ON THE RELATIONSHIP OF LIVER GLUCOSE-6-PHOSPHATASE TO THE PROLIFERATION OF ENDOPLASMIC RETICULUM IN PHENOBARBITAL INDUCTION

The differentiated effects of phenobarbital treatment on liver microsomal enzymes have been further studied. The relationship between the resulting decrease in the specific glucose-6-phosphatase activity and the enhancement of formation of endoplasmic reticulum membranes with high drug-hydroxylating activity has been investigated with biochemical and histochemical methods. Biochemically and histochemically demonstrable glucose-6-phosphatase activity was found to be present in all endoplasmic reticulum membranes, including the phenobarbital-induced smooth-surfaced proliferates, even though there was an over-all decrease in activity. Actinomycin D did not inhibit the decrease in glucose-6-phosphatase activity. The findings are discussed with reference to the enzyme-membrane relationship in phenobarbital induction.     

Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.     

FURTHER STUDIES ON THE INDUCTION OF THE DRUG-HYDROXYLATING ENZYME SYSTEM OF LIVER MICROSOMES

Further studies of the induction of the liver microsomal drug-hydroxylating enzyme system by pretreatment of rats with various drugs are presented. The phenobarbital-induced increase in the microsomal content of CO-binding pigment and in the activities of TPNH-cytochrome c reductase and the oxidative demethylation of aminopyrine is proportional, within certain limits, to the amount of phenobarbital injected. Removal of the inducer results in a parallel decrease in the levels of CO-binding pigment, TPNH-cytochrome c reductase, and aminopyrine demethylation. Other inducing drugs have been investigated and shown to act similarly to phenobarbital. The early increase in these enzymes is found in the microsomal subfraction consisting of rough-surfaced vesicles, whereas repeated administration of the inducing drug results in a concentration of the enzymes in the smooth-surfaced vesicles. The phenobarbital-stimulated formation of endoplasmic membranes is reflected in increased amounts of the various microsomal phospholipid fractions as revealed by thin layer chromatography. There is no significant difference between the stimulated rates of P_i³² incorporation into phospholipids of the two different microsomal subfractions in response to phenobarbital treatment. The drug-induced enzyme synthesis is unaffected by adrenalectomy.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS : I. The Site of Activity of Acyltransferases Involved in Synthesis of the Membrane Phospholipid

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.     

PHENOBARBITAL-INDUCED SYNTHESIS OF THE MICROSOMAL DRUG-METABOLIZING ENZYME SYSTEM AND ITS RELATIONSHIP TO THE PROLIFERATION OF ENDOPLASMIC MEMBRANES : A Morphological and Biochemical Study

Liver microsomes, isolated from rats which had been treated with phenobarbital in vivo, were found to exhibit increased activities of oxidative demethylation and TPNH-cytochrome c reductase and an increased amount of CO-binding pigment. Simultaneous administration of actinomycin D or puromycin abolished the phenobarbital-induced enzyme synthesis. Increased rate of P_i³² incorporation into microsomal phospholipid was the first sign of phenobarbital stimulation and appeared 3 hours after a single injection of this drug. Microsomes were divided into smooth-surfaced and rough-surfaced vesicle fractions. The fraction consisting of smooth-surfaced vesicles exhibited the greatest increase in protein content and oxidative demethylation activity after phenobarbital administration in vivo. Ultrastructural studies revealed that drug treatment also gave rise to proliferation of the endoplasmic reticulum in the hepatic parenchymal cells, first noticed after two phenobarbital injections. The phenobarbital-induced synthesis of the metabolizing enzymes is discussed with special reference to the relationship to the stimulated synthesis of the endoplasmic membranes.     

ON THE MECHANISM OF DRUG HYDROXYLATION IN RAT LIVER MICROSOMES

The TPNH- and O₂-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed.     

Differential time-course of induction of rat liver gamma-glutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, Golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis

This study was conducted to follow as a function of time the activity of gamma-glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg-1 body weight). Gamma-glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P-450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP-glucuronosyltransferase activity was not greatly altered by PB under the same conditions. The comparison of two-dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital-treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 x 10(3)) of hepatic gamma-glutamyltransferase was provisionally identified as a collection of polypeptide which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma-glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced by appearance of new gamma-glutamyltransferase isoforms in plasma membranes. We demonstrate that after a single injection of PB, gamma-glutamyltransferase activity increases simultaneously with some drug-metabolizing enzymes, such as total cytochrome P-450 but not with others, such as UDP-glucuronosyltransferases.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Δ^I-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.     

Induction of cytochrome P-450-dependent hydroxylases in primary monolayer cultures of rat hepatocytes

Hydroxylation of androstenedione was studied in rat hepatocytes in primary monolayer culture following induction with phenobarbital. Six days after addition of phenobarbital and seven days after isolation of cells from liver, a maximal induction of total androstenedione hydroxylation of 5–6 times was seen at a phenobarbital concentration of 1·10⁻⁴ M. The 6β-, 7α- and 16α-hydroxylase activities showed different responses towards phenobarbital in agreement with the contension that different forms of cytochrome P-450 with different sensitivity towards phenobarbital participate in hepatic steroid hydroxylation. These results were obtained with cells supplemented with 1% (v/v) rat serum. The present cell culture system should be suitable for in vitro studies on mechanisms of induction of cytochrome P-450-dependent enzymes in normal liver cells.     

A MORPHOMETRIC STUDY OF THE REMOVAL OF PHENOBARBITAL-INDUCED MEMBRANES FROM HEPATOCYTES AFTER CESSATION OF TREATMENT

It is well known that phenobarbital (PB) treatment produces an increase in the amount of cytoplasmic membranes of hepatocytes, with a parallel enhancement in the activity of drug-metabolizing enzymes. However, little is known about how the induced membranes are removed after the drug treatment is stopped. To consider this problem, the recovery of rat hepatocytes from PB induction (five daily injections, 100 mg/kg) was followed morphometrically. Treatment with PB produced a cellular enlargement (26%) due to increases in the volume of the cytoplasmic matrix (20%) and the volume (100%) and surface area (90%) of the smooth-surfaced endoplasmic reticulum (SER). The volume of the nuclei and the surface area of the Golgi apparatus were also increased, but no changes were detected in the volumes of the mitochondria or peroxisomes. The SER membranes induced by the PB were removed within 5 days after the end of the treatment period. During this period of membrane removal, we observed an increase in the volume (800%) and number (96%) of autophagic vacuoles without a change in dense bodies. A morphometric analysis of the content of the autophagic vacuoles showed that the endoplasmic reticulum membranes were preferentially removed, and from this we conclude that the formation of autophagic vacuoles was not a random process. Our findings show that the removal of excess cytoplasmic membranes is associated with an increase in autophagic activity and thus demonstrates the presence of a specific cellular mechanism which may be responsible for the bulk removal of PB-induced membranes.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS : II. The Site of Phospholipid Synthesis in the Initial Phase of Membrane Proliferation

The specific activity of the acyltransferases of smooth microsomes of rat liver rose threefold by 12 h after injection of phenobarbital, while the activity of the acyltransferases of the rough microsomes rose slightly to peak at 3–4 h, and subsequently fell. The latter rise was abolished by treatment of the animal with actinomycin D or puromycin, while that of the smooth microsomes was unaffected. Incorporation of [¹⁴C]glycerol into phospholipid of smooth microsomes was elevated 100% by phenobarbital, while that of the rough microsomes was elevated 15%, and this could be accounted for by exchange between the microsomal phospholipids. The phospholipid/protein ratio of the smooth microsomes rose 1.5 times 3–4 h after injection of phenobarbital, while that of the rough microsomes fell slightly. The specific activity of NADPH cytochrome c reductase and NADPH diaphorase rose first in the rough microsomes, and subsequently in the smooth microsomes at a time coinciding with the return of the phospholipid/protein ratio to the control level. The rise in phospholipid/protein ratio was unaffected by actinomycin D or puromycin. These results indicate that the proliferating smooth membranes are the site of phospholipid synthesis, and that the phospholipid/protein ratio of these membranes may change independently.     

Dietary orotic acid accentuates the hepatic response to phenobarbital in rats.

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.     

Phenobarbital induction of egasyn: availability of egasyn in vivo determines glucuronidase binding to membrane.

Murine egasyn, a protein which stabilizes the binding of β-glucuronidase to microsomal membranes, was induced 1.9 fold in liver by phenobarbital treatment. Accompanying this increase was an alteration of the subcellular distribution of liver β-glucuronidase, although total glucuronidase activity remained constant. In control mice 32.6 ± 4.6% of the activity was microsomal, while after four days of phenobarbital treatment 50.5 ± 3.1% was microsomal. Thus, the availability of egasyn appears to be an important factor in determining the proportion of glucuronidase distributed to either microsomes or lysosomes.     

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. II. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes.

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components. It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction. A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.     

Adoptive Infusion of Tolerogenic Dendritic Cells Prolongs the Survival of Pancreatic Islet Allografts: A Systematic Review of 13 Mouse and Rat Studies

Objective

The first Phase I study of autologous tolerogenic dendritic cells (Tol-DCs) in Type 1 diabetes (T1D) patients was recently completed. Pancreatic islet transplantation is an effective therapy for T1D, and infusion of Tol-DCs can control diabetes development while promoting graft survival. In this study, we aim to systematically review islet allograft survival following infusion of Tol-DCs induced by different methods, to better understand the mechanisms that mediate this process.

Methods

We searched PubMed and Embase (from inception to February 29^th, 2012) for relevant publications. Data were extracted and quality was assessed by two independent reviewers. We semiquantitatively analyzed the effects of Tol-DCs on islet allograft survival using mixed leukocyte reaction, Th1/Th2 differentiation, Treg induction, and cytotoxic T lymphocyte activity as mechanisms related-outcomes. We discussed the results with respect to possible mechanisms that promote survival.

Results

Thirteen articles were included. The effects of Tol-DCs induced by five methods on allograft survival were different. Survival by each method was prolonged as follows: allopeptide-pulsed Tol-DCs (42.14±44 days), drug intervention (39 days), mesenchymal stem cell induction (23 days), genetic modification (8.99±4.75 days), and other derivation (2.61±6.98 days). The results indicate that Tol-DC dose and injection influenced graft survival. Single-dose injections of 10⁴ Tol-DCs were the most effective for allograft survival, and multiple injections were not superior. Tol-DCs were also synergistic with immunosuppressive drugs or costimulation inhibitors. Possible mechanisms include donor specific T cell hyporesponsiveness, Th2 differentiation, Treg induction, cytotoxicity against allograft reduction, and chimerism induction.

Conclusions

Tol-DCs induced by five methods prolong MHC mismatched islet allograft survival to different degrees, but allopeptide-pulsed host DCs perform the best. Immunosuppressive or costimulatory blockade are synergistic with Tol-DC on graft survival. Multiple injections are not superior to single injection. Yet more rigorously designed studies with larger sample sizes are still needed in future.     

The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis

1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of (32)P into the different phospholipid constituents of microsomal membranes. 7. Nascent (14)C-labelled protein with the highest specific activity was recovered in the ;heavy' rough membrane fraction of microsomes, whereas little (14)C was associated with ;free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7-8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.     

Endoplasmic reticulum as a site of phenylpropanoid and flavonoid metabolism in hippeastrum

The nature of bound forms of enzymes of phenylpropanoid and flavonoid metabolism have been investigated in Hippeastrum CV Dutch Red Hybrid. Particulate components of petal homogenates were fractionated on sucrose gradients and the EDTA shift method was employed to characterize membranes of the endoplasmic reticulum. In magnesiumcontaining gradients, a portion of phenylalanine ammonia lyase, chalcone synthase, glucosyl transferase, and all of the trans-cinnamate 4-monooxygenase and NADH Cytochrome c reductase (the last an endoplasmic reticulum marker) were associated with membranes equilibrating at 1.18 specific gravity. In gradients lacking magnesium and containing EDTA, the above activities—except chalcone synthase, which was lost—and protein were diminished at 1.18 specific gravity and enhanced at lower densities characteristic of membranes of the smooth endoplasmic reticulum. These results are consistent with the contention that endoplasmic reticulum is a site of phenylpropanoid and flavonoid metabolism in Hippeastrum.