EUE active components geniposide and aucubin regulate palmitate-induced cell death.

<p>Cells were treated with 500 µM palmitate in the presence or absence of 2.5, 5, or 10 µg/mL aucubin or geniposide for 24 hours. Cell viability (A) and caspase-3 activity (B) were analyzed. Immunoblotting was performed with antibody against active caspase-3, caspase-9, or β-actin (C). Cells were incubated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 0, 6, 12, 24, or 48 hours. Cell viability (D) and caspase-3 (E) activity were analyzed. Cells were incubated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 or 48 hours. Immunoblotting was performed with antibody against active caspase-3, caspase-9, or β-actin (F). Cells were incubated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 hours and stained with Hoechst (G). Cells were incubated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 or 48 hours. Immunoblotting was carried out with antibody against BAX, cathepsin B, LAMP-1, or tubulin (H). Cathepsin B activity in the medium was measured (I). Cells were incubated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 hours. Immunostaining was performed with anti-LAMP-1 antibody and subsequently with anti-cathepsin B antibody. The degree of overlap in staining was quantified (J). ^*<i>p</i><0.05, significantly different from palmitate-treated condition Pal.; palmitate.</p

EUE reduces hepatic lipotoxicity in rats fed a high-fat diet.

<p>Rats were given a normal diet or a high-fat diet with or without 0.25, 0.5, or 1 g/kg EUE for 10 weeks, and serum and livers were harvested. Liver tissues were loaded with 5 µM dihydroethidium and fluorescence image acquisition was performed (A). Liver tissue was subjected to lipid peroxidation assay (B), caspase-3 activity assay (C), and immunoblotting with antibody against caspase-3, -9, or β-actin (D). Serum levels of AST and ALT were <b>measured (E)</b>. Following subcellular fractionation, immunoblotting with antibody against BAX, t-Bid, PDI, COX II, or LAMP-1 was performed (F). ^*<i>p</i><0.05, significantly different from high-fat diet. HFD; high fat diet, EUE<i>; Eucommia ulmoides Oliver extract.</i></p

EUE regulates palmitate-reduced lysosomal activity.

<p>Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE for 24 hours followed by exposure to 5 µM LysoTracker and image acquisition (A). Lysosomal fluorescence was quantified (A; lower). Lysosomal V-ATPase activity was measured as described in Materials and Methods (B). Acridine orange solution and valinomycin were added to cell monolayers and intravesicular H⁺ uptake was initiated by the addition of Mg-ATP (C); fluorescence was quantified at 24 hours (C; right). Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE for 0, 6, 12, 24, or 48 hours, and levels of α-galactosidase, α-mannosidase, and acid phosphatase were measured (D). ^*<i>p</i><0.05, significantly different from palmitate-treated condition. DIC; differential interference contrast microscopy, Pal.; palmitate, EUE<i>; Eucommia ulmoides</i> Oliver extract.</p

EUE protects against palmitate-induced cell death through the regulation of lysosomal BAX localization.

<p>Cells were treated with 1 µM etoposide, 1 µM staurosporine, or 500 µM palmitate with or without 100 µg/mL EUE. Cell viability was assessed after 24 hours (A). Immunoblot analysis of the lysosomal fraction was performed with antibody against BAX, Hsp60, or LAMP-1 (B). Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE. After 24 or 48 hours, cell lysate and lysosome fractions were subjected to immunoblotting with antibodies against BAX, t-Bid, PDI (an ER marker protein), COXII (a mitochondrial marker protein), or LAMP-1 (a lysosomal marker protein). (C). Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE. After 24 hours, immunostaining was performed with antibodies against BAX or LAMP-1 (D). The overlapping pattern of fluorescence was quantified (D; right). ^*<i>p</i><0.05, significantly different from palmitate-treated condition, Pal.; palmitate, EUE<i>; Eucommia ulmoides</i> Oliver extract, DIC; differential interference contrast microscopy.</p

EUE active components geniposide and aucubin enhance lysosomal enzyme activation.

<p>Cells were treated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 hours. Lysosomal V-ATPase activity was measured as described in Materials and Methods (A). Cells were treated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 24 hours followed by exposure to 5 µM LysoTracker and image acquisition. The fluorescence was quantified (B). Acridine orange solution and valinomycin were added to cell monolayers and intravesicular H⁺ uptake was initiated by the addition of Mg-ATP (C); the fluorescence was quantified at 24 hours (C; right). Cells were treated with 500 µM palmitate in the presence or absence of 10 µg/mL aucubin or geniposide for 0, 12, 24, or 48 hours and the level of α-galactosidase, α-mannosidase, or acid phosphatase was measured (D). ^*<i>p</i><0.05, significantly different from palmitate-treated condition. Con; control, Pal.; palmitate.</p

Lysosomal V-ATPase inhibitor bafilomycin blocks the effect of EUE on lysosomal BAX location and cell death.

<p>Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE after pretreatment with 1 µM bafilomycin for 24 hours. Lysosomal V-ATPase activity was measured (A). Acridine orange solution and valinomycin were added to cell monolayers and intravesicular H⁺ uptake was initiated by the addition of Mg-ATP (B); the fluorescence was quantified at 24 hours (B; right). Cell viability assay (C) and caspase-3 activity analysis (D) were performed. Immunostaining was performed with anti-BAX or LAMP-1 antibody and the co-localized BAX was quantified as the percent of lysosomal-translocated BAX (E). Immunoblot analysis of lysosome fractions with antibody against BAX, t-Bid, PDI, COX II, or LAMP-1 (F). ^*<i>p</i><0.05, significantly different from EUE-treated condition in the presence of palmitate. Con; control, Pal.; palmitate, EUE<i>; Eucommia ulmoides</i> Oliver extract, Bafi<i>; Bafilomycin.</i></p

EUE regulates palmitate-induced lysosomal BAX localization.

<p>Cells were treated with 500 µM palmitate. After 24 hours, fat accumulation in cells was stained by Oil Red O (A, left). Cells were treated with 500 µM palmitate in the presence or absence of 25, 50, or 100 µg/mL EUE for 24 hours and cell viability was analyzed (A). Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE for 0, 6, 12, 24, or 48 hours. Cell viability (B) or caspase-3 activity (C) was analyzed and immunoblotting with anti-caspase-3, caspase-9, or β-actin antibody was performed (D). Immunoblotting of lysosome and cytosol fractions was performed with antibody against cathepsin B, LAMP-1, or tubulin (E). Cathepsin B activity in the medium was measured (F). Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE for 24 hours. Hoechst staining (G) and cathepsin B immunostaining (H) were performed, and the diffuse staining pattern was quantitatively analyzed (H; lower). Co-immunostaining of cathepsin B and LAMP-1 was performed (I), and the overlap of staining was quantified (right). ^*<i>p</i><0.05, significantly different from palmitate-treated condition, Pal.; palmitate, EUE<i>; Eucommia ulmoides</i> Oliver extract.</p

<i>Eucommia ulmoides</i> Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation

<div><p><i>Eucommia ulmoides</i> Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of <i>Eucommia ulmoides</i> Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by <i>in vitro</i> and <i>in vivo</i> studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia.</p></div

<i>E. ulmoides</i> Oliver extract regulates ER stress and hepatic lipid accumulation and enhances lysosome activity in high-fat-diet rats.

<p>Rats were fed a normal or high-fat diet with 0, 0.25, 0.5, or 1 g/kg EUE for 10 weeks, after which livers were isolated. (A) Immunoblotting was performed with antibodies against GRP78, PERK, p-PERK, CHOP, IRE1-α, p-eIF2α, eIF2α, or β-actin. (B) Lysosome fractionation was performed using liver samples, and the activities of α-mannosidase, β-glucuronidase, and β-galactosidase were subsequently determined. (C) Livers were stained with Oil Red O dye, and images were obtained at 200X magnification to observed hepatic fat accumulation. (D) Triglyceride and cholesterol levels were measured in both the liver and plasma. (E) Immunoblotting was performed with ApoA1 or ApoB antibodies using liver and plasma samples. ^*<i>p</i><0.05, significantly different from the control group at each corresponding time point. CV, central vein; Con, control; HFD, high-fat-diet; EUE, <i>E. ulmoides</i> Oliver extract.</p

Bafilomycin reverses <i>E. ulmoides</i> Oliver extract-induced regulation of ER stress and lipid accumulation.

<p>(A) Cells were treated with 300 μM palmitate and 100 μg/mL EUE, 10 μg/mL aucubin, or 10 μg/mL geniposide in the presence or absence of 10 nM bafilomycin for 12 hours. Immunoblotting was performed using antibodies against GRP78, PERK, p-PERK, CHOP, IRE1-α, p-eIF2α, eIF2α, or β-actin. (B) Images were obtained at 200X magnification for determination of fat accumulation by Oil Red O staining. (C) Cell lysates and media were immunoblotted with anti-ApoA1 or anti-ApoB. (D) Triglycerides and cholesterol levels in lysates or media were measured as described in the Materials and Methods. ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone. Values are the mean±SE of three independent experiments. Con, control; Pal, palmitate; EUE, <i>E. ulmoides</i> Oliver extracts; Au, aucubin; Geni, geniposide; Bafi, Bafilomycin; CBB, Coomassie brilliant blue staining.</p