Cytokeratin inclusions in alcoholic liver disease and their relation to the amount of alcohol intake

ABSTRACT: In the present study, the frequency and the distribution pattern of immunoreactive hepatocytic cytokeratin (CK) inclusions was investigated using the monoclonal antibody (MAb) CAM 5.2 detecting CKs 8, 18 and 19. The CK antigenicity of the inclusions was confirmed on frozen sections with MAbs for the CKs 7, 8, 17, 18 and 19. The frequency of hepatocytic CK aggregates was compared to the presence of non-alcoholic and alcoholic liver disease (ALD) as well as to the average all-year daily ethanol intake of 195 consecutive males subjected to medicolegal autopsy. Hepatocytic CK inclusions were infrequent in patients with normal liver histology, portal fibrosis and/or portal inflammation. In ALD, however, inclusions were observed in 35.6% of patients with fatty liver, in 63.2% of patients with alcoholic hepatitis, in 30.8% of patients with bridging fibrosis and in 60.0% of patients with liver cirrhosis. In all specimens studied, the inclusions were reactive only for CKs 8 and 18, CKs 7, 17, and 19 being unreactive. The frequency of inclusion bodies increased, paralleling increasing average all-year daily alcohol consumption. Compared to non-drinkers, a daily intake of between 40 and 80 g of absolute alcohol was associated with a statistically significantly (relative risk, RR=6.6) increased risk of formation of aggregates. Similarly, daily consumption exceeding 80 g was related to increased (RR=6.0) frequency of CK aggregates. The frequency of full-blown “classical” Mallory bodies (MBs) was, however, increased (RR=8.9) only in patients with a daily intake exceeding 160 g. These results indicate that CK inclusions are unequivocally related to the presence of ALD in longitudinal autopsy series. Their frequency in autopsy series was, however, significantly lower than their frequency in previous studies comprising hospitalised patients diagnosed as having alcoholic liver disease. Furthermore, our results suggest that a daily ethanol intake between 40 and 80 g can increase the risk of appearance of smaller CK inclusions, although the risk of occurrence of “classical” full-blown MBs was associated only with heavy drinking exceeding 160 g of absolute alcohol daily.     

Trehalose activates autophagy and decreases proteasome inhibitor‐induced endoplasmic reticulum stress and oxidative stress‐mediated cytotoxicity in hepatocytes

Aim

Endoplasmic reticulum stress is associated with the pathophysiology of various liver diseases. Endoplasmic reticulum stress mediates the accumulation of abnormal proteins and leads to oxidative stress, cytoplasmic inclusion body formation, and apoptosis in hepatocytes. Autophagy is a bulk degradation pathway for long‐lived cytoplasmic proteins or damaged organelles and is also a major degradation pathway for many aggregate‐prone and disease‐causing proteins. We previously reported that rapamycin, a mammalian target of rapamycin inhibitor, activated autophagy and decreased proteasome inhibitor‐mediated ubiquitinated protein accumulation, cytoplasmic inclusion body formation, and apoptosis in hepatocytes. Trehalose is a non‐reducing disaccharide that has been shown to activate autophagy. It has been reported to decrease aggregate‐prone proteins and ameliorate cytotoxicity in neurodegenerative disease models. However, the effects of trehalose in hepatocytes are unclear.

Methods

We show here that trehalose activated autophagy and reduced endoplasmic reticulum stress, cytoplasmic inclusion body formation, and apoptosis in proteasome inhibitor‐treated liver‐derived cultured cells.

Conclusion

To our knowledge, this is the first report showing that trehalose activates autophagy and has cytoprotective effects in hepatocytes. Our findings suggest that trehalose can become a therapeutic agent for endoplasmic reticulum stress‐related liver diseases.     

Argyrophilic nucleolar organizer regions in neoplastic and non-neoplastic hepatocytes bearing Mallory bodies

ABSTRACT— The proliferative activity of Mallory bodies (MB)-positive hepatocytes (neoplastic and non-neoplastic) was examined by counting the argyrophilic nucleolar organizer regions (AgNORs). Among 19 cases of hepatocellular carcinoma, the mean number of AgNORs was lower in the MB-positive carcinoma cells than in the negative ones in nine cases, higher in six, and there was no difference in four. In non-neoplastic cases (seven cases of advanced primary biliary cirrhosis and seven cases of alcoholic or nutritional liver injury), the mean number of AgNORs was lower in the MB-positive hepatocytes than that in the negative ones in eight cases, and approximately equal in number in six cases. These findings imply that MB formation does not directly represent the level of proliferative activity of hepatocytes, regardless of whether they are malignant or not.     

Unusual intracytoplasmic inclusions in acute myeloblastic leukemia

Unusual intracytoplasmic inclusions within early granulocyte precursor cells from a patient with acute myeloblastic leukemia (AML) are described. Based upon their staining characteristics and electron- and light-microscopic appearance, the inclusions are distinctly different from any previously described. The inclusions display a variety of shapes, including rectangles, squares, circles, ovals, and irregular, globular forms. Most of the inclusions are refractile and crystal-like. The possible composition of these inclusions is discussed. They are compared with inclusions previously described within leukemic and granulocytic cells.     

Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the International Society of Amyloidosis

A system of amyloid fibril nomenclature based on the chemical identity of the amyloid fibril forming protein is recommended. This system has been in use for approximately 40 years, but current literature remains confused with clinical and histochemical designations used when the amyloid disease processes were poorly understood. To be designated an amyloid fibril protein, the protein must occur in tissue deposits and exhibit affinity for Congo red and green birefringence when viewed by polarisation microscopy. Furthermore, the protein must have been unambiguously characterised by protein sequence analysis (DNA sequencing in the case of familial diseases). Current nomenclature lists of 27 human and nine animal fibril proteins are provided together with a list of eight inclusion bodies that exhibit some of the properties of amyloid fibrils.     

Secretion of amyloidogenic gelsolin progressively compromises protein homeostasis leading to the intracellular aggregation of proteins

Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF. Amyloidogenesis is observed only in tissues synthesizing human D187N gelsolin, despite the presence of full-length D187N gelsolin and its 68-kDa cleavage product in blood—demonstrating the importance of local synthesis in FAF. Loss of muscle strength was progressive in homozygous D187N gelsolin mice. The presence of misfolding-prone D187N gelsolin appears to exacerbate the age-associated decline in cellular protein homeostasis (proteostasis), reflected by the intracellular deposition of numerous proteins, a characteristic of the most common degenerative muscle disease of aging humans, sporadic inclusion body myositis.     

Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin

Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.Aging is universally associated with a global decline in cellular function (1–3). Due to the multiplicity of mechanisms that undergo aging-related dysfunction, its mechanistic basis, or “senescence factor,” has been difficult to pinpoint. Several studies have provided key insight into the identities of senescence factors by studying the asymmetric segregation of damage in single-cell organisms that rejuvenate the emerging generation by preventing the inheritance of damaged factors such as DNA, lipids, and proteins (1, 4, 5). In particular, a number of seminal studies have demonstrated that bacteria and yeast use a complex and multifaceted machinery to prevent the inheritance of damaged and aggregated proteins by the new generation by restricting them to the older lineage during cell division (1, 6, 7).Although the precise mechanism for asymmetric inheritance of aggregates has been a matter of much debate (1, 7), the emerging model is that the spatial arrangement of misfolded proteins into quality control-associated IB (inclusion body)-like structures plays an essential role in asymmetric inheritance (1, 7). A key property of some quality-control IBs and other IB-like structures, which allows the cell to retain them in a specific lineage during mitosis, is their association and interaction with cellular organelles and cytoskeleton. In bacteria, for example, aggregated proteins are collected at the old pole of a dividing cell (5). A similar mechanism has been proposed in fission yeast (8). In the budding yeast Saccharomyces cerevisiae, several detailed studies demonstrated interdependence between asymmetric inheritance and the maintenance of the actin cytoskeleton and, in particular, the polarisome complex, which anchors the actin cytoskeleton in the emerging bud (9). Compromising the architecture of the actin cytoskeleton, even for a single generation, leads to a decrease in the replicative lifespan of the emerging cells (4).Yeast cells manage the triage of protein misfolding and aggregation by spatially partitioning subpopulations of misfolded proteins to several membrane-less cytosolic quality-control IBs (10, 11). Upon misfolding, substrates localize to transient stress foci (SFs), which concentrate chaperones, holdases, and disaggregases (7). SFs are dynamic IBs that form in response to acute stress and may participate in the triage decision of whether to refold, degrade, or aggregate misfolded proteins (7). As misfolded substrates accumulate, and especially in response to external stresses, they accumulate in two quality-control IBs: Proteins targeted for degradation are directed to the JUNQ compartment and proteins that are targeted for active aggregation are directed to an insoluble IPOD compartment (10). Recently, we have demonstrated a new vital role for these inclusions in asymmetric inheritance of aggregates (or spatial quality-control/replicative rejuvenation) in yeast (7). The IPOD and JUNQ are selectively retained in the mother cell during budding. This asymmetry is due to the fact that IPOD and JUNQ are tethered to organelles. Critically, proteins that were trapped in SFs, and therefore failed to migrate to IPOD or JUNQ in time for mitosis, were passed on to successive generations of daughter cells (7).Although a similar replicative rejuvenation mechanism in multicellular organisms has been sought after for some time, only a few seminal studies have found evidence for asymmetric aggregate segregation during mammalian or Drosophila mitosis (12, 13, 14). The mechanism for directing misfolded proteins to different inclusions structures, however, appears to be at least partially conserved from yeast to mammals. In previous work, we have demonstrated the existence of distinct IBs in human cultured cells: a JUNQ-like IB and an IPOD-like IB (from here on JUNQ and IPOD) (15, 16). The JUNQ contains mobile aggregates and accumulates chaperones, such as Hsp70, and active proteasomes (15). The IPOD sequesters insoluble amyloid aggregates from the rest of the cytosol (15). In striking similarity to the yeast JUNQ, the properties of the mammalian JUNQ are highly sensitive to stress. Under low-stress conditions, the JUNQ is a dynamic liquid phase compartment with high-degradation capacity (11, 17). Increased exposure to misfolding stress or localization of disease-associated proteins to the JUNQ instead of the IPOD leads to the maturation of the JUNQ into a less dynamic solid phase compartment, inhibiting degradation and eventually killing the cell (15, 18).We set out to examine the mechanism of mitotic inheritance of misfolded proteins and aggregates in mammalian cells. Using long-term 4D imaging (19), we demonstrate asymmetric inheritance of JUNQ IBs during mitosis. Although the IPOD, containing amyloidogenic proteins, is frequently inherited by the same cell as the JUNQ, it is sometimes misinherited. Similar to yeast, we observe SFs in mammalian cells, which fail to be retained asymmetrically. We show that the inheritance of the JUNQ is mediated by the association of the JUNQ with the cytoskeleton. The misfolded proteins in the JUNQ are confined by a network of vimentin intermediate filaments and sometimes also actin (20). Whereas in yeast the JUNQ and IPOD are both tethered to organelles, the mammalian IPOD does not appear to specifically associate with the cytoskeleton or the MTOC (Microtubule Organizing Center). Therefore, a critical difference between yeast and mammalian asymmetry mechanisms may be a reduced ability to maintain the partitioning of insoluble amyloid aggregates. Finally, we show that replicative rejuvenation may confer a slight fitness advantage, under certain conditions, on the daughter cell that fails to inherit a JUNQ. In addition to uncovering a novel replicative rejuvenation mechanism in higher eukaryotes, our study suggests that vimentin establishes an axis of mitotic polarity in mammalian cells.     

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.     

Amyloid fibril protein nomenclature: 2012 recommendations from the Nomenclature Committee of the International Society of Amyloidosis

The Nomenclature Committee of the International Society of Amyloidosis (ISA) met during the XIIIth International Symposium, May 6–10, 2012, Groningen, The Netherlands, to formulate recommendations on amyloid fibril protein nomenclature and to consider newly identified candidate amyloid fibril proteins for inclusion in the ISA Amyloid Fibril Protein Nomenclature List. The need to promote utilization of consistent and up to date terminology for both fibril chemistry and clinical classification of the resultant disease syndrome was emphasized. Amyloid fibril nomenclature is based on the chemical identity of the amyloid fibril forming protein; clinical classification of the amyloidosis should be as well. Although the importance of fibril chemistry to the disease process has been recognized for more than 40 years, to this day the literature contains clinical and histochemical designations that were used when the chemical diversity of amyloid diseases was poorly understood. Thus, the continued use of disease classifications such as familial amyloid neuropathy and familial amyloid cardiomyopathy generates confusion. An amyloid fibril protein is defined as follows: the protein must occur in body tissue deposits and exhibit both affinity for Congo red and green birefringence when Congo red stained deposits are viewed by polarization microscopy. Furthermore, the chemical identity of the protein must have been unambiguously characterized by protein sequence analysis when so is practically possible. Thus, in nearly all cases, it is insufficient to demonstrate mutation in the gene of a candidate amyloid protein; the protein itself must be identified as an amyloid fibril protein. Current ISA Amyloid Fibril Protein Nomenclature Lists of 30 human and 10 animal fibril proteins are provided together with a list of inclusion bodies that, although intracellular, exhibit some or all of the properties of the mainly extracellular amyloid fibrils.     

Immuoelectron microscopic localization of actin in normal and cholestatic rat hepatocytes

The immunoelectron microscopic localization of actin in hepatocytes was studied in normal, common bile duct-ligated, and phalloidin treated rats. We used the low temperature embedding procedure with Lowicryl K4M and protein A-gold technique was applied to demonstrate the localization of actin. The tissues embeded in Lowicryl K4M at low temperature provided good preservation of ultrastructure and antigenicity of actin. In control rats bile canaliculi were clearly visualized and most of the gold particles were observed on the bile canalicular microvilli, at the pericanalicular ectoplasm, at the cell border, and on the sinusoidal microvilli. In common bile duct-ligated rats and phalloidin treated rats, pericanalicular ectoplasm was thickened and many gold particles were seen there. This work was supported in part by a grant 62480198 from the Ministry of Education, Japan.     

3,4-Methylenedioxymethamphetamine (ecstasy)-induced hepatotoxicity: effect on cytosolic calcium signals in isolated hepatocytes

Abstract: Aims/Background: Hepatocellular damage has been reported as a consequence of 3,4-methylenedioxymethamphetamine (MDMA) intake. However, little is known about the cellular mechanisms involved. The present study was undertaken to evaluate the effects of MDMA on cell viability as well as free calcium levels ([Ca²⁺]_i) in short-term cultured hepatocytes. Reduced glutathione (GSH), adenosine-5′-triphosphate (ATP) and lipid peroxidation were investigated to evaluate the toxic effect of MDMA, in vitro, using freshly isolated rat hepatocytes. Methods: In order to measure cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), rat hepatocytes were loaded with the Ca²⁺ indicator fura-2-acetoxymethylester (fura-2-AM). Results: A sustained rise of ([Ca²⁺]_i) after incubation with MDMA was the most noteworthy finding. In Ca²⁺-free medium, MDMA caused a reduced increase of ([Ca²⁺]_i). On the other hand, MDMA (0.1–5 mM) induced a concentration-dependent and time exposure-dependent GSH and ATP depletion. Although it did not reach statistical significance, GSH deficits were accompanied by a tendency to increase lipid peroxidation 3 h after MDMA incubation. Conclusions: The above data suggest that the marked rise of ([Ca²⁺]_i) and subsequent ATP and GSH depletion can lead to a rapid decrease in cell viability.     

Dietary restriction initiated late in life can restore age-related changes in protein metabolism

Dietary restriction (DR) from weaning or young adult stages in laboratory animals throughout their usual life span extends maximum lifespan and retards the development of a broad spectrum of pathophysiological changes. However, the effects of DR when performed at middle age or later have not been well investigated. We previously reported that short-term (about 2.5 months) DR in early senescence reduced the amount of altered proteins and shortened the half-life of proteins. Furthermore, our previous studies in rats demonstrated DR initiated later in life reduces the content of oxidatively modified proteins, measured as protein carbonyls, in mitochondria from old rats. In addition, we recently found that the age-related decline in the activities of proteasomes that have been implicated in removal of altered proteins was restored by DR. Thus, our findings suggest that DR initiated even relatively late in life can restore an animal's youthful condition, reducing the accumulation of altered proteins.     

Stimulation of protein synthesis in isolated hepatocytes by somatomedin

Insulin (1 mU/ml) stimulated the incorporation of 14C-leucine into trichloroacetic acid-precipitable material by isolated hepatocytes from normal and hypophysectomized adult rats and 12-day-old rabbits. Somatomedin (200 ng/ml) purified from human plasma had an insulinlike effect in hepatocytes from hypophysectomized rats and baby rabbits but not from normal rats. This study suggests that, as well as being a site of somatomedin synthesis, the liver may be a target organ for this hormone.     

Sulphonylureas in vitro do not alter insulin binding or insulin effect on amino acid transport in rat hepatocytes

Summary The effects of four sulphonylureas (gliclazide, glibenclamide, chlorpropamide and glipizide) on insulin binding and insulin action were studied in vitro using primary cultured rat hepatocytes. Cells were cultured for 20 h in the absence or presence of the sulphonylurea. The binding of insulin to rat hepatocyte monolayers was not altered in cells previously exposed to gliclazide at 0.7, 7.0 or 70 g/ml; and to glibenclamide, chlorpropamide, or glipizide at 0.1, 1.0 and 10 g/ ml. Insulin-induced down regulation was not affected by a simultaneous exposure of hepatocyte monolayers to any of the four agents. The stimulatory effect of insulin on -aminoisobutyric acid uptake by the cells was not modified following exposure to the drugs. These studies indicate that the sulphonylureas tested do not have a direct effect on insulin receptors in hepatocytes; and that, in vitro, they do not alter the post-receptor events involved in the insulin-induced stimulation of amino acid transport in these cells.     

Effect of ethanol on adenosine triphosphate,cytosolic free calcium,and cell injury in rat hepatocytes

The events implicated in the early phases of acute ethanol-induced hepatocyte injury and their relation with the nutritional status of the liver are not clearly defined. We aimed to determine the effect of ethanol on ATP and cytosolic free Ca²⁺ in hepatocytes isolated from fed or fasted rats. Cell injury was assessed by LDH release and trypan blue uptake, ATP by [³¹P]NMR spectroscopy, and cytosolic free Ca²⁺ with aequorin. In control conditions, cells from fasted animals had a lower ATP level (–52%) and a higher cytosolic free Ca²⁺ (+101%) than did those isolated from fed animals. Ethanol caused a dose-dependent cell injury in both groups. At all ethanol doses, greater damage occurred when using hepatocytes isolated from fasted rats. In both groups, a dose-dependent decrease in ATP content and a rise in cytosolic free Ca²⁺ were seen. The magnitude of these changes were significantly greater in the fasted group. In conclusion, these data showed that fasting affects the energy status and cytosolic free calcium level in hepatocytes; ethanol causes a dose-dependent cell injury that occurs in association with a fall in ATP and a rise in cytosolic free Ca²⁺ levels. The nutritional status of an animals is an important determinant of the severity of ethanol-induced damage to liver cells.     

Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization

Abstract: Besides its presence in the pineal gland, melatonin has been found in a variety of other tissues as well. The indoleamine also has been identified in invertebrates including an unicellular organism where it exhibits a diurnal rhythm. Although melatonin is mainly known for its effects on seasonal reproduction and endocrine physiology, there is evidence showing that this ubiquitously acting hormone is also a potent free radical scavenger, thereby providing protection from oxidative attack to DNA and other biomolecules. Through the years, melatonin was thought to be exclusively cytosolic. However, careful examination of some of these pioneering reports revealed a nuclear localization of melatonin in different tissues including the retina and Harderian glands. Using a very sensitive immunocytochemical method, we have also found that melatonin is located in the nucleus of many cells where it may bind to nuclear components. The use of cell fractionation studies followed by radioimmunoassay confirmed these results. The administration of exogenous melatonin resulted in a marked increase in the nuclear melatonin content without a concomitant change in the cytosolic fraction. In addition to its ability to scavenge free radicals, its location in the nucleus suggests possible genomic actions.