Lipid Droplets In Hepatic Stellate Cells Research Articles

New Perspectives on Chinese Medicine in Treating Hepatic Fibrosis: Lipid Droplets in Hepatic Stellate Cells.

Hepatic fibrosis (HF) is a wound healing response featuring excessive deposition of the extracellular matrix (ECM) and activation of hepatic stellate cells (HSCs) that occurs during chronic liver injury. As an initial stage of various liver diseases, HF is a reversible pathological process that, if left unchecked, can escalate into cirrhosis, liver failure, and liver cancer. HF is a life-threatening disease presenting morbidity and mortality challenges to healthcare systems worldwide. There is no specific and effective anti-HF therapy, and the toxic side effects of the available drugs also impose a heavy financial burden on patients. Therefore, it is significant to study the pathogenesis of HF and explore effective prevention and treatment measures. Formerly called adipocytes, or fat storage cells, HSCs regulate liver growth, immunity, and inflammation, as well as energy and nutrient homeostasis. HSCs in a quiescent state do not proliferate and store abundant lipid droplets (LDs). Catabolism of LDs is characteristic of the activation of HSCs and morphological transdifferentiation of cells into contractile and proliferative myofibroblasts, resulting in the deposition of ECM and the development of HF. Recent studies have revealed that various Chinese medicines (e.g., Artemisia annua, turmeric, Scutellaria baicalensis Georgi, etc.) are able to effectively reduce the degradation of LDs in HSCs. Therefore, this study takes the modification of LDs in HSCs as an entry point to elaborate on the process of Chinese medicine intervening in the loss of LDs in HSCs and the mechanism of action for the treatment of HF.

HIF-1α-upregulated lncRNA-H19 regulates lipid droplet metabolism through the AMPKα pathway in hepatic stellate cells

Activation of hepatic stellate cells (HSCs) is a central event in the pathogenesis of liver fibrosis and is characterized by the disappearance of lipid droplets. Although the exogenous supplementation of lipid droplet content can effectively reverse the activation of HSCs, the underlying molecular mechanisms are largely unknown. In our current study, we sought to investigate the role of lncRNA-H19 in the process of lipid droplets disappearance and to further examine the underlying molecular mechanisms. We found that the lncRNA-H19 level was increased in CCl4-induced fibrotic liver, which activated HSCs. Further research showed that hypoxia inducible factor-1α (HIF-1α) significantly increased lncRNA-H19 expression by binding to the lncRNA-H19 promoter at two hypoxia response element (HRE) sites located at 492–499 and 515–522 bp. Importantly, lncRNA-H19 knockdown markedly inhibited HSC activation and alleviated liver fibrosis, indicating that lncRNA-H19 may be a potential target for anti-fibrosis therapeutic approaches. Moreover, lncRNA-H19 knockdown could reverse the lipid droplet phenotype of activated HSCs, inhibiting the phosphorylated AMPKα-mediated lipid oxidation signaling pathway. The AMPK agonist AICAR promoted AMPKα phosphorylation and abrogated lipid droplets restoration in HSCs transfected with the lncRNA-H19 knockdown plasmid. Experimental molecular analysis showed that lncRNA-H19 triggered AMPKα to interact with LKB1 and resulted in AMPKα phosphorylation, which accelerating lipid droplets degradation and lipid oxidation. Taken together, our results highlighted the role of lncRNA-H19 in the metabolism of lipid droplets in HSCs, and revealed a new molecular target for alleviating liver fibrosis.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF HEPATIC STELLATE CELLS PRE AND POST-TRANSPLANTED HUMAN LIVER

Background: Hepatic stellate cells (HSCs) regulate vitamin A metabolism and play a vital role during the activation of the immune response. During liver damage, HSCs transform to myofibroblast-like cells, leading to loss of their lipid content and synthesis of extracellular matrix (ECM) inducing liver fibrosis. Objective: The present study aimed to establish the correlation between HSCs activity within different areas of hepatic tissue and the degree of liver fibrosis in individuals with normal liver, hepatitis C virus infected patients and post-transplanted liver. Patients and methods: The study involved thirty four cases from the international medical center classified into three groups: group 1 included ten healthy individuals as control, group 2 included twelve patients with chronic hepatitis C virus and liver cirrhosis and group 3 included twelve patients with post-transplanted liver due to liver previous hepatitis C virus infection and hepatic cirrhosis subdivided into group 3a five patients who received antihepatitis C treatment, and group 3b seven patients who didn’t receive antihepatitis C treatment. We used H&E and Masson trichrome stains, immunohistochemical detection of a-smooth muscle actin (a-SMA), Glial Fibrillary acidic protein (GFAP) and transmission electron microscopy (TEM). Results: H&E stain revealed hepatic tissue with preserved architecture in group 1, disrupted architecture and areas of feathery degeneration in group 2, normal tissue histology in group 3a and moderate cellular infiltration in group 3b. Masson trichrome stain revealed normal collagen fibers distribution in group1, advanced fibrosis in group 2, no fibrotic changes in group 3a and few foci of bridging fibrosis in group 3b. Immunohistochemical analysis revealed increased expression of α-SMA in group 2 and group 3b in comparison to group 1 and group 3a. Immunohistochemical analysis revealed increased expression of GFAP in group 3b in comparison to group 3a, group 2 and group 1. TEM showed increase in the fibrous tissue and degeneration in the ultrastructure of the hepatocyte with few lipid droplets in HSCs of group 2 compared to group 1. Conclusion: The hepatic stellate cells play an important role in the fibrosis of the liver damaged by hepatitis C virus and in the post-transplanted liver which not treated from HCV.

DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis.

Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver. We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage. Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [(3)H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression. Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis.

Abstract 181: Hepatic retinoid signaling in mouse hepatocarcinogenesis models.

Abstract Retinoids are known to possess anti-cancer effects and a clinical trial of a synthetic retinoid for hepatocellular carcinoma (HCC) is ongoing recently in Japan. Hepatic retinoid metabolism is usually impaired during development of liver diseases. Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver diseases and retinoid stores are progressively lost from HSCs leading to hepatic fibrosis, cirrhosis, and HCC. In the present study, we employed two types of genetically-modified mice for investigating hepatic retinoid metabolism and signaling in diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis models. One is lecithin:retinol acyltransferas (LRAT) knockout (KO) mouse which lacks HSC lipid droplets and has much less hepatic retinoid stores, and the other is a transgenic mouse which expresses dominant negative form of retinoic acid receptor (dnRAR) to block retinoid signaling in the liver. Male 15 day-old mice were given intraperitoneal injections of DEN (25 mg/kg body weight). Eight-month-old DEN-treated Lrat KO mice showed lower liver tumor incidence and dnRAR transgenic mice showed higher incidence than that of respective control mice. In the acute phase after DEN injection, dnRAR transgenic mice exhibited higher hepatic levels of inflammatory cytokines. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN-bioactivation, and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Our results indicate that Lrat KO mice, instead of almost no hepatic retinoid storage, are less susceptible to DEN-induced hepatocarcinogenesis due to increased hepatic retinoid signaling which is accompanied by higher expression of p21 in the liver. In conclusion, retinoid signaling plays a key role in these hepatocarcinogenesis models. Citation Format: Yohei Shirakami, William S. Blaner, Masahito Shimizu, Hisataka Moriwaki, Mitsuru Seishima. Hepatic retinoid signaling in mouse hepatocarcinogenesis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2013-181

Abstract 4428: Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in mice lacking hepatic retinoid storage

Abstract Retinoids are known to possess anti-cancer effects and a clinical trial of a synthetic retinoid for hepatocellular carcinoma (HCC) is ongoing recently in Japan. Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma (HCC). Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15 day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body weight). Eight months later Lrat KO mice showed significantly less liver tumor development compared to WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN-bioactivation, and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4428. doi:1538-7445.AM2012-4428

Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin:retinol acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation‐related protein (ADRP)

Hepatic stellate cells (HSCs) store retinoids and triacylglycerols in cytoplasmic lipid droplets. Two prominent features of HSC activation in liver fibrosis are loss of lipid droplets along with increase of alpha-smooth muscle actin (alpha-SMA), but the link between these responses and HSC activation remains elusive. In non-adipose cells, adipose differentiation-related protein (ADRP) coats lipid droplets and regulates their formation and lipolysis; however its function in HSCs is unknown. Here, we observed, in human liver sections or primary HSC culture, ADRP localization to lipid droplets of HSCs, and reduced staining coincident with loss of lipid droplets in liver fibrosis and in culture-activated HSCs, consistent with HSC activation. In the LX-2 human immortalized HSCs, with scant lipid droplets and features of activated HSCs, we found that the upregulation of ADRP mRNA by palmitate is potentiated by retinol, accompanied by increased ADRP protein, generation of retinyl palmitate, and lipid droplet formation. ADRP induction also led to decreased expression of alpha-SMA mRNA and its protein, while ADRP knockdown with small interfering RNA (siRNA) normalized alpha-SMA expression. Furthermore, ADRP induction by retinol and palmitate resulted in decreased expression of collagen I and matrix metalloproteinase-2 mRNA, fibrogenic genes associated with activated HSCs, while increasing matrix metalloproteinase-1 mRNA; ADRP knockdown with siRNA reversed these changes. Tissue inhibitor of metalloproteinase-1 was not affected. Thus, ADRP upregulation mediated by retinol and palmitate promotes downregulation of HSC activation and is functionally linked to the expression of fibrogenic genes.

Raman imaging and lipidomic analysis of lipid droplets in (activated) hepatic stellate cells

In eukaryotic cells the excess of hydrophobic molecules is stored in special organelles named lipid droplets (LDs). These droplets contain triacylglycerides, cholesteryl esters and/or retinyl esters, depending on the function of the cell in which they reside. Retinyl esters, the storage form of vitamin A, are mainly stored in LDs in hepatic stellate cells (HSC), which comprise about 5–10% of total liver cells.

Assessing the roles of various retinoid‐metabolizing CYP enzymes in liver disease

Development of liver disease (LD) is associated with progressive depletion of hepatic stellate cell (HSC) retinoid‐storing lipid droplets. It is not clear whether this loss is the result of increased hepatic mobilization of retinol or increased retinoid catabolism by hepatic cytochrome P450 (CYP) enzymes. Wild type (WT) mice were treated with CCl4 as a model of experimentally induced hepatic fibrosis (EIHF). We did not observe any alterations in the expression of genes encoding proteins involved in retinoid transport in the livers of WT mice in response to CCl4. However, we did find that EIHF is accompanied by an approximate 4‐fold elevation in CYP26A1 and CYP2S1 mRNA levels. Because retinol uptake and storage in the liver involves both hepatocytes and HSCs, we were interested in understanding how each cell type may contribute to the progression of LD. We isolated hepatocytes and HSCs from the livers of WT mice and generated CYP expression profiles for each cell type. HSCs have approximately 50‐fold higher levels of CYP2S1 mRNA relative to hepatocytes, whereas CYP26A1 is highly expressed in hepatocytes. The high expression of CYP2S1 in HSCs and its induction in EIHF provides evidence for a role of this cytochrome in the progression of LD. Thus, the loss of HSC lipid droplets in LD may arise from increased CYP‐mediated retinoid catabolism and not increased retinol mobilization from the liver.Grant Funding SourceR01 DK079221

M1580 Loss of Hepatic Stellate Cell Lipid Droplets Does Not Contribute to Stellate Cell Activation and Liver Fibrosis

M1580 Loss of Hepatic Stellate Cell Lipid Droplets Does Not Contribute to Stellate Cell Activation and Liver Fibrosis

Retinoid storage in hepatic stellate cells

The liver is the main site of retinoid storage in the body. Within the liver, retinyl esters are hydrolyzed to retinol within hepatocytes and then may be secreted from the liver into the circulation bound to retinol‐binding protein (RBP) or may be re‐esterified by lecithin:retinol acyl transferase (LRAT) to form retinyl ester. Retinyl esters are then stored within the lipid droplets of hepatic stellate cells. We have previously reported that LRAT −/− mice have impaired absorption and storage of retinyl esters and have hepatic stellate cells devoid of their characteristic lipid droplets. Immunohistochemical studies of mouse liver using a monoclonal antibody against LRAT show expression primarily in hepatic stellate cells. We propose that LRAT is necessary for the formation of the lipid droplet by the stellate cell. To determine how retinoids are released from the lipid droplet, histologic examination of livers of RBP−/− mice was performed. In 18 month‐old RBP−/− mice, hepatic stellate cell lipid droplets were markedly enlarged. In 31 month‐old RBP−/− mice, numerous lipogranulomas, composed of lipid laden macrophages, were identified. EPON‐embedded thin sections and electron microscopy showed that the lipogranulomas were centered around hepatic stellate cells. Minimal steatosis of the RBP−/− livers was present. We propose that in the absence of RBP, hepatic stellate cell lipid droplets become distended and leak lipid eliciting a granulomatous reaction. The findings suggest that LRAT is necessary for lipid droplet formation and RBP is involved in mobilization of retinyl esters from lipid droplets in hepatic stellate cells. This work was supported by grants R01 DK061310, DK068436 and DK079221.

脂肪肝牛における血清中ビタミンA濃度と肝臓星細胞の関連性

Vitamin A (VA) is stored mainly in lipid droplets in hepatic stellate cells (HSCs) of the liver. Apparently, when the level of serum VA drops below the basic-requirement level, lipid droplets in an HSC either decrease in number or disappear altogether. Fatty-liver disease (fat-cow syndrome), which is common in periparturient cattle and makes the animals highly susceptible to ketosis, is a major cause of significant economic loss. In the past, some reports have dealt with the relationships between VA serum concentrations and pathological changes in cattle with fatty-liver disease. The present study aims to determine the correlation between morphological changes in HSCs and VA serum concentration in both normal (control) cattle (n= 5) and cattle with fatty-liver disease (experimental group). In the experimental group (n= 15), the number of HSCs per unit area decreased significantly with the disappearance of lipid droplets and collagen from the perisinusoidal space. The basementmembrane of the subsinusoidal endothelium, however, became significantly thicker than in the control group because of an accumulation of both cellular and noncellular substance in situ. Moreover, in the experimental group, nuclei and lipid droplets in HSCs were comparatively small. In the control group, on the other hand, lipid droplets in HSCs were notably large. Numbers of lipid droplets, VA serum concentrations and HSC formation in the controlgroup increased significantly. These findings clearly show correlation between VA serum concentration and HSC numbers and indicate the need to maintain blood CA concentrations in cattle with fatty-liver disease.

Nonparenchymal cells in chronically hyperinsulinemic liver acini of diabetic rats, with special regard to hepatic stellate cells

Background/Aims: An increase in proliferative activity and other distinct hepatocellular alterations — resembling preneoplastic foci and progressing to hepatocellular tumors — have been shown to develop in liver acini draining the blood from islets of Langerhans, transplanted through the portal vein into the liver of streptozotocin-diabetic rats. Methods: Altered and unaltered liver acini were investigated for possible changes in hepatic stellate cells 4–76 days after islet transplantation. Results: Corresponding to a significant increase in the hepatocellular volume, the volume density of total non-parenchymal cells was significantly reduced in altered compared to unaltered liver acini. With regard to the total nonparenchymal cell volume, the hepatic stellate cell fraction was not different, whereas the fraction of Kupffer cells was significantly reduced and the fraction of sinusoidal endothelial cells was significantly increased in altered compared to unaltered liver acini, respectively. The volume density as well as the single volume of the hepatic stellate cell mitochondria increased significantly in altered compared to unaltered liver acini. Hepatic stellate cell lipid droplets did not show significant differences between altered and unaltered liver acini. In situ hybridization for hepatocyte growth factor mRNA showed no differences in intensity of the specific signals in hepatic stellate cells of altered versus unaltered liver acini. The transplanted islets were negative for hepatic growth factor mRNA. Conclusions: The results suggest that hepatic growth factor production by hepatic stellate cells or by islet cells is not relevant to hepatocellular proliferative activity in altered liver acini.