Ki M. Mak

Phosphatidylcholine Protects Against Fibrosis and Cirrhosis in the Baboon

BACKGROUND/AIMS Polyunsaturated soybean lecithin (55%-60% phosphatidylcholine [PC]) protects against fibrosis in alcohol-fed baboons. The present study was undertaken to determine whether PC is the active agent. METHODS Virtually pure PC (equivalent to that contained in the lecithin) was administered for up to 6.5 years with or without alcohol, and the results were compared with those of unsupplemented groups. RESULTS Control livers remained normal, whereas 10 of 12 baboons fed alcohol without PC developed septal fibrosis or cirrhosis with transformation of 81% +/- 3% of the hepatic lipocytes to collagen-producing transitional cells. By contrast, none of the eight animals fed alcohol with PC developed septal fibrosis or cirrhosis, and only 48% +/- 9% of their lipocytes were transformed, indicating that PC was indeed the protective compound. Ethanol feeding also resulted in decreased liver phospholipids and PC, and both were corrected by the supplementation. Furthermore, PC stimulated collagenase activity in cultured lipocytes. This PC consisted of several species, mainly dilinoleoylphosphatidylcholine (40%-52%) and palmitoyl-linoleoylphosphatidylcholine (23%-24%). Only dilinoleoylphosphatidylcholine duplicated the effect of the PC on collagenase. Other species of PC, phosphatidylethanolamine, free fatty acids, or choline were without effect. CONCLUSIONS PC prevents alcohol-induced fibrosis and cirrhosis in nonhuman primates, and dilinoleoylphosphatidylcholine appears to be the active species, possibly by promoting collagen breakdown.

Dilinoleoylphosphatidylcholine decreases LPS-induced TNF-α generation in Kupffer cells of ethanol-fed rats: respective roles of MAPKs and NF-κB

Activation of Kupffer cells by lipopolysaccharide (LPS) after ethanol feeding results in overproduction of TNF-alpha, leading to liver injury. Since dilinoleoylphosphatidylcholine (DLPC) protects against liver injury and has antioxidant properties, we investigated whether it alters LPS signaling leading to decreased TNF-alpha production. Kupffer cells were isolated from rats fed alcohol-containing or isocaloric control diets for 3 weeks. With ethanol, cytochrome P4502E1 was upregulated. When stimulated with LPS in culture, Kupffer cells released more TNF-alpha compared to control rats; DLPC diminished the increase. It also reduced ERK1/2 and p38 phosphorylation as well as NF-kappaB activation with decreased nuclear p65 and increased cytosolic IkappaB-alpha expression. ERK1/2 and NF-kappaB activation were abolished by the ERK1/2 inhibitor PD098059. The p38 inhibitor SB203580 abolished p38 activation without affecting NF-kappaB. Both inhibitors reduced TNF-alpha generation. Thus, DLPC diminishes LPS-dependent TNF-alpha generation by inhibiting p38 and ERK1/2 activation; the latter leads to decreased NF-kappaB activation.

Leptin enhances α1(I) collagen gene expression in LX‐2 human hepatic stellate cells through JAK‐mediated H2O2‐dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal‐regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase‐1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX‐2 human HSCs, leptin enhances the levels of α1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK‐mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2‐dependent mechanism. AG490 and catalase also prevented leptin‐stimulated α1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed α1(I) collagen promoter activity, resulting in mRNA down‐regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down‐regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half‐life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2‐dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates α1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin‐induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis. J. Cell. Biochem.

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 α‐smooth muscle actin (α‐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 α‐SMA mRNA and its protein, while ADRP knockdown with small interfering RNA (siRNA) normalized α‐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. J. Cell. Physiol. 223:648–657, 2010.

Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-α generation in Kupffer cells of ethanol-fed rats

We previously reported that dilinoleoylphosphatidylcholine (DLPC) decreases lipopolysaccharide-induced TNF-alpha generation by Kupffer cells of ethanol-fed rats by blocking p38, ERK1/2, and NF-kappaB activation. Here we show that DLPC also decreases TNF-alpha induction by acetaldehyde, a toxic metabolite released by ethanol oxidation. Acetaldehyde induces TNF-alpha generation with a maximal effect at 200 microM and activates p38 and ERK1/2; the latter in turn activates NF-kappaB. This effect is augmented in Kupffer cells of ethanol-fed rats, with upregulation of cytochrome P4502E1 by ethanol. DLPC decreases TNF-alpha generation by blocking p38, ERK1/2, and NF-kappaB activation. Likewise, SB203580, which abolishes p38 activation, and PD098059, which abrogates ERK1/2 and NF-kappaB activation, diminish TNF-alpha generation. Since increased TNF-alpha generation plays a pathogenic role in alcoholic liver disease, the DLPC action on Kupffer cells may explain, in part, its beneficial effects on liver cell injury after ethanol consumption.

Adipose Differentiation‐Related Protein Is a Reliable Lipid Droplet Marker in Alcoholic Fatty Liver of Rats

BACKGROUND Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that coats cytoplasmic lipid droplets. The present study evaluated whether alcohol feeding enhances ADRP expression and whether ADRP is a lipid droplet marker in alcoholic fatty liver of rats. Because medium-chain triglycerides (MCT) reduce alcoholic hepatosteatosis, their effects on ADRP were also evaluated. METHODS Fatty liver was induced in rats by the consumption of the Lieber-DeCarli alcohol liquid diet with or without replacement of long-chain triglycerides (LCT) by MCT (32% of calories). Immunohistochemical staining for ADRP was performed in formalin-fixed, paraffin-embedded liver sections. ADRP immunostaining was quantified by image analysis. Triacylglycerol was measured chemically. ADRP mRNA and protein were analyzed by real-time polymerase chain reaction and western blot, respectively. Double staining technique was performed to distinguish ADRP from glycogen in hepatocytes. RESULTS Alcohol feeding for 21 days increased ADRP staining in the centrilobular and mid zonal regions of the liver lobules coincident with fat deposition in the liver. Replacing LCT in the alcohol diet with MCT diminished ADRP immunostaining in parallel with reduced steatosis. MCT also attenuated the up-regulation of ADRP mRNA and protein after alcohol. In steatotic hepatocytes ADRP selectively stained the surface of macrovesicular and microvesicular lipid droplets. ADRP immunostaining quantitatively correlated with hepatic triacylglycerol levels, validating ADRP as a reliable lipid droplet marker. Compared with hematoxylin and eosin stains, ADRP was more sensitive in detecting microvesicular lipid droplets. ADRP immunostaining also distinguished lipid droplets from glycogen, as demonstrated by double staining for ADRP and glycogen. CONCLUSIONS Alcohol induction of fatty liver enhances ADRP expression and MCT oppose the alcohol effects. ADRP is a reliable and sensitive marker for lipid droplets in alcoholic fatty liver. ADRP immunostaining permits quantification of fatty change in hepatocytes and can be used as an ancillary technique in assessing the efficacy of diets or drugs against hepatosteatosis.

Dilinoleoylphosphatidylcholine selectively modulates lipopolysaccharide-induced Kupffer cell activation

Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, protects against alcoholic and non-alcoholic liver injury. Because Kupffer cells mediate liver injury, we hypothesized that PPC may modulate their activation. The activation of Kupffer cells by lipopolysaccharide (LPS) leads to an enhanced production of cytokines. Among these, tumor necrosis factor-alpha(TNF-alpha) exerts mainly a hepatotoxic effect, whereas interleukin-1beta (IL-1beta) appears to be hepatoprotective. The present study evaluated whether dilinoleoylphosphatidylcholine (DLPC), the main component of PPC (40% to 52%), affects LPS-induced Kupffer cell activation in vitro. For comparison, palmitoyl-linoleoylphosphatidylcholine (PLPC), the other major component of PPC (23% to 24%), and distearoylphosphatidylcholine (DSPC), the saturated counterpart of DLPC, were also tested. Rat Kupffer cells were cultured in serum-free RPMI-1640 medium containing 10 micromol/L of either DLPC, PLPC, or DSPC in the presence or absence of LPS (1 microg/mL). After 20 hours in culture, the media were collected for cytokine measurements by enzyme-linked immunosorbent assays. LPS significantly stimulated TNF-alpha and IL-1beta production by 62% and 328%, respectively. Treatment of Kupffer cells with LPS plus DLPC decreased the production of TNF-alpha by 23% (12.17+/-1.83 pg/ng DNA vs 15.72 +/-2.74 pg/ng DNA, P < .05, n = 6) and increased that of IL-1beta by 17% (1.80 +/- 0.16 pg/ng DNA vs 1.54 +/- 0.08 pg/ng DNA, P< .05, n = 6). No effect of PLPC or DSPC on LPS-induced TNF-alpha or IL-1beta generation was observed, thereby illustrating the selective effect of DLPC in this process. Thus DLPC selectively modulates the LPS-induced activation of Kupffer cells by decreasing the production of the cytotoxic TNF-alpha while increasing that of the protective IL-1beta. This dual action of DLPC on cytokines may provide a mechanism for the protective effect against liver injury, but its significance still needs to be determined by in vivo studies.

Isolation and culture of myofibroblasts from rat liver.

Abstract Successful isolation and culture of myofibroblasts from rat liver is described for the first time. After collagenase digestion of livers, obtained from rats fed regular laboratory chow diet as well as control and vitamin A-supplemented liquid diets, cells were separated on a Percoll density gradient. Cells characterized as myofibroblasts by electron microscopy were cultured in a Dulbeccos modified Eagles medium. Characteristic features of myofibroblasts (abundant myofibrils) with dense bodies, indented nucleus, basal lamina-like structure, capacity to synthesize types I, III, and IV collagens and laminin were demonstrated in both primary and secondary cultures.

Metabolism of ethanol in rat gastric cells and its inhibition by cimetidine

BACKGROUND/AIMS Several studies have shown that the stomach has sufficient alcohol dehydrogenase activity to metabolize a significant amount of alcohol and that cimetidine depresses this alcohol dehydrogenase activity. However, both gastric metabolism of ethanol and its inhibition by cimetidine remain controversial. Given the difficulty in assessing gastric metabolism of ethanol in vivo, this subject was investigated in vitro. METHODS Cultured rat gastric epithelial cells were incubated with 200 mmol/L [1-14C]ethanol for 90 minutes with and without cimetidine (0.1-1 mmol/L) or omeprazole (1 mmol/L). The quantity of ethanol oxidized by gastric cells was measured by the amount of acetate produced using ion exchange chromatography. RESULTS The majority of cells at confluency had typical features of mucous cells. The gastric cells metabolized significant amounts of ethanol, sufficient to account for in vivo first-pass metabolism of ethanol in rats. Cimetidine, but not omeprazole, reduced ethanol metabolism by 39.9% +/- 4.9% (P < 0.01), an inhibition comparable with that previously reported for first-pass metabolism in vivo. CONCLUSIONS Gastric cells in tissue culture are capable of significant ethanol oxidation, the in vitro rates are sufficient to account for first-pass metabolism of ethanol in vivo, and cimetidine inhibits ethanol metabolism in tissue culture, an effect that parallels its decrease of first-pass metabolism in vivo.

Type V Collagen in Health, Disease, and Fibrosis

Type V collagen (COLV) is a regulatory fibril‐forming collagen. It has at least three different molecular isoforms—α1(V)2α2(V), α1(V)3, and α1(V)α2(V)α3(V)—formed by combinations of three different polypeptide α chains—α1(V), α2(V), and α3(V). COL V is a relatively minor collagen of the extracellular matrix (ECM). Morphologically, COLV occurs as heterotypic fibrils with type I collagen (COLI), microfilaments, or 12‐nm‐thick fibrils. COLV is synthesized in various mesenchymal cells and its gene expression is modulated by TGF‐β and growth factors. While resistant to digestion by interstitial collagenases, native and denatured COLV are degraded by metalloproteinases and gelatinases, thereby promoting ECM remodeling. COLV interacts with matrix collagens and structural proteins, conferring structural integrity to tissue scaffolds. It binds matrix macromolecules, modulating cellular behavior, and functions. COLV co‐assembles with COLI into heterotypic fibrils in the cornea and skin dermis, acting as a dominant regulator of collagen fibrillogenesis. COLV deficiency is associated with loss of corneal transparency and classic Ehlers–Danlos syndrome, while COLV overexpression is found in cancer, granulation tissue, inflammation, atherosclerosis, and fibrosis of lungs, skin, kidneys, adipose tissue, and liver. COLV isoform containing the α3(V) chain is involved in mediating pancreatic islet cell functions. In the liver, COLV is a minor but regular component of the ECM. Increases in COLV are associated with both early and advanced hepatic fibrosis. The neoepitopes of COLV have been shown to be a useful noninvasive serum biomarker for assessing fibrotic progression and resolution in experimental hepatic fibrosis. COLV is multifunctional in health, disease, and fibrosis. Anat Rec, 299:613–629, 2016.