Search

Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes. NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.

Background: It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation., Methods: HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM)., Results: First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive., Conclusions: Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans., (Copyright © 2016 by the Research Society on Alcoholism.)

The VLDL transport vesicle (VTV) mediates the transport of nascent VLDL particles from the ER to the Golgi and plays a key role in VLDL-secretion from the liver. The functionality of VTV is controlled by specific proteins; however, full characterization and proteomic profiling of VTV remain to be carried out. Here, we report the first proteomic profile of VTVs. VTVs were purified to their homogeneity and characterized biochemically and morphologically. Thin section transmission electron microscopy suggests that the size of VTV ranges between 100 nm to 120 nm and each vesicle contains only one VLDL particle. Immunoblotting data indicate VTV concentrate apoB100, apoB48 and apoAIV but exclude apoAI. Proteomic analysis based on 2D-gel coupled with MALDI-TOF identified a number of vesicle-related proteins, however, many important VTV proteins could only be identified using LC-MS/MS methodology. Our data strongly indicate that VTVs greatly differ in their proteome with their counterparts of intestinal origin, the PCTVs. For example, VTV contains Sec22b, SVIP, ApoC-I, reticulon 3, cideB, LPCAT3 etc. which are not present in PCTV. The VTV proteome reported here will provide a basic tool to study the mechanisms underlying VLDL biogenesis, maturation, intracellular trafficking and secretion from the liver.

Phase contrast electron microscopy utilizing phase plates has been considered suitable for high-contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high-contrast gain even for strong phase objects. This result clearly indicates that phase contrast electron microscopy can be effectively used not only for weak phase objects but also for strong phase objects in biology.

The structure and functional activity of the centrosome was analyzed in hepatocytes of 5-day old mice, as well as the lengths of Golgi complex cistemae. In the early postnatal development of mice, the liver was represented by two types of hepatocytes: in the first type hepatocytes, the centrosome was active as an organizing center of microtubules, while in the second type hepatocytes, it was inactive. It was proposed that during ontogenesis the centrosome is inactivated as an organizing center of microtubules and activated as an organizing center of intermediate filaments characteristic for differentiated hepatocytes of adult liver. Morphometry of the Golgi complex has shown that Golgi cisternae in the cell center area of early postnatal hepatocytes were longer than in the adult hepatocytes and comparable to those in G1-phase hepatocytes of regenerating liver. The possibility of relations between the differences in the Golgi complex morphology and ontogenetic changes in the functional activity of centrosomes is discussed.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine-phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [gamma-32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G/E fractions. Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Src-containing signalling platforms.

As we have observed previously, rat liver Golgi complexes are very useful cell organelle in investigating the effectiveness of various drugs with cytoprotective or normalizing activities in streptozotocin (STZ)-induced diabetes. The diabetes-associated biochemical and morphological alterations in the organelle were investigated in four groups of rats: control and STZ-diabetic (C and D groups) and compared with the same groups treated with 1.5 mM metavanadate administered as drinking solutions in 0.09 M NaCl over 7 days (C+V and D+V groups). Apart from the untreated group C, a decrease of body weight during the experiment was noted in the remaining three groups, reaching a statistically significant level in the diabetic groups (c. 15%). Fluid and food intake were statistically significantly (p<0.001) limited in both the vanadium treated groups. In the diabetic group treated with metavanadate, the free blood sugar level decreased, but euglycemia was not achieved. In groups C+V, D and D+V, the activity of Golgi marker enzyme, i.e. galactosyltransferase (GalT), was statistically lower as compared with group C (p<0.001). The treatment of diabetic rats with 1.5 mM NaVO3 [V(V)I in 0.09 M NaCl as a drinking solution during 7 days did not normalize the yield of Golgi membrane preparations or the Golgi marker enzyme activity. Electron microscopy revealed marked ultrastructural changes triggered by the employed vanadium compound. A striking change was seen in the presence of giant intracytoplasmic vacuoles. These alterations were seen in both experimental groups, i.e. C+V and D+V. Group C+V showed more advanced ultrastructural changes, what was expressed in a poorer state of mitochondrial membranes, a greater number of vesicular structures and less frequently seen Golgi structures. In spite of the fact that the animals were exposed to two compounds with a strong biological activity, the group of diabetic rats treated with metavanadate (D+V) showed no such advanced changes: more numerous Golgi structures were noted and their form was practically ring-like, i.e. characteristic for this group of organelles.

The paper presents the effect of one-week 3mM sodium orthovanadate (Na3VO4) oral treatment of control and streptozotocin[STZ]-diabetic rats. The body weight decreased as compared with untreated control (C group) in both vanadate treated groups (C + V and D + V) and in diabetic untreated rats (D group)--in all cases p < 0.01. A similar tendency was demonstrated by the weight of the livers, which was statistically significant lower than in the controls (p < 0.01). The fluid and food intake were lower in comparison with control vanadium treated groups, in D + V as compared with D it was limited, however, not achieved control level. A high mortality rate, approx. 67%, after the administration of streptozotocin and vanadate (D + V group) was noted; such result had never been previously found within all study groups of rats. But the surviving rats show very good decreased (60%) free blood sugar levels, however euglycaemia was not achieved. The activity of galactosyltransferase, the Golgi complex marker enzyme in group D, was statistically lower than the controls (p < 0.001). Treatment of STZ-diabetic rats with orthovanadate did not increase the enzyme activity toward control level, in both diabetic groups (treated and untreated with Na3VO4) similar dispersion of individual results was found. Morphological study demonstrated, for the first time, no larger cellular lesion in C + V group. The Golgi complex was well developed; showed several cisterns at the trans side, which were grossly distended and contained electron-lucid floccular material. In D + V group typical, cylindrical forms of Golgi complexes predominated. These structures consisted of 3-4 almost practically non-distended cisterns. Also in this case, large, electron-dense vesicles were noted in the vicinity. In this group, small in size, myelin-like structures were also found. These structures might indicate a relatively small, but nevertheless clear damage of the internal membrane system. The external cistern of the cylindrical forms of Golgi complexes, which corresponded the trans side, was often markedly distended and formed a vacuole-like structure filled with electron lucent material; the structure itself sometimes looked empty. Multi-vesicular structures were observed also in this case, but they were seen much more rarely.

Twenty years ago, we detected the interdependence between structure and function of rat liver Golgi complexes that are characteristic for streptozotocin diabetes, which served us in further investigations as a useful indicator of the effectiveness of drugs we were testing. This work presented results obtained in eight groups of rats (four control and four diabetic) that were administered orally either bis(maltolato)oxovanadium(IV) [BMOV] or maltol alone. The activities of the rat liver Golgi marker enzyme, galactosyltransferase [GalT], as well as the morphology of Golgi complexes were studied in situ using an electron microscope; parallel estimations of vanadium concentration and phospholipid percentage were made in Golgi-rich preparations isolated from the liver. Our main findings were normalization in diabetic animals orally treated with 1.8 mmol BMOV in 0.09 mol NaCl solutions over seven days, which demonstrated an accompanying increase in phosphatidic acid (PA) percentage (p < 0.05) compared to controls. In the diabetic groups, Pearson's test showed a positive double correlation between GalT activity, vanadium concentration, and PA percentage in Golgi-rich membrane preparations from the liver. Additionally, a negative correlation was found between vanadium concentration and phosphatidylcholine percentage in the fractions.

The authors studied the effect of a short (2 days) oral treatment of rats with bis(kojato)oxovanadium (IV) [VO(ka)2] as 1.8 mmol liquid solution on the biochemical activity and morphology of liver Golgi complexes (Group pVC). Such a short treatment induced greater changes than a longer (1 week) application of the same vanadium compound, what had been observed previously. Especially the Golgi marker enzyme activity (GalT) was the highest among all the investigated groups, and additionally, the greatest dispersion of result was obtained in this group. This group of animals showed twisted Golgi complexes, which--apart from 2-3 narrow cisterns--often contained 1-2 grossly distended cisterns filled with clear, floccular contents. The fairly long cisterns were irregular in shape and were often bent, forming ring-like structures. In the second experiment, we studied the effect of the ligand alone (kojic acid) (Group C+ka2) employed in the same way as in the case of the previously used vanadium complex (time and concentration). The biochemical parameters (body and liver weight, liquid and food intake, blood sugar level and GalT activities) were the same as in the untreated control group (C). Contrary to the biochemical findings, the morphology of Golgi complexes changed in effect of 3.6 mmol kojic acid application (the same application, time and concentration as in the whole complex with vanadium) over a 1-week period, manifesting the stimulation of exocytosis (in the trans region and directed toward plasma membrane), with the cisterns of Golgi dictyosomes being rounded or oval in more than 85% of cases. In this group, electron microscopy revealed the presence of two types of Golgi complexes, namely 2-3 short, slightly arched cisterns grossly distended at both ends and filled with clear, floccular material, as well as vacuoles with the same contents, which were visible in the vicinity of the cisterns. The other type, which was observed less frequently, was represented by Golgi complexes formed by haphazardly twisted cisterns.

Intermediate filaments are one of the three major cytoskeletons. Some roles of intermediate filaments in cellular functions have emerged based on various diseases associated with mutations of cytokeratins. However, the precise functions of intermediate filament are still unclear. To resolve this, we manipulated intermediate filaments of cultured cells by expressing a mutant cytokeratin. Arginine 89 of cytokeratin18 plays an important role in intermediate filament assembly. The expression of green fluorescent protein-tagged cytokeratin18 arg89cys induced aggregations and loss of the intermediate filament network composed of cytokeratins in liver-derived epithelial cells, Huh7 and OUMS29, but only induced the formation of cytokeratin aggregates and did not affect the intermediate filament network of endogenous vimentin in HEK293. The expression of this mutant affected the distribution of Golgi apparatus and the reassembly of Golgi apparatus after perturbations by nocodazole or brefeldin A in both Huh7 and OUMS29, but not in HEK293. Our data show that loss of the original intermediate filament network, but not the existence of cytokeratin aggregates, induces redistribution of the Golgi apparatus. The original intact intermediate filament network is necessary for the organization of Golgi apparatus., (Copyright 2003 Wiley-Liss, Inc.)

The authors describe the influence of vanadyl sulphate on liver Golgi complexes in control and streptozotocin (STZ)-diabetic rats. VOSO4, one of inorganic vanadium compounds widely used in animal models and human diabetes, acts as an insulin-mimetic drug and is relatively well known as a complex activated or inhibited on many enzymes involved in carbohydrate or lipid metabolic pathways. A relatively small in scope investigation was performed on subcellular levels, while changes of Golgi complexes under vanadium influence have not been described with the exception of our previous investigations with four organic derivatives. This paper presents the action of vanadyl sulphate used in 3mM in 0.5% NaCl as a drinking solution for 7 days on control and STZ-diabetic rat liver Golgi complexes. Changes induced by this vanadium compound were greater in the controls as compared to the diabetic rats, what was true for both biochemical and morphological data. Physiological and biochemical analyses showed a partial normalization of the investigated parameters in diabetic animals after short time treatment with vanadyl ions, although STZ-diabetic, vanadium treated rats were affected by two types of adverse effects exterted by these compounds. The controls manifested more numerous and advanced subcellular changes. The moderately developed Golgi apparatus showed no major changes. In the control group, subcellular changes were seen sporadically. More extended Golgi complexes showed certain anomalies.

Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Among the previously studied organic vanadium derivatives showing an anti-diabetic action, we investigated a new complex, bis(2,2'-bipyridine)oxovanadium(IV) sulphate. We tested its ability to normalise parameters previously described for streptozotocin (STZ)-diabetes, such as lower yields of Golgi-rich membrane fraction isolation, decreased activity of Golgi membrane marker enzyme - galactosyltransferase (GalT) - and altered morphology of rat liver Golgi complexes. Oral application as a drinking solution of 1.8 mmol bis(2,2'-bipyridine)oxovanadium(IV) (dissolved in 0.09 M NaCl) caused a similar dispersion of GalT activities in both vanadium treated groups, control and diabetic. Very low activities of the enzyme (characteristic for untreated diabetes) we found only in approximately 35 % of the STZ-diabetic rats treated with the new vanadium compound. The morphology of liver Golgi complexes in diabetic rats treated with bis(2,2'-bipyridine)oxovanadium(IV) sulphate was improved, which manifested itself in the reappearance of vacuoles with VLDL and coated and uncoated secretory vesicles. In view of biochemical and morphological parameters of normalised diabetic rat liver Golgi apparatus, the new vanadium complex was more effective than bis(oxalato)oxovanadium(IV) or bis(kojato)oxovanadium(IV), but in our experimental model, the best anti-diabetic, orally applied drug was the bis(maltolato)oxovanadium(IV) previously investigated.

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.

In this study oral treatment with bis(kojato)oxovanadium(IV) solution was administered twice. The first, short vanadium treatment (so called pretreatment) was used to accustom animals to the flavour of this liquid. After 2-2.5 weeks the second treatment, in high concentration and for a longer time served as a "drug". The physiological parameters such as weights of animals and their livers, reduction of liquid and food intake were lower than in control. Activity of Golgi marker enzyme GalT was significantly lower in both vanadium treated groups (p < 0.01), but in diabetic vanadium treated group it was higher, albeit not significantly, than in control vanadium treated rats. The morphology of Golgi complexes in diabetic rats on prolonged vanadium treatment was similar to that in the one week treated rats (see Part I). Rounded stacks of cisternae characteristic of untreated streptozotocin (STZ)-diabetes were also seen in vanadium treated diabetic rats, but in comparison with untreated diabetic livers, the secretory activities of Golgi complexes were preserved or even stimulated.

Oral treatment with maltol or bis(maltolato)oxovanadium(IV) [BMOV] alters the biochemical activity of the rat liver Golgi marker enzyme, i.e., galactosyltransferase (GalT), and the organelle morphology in a relatively short time. Four groups of rats were investigated: control (C), treated with BMOV for 2 days (pVC), treated with BMOV for 7 days (C+V), and treated with maltol alone for 7 days (C+M). All drugs were administered as drinking solutions. These conditions were used, because normalization of galactosyltransferase activity (GalT) and morphology of rat liver Golgi complexes were previously found by us in streptozotocin-induced diabetes. In this paper, we present the influence of BMOV or maltol alone (as a vanadium ligand in BMOV compound) on rat liver Golgi complexes. The lowest statistically significant enzyme activity, in comparison with three other groups of rats (p < 0.01), was found in rats treated with BMOV solution for two days (pVC). Liver Golgi complexes in these rats showed relatively slight changes as compared with controls. The activity of GalT was similar to controls of the C+V and C+M groups. Morphological examinations of the Golgi apparatus in rats treated with vanadium salts revealed a slightly increased secretory activity. In response to various agents used in experiments, the Golgi complexes were generally reduced in size, except for the (C+M) group. Not only cisternae, but also vacuoles and associated vesicles on both sides of stacks were reduced in almost all Golgi structures. Ultrastructural findings were generally in agreement (except for pVC group) with biochemical results (yields of liver Golgi-rich fractions, activity of galactosyltransferase) obtained in the same rats.

In comparison with untreated control, reduced body and liver weights were found in two groups of rats (such as control and STZ-diabetic) treated orally with bis(kojato)oxovanadium(IV) solution. Free blood sugar in STZ-diabetic rats was lower by about 38%, but did not achieve euglycemic values. Yields of Golgi-rich fraction were lower than those in untreated controls, similar to the activity of galactosyl transferase (GalT) in both vanadium treated groups (control and diabetic). Under electron microscope in the control kojate-treated group, subcellular changes were observed. The morphology of Golgi apparatus was typical, resembled that in untreated animals. In diabetic animals treated with kojate subcellular changes were less severe. Golgi apparatus was usually semicircular or arched in shape similar to that observed previously in diabetic untreated rats.

A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smooth endoplasmic reticulum, and the rough endoplasmic reticulum. Total microsomes were prepared from rat liver by differential centrifugation and resuspended in 20% iodixanol. The microsomal suspension was then layered between a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h. The microsomes distributed in four visible bands. The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyltransferase (Golgi marker) NADPH-cytochrome c reductase (endoplasmic reticulum marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Golgi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1. 090 g/ml), which contains 68% of the galactosyltransferase activity, consists of vesicles derived from the Golgi. The second broad band in the middle of the tube (density 1.130-1.160 g/ml), which contains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1. 220 g/ml) appear to contain two subfractions of vesicles derived from the rough endoplasmic reticulum., (Copyright 1999 Academic Press.)

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo., (Copyright 1999 Academic Press.)

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.

Activated Kupffer cells and macrophages accumulate in necrotic areas in the liver. Osteopontin, an extracellular matrix with RGD sequence, has been shown to act as a chemokine that can induce monocyte migration. The possibility that osteopontin can play a role in infiltration of both cells into hepatic necrotic areas was investigated in rats. Northern blot analysis revealed that osteopontin mRNA expression was minimal in Kupffer cells and hepatocytes immediately after isolation from normal rats, but slight in hepatic stellate cells assumed nearly quiescent in function after 3 days of culture on plastic dishes. When rat received carbon tetrachloride, liver necrosis developed between 1 and 3 days following the intoxication. In these rats, osteopontin mRNA expression assessed by quantitative competitive RT-PCR was increased in the liver later than 1 day with its peak at 2 days following the intoxication. Kupffer cells and hepatic macrophages and hepatic stellate cells isolated from such liver showed marked expression of osteopontin mRNA on Northern blotting. Immunohistochemical examination disclosed that osteopontin was stained in macrophages including Kupffer cells and stellate cells in the necrotic areas. On electron microscopy, osteopontin stains were present in the Golgi apparatus in these cells. Recombinant human osteopontin promoted migration of Kupffer cells isolated from normal rats and cultured in a Transwell cell culture chamber in a dose-related manner. We conclude that activated Kupffer cells and hepatic macrophages and stellate cells express osteopontin. These cells might contribute to the infiltration of Kupffer cells and macrophages into hepatic necrotic areas by expressing osteopontin., (Copyright 1999 Academic Press.)

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5'-adenylylimidodiphosphate, and 100 microM but not 20 microM vanadate. Motility was also blocked by GTPgammaS or A1F4- but was insensitive to A1C13, NaF, staurosporin, or okadaic acid. The targets for GTPgammaS and A1F4- were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low density lipoprotein (VLDL), increased low density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by > 70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of d approximately 1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of d < 1.21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus downregulating the expression of the LDL receptor.

Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway.

The Golgi apparatus is a membrane bound organelle involved in synthesis of N-linked oligosaccharides which are trimmed and then lengthened by a series of sugar transferases adding N-acetylglucosamine, galactose and sialic acid in sequence. We previously published qualitative work which localized Galbeta1,4GlcNAc alpha2,6 sialyltransferase of rat hepatocytes to the trans cisternae and the trans Golgi network. We now report the use of combined stereological and immunoelectron microscopical techniques for mapping the Golgi stack composition and distribution of sialyltransferase protein in rat hepatocytes. The Golgi stack showed substantial variation in composition consisting of 1, 2, 3, 4, or 5 cisternae with an average of 2.5 cisternae. Sialyltransferase labeling was mainly located in the central cisternae of the Golgi stacks irrespective of whether the stacks were oriented in a cis/trans direction using morphological criteria. Only 20% of the total sialyltransferase labeling was present in the transmost cisterna and 2% in the trans Golgi Network. The low labeling in the transmost cisterna was essentially due to the presence of a sialyltransferase negative cisterna. These data emphasize the importance of quantitation in obtaining a representative picture of Golgi enzyme distribution in three dimensions. They indicate that central cisternae, rather than the transmost cisterna and TGN, function in sialylation along the secretory pathway of rat hepatocytes.

The relation between bis(maltolato)oxovanadium(IV) (BMOV) influencing the biochemical activity of rat liver Golgi apparatus and the morphology of this organelle was studied in normal and streptozotocin-diabetic rat livers. Ultrastructural examinations revealed marked differences in the morphology of Golgi apparatus in three groups of animals. In the control rats treated only with 0.5% NaCl we did not find any biochemical and morphological changes. Marked changes were found in the rat liver after 1.8 mmol BMOV in 0.5% NaCl (as drinking solution) applied for 7 days, so-called "control" group for vanadium. In this group Golgi apparatus seemed shorter than in the diabetic animals. Finally, the same treatment of rats with previously induced SZ-diabetes, showed relatively small morphological alterations. The ultrastructural observation was compatible with the activity of galactosyltransferase (GalT), the Golgi marker enzyme. In diabetic rats treated with BMOV the activity of this enzyme was almost the same as in controls. Summing up dramatic alterations, previously found in diabetic-untreated rats [22], normalized after orally applied BMOV solution, even after a short time.

We evaluated cytotoxic effects of different unconjugated and glycine- and taurine-conjugated bile salts (BS) against bile duct epithelial cells in isolated bile ductule fragments and isolated perfused rat liver. Ultrastructural morphometric studies were performed in polarized rat bile ductule fragments exposed in vitro to increasing concentrations (10-100 micromol/L) of lithocholate (LCA), deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), ursodeoxycholate (UDCA), their taurine-conjugates, and glycoconjugates of cholic (GCA) or chenodeoxycholic acid (GCDCA) for 20, 30, or 75 minutes. To evaluate the cytotoxicity of unconjugated hydrophobic bile salts against biliary epithelium (BDE) in the whole liver, livers were isolated from rats with impaired taurine-conjugation capacity (beta-alanine treatment) and perfused for 70 minutes with 2 micromol/min LCA (n = 6), CDCA (n = 6), CA (n = 6), or 0.5 micromol/min tauro-LCA (n = 4). In isolated bile ductule fragments, hydrophobic unconjugated bile salts (LCA, CDCA, DCA) induced a marked damage of intracellular organelles, mainly mitochondria. The damage started at a concentration of 10 micromol/L and became prominent at concentrations higher than 50 micromol/L. No damage of the apical and basolateral membrane was seen and tight junctions appeared intact. UDCA, taurine and glycoconjugated bile salts failed to induce any evident ultrastructural alteration. In taurine-depleted isolated livers, perfused with LCA, CDCA, or CA, bile duct epithelial cells showed no evidence of intracellular damage, despite the increased biliary excretion of unconjugated BS. Marked alterations of the apical cell membrane were seen only in livers perfused with LCA and in isolated segments of the biliary epithelium. In contrast with biliary epithelium, hepatocytes showed prominent subcellular damage with CA and CDCA, and profound alterations of the canalicular membrane with LCA and tauro-LCA. We have shown that, in vitro, BDE cells are not damaged by taurine- or glycine-conjugated BS, but they are very sensitive to cytotoxicity of hydrophobic unconjugated BS. Such sensitivity is not present in the whole liver, probably because of the specificity of BS transport processes, the microvascular architecture of the bile ductal system, and the presence in bile of a physiological surfactant, such as phospholipids.

Recent studies have shown the insulin-like effect of vanadyl sulphate or sodium ortho (or meta-)vanadate administered orally to rats. Toxicity of these drugs and reluctance by the animals to drink the solutions and take food, concerning the amelioration of some diabetes syndrome discussed in 1994 by Domingo et al. (1), McNeill et al. (2) and Wiliams and Malabu (3), prompted us to investigate a new vanadate complex: disodium bis(oxalato)oxovanadate (IV), Na2[VO(OX)2]H2O. The main object of the experiment was to study whether this complex administered as 3 mmol/l solution in 0.5% NaCl during 7 days could act on the subcellular level and influence the activity of liver Golgi membrane galactosyltransferase activity. Free blood sugar level was lowered (but was still higher than in the control group) in diabetic rats after seven days of vanadate action and was accompanied by lowered, however not statistically significant, serum triglyceride levels. The yields of isolated Golgi-rich membrane fractions were about half of the level in diabetic groups (untreated and treated with vanadium) compared with the control groups. Purity of these membrane fractions, expressed as nmol Gal transferred per mg of proteins and per h, was the same in four groups investigated and showed the possibility to compare them. Activity of galactosyltransferase calculated in nmol Gal transferred per 1 g of liver and per 1 h or per whole liver in the same time (as a possibility of glycosylation of the secretory and membrane glycoproteins) was lower in both diabetic groups. However, after vanadium treatment (D+V group), the activity was higher than in untreated diabetic rats (D group) in three of five investigated animals. Vanadyl-oxalate complex did not normalize in a statistically significant manner the enzyme activity which was significantly lower in diabetes than in control. This is similar to insulin influence on the galactosyltransferase activity reported previously by Kaczmarski et al. in 1981 (4) and Kordowiak et al. in 1981 (5).

This paper presents yields of Golgi-rich membrane isolation, the activity of galactosyltransferase (GalT), the marker enzyme of Golgi apparatus as well as the morphology of the organelle from the livers in situ, in two groups of rats. One group consisted of control rats injected twice intraperitoneally with LEPK. Second group consisted of rats injected with LEPK and additionally after 24hrs given streptozotocin (SZ) to induce experimental diabetes. The results were compared with our previous investigations in control and diabetic rats. In the latter the activity of GalT was diminished, therefore diminishing glycosylation ability, and destructing Golgi apparatus morphology. This experiment shows that two-fold injection of LEPK prior to SZ does not prevent from changes in such biochemical parameters as free blood glucose level, yield of liver Golgi membranes isolation or total activity of GalT. For the first time in c. 30% of investigated rats the inactive enzyme of Golgi apparatus was found in the rats treated with LEPK+SZ. Morphological investigations of liver Golgi apparatus in rats treated with LEPK show slightly increased secretory activity with similar to untreated control rats morphological structure of this organelle. In the rats treated with LEPK and SZ the same morphological changes as in diabetic liver were found, however, such dramatic alterations as in SZ-diabetic rats were never found, irrespective of active or inactive GalT.

Albumin immunoreactivity in the liver was examined on days 2, 5 and 10 after two-thirds partial hepatectomy by light and ultrastructural immunoperoxidase methods and the ultrastructural area of the rough endoplasmic reticulum (ER) in hepatocytes was measured. Albumin immunoreactivity was seen in the rough ER and Golgi apparatus of all hepatocytes in the hepatectomized liver and ultrastructural analysis showed a significantly greater area of rough ER on day 5 than on days 2 or 10. Albumin mRNA was studied by the in situ hybridization technique using radioisotopes and their numbers were determined visually. Albumin mRNA was present as grains in all hepatocytes and the grains varied in number during regeneration of the liver, being more abundant on day 5 than on days 2 or 10. The activity of [3H]-leucine incorporated into albumin synthesis, an indicator of translational activity, was higher on days 5 and 10 than on day 2 and was highest on day 5. In conclusion, albumin synthesis varied during liver regeneration after partial hepatectomy, being reduced at the peak of cell proliferation on day 2 and being most active on day 5.

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Mature male rainbow trout (Oncorhynchus mykiss) were exposed for 28 days to 0, 1, 5, and 20 micrograms/liter disulfoton, i.e., to concentrations well below any macroscopically visible effect due to the primary acute toxic mechanism of acetylcholine esterase inhibition. In an attempt to reveal sublethal injury of disulfoton in rainbow trout, ultrastructural and stereological parameters were recorded in the liver as the central organ of xenobiotic metabolism in fish. Quantitative methods were definitely not able to replace qualitative techniques because, except for mitochondria, peroxisomes, and hepatocellular lipid inclusions, stereological analysis revealed only insignificant variations of hepatocellular components, whereas hepatocytes displayed a complex pattern of numerous delicate qualitative alterations. Effects were most evident within cisternae of the rough endoplasmic reticulum (RER), thus suggesting modifications of protein metabolism. Structural alterations included degenerative effects such as dilation and vesiculation of RER cisternae, formation of concentric RER arrays and augmentation of smooth endoplasmic reticulum, dilation of Golgi cisternae, and the development of cytoplasmic myelinated bodies as well as stacks of membranous material within mitochondria. Structural integrity and augmentation of peroxisomes and mitochondria as well as increased activity of the Golgi system were indicative of adaptive/compensative reactions following disulfoton treatment. In fact, adaptive effects seemed more pronounced than degenerative phenomena resulting in only minor disturbances in hepatocyte structure following disulfoton exposure. Because most effects had to be classified as unspecific responses to environmental or xenobiotic stressors, no distinct mode of sublethal action can be suggested for disulfoton.

Diethylmaleate is an organic anion secreted into bile as a glutathione conjugate. Its transport by the hepatocyte is associated with dilatation of the Golgi apparatus and the appearance of small vesicles in the pericanalicular area. It has been speculated that the Golgi apparatus could play a role in the intracellular transport and/or the biliary canalicular secretion of diethylmaleate. The purpose of this work was to determine whether the alterations in the Golgi apparatus and the pericanalicular vesicles could mediate the canalicular secretion of diethylmaleate. Diethylmaleate biliary secretion and diethylmaleate-induced bile flow were measured in Sprague-Dawley rats, and in TR- rats which have an inherited defect in the excretion into bile of organic anions, including glutathione conjugates. Livers of both Sprague-Dawley and TR-rats were examined by electron microscopy, to characterize the changes in intracellular organelles. In Sprague-Dawley rats, as previously described, diethylmaleate administration was associated with an increase in bile flow, which was parallel in time to the secretion into bile of diethylmaleate conjugates. Electron microscopic examination of the liver after diethylmaleate administration showed dilatation of the Golgi saccules. In contrast, in TR- rats, the increase in bile flow and the secretion of diethylmaleate conjugated were nearly absent. Nevertheless, electron microscopic examination showed a dilatation of the Golgi saccules similar to that observed in Sprague-Dawley rats. TR- rats, in addition to the changes in the Golgi apparatus, had marked dilatation of the endoplasmic reticulum. These results show that biliary secretion of diethylmaleate conjugates was severely impaired in TR- rats, in spite of a dilatation of the Golgi apparatus and of the endoplasmic reticulum. We conclude that it is unlikely that the alterations in the Golgi apparatus (and the endoplasmic reticulum) induced by diethylmaleate play a role in the canalicular secretion of diethylmaleate. We do not exclude the possibility that these organelles could play a role in intracellular transport of this compound. Alternatively, these alterations could be due to a "toxic" effect of diethylmaleate accumulation in hepatocytes.

Background: In a previous study of the hepatocyte Golgi apparatus 24 hr following the induction of the acute phase response to inflammation, it was found that the predominant secretory content was a granulofilamentous material (GFM), rather than lipoprotein particles (LP) as in controls (Thorne-Tjomsland and Jamieson, 1995, Anat. Rec., 241:439-450). The present study aimed to determine when and how this switch occurs and to monitor the Golgi-lysosome region of hepatocytes in general during early inflammation., Methods: Rats were injected with turpentine oil as an inflammatory agent. At 1, 2, 6, 12, and 16 hr after turpentine-injection (TI), the livers were prefixed by perfusion with cacodylate-buffered glutaraldehyde, postfixed with ferrocyanide reduced osmium, and processed for thin section transmission electron microscopy., Results: LP were processed at all time points between 1 and 16 hr post-TI, and at 1 and 2 hr post-TI, the Golgi stacks were engorged with LP and therefore apparently processed more of these particles than in controls. A GFM was processed at later time points, starting at 12 hr post-TI. At the two time points when LP and GFM were co-processed, these materials were frequently seen sorted from each other within the trans-saccule and/or the trans-Golgi network (TGN). At the level of the TGN, these materials were packaged into secretory vesicles which typically contained LP and/or GFM. Massive depletion of cytoplasmic glycogen was observed primarily at 1, 2, and 6 hr following TI. Concomitantly and temporarily, glycogen accumulated within Golgi-associated lysosome-like bodies with extensive appendages., Conclusions: The switch in Golgi apparatus secretory content from LP to GFM occurred at around 12 hr post-TI, but was not discrete. The initial appearance of the GFM coincided with the initial increase in the serum of hepatocyte-derived acute phase reactants (see review Schreiber et al., 1989, Ann. NY Acad. Sci., 557: 61-85). An additional and surprising finding of the present study was that significant changes occurred in the Golgi-lysosome region much prior to the onset of acute phase reactant synthesis: 1 and 2 hr post-TI, the Golgi processed above-normal amounts of LP and Golgi-associated, lysosome-like bodies sequestered glycogen.

The formation of chylomicrons by the intestine is important for the absorption of dietary fats and fat-soluble vitamins (e.g., retinol, alpha-tocopherol). Apo B plays an essential structural role in the formation of chylomicrons in the intestine as well as the VLDL in the liver. We have developed genetically modified mice that express apo B in the liver but not in the intestine. By electron microscopy, the enterocytes of these mice lacked nascent chylomicrons in the endoplasmic reticulum and Golgi apparatus. Because these mice could not form chylomicrons, the intestinal villus enterocytes were massively engorged with fat, which was contained in cytosolic lipid droplets. These mice absorbed D-xylose normally, but there was virtually no absorption of retinol palmitate or cholesterol. The levels of alpha-tocopherol in the plasma were extremely low. Of note, the absence of chylomicron synthesis in the intestine did not appear to have a significant effect on the plasma levels of the apo B-containing lipoproteins produced by the liver. The mice lacking intestinal apo B expression represent the first genetic model of defective absorption of fats and fat-soluble vitamins and provide a useful animal model for studying nutrition and lipoprotein metabolism.

We studied the kinetics of the biosynthetic transport of lysosome-associated membrane glycoprotein-1 (lamp-1) to the endocytic compartments in cultured rat hepatocytes. Newly synthesized lamp-1 (NS-lamp-1) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 13 min. From the trans-Golgi, at least 25% of NS-lamp-1 was delivered to the cell periphery: to the cell surface and early endosomes with t1/2 s of 32 and 33 min, respectively. A comparison of the kinetics of the biosynthetic transport of lamp-1 to both compartments demonstrated that NS-lamp-1 takes two peripheral routes from the Golgi apparatus; it is delivered to early endosomes directly and after reaching the cell surface. A major portion of NS-lamp-1 follows a direct intracellular pathway to late endosomes (t1/2 = 45 min) and subsequently to lysosomes (t1/2 = 85 min). The kinetic data of the biosynthetic transport to these endocytic vacuoles suggested that a significant fraction of NS-lamp-1 returns to the late endosomes immediately after its arrival at lysosomes and that there is a unique retrograde delivery of NS-lamp-1 from late to early endosomes prior to its transport to lysosomes. Thus, in cultured rat hepatocytes, the lamp-1 biosynthetic and the endocytic pathways converge at the three distinctive points. Late endosomes are centrally situated in the complex biosynthetic route of lamp-1.

Primary pig hepatocytes form a tissuelike structure in an in vitro culture model that has provision for three-dimensional cell orientation, cell aggregation, decentralized cell perfusion with low metabolite gradients, integral oxygenation, and nonparenchymal cell coculture. Scanning electron microscopy (SEM) has shown that hepatocytes spontaneously form aggregates in a three-dimensional structure between and on the surface of artificial capillaries. Transmission electron microscopy (TEM) has shown that after 7 weeks of in vitro perfusion, the cell ultrastructure remains similar to that of the parenchyma in vivo. Golgi complexes, active membrane processes, reorganization of cell junctions, and bile canaliculi-like intercellular spaces were demonstrated.

The occurrence of individual G proteins within Golgi membranes from liver and from adrenal medulla were analyzed by Western blotting. Two splice variants of G alpha s and also G alpha i-3 were found in both tissues. Additionally, G alpha i-2, its 43-kDa splice variant and G alpha o were strongly labeled in Golgi of adrenal medulla. Golgi preparations of liver contained comparable quantities of G alpha i-2 and a second variant of 46 kDa, but no G alpha o was detected. Immunoelectron microscopic studies of the above Golgi preparations showed that both G alpha i-2 and G alpha i-3 are localized at multivesicular structures identified as Golgi complexes. The occurrence of two stimulatory G proteins (G alpha s-S and G alpha s-L) and three or four alpha subunits of the Gi/Go type as components of the Golgi membrane support the notion that formation of different vesicle types might be regulated by individual stimulatory and inhibitory G proteins.

Lipid metabolism takes place in the Golgi apparatus, but at a higher rate in female than in male rats. I therefore examined the Golgi apparatus by morphometric means for differences between the sexes at the light- and electron-microscopic level. The Golgi apparatus was stained in situ by a zinc-iodide-osmium method. The counts of the Golgi apparatus in cross-sections in female hepatocytes by light microscopy were approximately twice that in male hepatocytes. Upon ovariectomy, these counts were greatly reduced but were reestablished after estrogen supplement. To clarify this phenomenon, three-dimensional reconstructions of the Golgi apparatus were produced from electron-microscopic images of serially cut 160-nm sections. The Golgi apparatus of both male and ovariectomized females had the shape of a small ring, whereas it took the form of a large elongated cylinder in normal females and in castrated males after treatment with estrogen. The numerical difference in Golgi apparatus counts by light microscopy of in males and females is, therefore, apparently attributable to the size and shape of the Golgi apparatus, and is controlled by the estrogen level.

Rat liver Golgi membranes were washed with KCl and urea, and a polyclonal antiserum that stained the Golgi complex by immunofluorescence microscopy was raised. A group of proteins of apparent molecular mass 500 kDa, 200 kDa and 100 kDa were identified by immunoblotting with the antiserum, and were enriched in the Golgi membrane fraction. These proteins were also localised to the Golgi by immunofluorescence microscopy with affinity-purified antibodies. They are integral membrane proteins, and protease digestion experiments suggested that they are not exposed on the cytoplasmic face of the Golgi membrane. Immunofluorescence microscopy showed that staining of the Golgi complex by antibodies to the 100 kDa Golgi protein can be demonstrated among a wide range of mammalian species. This conservation may point to an important structural or functional role for the molecule. When the 100 kDa protein was reduced with dithiothreitol it was no longer recognised by the anti-Golgi antiserum. During phase separation in Triton X-114 the 100 kDa protein partitioned into the aqueous phase, rather than into the detergent phase, suggesting that it has a large luminal domain of hydrophilic amino acids.

The intrahepatic distribution of apolipoprotein E has been assessed by immunogold labeling of cryosections as well as by Western blotting of organelles isolated from liver homogenates. Both techniques supported the prior analytical fractionation studies of Wong (1989) who concluded that intrahepatic apoE was largely endosomal. All endosomal components decorated by gold particles indicative of apoE antigenicity in cryosections appeared filled with lipoprotein-like particles thereby accounting for this prominent morphological feature of isolated liver endosomes. The distribution of gold particles about the hepatic Golgi apparatus revealed a high content of apoE in closely apposed endosomes, ca. 400 nm in diameter, double labeled for apoE and internalized HRP. Remarkably, apoE (but not internalized HRP) was also observed within saccular distensions of all saccules of stacked Golgi cisternae but absent from the flattened saccular components as was also observed for apoB. This contrasted with albumin, the major secretory protein, which was uniformly distributed throughout the hepatic Golgi apparatus. These observations support a growing body of evidence for intra-Golgi sorting of secretory material in hepatic Golgi apparatus. The lack of any immunoreactive apoE or albumin in small 70-90 nm vesicles about the Golgi cisternae suggests limits to current models of vesicle-mediated intra-Golgi transport.

Previous studies have demonstrated that there is a change in the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer alpha 2,3-ST) when the lipid composition of the Golgi membrane is altered (Kadowaki, Grant, and Williams, 1993. J. Lipid Res. 34: 905-914). To understand the basis of this phenomenon, the molecular species specificity of rat liver LacCer alpha 2,3-ST was determined under conditions in which the phospholipid class composition of the Golgi membrane was changed to resemble that of cultured mouse neuroblastoma NB2a cells, a cell line in which LacCer alpha 2,3-ST exhibits no molecular species specificity. The change in the lipid composition of the Golgi membrane was accomplished by incubating the Golgi membrane vesicles with nonspecific lipid transfer protein and a 10-fold excess of liposomes prepared with various proportions of purified rat liver lipids. The molecular species specificity of LacCer alpha 2,3-ST was also determined under conditions where the phospholipid molecular species composition but not the phospholipid class composition of the Golgi membrane was changed, and in which both the phospholipid class and molecular species compositions were changed by using liposomes prepared with lipids purified from a mouse brain tumor (ependymoblastoma). When using liposomes prepared with rat liver lipids, a change in the phospholipid class composition of the Golgi membrane to a composition similar to that of NB2a cells increased rather than decreased the molecular species specificity of rat liver LacCer alpha 2,3-ST.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.

In practice it has been possible to separate membrane particles of different origins but of similar chemical composition by preparative free-flow electrophoresis. Examples include the vacuolar (tonoplast) and plasma membranes of plants and membranes derived from the cis and trans regions of the rat liver Golgi apparatus. Yet, when analyzed for intrinsic molecules that might contribute to significant differences in surface charge, the separated membranes were surprisingly similar. As more information was generated, it became apparent that the membranes with greatest electrophoretic mobility (i.e. lysosomes, rightside-out tonoplast vesicles and membranes from the trans region of the Golgi apparatus), where those membranes with an inherent ability to acidify their interiors. By so doing, the vesicles generate a membrane potential, negative outside, which might serve as a basis for enhanced electrophoretic mobility. To test the hypothesis, tonoplast membranes were incubated with ATP to drive proton import or with monensin to dissipate the ATP-supported proton gradient. With ATP, mobility was enhanced. Also, when ATP-treated vesicles were analyzed in the presence of monensin, the ATP effect on mobility was reversed. Similarly with Golgi apparatus, mobility of the most electrophoretically mobile portions of the separation was enhanced by ATP and the ATP effect was reversed with monensin. A trans origin of the vesicles was verified by assay of the trans Golgi apparatus marker, thiamine pyrophosphatase. Finally, incubation with ATP (and reversal by monensin) was employed as an aid to the free-flow electrophoretic separation of kidney endosomes from complex mixtures. These lysosomal derivatives also are capable of acidification of their interiors in an ATP-dependent process and of generating, at the same time, a negative (outside) membrane potential. The findings provide both an experimental basis to enhance membrane separations by preparative free-flow electrophoresis and, at the same time, a theoretical basis to help explain why certain membranes of very similar overall chemical composition may be separated by electrophoretic methods.

Studies in hepatoma cells and hepatocytes have revealed that the biogenesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well differentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to cell surface contact sites of attached cells. We demonstrate that a fluorescent head group-labeled lipid analog, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertion into the plasma membrane of HepG2 cells at 4 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that the MLV are a target for plasma membrane-derived lipids. Furthermore, also the Golgi apparatus is involved in the formation of the vesicles. After initial accumulation of the fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (C6-NBD)-ceramide in the Golgi apparatus at 37 degrees C, prolonged incubation at 37 degrees C results in the appearance of NBD fluorescence in the microvilli-lined vesicles. The transport route for the Golgi-derived material to the developing bile canalicular vesicle is not an indirect pathway, i.e. involving transcytosis via the basolateral plasma membrane. This could be demonstrated by including bovine serum albumin (BSA) in the incubation media, a lipid scavenger that will remove any C6-NBD-lipids exposed at the basolateral membrane. At these conditions, lipid trafficking between the Golgi complex and MLV still occurred. We further demonstrate that the targeting from the Golgi apparatus to the bile canaliculus is also operational in isolated human hepatocytes. The latter results suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully differentiated cells.

Using weakly basic amines, we investigated the step at which the secretion kinetics of concanavalin-A-retained and nonretained alpha 1-acid glycoprotein glycoforms diverge in isolated rat hepatocytes. Both chloroquine and primaquine, whose action on protein secretion is targeted to terminal domains of the Golgi apparatus, cancelled the kinetic difference without influencing carbohydrate chain sialylation. To test for a possible interaction of alpha 1-acid glycoprotein with Golgi membranes, we also permeabilized control and primaquine-treated hepatocytes, as well as purified Golgi preparations, with saponin. In each case, we found that alpha 1-acid glycoprotein was associated with Golgi membranes, the association being more marked in primaquine-treated cells than in control cells. Membrane-bound alpha 1-acid glycoprotein appeared to be preferentially retained on concanavalin A. Such retention could account for the divergent secretion kinetics of alpha 1-acid glycoprotein glycoforms.

Liver cells of rats given a single insulin dose at birth (imprinted) do not alter from the controls at 1 week postnatal. When hormone treatment occurs at 1 week postnatal without any prior administration of it, an increase in glycogen and lipid with nuclear deposition of the latter can be seen. The cell surface displays a dense layer, and increased micropinocytosis is accompanied by an increase in the number of coated pits and vesicles. Liver cells of imprinted animals treated repeatedly at 1 week postnatal have much more glycogen and lipid, and more prominent endoplasmic reticulum but a less mature Golgi apparatus. The dense substance of the cell surface is missing and the signs of endocytosis are rarely seen. These alterations indicate the influence of imprinting on hormone binding and possibly on the next steps after interaction in the cell.