DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

THE DEVELOPMENT OF GOLGI COMPLEXES AND THEIR DEPENDENCE UPON THE NUCLEUS IN AMEBAE

The production of Golgi complexes was investigated in Amoeba proteus by introducing a nucleus into cells that had been enucleated for 5 days. Golgi complexes were not detected in 5 day enucleates, nor were they observed in amebae fixed 15 min after renucleation. Samples taken at longer intervals after the introduction of a nucleus exhibited an increase in the size and abundance of Golgi complexes. Small curved smooth cisternae, some of which were aligned in parallel to form small Golgi complexes, were observed 30 min after the operation. Aggregations of small Golgi complexes increased in number in amebae fixed 1 to 6 hr after renucleation. Golgi complexes of normal size were present 6 hr after the operation and became more abundant in samples fixed 12 hr, and 1, 2, and 3 days after renucleation. The possible participation of the granular endoplasmic reticulum in the development of Golgi complexes was suggested by two observations. First, the Golgi complexes in renucleates contained a dense material similar to the content of the endoplasmic reticulum in enucleates and early renucleates. Second, examples of continuity between the endoplasmic reticulum and Golgi cisternae were present in renucleates. The possibility that Golgi complexes can be produced in the absence of preexisting Golgi complexes is discussed.     

THE EFFECTS OF ENUCLEATION ON THE CYTOPLASMIC MEMBRANES OF AMOEBA PROTEUS

The dependence of cytoplasmic membranes upon the nucleus was studied by examining enucleated amebae with the electron microscope at intervals up to 1 wk after enucleation. Amebae were cut into two approximately equal parts, and the fine structure of the enucleated portions was compared with that of the nucleated parts and starved whole cells which had been maintained under the same conditions. Golgi bodies were diminished in size 1 day after enucleation and were not detected in cells enucleated for more than 2 days. The endoplasmic reticulum of enucleated cells appeared to increase in amount and underwent changes in its morphology. The sparsely scattered short tubules of granular endoplasmic reticulum present in unmanipulated amebae from stock cultures were replaced in 1–3-day enucleates by long narrow cisternae. In 3–7-day enucleates, similar cisternae of granular endoplasmic reticulum encircled areas of cytoplasm partially or completely. It was estimated that in most cases hundreds of these areas encircled by two rough membranes were formed per enucleated cell. The number of ribosomes studding the surface of the endoplasmic reticulum decreased progressively with time after enucleation. In contrast, the membranes of nucleated parts and starved whole cells did not undergo these changes. The possible identification of membrane-encircled areas as cytolysomes and their mode of formation are considered. Implications of the observations regarding nuclear regulation of the form of the Golgi apparatus and the endoplasmic reticulum are discussed.     

ESTROGEN-INDUCED CYTODIFFERENTIATION OF THE OVALBUMIN-SECRETING GLANDS OF THE CHICK OVIDUCT

The histological, ultrastructural, and biochemical changes occurring during hormone-induced cytodifferentiation of the ovalbumin-secreting glands in the chick oviduct have been studied. Marked perivascular edema is an initial response of the immature oviduct stroma to diethylstilbestrol administration and is accompanied by an interstitial migration of mononuclear cells. Mitotic activity in the immature mucosal epithelium increases within 24 hr, and glands begin to develop on days 2–4 as budlike invaginations into the subepithelial stroma. An immediate intracellular effect of the hormone is aggregation of previously dispersed ribosomes. Ribosomal zones in the nucleolus gain prominence, and there is a progressive development of rough endoplasmic reticulum in the epithelial cells. Extensive profiles of endoplasmic reticulum are present in the gland cells by day 6. Fine apical progranules appear in the epithelial cells on day 2, and ovalbumin can be measured immunochemically by day 3 at about the same time that new species of nuclear RNA have been identified. Ovalbumin granules form within condensing vacuoles in the Golgi zone and begin to be released into the lumina of the gland acini at about day 6 of the treatment.     

IN VITRO STIMULATION OF ENZYME SECRETION AND THE SYNTHESIS OF MICROSOMAL MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis: intact patches of membrane may be internalized and then reutilized within the cell; alternatively these membranes may be either disassembled to subunits or degraded. In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex. In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0.0005 M puromycin at high ionic strength followed by mild (pH 7.8) alkaline extraction with 0.2 M NaHCO₃. Such treatments release the majority of nonmembrane components of both microsomal fractions (i.e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation. The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0.0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated. In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased. In contrast, the synthesis of microsomal membrane proteins was unchanged. These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle.     

CYTOLOGICAL ALTERATIONS RELATED TO STIMULATION OF THE ZONA GLOMERULOSA OF THE ADRENAL GLAND

The structure of the zona glomerulosa of the rat adrenal gland stimulated by sodium restriction has been studied by light and electron microscopy. The major changes observed during the course of the experiment in stimulated glands involve cytoplasmic droplets, mitochondria, and the endoplasmic reticulum. There is a progressive decrease in the number of cytoplasmic droplets of low electron opacity. Numerous, greatly elongated mitochondria containing parallel arrays of tubules are noted. These tubules extend from within the mitochondria through gaps in the mitochondrial-limiting membranes into the cytoplasm. In addition, amorphous intramitochondrial deposits, possibly aldosterone precursors, are seen. Increased amounts of smooth-surfaced endoplasmic reticulum, often showing complex arrangements, are another feature of the stimulated zona glomerulosa. Other alterations include the presence of large numbers of dense bodies as well as cytoplasmic droplets of high electron opacity. These observations are discussed in relation to the biosynthesis of aldosterone.     

Organelle Membranes from Germinating Castor Bean Endosperm: II. ENZYMES, CYTOCHROMES, AND PERMEABILITY OF THE GLYOXYSOME MEMBRANE

Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

Effect of retinol deficiency on the activity and solubility of various enzymes of the endoplasmic reticulum membranes in the rat liver

Solubilization by sodium deoxycholate and trypsin of some metabolic enzymes of unrelated compounds associated with endoplasmic reticulum membranes was carried out. The effects of urea, butanol and detergents on the retinol content in the membranes were studied. It was shown that retinol deficiency causes changes in the interactions of NADH-arylesterase with microsomal membrane components that are manifested in the decrease of the activating effect of butanol and low detergent concentrations on the NADH-reductase activity as well as in the increase in the damaging effect of urea and high detergent concentrations on the enzyme activity. Under conditions of retinol deficiency, the degree of solubilization of NADH-reductase, hydroxylase and arylesterase in the presence of sodium deoxycholate is enhanced. After treatment of liver microsomes of retinol-deficient animals with trypsin or with a trypsin-sodium cholate mixture, the content of these enzymes in the supernatant becomes much greater than that in liver microsomes of vitamin A-deficient rats. It is assumed that retinol deficiency causes of weakening of hydrophobic interactions within the membrane as well as partial translocation of the enzymes from the hydrophobic to the hydrophilic layer.     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.     

THE STRUCTURE OF SOME CYTOPLASMIC COMPONENTS OF PLANT CELLS IN RELATION TO THE BIOCHEMICAL PROPERTIES OF ISOLATED PARTICLES

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported.     

EFFECT OF DEHYDROASCORBIC ACID ON THE ISLETS OF LANGERHANS OF THE RAT PANCREAS

Rat pancreatic islets have been studied following successive daily administration of dehydroascrobic acid (DHA) and during the recovery phase following 3 daily injections. One injection of DHA produces degranulation of B cells seen in the light microscope as a loss of aldehyde fuchsin positivity. In the electron microscope the B cells appear to have secretory granules accumulated subjacent to the plasma membranes. Following 2 and 3 daily injections, B cells evidence alterations in the organization of the granular endoplasmic reticulum and mitochondria, and secretory granules are scant but when present are subjacent to the plasma membrane. After 5 to 7 days' recovery few secretory granules remain in B cell cytoplasm, but the cells have prominent granular ER and a Golgi apparatus with numerous prosecretory granules. The primary effect of DHA is an exaggerated secretory response of B cells, which is intensified with subsequent injections. Necrosis of B cells as produced by alloxan is not seen.     

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.     

Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis.

Early stages of rat thymocyte apoptosis measured as annexin-V positive events and induced by methylprednisolone (MPS), etoposide, and thapsigargin, showed a sequential increase in nitric oxide (NO) production by mitochondrial and endoplasmic reticulum membranes. Thapsigargin induced the highest NO production, a sevenfold increase as compared with untreated thymocytes, in mitochondrial and microsomal membranes. MPS and etoposide were equally effective in increasing NO production by mitochondrial membranes by a factor of 4-5, with only a slight increase in NO production by endoplasmic reticulum membranes. Western blot analysis of both types of membrane indicated that a nitric oxide synthase (NOS) isoenzyme is present in mitochondrial membranes and reacts with antibodies to i-NOS (type II), while reactivity to antibodies to e-NOS (type III) was restricted to endoplasmic reticulum. The participation of endoplasmic reticulum during apoptosis was further determined by alterations in UDP-Glucosyltransferase (UDP-GT) and NADPH cytochrome P450 reductase. Increased UDP-GT activity was observed after thapsigargin treatment, and no changes were found after treatment with etoposide or MPS. NADPH cytochrome P450 reductase activity markedly decreased during apoptosis, being stronger after thapsigargin treatment. The latest stage of the apoptotic process was measured by caspase activities. Caspase 3 activity was markedly increased by the three apoptosis inducers; caspase 6 was only activated by MPS and etoposide, while caspase 8 was not activated by any of these inducers. It is clear that mitochondria and endoplasmic reticulum are involved in thapsigargin induced thymocyte apoptosis. Meanwhile, other thymocyte apoptotic pathways, such as those induced by MPS or etoposide, seem to centrally involve mitochondria but not endoplasmic reticulum.     

Structure-function relationships during preovulatory development of porcine follicles following equine chorionic gonadotropin stimulation.

Porcine follicular maturation begins by recruitment from a continually proliferating pool of small antral follicles; those receiving the appropriate stimulus differentiate rapidly through a series of structural and functional changes. Such ovarian activity can be induced in prepubertal gilts with a single injection of equine chorionic gonadotropin (eCG). Average follicular diameter in eCG treated females increased from approximately 2 mm before stimulation to 3.5 mm by 24 hr after injection, with subsequent growth to ovulatory size (8 or 9 mm) by 96 hr. Both theca and granulosa layers increased in thickness and complexity, and a prominent capillary bed evolved immediately outside the basement membrane separating the two layers. Cytoplasmic organelles associated with increased metabolic activity and steroidogenesis proliferated within the first 24 hr. Progressive changes included increasing amounts of lipid and rough and smooth endoplasmic reticulum, with the latter occurring in vesicular or lamellar forms and as lipid-associated whorls. Bizarre mitochondrial forms also appeared, often associated with lipids. The amount and proportion of rough and smooth endoplasmic reticulum shifted dramatically as follicles matured. By 24 hr, rough endoplasmic reticulum in thecal cells increased from 4.2 to 7% of cell volume, while the amount in granulosa cells increased from less than 3.5% to more than 10%; the quantity remained relatively constant in the theca but declined to prestimulation values in the granulosa layer. Rough endoplasmic reticulum predominated over smooth in the first 24 hr following stimulation but the proportions were then reversed, so that more than 10% of both layers was composed of smooth endoplasmic reticulum by the time ovulation was imminent. Some follicles had or were in the process of ovulating by 96 hr. Their walls were collapsed into prominent folds with the two cell types beginning to mix. Slight undulations and some regions of discontinuity were observed in basement membranes of large unovulated follicles at this time. In specimens collected at 96 hr poststimulation and processed for retention of lipid, lipid-like material was noticeable in the extracellular matrix surrounding cells that contained organelle configurations suggestive of steroidogenesis.     

LONG-TERM ORGAN CULTURE OF THE SALAMANDER HEART

Beating salamander hearts were maintained in tissue culture for periods ranging from 1 to 6 months. After 1, 3, or 6 months of culture, six hearts, along with six control hearts, were fixed for electron microscopy. In control tissue, the sarcoplasmic reticulum usually demonstrated the normal pattern of paired, linearly arranged membranes, although in some cases, the reticulum showed a variation from these membranes to a series of small vesicles. There was no evidence of a T-system of tubules in any of the material examined. Desmosome-Z band complexes were observed in almost all sections of both control and experimental material. A possible role of these complexes in the excitation-contraction mechanism is discussed. In 3 month cultured material, alterations in normal myofibrillar pattern occurred. Small segments of myofibrils branched from one Z band to join the Z band of an adjacent myofibril, or appeared to be fraying out into the sarcoplasm. In 6 month cultured material, myofibrils were fragmented into short segments from which myofilaments frayed out into the sarcoplasm. This filamentous material may be remnants of myofilaments. Despite the morphological changes in myofibrils, the heart pulsation rate, established at the beginning, was maintained throughout the culture period. It is suggested that the alterations, observed in the experimental material, occurred in elements not essential for heart beat maintenance, or that these alterations have not yet progressed to a critical point of affecting the heart beat.     

RADIOAUTOGRAPHIC VISUALIZATION OF THE INCORPORATION OF GALACTOSE-3H AND MANNOSE-3H BY RAT THYROIDS IN VITRO IN RELATION TO THE STAGES OF THYROGLOBULIN SYNTHESIS

Rat thyroid lobes incubated with mannose-³H, galactose-³H, or leucine-³H, were studied by radioautography. With leucine-³H and mannose-³H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-³H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-³H and mannose-³H, but has no detectable effect on galactose-³H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-³H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-³H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.     

Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins

The transfer of triglyceride from sites of synthesis in the endoplasmic reticulum to cytoplasmic lipid droplets and nascent VLDL (very low density lipoproteins) in rat liver in vivo has been examined with [3H]glycerol, cell fractionation, and electron microscopy. Rates of mass transfer of newly synthesized triglyceride were estimated from the specific radioactivity of triglyceride present in microsomal membranes and the radioactivity observed in recipient triglyceride pools. Fasting decreased the transfer of triglyceride to nascent VLDL without affecting transfer to lipid droplets. Stimulation of triglyceride synthesis with 2-tetradecylglycidic acid (TDGA) increased transfer of triglyceride to nascent VLDL 5-fold, and to lipid droplets 14-fold, 1 hr after TDGA administration. Triglyceride transfer to nascent VLDL was increased 6-fold, and to lipid droplets 37-fold, above control rates 6 hr following TDGA treatment, indicative of saturation of triglyceride assembly into nascent VLDL and storage of excess triglyceride in lipid droplet reservoirs. These liver triglyceride pools were concurrently expanded and electron microscopy demonstrated more abundant VLDL particles in the endoplasmic reticulum together with a proliferation of lipid droplets in hepatocytes. TDGA progressively decreased hepatic sn-glycerol-3-phosphate in fasting rats while triglyceride synthesis increased, indicating that sn-glycerol-3-phosphate does not limit the rate of triglyceride synthesis in this metabolic state. Results implicate triglyceride transfer from endoplasmic reticulum membranes to nascent VLDL as a regulated determinant of hepatic VLDL assembly and VLDL triglyceride secretion in vivo.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.