or not liver regeneration is facilitated by dehydroepiandrosterone (DHEA) after partial (70%) hepatectomy in rats. Treatment with DHEA (300 mg/kg body weight) did not cause any significant increase in the expression ratio of proliferating cell nuclear antigen (PCNA) in sham-operated controls; however, in partially hepatectomized rats it caused a significant increase in the ratio in hepatocytes 24 and 36 hr after hepatectomy. In partially hepatectomized rats, DHEA treatment significantly accelerated the restoration of liver 48, 60, and 72 hr after partial hepatectomy. The restoration rate in DHEA-treated hepatectomized rats at 72 hr was 1.3-fold greater than in partially hepatectomized controls. Treatment with androstenedione (300 mg/kg body weight), the first metabolite of DHEA, did not cause any significant increase in the expression of PCNA in either sham-operated controls or partially hepatectomized rats. These results indicate that DHEA itself promotes the liver regenerative process after partial hepatectomy in rats.     

HEPATOCYTIC PROLIFERATION IN NORMAL RATS AFTER MULTIPLE EXCHANGE TRANSFUSIONS WITH BLOOD FROM PARTIALLY HEPATECTOMIZED RATS

Either three or four (but not one or two) consecutive exchange transfusions (75–85% washout of blood) of rats with intact livers, utilizing blood from donors partially hepatectomized 24 hr earlier, results in increased incorporation of ³H-thymidine into hepatic DNA and hepatocytic nuclei 20 hr after the first transfusion. Exchange transfusion with blood from sham-hepatectomized animals does not produce this effect.     

Effect of pregnenolone-16alpha-carbonitrile, a microsomal enzyme inducer, on the regenerating rat liver.

PCN, a microsomal enzyme inducer, given orally (10 mg in 1 ml water twice daily for 5 days), increased liver weight and mitotic activity in intact as well as in partially hepatectomized rats. Electron microscopy revealed SER proliferation in the hepatocytes of animals treated with PCN alone. Accumulation of SER membranes was also evident in the liver cell cytoplasm of untreated, partially hepatectomized rats; it was however, more pronounced in the hepatocytes of partially hepatectomized animals given PCN. These results indicate that the steriod has a marked effect on the regeneration rat liver.     

The effect of alpha-melanophor-stimulating hormone on liver regeneration and incorporation of amino acid in rats' liver protein.

Following 25 mug/day synthetic alpha-MSH administration, the liver regeneration of partially hepatectomized rats proved to be increased. The hormone treatment resulted in an enhanced alanine incorporation of the liver proteins, but this effect was uncertain on partially hepatectomized rats. Due to the hormone treatment the low liver protein content of the operated rats became normal. The pseudocholinesterase activity of the liver homogenate of alpha-MSH treated rats was also elevated. On the basis of these experiments authors are supposing some protein synthesis increasing effect of synthetic alpha-MSH.     

Effects of ionizing radiation and partial hepatectomy on messenger RNA synthesis.

Newly synthesized messenger RNA, as measured by a 40 min uptake of the radioactive precursor (6-14C) orotic acid, was studied in the regenerating livers of non-irradiated and gamma-irradiated (1800 rad) adrenal-intact and adrenalectomized rats 24 aand 48 hours after partial hepatectomy. Two groups of rats, one with and one without adrenal glands, were each divided into four subgroups: (1) control rats, (2) irradiated rats, (3) partially hepatectomized rats and (4) irradiated, partially hepatectomized rats. The radioactive profile of polyribosome formation and distribution was determined by sucrose density gradient centrifugation (10--40 per cent). The result of this study indicates that ionizing radiation decreases the synthesis of newly formed messenger RNA in re generating livers of adrenal-intact rats. However, adrenalectomy largely abolished that inhibition. These data suggest that the decrease in messenger RNA synthesis may be explained by the disturbance of adrenal hormones induced by partial hepatectomy and ionizing radiation.     

EFFECT OF AN INHIBITING FACTOR ISOLATED FROM RAT LIVER ON DNA POLYMERASES IN REGENERATING RAT LIVER

We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G₁—S transition, and reduced in vivo [³H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented α DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on α DNA polymerase was detected. LIF did not affect β DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Labeling of non poly(A) associated RNA in the rabbit cerebral cortex 24 hours after a single electroconvulsive shock

The labeling pattern of non poly(A) associated (poly(A)^–) RNA of rabbit cerebral cortex was studied 24 hr after a single electroconvulsive shock (ECS). The animals were injected subarachnoidally with [³H]uridine and sacrificed 1 hr later. The fractionation pattern of labeled nuclear poly(A)^– RNA in the cerebral cortex of ECS treated animals was identical to that of the controls. However, microsomal poly(A)^– RNA from the treated animals showed an increased labeling of 18S ribosomal RNA. Also 28S RNA displayed a higher labeling but the effect was not statistically significant. These results indicate a more efficient production of ribosomal RNA in the late post-ECS period which might be in relationship with an increased activity of brain protein synthesis machinery.     

EFFECT of DOUBLE PARTIAL HEPATECTOMIES AT VARIOUS INTERVALS ON [3H]THYMIDINE INCORPORATION INTO RAT LIVER DNA

At various intervals after a 34% hepatectomy, another 34% (50% of the remnant) hepatectomy was performed on rats, and the [³H]thymidine incorporation into the DNA of remaining liver cells was measured 24 hr after the first operation. the values of [³H]thymidine incorporation into liver DNA of rats hepatectomized doubly (34% and 34%) at 6, 8 and 10 hr intervals were greater than the sum of the value of rats which received a single 34% hepatectomy at the start and those of rats which received a single 68% hepatectomy at 6, 8 and 10 hr, respectively.     

Effect of chromium(III) on nucleolar RNA synthesis

The enhancement of hepatic nucleolar RNA synthesis induced by Cr(III) in partially hepatectomized rats and its mechanisms are described. Cr(III)-administered (0.5 mg Cr/kg, ip) and then partially hepatectomized rats were significantly enhanced in the hepatic nucleolar RNA synthesis at the very early stage of liver regeneration. This enhancement was caused both by the induction of newly found nucleolar Cr-bound protein of 70 kD (Cr-p70) and by the activation of nucleolar chromatin, both of which arose from nuclear accumulation of Cr together with partial hepatectomy. Studies on the mechanism of this enhancement indicated that the Cr-p70 bound to the activated nucleolar chromatin and loosened its higher-order structure, resulting in an increase of the B-form fraction of chromatin DNA. The degree of this loosening well correlated with the amount of Cr-p70 bound to chromatin and also with the extent of elevation of RNA synthesis. Some molecular species of nonhistone proteins in chromatin were found to play an important role in the interaction to Cr-p70. These results suggest a possibility that the action of Cr is involved in cell proliferation process.     

Increase in liver glucose transporter mRNA levels during rat liver regeneration

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.     

Pretreatment with recombinant human interleukin 2 (rhIL-2) Up-regulates PCNA-positive cells after partial hepatectomy in rat liver

We prepared recombinant human interleukin-2 (rhIL-2) and studied its pretreated influence on liver regeneration and the blood profile in partially (67%) hepatectomized (PH) male Sprague-Dawley rats. Rats were injected in the tail vein with rhIL-2 three times per day for 3 consecutive days and 67% underwent a partial hepatectomy (PH). Five days after the PH, liver tissue and blood samples were analyzed for liver regeneration and hematological changes. The weight of the liver in the rhIL-2 pretreated groups increased in a dose-dependent manner; with the highest treatment (24 × 10⁴ IU/kg), the maximum liver weight of 88.6% was exhibited. The control group showed a gradual increase to 76.3% of the original liver weight. A histological analysis of the liver showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells in rhIL-2 pretreated rat livers. The rate of hepatocyte proliferation also increased significantly in primary cultured rat liver cells following rhIL-2 treatment. These results suggest that pretreatment with rhIL-2 may play adjuvant roles in liver regeneration after PH.     

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-(14)C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed.     

Ribosomal RNA synthesis in liver of adrenalectomized rats after partial hepatectomy

The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized — partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Thymidine metabolism in regenerating rat liver one to two hours after partial hepatectomy

1. When [(3)H]thymidine was injected intravenously into rats in amounts up to 40mg/kg body wt. and the (3)H radioactivity in the livers measured at 30min, saturation kinetics for thymidine uptake were not found. If the animals were examined 3 min after intravenous injection, saturation could be attained in normal rats with 12mg of thymidine/kg and in partially hepatectomized rats with 4mg/kg. At concentrations of thymidine close to saturation, no differences were found in rate or amount of uptake/g of liver between normal and partially hepatectomized rats 1-2h after operation. 2. Perfusion techniques were used to compare thymidine uptakes in the two sets of rats at concentrations up to 40mum-thymidine. Uptakes with tracer amounts of thymidine after 30min were identical in vivo and in the perfusion studies and were twice as great in livers from partially hepatectomized rats with concentrations up to 40mum-thymidine. 3. At 1.5h after operation there was nearly twice as much beta-aminoisobutyrate present per g of liver from partially hepatectomized as compared with normal rats.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

PERSISTENCE OF ALTERED RNA SYNTHESIS IN RAT CEREBRAL CORTEX 12h AFTER A SINGLE ELECTROCONVULSIVE SHOCK

The effect of electroshock (ECS) on RNA synthesis in nuclei and cytoplasm of rat cerebral cortex was examined using a double label technique by intraventricular injection of [³H] and [¹⁴C]orotate. At t h after ECS, the incorporation into nuclear RNA was 80% of the control rate and the appearance of newly synthesized RNA in the cytoplasm was only 27.6%. Analysis on composite polyacrylamide-agarose gels of purified RNA showed that the ³H/¹⁴C ratio of each gel slice slowly increased with decreasing M.W. of the RNA. This has been interpreted as an inhibition in the rate of processing of nuclear RNA. When the nuclear RNA was subjected to denaturation with 50% dimethyl sulphoxide (DMSO) this effect was enhanced. In a similar experiment, rats were injected, treated to ECS and killed 12 h later. The overall incorporation into nuclear and cytoplasmic RNA was increased to 174%, and 137.5% respectively. Analysis on gels showed very little variation in the ³H/¹⁴C ratio of the steady state levels of nuclear RNA. They compared well with a control experiment where rats were injected with [³H] and [¹⁴C]orotate as described above but no ECS was applied to the [¹⁴C] labelled animals. However a 1 h pulse label given 11 h after ECS treatment revealed that the rate of incorporation into nuclear RNA still showed a decrease of 81% of the control. The nuclear RYA fractionated on gels clearly showed that the inhibition of the processing rate of nuclear RNA was still occurring. This effect was again magnified on denaturation of the RNA with DMSO. This suggests that ECS may disturb RNA metabolism in nervous tissue for much longer periods than previously realised.     

Effect of an inhibiting factor isolated from rat liver on DNA polymerases in regenerating rat liver.

We partially purified an inhibitory factor (LIFE), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G1--S transition, and reduced in vivo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented alpha DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on alpha DNA polymerase was detected. LIF did not affect beta DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Beryllium toxicity. The selective inhibition of casein kinase 1.

1. Cyclic AMP-independent casein kinase 1 in liver cytoplasm and nuclei was inhibited by Be2+ in vitro (Ki 2.5 microM and 29 microM respectively). Casein kinase 2 (phosvitin kinase) and cyclic AMP-dependent protein kinase were unaffected. 2. The inhibition of casein kinase 1 by Be2+ was competitive with respect to the protein substrate; at non-saturating concentrations of casein, inhibition was non-competitive with respect to ATP. 3. In rats given LD50 doses of Be2+ 24 h before death, the activities of cytoplasmic and nuclear casein kinase 1 in livers from partially hepatectomized animals were diminished approx. 50%; with intact rats, nuclear casein kinase 1 was inhibited at concentrations of casein less than the Km.