Cytoplasm Of Hepatocytes Research Articles

Nuclear Factor Related to KappaB Binding Protein (NFRKB) Is a Telomere-Associated Protein and Involved in Liver Cancer Development

Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism activated in 10-15% of human cancers. Although significant progress has been made, the key regulators of the ALT pathway and its role in cancer development remain elusive. Bioinformatics methods were used to predict novel telomere-associated proteins (TAPs) by analysis of large-scale ChIP-Seq data. Immunostaining and fluorescence in situ hybridization experiments were applied to detect the subcellular location of target genes and telomeres. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the expression of targeting genes. Overall survival (OS) analyses were used to evaluate the relationship between gene expression and survival time; immunohistochemistry was used to detect the distribution of target genes in liver cancer tissues. We found that nuclear factor related to kappaB binding protein (NFRKB), a metazoan-specific subunit of the INO80 complex, can associate with telomeres in human ALT cells. Loss of NFRKB induces dysfunction of telomeres and less PML bodies in U2OS cells. In addition, NFRKB is low/moderately expressed in cytoplasm of normal hepatocytes but heavily accumulating in the nucleus of liver cancer cells. Finally, the high expression of NFRKB is associated with short OS time and poor prognosis. NFRKB is a TAP and protects telomeres from DNA damage in ALT cells. It is highly expressed in hepatocellular carcinoma (HCC) cells and predicts a poor prognosis. NFRKB may be a promising prognostic biomarker for the treatment of HCC in the future.

Interaction between cadherins, vimentin, and V-set and immunoglobulin domain containing 1 in gastric-type hepatocellular carcinoma.

In hepatocellular carcinomas (HCCs), the role of the cell surface protein V-set and immunoglobulin domain containing 1 (VSIG1), which is known as a specific marker of the gastric mucosa and testis, has not yet been determined. We examined VSIG1 immunohistochemical (IHC) expression in 105 consecutive samples provided by HCC patients, along with the IHC expression of three of the biomarkers known to be involved in the epithelial-mesenchymal transition (EMT): vimentin (VIM), and E- and N-cadherin (encoded by CDH1 and CDH2 genes). IHC subcellular localization of thyroid transcription factor 1 (TTF1), in which nuclear-to-cytoplasmic translocation is known to cause a lineage shift from lung to gastric-type adenocarcinoma, was also checked. The obtained data were validated using the miRNET program. In the examined HCC samples, VSIG1 expression was observed in the cytoplasm of normal hepatocytes and downregulated in 47 of the 105 HCCs (44.76%). In 29 cases (27.62%), VSIG1 was co-expressed with cytoplasmic TTF1. VSIG1 expression was positively correlated with both E-cadherin and N-cadherin and negatively correlated with VIM (p < 0.0001). The VSIG1+/E-cadherin+/N-cadherin-/VIM phenotype was seen in 13 cases (12.4%) and was characteristic of well-differentiated (G1/2) carcinomas diagnosed in pT1/2 stages. Like pulmonary carcinomas, simultaneous cytoplasmic positivity of HCC cells for VSIG1 and TTF1 may be a potential indicator of a lineage shift from conventional to gastric-type HCC. The E-cadherin/VSIG1 complex can help suppress tumor growth by limiting HCC dedifferentiation. The miRNET-based interaction between VSIG1/VIM/CDH1/CDH2 genes might be interconnected by miR-200b-3p, a central regulator of EMT which also targets VIM and VSIG1.

Early Nutrition with Different Diets Composition versus Fasting on Immunity-Related Gene Expression and Histomorphology of Digestive and Lymphoid Organs of Layer-Type Chicks.

Simple SummaryWith the continuous improvement in the progress of poultry industry, a better understanding of the avian immune system is necessary. A prolonged holding period (36–72 h), along with a delay in access to feed and/or water post-hatching, has a negative influence on performance, intestinal histomorphology, and the immune system development of chicks. Therefore, the present study aimed to investigate the effect of early feeding with different diet composition or delayed feeding during the first 72 h of chick’s life on the expression of immunity-related genes and histomorphology of digestive and lymphoid organs of layer-type chicks. Early nutrition post-hatching had no negative effect on the development of the lymphoid and digestive organs in chicks. Histomorphological examination revealed an increase in cortex and cortex:medulla of thymus and bursa in the early fed groups compared to the fasted ones, with resultant impacts on the primary lymphoid organs. Higher germinal center areas and white pulp of the spleen were recorded in the early fed chicks, implying augmented proliferation and maturation of B cells in the secondary lymphoid organs. In the liver, a strong positive reaction to Best’s carmine stain in the early fed groups, indicating that the liver of these chicks had numerous glycogen granules in the cytoplasm of hepatocytes. The expression levels of splenic-immunity related genes were up-regulated in most of the early fed chicks at 14 day of age. Our findings suggested that early feeding post-hatch can modify the splenic-immunity related genes and modulate the histomorphology of the digestive (liver and proventriculus) and lymphoid organ in layer-type chicks during the early life post-hatching.Early feeding post-hatching (EFPH) can impact the immune response and modify the immunity-related gene expression. Therefore, we aimed to examine the effects of EFPH with different diets composition versus fasting during the first 72 h of chick’s life on the histomorphological structures of the liver, proventriculus, central and peripheral lymphoid organs, and immunity-related genes in layer-type chicks during the brooding period. A total of 400 chicks were randomly allotted into 4 groups with 4 replicates each. The experimental groups during the first 72 h of life were: feed and water deprivation (control, T1), feeding a starter layer diet (20% CP and 11.84 MJ/kg ME, T2), feeding a starter layer diet contained 3% molasses in its composition (20% CP and 11.81 MJ/kg ME; T3), and feeding a starter broiler diet (23% CP and 12.68 MJ/kg ME, T4). After the first 72 h of chick’s life, all chicks were fed ad libitum the T2 diet. EFPH had no negative effect on the development of the lymphoid or digestive organs in chicks. Greater relative weights of the spleen and bursa of Fabricius (p < 0.05) were observed in the early fed chicks compared to control at day 14 of age. Histomorphological examination revealed an increase (p < 0.05) in thymus cortex and cortex:medulla in the T3 and T4 groups compared to the fasted ones at day 28 of age. Pelicae height, follicular width, cortex, and cortex:medulla of bursa were improved (p < 0.01) in the fed groups compared to fasted chicks, with resultant influences on the primary lymphoid organs. Compared to control, higher germinal center areas and white pulp of the spleen (p < 0.05) were recorded in the early fed chicks, implying augmented proliferation and maturation of B cells in the secondary lymphoid organs. In the liver, a strong positive reaction to Best’s carmine stain in the early fed groups, indicating that the liver of these chicks had numerous glycogen granules or greater glycogen density in the cytoplasm of hepatocytes. There was a significant enhancement in the proventriculus mucosal and gland thickness, as well as fold height (p < 0.05) in the early fed chicks. The expression levels of splenic Toll-like receptor 2, interleukin 4, tumor necrosis factor α, and interferon gamma were up-regulated (p < 0.01) in most of the early fed chicks (T2, T3, and T4) compared to fasted ones at 14 day of age. In conclusion, EFPH could modify the splenic-immunity related genes and modulate the histomorphology of the digestive (liver and proventriculus) and lymphoid organs in layer-type chicks during the brooding period.

Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Blueberry-derived exosomes-like nanoparticles ameliorate nonalcoholic fatty liver disease by attenuating mitochondrial oxidative stress.

Accumulating evidence indicates that mitochondrial dysfunction and oxidative stress play a pivotal role in the initiation and progression of nonalcoholic fatty liver disease (NAFLD). In this study, we found that blueberry-derived exosomes-like nanoparticles (BELNs) could ameliorate oxidative stress in rotenone-induced HepG2 cells and high-fat diet (HFD)-fed C57BL/6 mice. Preincubation with BELNs decreased the level of reactive oxygen species (ROS), increased the mitochondrial membrane potential, and prevented cell apoptosis by inducing the expression of Bcl-2 and heme oxygenase-1 (HO-1) and decreasing the content of Bax in rotenone-treated HepG2 cells. We also found that preincubation with BELNs accelerated the translocation of Nrf2, an important transcription factor of antioxidative proteins, from the cytoplasm to the nucleus in rotenone-treated HepG2 cells. Moreover, administration of BELNs improved insulin resistance, ameliorated the dysfunction of hepatocytes, and regulated the expression of detoxifying/antioxidant genes by affecting the distribution of Nrf2 in the cytoplasm and nucleus of hepatocytes of HFD-fed mice. Furthermore, BELNs supplementation prevented the formation of vacuoles and attenuated the accumulation of lipid droplets by inhibiting the expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC1), the two key transcription factors for de novo lipogenesis in the liver of HFD-fed mice. These findings suggested that BELNs can be used for the treatment of NAFLD because of their antioxidative activity.

A Quantitative Study on the Protective Effects of Resveratrol against Bisphenol A-induced Hepatotoxicity in Rats: A Stereological Study.

Background: Bisphenol A (BPA) is a widely used chemical with toxic effects on the liver. Resveratrol (RES) is an herbal compound with protective properties. This study aimed to investigate the protective effects of RES on the liver in rats exposed to BPA.Methods:This study was conducted in 2018 in Shiraz, Iran. Thirty Sprague-Dawley male rats were divided into five groups: a control group (distilled water), a sham group (olive oil as a BPA solvent), a BPA group (50 mg/kg), an RES group (100 mg/kg), and a RES+BPA group (50 mg/kg+100 mg/kg). Olive oil, BPA, and RES were administered to the animals via gavage for eight weeks. After eight weeks, the animals’ livers were removed, and stereological measurements were made to obtain the total liver volume, portal triad volumes, hepatocyte nucleus and cytoplasm volumes, hepatocyte numbers, sinusoidal space volumes and lengths, and Kupffer cell (KC) numbers. The data were analyzed using the one-way analysis of variance testResults: The hepatocyte number, the total liver volume, and hepatocyte nucleus and cytoplasm volumes in the BPA group decreased by 41% (P<0.001), 18% (P<0.001), 32% (P=0.030), and 37% (P=0.014), respectively. The number of KCs and the length of sinusoids in the BPA group were increased compared with all the other groups (P<0.001). Our histological study revealed vacuolization, sinusoidal space dilatation, and congestion in the BPA group.Conclusion: In this study, the RES group, compared with the BPA group, exhibited a decrease in the total volume and length of sinusoids and the number of KCs. Additionally, the RES group showed an increase in the total liver volume, hepatocyte nucleus and cytoplasm volumes, portal triad volumes, and hepatocyte numbers after oral administration

Clofibric acid increases molecular species of phosphatidylethanolamine containing arachidonic acid for biogenesis of peroxisomal membranes in peroxisome proliferation in the liver

The biogenesis of peroxisomes in relation to the trafficking of proteins to peroxisomes has been extensively examined. However, the supply of phospholipids, which is needed to generate peroxisomal membranes in mammals, remains unclear. Therefore, we herein investigated metabolic alterations induced by clofibric acid, a peroxisome proliferator, in the synthesis of phospholipids, particularly phosphatidylethanolamine (PE) molecular species, and their relationship with the biogenesis of peroxisomal membranes. The subcutaneous administration of clofibric acid to rats at a relatively low dose (130 mg/kg) once a day time-dependently and gradually increased the integrated perimeter of peroxisomes per 100 μm2 hepatocyte cytoplasm (PA). A strong correlation was observed between the content (μmol/mg DNA) of PE containing arachidonic acid (20:4) and PA (r2 = 0.9168). Moreover, the content of PE containing octadecenoic acid (18:1) positively correlated with PA (r2 = 0.8094). The treatment with clofibric acid markedly accelerated the formation of 16:0–20:4 PE by increasing the production of 20:4 and the activity of acyl chain remodeling of pre-existing PE molecular species. Increases in the acyl chain remodeling of PE by clofibric acid were mainly linked to the up-regulated expression of the Lpcat3 gene. On the other hand, clofibric acid markedly increased the formation of palmitic acid (16:0)–18:1 PE through de novo synthesis. These results suggest that the enhanced formation of particular PE molecular species is related to increases in the mass of peroxisomal membranes in peroxisome proliferation in the liver.

EMBRYOGENESIS, LEUKOCYTIC PROFILE AND LIVER MORPHOLOGY OF PREGNANT RATES UNDER THE ACTION OF CITRIC ACID

The article presents the results of studies on the effects of citric acid on the female’s body. The studies were performed on white laboratory rats of the Wistar line, which at the age of 3 - 3.5 months with a body weight of 192 - 210 g were divided into two groups of 4 individuals in each. The animals of the experimental group were fed by citric acid at a concentration of 80 mg/l of drinking water during the period physiological and puberty, fertilization and pregnancy. A control group of female rats was fed by water. The animals had free access to drinkers and food. Indicators of embryogenesis, leukocyte profile of blood and body weight of animals, weighting factor, histological changes of the liver were determined.&#x0D; Female rats were found to have no abortions or premature births, however, the number of corpora lutea of pregnancy decreased by 19.2%, although there was no significant difference in control. The number of implantation sites per female was significantly lower by 28.8%. The mortality rate of embryos was 15.8%, which is 3.8 times higher than in intact animals. Regarding white blood cells, the analysis of the results indicates a tendency to increase the percentage of eosinophils in 2 times in the relation to the animals in the control group on the background of a probable increase in leukocytes by 42.4%.&#x0D; The applied dose of citric acid caused certain changes in the macro- and microstructure of the liver. It is macroscopically established that under the influence of citric acid the organ is slightly enlarged, sluggish consistency and changed its color from light red to light brown. The relative weighting factors of the liver are probably higher in the relation to the control (52.12 ± 8.16 g/kg vs 43.38 ± 1.96 g/ kg).&#x0D; The results of morphological studies indicate a partial violation of the lamellar structure of the organ in the centro-lobular region, which is due to turbid swelling of the cytoplasm of hepatocytes. In most cells, the contours are blurred, the cytoplasm is poorly stained, their nuclei are weakly basophilic, and hepatocytes with a lysed nucleus in a necrobiosis condition also occur. However, among dystrophic altered cells are clearly isolated hepatocytes with a uniformly colored basophilic cytoplasm, nucleus, rich in chromatin, which indicated the activation of reparative processes. Kupffer cells in this area are mostly round, located in the lumen of the sinusoid. Therefore, occurs a violation of lipoprotein complexes of cells and increase the permeability of cell membranes, which is reflected in the development of protein dystrophy with varying degrees of severity. Activation of reparative processes of hepatocytes is also determined.

BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of β-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis.

BACKGROUNDBIR repeat-containing ubiquitin conjugating enzyme (BRUCE) is a liver tumor suppressor, which is downregulated in a large number of patients with liver diseases. BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine (DEN)-dependent acute liver injury and carcinogenesis. While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma (HCC), DEN exposure alone does not induce robust hepatic fibrosis. Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC. AIMTo investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development.METHODSMale C57/BL6/J control mice [loxp/Loxp; albumin-cre (Alb-cre)-] and BRUCE Alb-Cre KO mice (loxp/Loxp; Alb-Cre+) were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression. RESULTSBy using a liver-specific BRUCE knockout (LKO) mouse model, we found that BRUCE deficiency, in conjunction with DEN exposure, induced hepatic fibrosis in both premalignant as well as malignant stages, thus recapitulating the chronic fibrosis background often observed in HCC patients. Activated in fibrosis and HCC, β-catenin activity depends on its stabilization and subsequent translocation to the nucleus. Interestingly, we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity of β-catenin in the three stages of carcinogenesis: Pre-malignancy, tumor initiation, and HCC. This suggests that BRUCE negatively regulates β-catenin activity during liver disease progression. β-catenin can be activated by phosphorylation by protein kinases, such as protein kinase A (PKA), which phosphorylates it at Ser-675 (pSer-675-β-catenin). Mechanistically, BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level. However, in BRUCE deficient mouse livers or a human liver cancer cell line, both PKA activity and pSer-675-β-catenin levels were observed to be elevated.CONCLUSIONOur data support a “BRUCE-PKA-β-catenin” signaling axis in the mouse liver. The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation of β-catenin. This study implicates BRUCE as a novel negative regulator of both PKA and β-catenin in chronic liver disease progression. Furthermore, BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA and β-catenin.

REC8 promotes tumor migration, invasion and angiogenesis by targeting the PKA pathway in hepatocellular carcinoma.

REC8 is a member of the cohesin family, and its abnormal activation has been detected in cancer cells. This study explored the role and possible mechanism of REC8 in hepatocellular carcinoma (HCC). A total of 40 pairs of HCC and adjacent tissues were collected, and the clinical significance of REC8 expression in HCC was evaluated. REC8 expression in human HCC tissues and HCC cell lines was investigated by quantitative real-time PCR, Western blotting, immunohistochemistry and immunofluorescence staining. The biological functions of REC8 in HCC cell lines were detected by wound-healing assay, Matrigel invasion assay and tube formation assay. The proteins interacting with REC8 were identified by mass spectrometry after immunoprecipitation screening. There was a correlation between the high expression of REC8 and positive alpha-fetoprotein levels. The expression level of REC8 protein in HCC tissues was higher than that in adjacent tissues. REC8 has mainly located in the nucleus of HCC tissue cells and HCC cell lines, but it was expressed in the cytoplasm of adjacent normal tissue cells and hepatocytes. The results of wound healing, transwell invasion and tubular formation assays indicated that the overexpression of REC8 accelerated the metastasis of HCC in vitro; however, metastasis was suppressed after REC8 was silenced by small interference RNA. A total of 57 differentially expressed proteins were identified by mass spectrometry, and it was found that REC8 and PKA RII-α staining was colocalized in the nucleus. The expression levels of MMP-9 and VEGF-C were decreased after treatment with the PKA inhibitor H89. Overall, REC8 promotes the migration, invasion and angiogenesis of HCC cells through the PKA pathway.

Effect of amino acid imbalance in the nutrition on the morphology of animal liver

The article considers the influence of various forms of amino acid imbalance in diets on morphological changes in the liver of laboratory animals. An imbalance of amino acids caused a number of similar changes in the liver of animals. Liver sections revealed that hepatocytes had smoothed borders and areas with signs of granular dystrophy. The sinusoids and inter-protein spaces were overflowed with blood moulds. Small groups of lymphohistiocytic cells were presented in the parenchyma. Violation of the liver microcirculation contributed to the violation of metabolic processes in hepatocytes. Most of the hepatocytes were filled with yellow-orange fat droplets and had atrophied cytoplasm with nuclei shifted to the periphery. There were single cells with glycogen granules as well. The content of nucleic acids in tissues is an important indicator of the intensity of protein synthesis. Thus, in liver sections of groups 1, 2, 3 and 4, the cytoplasm of hepatocytes was less colored than in the control group, which indirectly indicates an increase in the intensity of protein synthesis in liver cells. Hemodynamic disorders in the form of dilated blood vessels and the appearance of stasis in groups 1, 2, 3 and 4 lead to difficulties in the penetration of amino acids to the sites of protein synthesis and, as a result, to a violation of metabolic processes in parenchymal liver cells.

Fowl Adenovirus Serotype 4 Induces Hepatic Steatosis via Activation of Liver X Receptor-α.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic virus that causes severe hepatic damage characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal necrosis in hepatocytes. Many aspects of FAdV-4 infection and pathogenesis, however, remain unknown. Here, we found that FAdV-4-induced hepatic injury is accompanied by the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in FAdV-4-infected chickens. Significant upregulation of adipose synthesis-related genes, such as liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding protein-1c (SREBP-1c), and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation- and lipid decomposition-related genes were observed in the infected chickens. FAdV-4 infection in cultured leghorn male hepatoma (LMH) cells caused similar signs of steatosis, with alterations in various lipogenesis-related genes. We eliminated the effect of LXR-α activation on FAdV-4-induced steatosis and found that treatment with an LXR-α antagonist (SR9243) and RNA interference (small interfering RNA targeting LXR-α [Si-LXR-α]) decreased the number of oil droplets and the accumulation of lipogenic genes, but treatment with an LXR-α agonist (T0901317) increased the number of oil droplets and the accumulation of lipogenic genes in the cells. Additionally, SR9243 treatment or Si-LXR-α transfection led to significant reductions in viral DNA level, protein expression, and virus production, whereas T0901317 treatment caused significant increases in viral DNA level, protein expression, and virus production. However, inhibition of SREBP-1c activity had no significant effect on virus production. Collectively, these results indicated that FAdV-4-induced steatosis involves activation of the LXR-α signaling pathway, which might be a molecular mechanism underlying the hepatic injury associated with FAdV-4 infection.IMPORTANCE Fowl adenovirus serotype 4 (FAdV-4) is an important hepatotropic adenovirus in chicken, but the underlying mechanism of FAdV-4-induced hepatic injury remains unclear. We report here that infection with FAdV-4 induced the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in the livers of chickens. FAdV-4-induced steatosis might be caused by a disrupted balance of fat metabolism, as evidenced by differential regulation of various lipase genes. The significant upregulation of liver X receptor-α (LXR-α) prompted us to investigate the interplay between LXR-α activation and FAdV-4-induced steatosis. Treatment with an agonist, an antagonist, or RNA interference targeting LXR-α in cultured leghorn male hepatoma (LMH) cells indicated that FAdV-4-induced steatosis was dependent upon LXR-α activation, which contributed to virus replication. These results provide important mechanistic insights, revealing that FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway and highlighting the therapeutic potential of strategies targeting the LXR-α pathway for the treatment of FAdV-4 infection.

Alterations of Gut Microbiota by Overnutrition Impact Gluconeogenic Gene Expression and Insulin Signaling.

A high-fat, Western-style diet is an important predisposing factor for the onset of type 2 diabetes and obesity. It causes changes in gut microbial profile, reduction of microbial diversity, and the impairment of the intestinal barrier, leading to increased serum lipopolysaccharide (endotoxin) levels. Elevated lipopolysaccharide (LPS) induces acetyltransferase P300 both in the nucleus and cytoplasm of liver hepatocytes through the activation of the IRE1-XBP1 pathway in the endoplasmic reticulum stress. In the nucleus, induced P300 acetylates CRTC2 to increase CRTC2 abundance and drives Foxo1 gene expression, resulting in increased expression of the rate-limiting gluconeogenic gene G6pc and Pck1 and abnormal liver glucose production. Furthermore, abnormal cytoplasm-appearing P300 acetylates IRS1 and IRS2 to disrupt insulin signaling, leading to the prevention of nuclear exclusion and degradation of FOXO1 proteins to further exacerbate the expression of G6pc and Pck1 genes and liver glucose production. Inhibition of P300 acetyltransferase activity by chemical inhibitors improved insulin signaling and alleviated hyperglycemia in obese mice. Thus, P300 acetyltransferase activity appears to be a therapeutic target for the treatment of type 2 diabetes and obesity.

Proteinuria converts hepatic heparan sulfate to an effective proprotein convertase subtilisin kexin type 9 enzyme binding partner

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

HBV Core Protein Is in Flux between Cytoplasmic, Nuclear, and Nucleolar Compartments.

ABSTRACTHepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.

Autophagy as a life support marker of isolated hepatocytes

AIM: The work aimed to reveal structural signs of autophagy in the cytoplasm of isolated hepatocytes in the dynamics of their cultivation.&#x0D; MATERIALS AND METHODS: The cultivated hepatocyte culture cell cycle was studied by flow cytofluorometry. The cells were cultured for 1, 24, and 48 hours. Morphometric analysis was performed using of the computer program Image J. The diameters of the nuclei and cytoplasm of hepatocytes, the volumes of nuclei and cytoplasm, and the nuclear-cytoplasmic ratio were determined. The concentration of intracellular organelles and autophagy was evaluated with magnification by 30000 times.&#x0D; RESULTS: The cell cycle arrest in the G0/G1 stage after 24 hours of hepatocyte cultivation and the preservation of their viability by hour 48 of the experiment without increase in the percentage of cells in the apoptosis stage were revealed. The decrease in the absolute count of cells was registered, as well as an increase in the nuclear-cytoplasmic ratio indicating a decrease in the proportion of hepatocyte cytoplasm in the course of cultivation. After 24 hours of cultivation, autophagosomes with fragments of cytoplasm, glycogen rosettes, and autolysosomes with partially degraded material were revealed in the cell cytoplasm. By hour 48 of the study, a significant decrease in the volume density of glycogen and mitochondria was noted, as well as an increase in basal autophagy in hepatocytes, with a prevalence of glycophagy and mitophagy.&#x0D; CONCLUSIONS: Autophagy maintains cellular homeostasis of isolated hepatocytes under standard culture conditions, as evidenced by a decrease in the volume density of glycogen and mitochondria, and an increase in basal autophagy in the hepatocyte cytoplasm. The findings indicate the contribution of autophagy to the survival of the primary culture of hepatocytes and can be used as an indicator of the adequacy of culturing conditions.

Assessment of cases of fatty liver disease using USG and CT scan

Background: Fatty Liver Disease (FLD) also known as Hepatic Steatosis (HS) is identified by the abnormal build-up of lipids particularly triglyceride in the cytoplasm of hepatocytes. The present study was conducted to assess fatty liver disease using USG and CT scan. Materials & Methods: 130 cases of fatty liver disease in both genders were thoroughly examined and underwent Ultrasonography (USG) and CT Scan. Results: Out of 130 patients, males were 78 and females were 52. FLD grading was grade I in 76, grade II in 34 and grade III in 20 patients. The difference was significant (P< 0.05). Liver CTHFN Mean HU in grade I was 42.6 HU, in grade II was 25.2 HU and in grade III was 3.8 HU. The difference was significant (P

Lipid Storage and Interferon Response Determine the Phenotype of Ground Glass Hepatocytes in Mice and Humans.

Background and AimsA histopathological hallmark of chronic hepatitis B virus (HBV) infection is the presence of ground glass hepatocytes (GGHs). GGHs are liver cells that exhibit eosinophilic, granular, glassy cytoplasm in light microscopy and are characterized by accumulation of HBV surface (HBs) proteins in the endoplasmic reticulum (ER). More important, GGHs have been accepted as a precursor of HCC and may represent preneoplastic lesions of the liver.MethodsHere we show that the reason for ground glass phenotype of hepatocytes in patients with chronic hepatitis B (CHB) and in HBs transgenic mice is a complex formation between HBs proteins and lipid droplets (LDs) within the ER.ResultsAs fat is a main component of LDs their presence reduces the protein density of HBs aggregates. Therefore, they adsorb less amount of eosin during hematoxylin-eosin staining and appear dull in light microscopy. However, after induction of interferon response in the liver LDs were not only co-localized with HBs but also distributed throughout the cytoplasm of hepatocytes. The uniform distribution of LDs weakens the contrast between HBs aggregates and the rest of the cytoplasm and complicates the identification of GGHs. Suppression of interferon response restored the ground glass phenotype of hepatocytes.ConclusionsComplex formation between HBs and LDs represents a very important feature of CHB that could affect LDs functions in hepatocytes. The strain specific activation of the interferon response in the liver of HBs/c mice prevented the development of GGHs. Thus, manipulation of LDs could provide a new treatment strategy in the prevention of liver cancer.

Involvement of PDL1 and Foxp3 Expression in Human Liver Transplantation Tolerance

Background: Operational tolerance after liver transplantation (LT) characterized by successful immunosuppression withdrawal (ISW) without rejection has been reported but its underlying mechanisms are unknown. Programmed cell death protein 1 (PD-1)/its ligand PD-L1 pathway, concomitant with forkhead box protein P3 (Foxp3), may play a specific role in operational tolerance after LT. Methods: Tolerance and control recipients are defined as recipients maintained without and with standard dose of immunosuppression (IS) without episodes of rejection for at least one year. Liver biopsies from 38 tolerant, 35 non-tolerant (including 16 samples that triggered reintroduction of IS and 19 samples after IS reintroduction) and 38 control LT patients were studied. Expression of PD-1, PD-L1, Foxp3, CD4 and CD8 were determined by immunohistochemical staining. Results: PD-1 was hardly detectable while PD-L1 was expressed in the cytoplasm of hepatocytes and Foxp3 was localized in the nucleus. CD4 and CD8 were expressed in the periportal area. Tolerant and control patients exhibited a higher PD-L1 level (median[interquartile]: 20.8[16.2-26] relative units (RU) and 19.8[11.2-30.8]RU vs. 13.3[8-19.3]RU; p=0.0008 and p=0.0036) and higher Foxp3/CD8 ratio (4[0.8-11.8] and 3[1-11.8] vs. 0[0-2.5]; p=0.0001 and p=0.0013) in compared to non-tolerant patients at the moment of triggering IS reintroduction; whereas CD4 expression was comparable between the three groups. A weak but significant correlation between PDL1 and Foxp3 was identified (r=0.2401, p=0.0211). Conclusion: PD-L1 and Foxp3 might concomitantly contribute to the regulation of immune-tolerant in LT patients. Further studies regarding PD-1/PD-L1 pathway may shed light on the mechanisms for operational tolerance after LT.

Spontaneous poisoning by Cestrum intermedium in dairy cattle

ABSTRACT: Acute hepatotoxicity caused by plants poisoning is responsible for economic losses in farm animals in Brazil. Reports of Cestrum intermedium natural poisoning in cattle are not commonly described in Rio Grande do Sul (RS). This study aimed to document an outbreak of spontaneous C. intermedium poisoning in dairy cattle in the Central-Eastern Mesoregion of RS. Three nine-month-old Holstein and Jersey heifers were affected after they were placed in a small paddock with shortage forage. In this area, specimens of C. intermedium Sendtn with signs of consumption were observed. Morbidity and lethality rates were 100% and clinical courses ranged from 9 to 12 hours. At post mortem examination of the three heifers, there was predominance of acute liver lesions. The liver was moderately enlarged and on the cut surface there was a marked accentuation of the lobular pattern and hemorrhage. Inside the rumen, partially digested C. intermedium Sendtn leaves were observed. The histological aspects of the liver were mostly centrilobular coagulative necrosis and hemorrhage, frequently extended to the midzonal region. The immunohistochemistry technique was performed, in which the polyclonal antibody caspase 3 was used in liver fragments. Moderate to marked immunolabeling was observed in the cytoplasm and nucleus of hepatocytes, predominantly on the periphery of areas of hepatic necrosis indicating cell apoptosis. The diagnosis of C. intermedium Sendtn poisoning in dairy cattle in this study was based on epidemiological, clinical and anatomopathological findings. Since the C. intermedium poisoning is uncommon in dairy cattle, we are describing it for the first time in the Central-Eastern Mesoregion of RS, and represents a differential diagnosis of other acute toxic liver diseases in cattle.