<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

<i>E. ulmoides</i> Oliver extract reduces hepatic lipid accumulation and levels of secreted apolipoprotein B.

<p>(A) Cells were treated with 300 μM palmitate in the absence or presence of 100 μg/mL EUE for 12 hours. Fat accumulation was determined by Oil Red O staining. Images of cells were obtained at 200X original magnification and used for quantitative analysis of cellular lipid deposition (right). ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone. (B) Cells were treated with 300 μM palmitate in the presence or absence of 100 μg/mL EUE for 0, 3, 6, 9, 12, 18, or 24 hours. Cell lysates and media samples were subjected to immunoblot analysis with anti-ApoA1 or anti-ApoB. CBB staining was performed as an equal loading control. (C) Cells were treated with 300 μM palmitate in the absence or presence of 100 μg/mL EUE for 0, 6, 12, 24, or 48 hours. Triglyceride and cholesterol levels were measured for both cell lysates and media alone. ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone at the corresponding time point. Pal, palmitate; EUE, <i>E. ulmoides</i> Oliver extracts; CBB, Coomassie brilliant blue.</p

Aucubin and geniposide reduce hepatic lipid accumulation and secretion of apolipoprotein B.

<p>(A) Cells were treated with 300 μM palmitate in the absence or presence of 10 μg/mL aucubin or geniposide for 12 hours. Fat accumulation was determined by Oil Red O staining. Images of cells were obtained at 200X original magnification and used for quantitative analysis of cellular lipid deposition (lower panel). ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone. (B) Cells were treated with 300 μM palmitate in the presence or absence of 10 μg/mL aucubin or geniposide for 0, 3, 6, 9, 12, 18, or 24 hours. Cell lysates and medium were immunoblotted with anti-ApoA1 or anti-ApoB, and CBB staining was performed separately as an equal loading control. (C) Cells were treated with 300 μM palmitate in the presence or absence of 10 μg/mL aucubin or geniposide for 0, 6, 12, 24, or 48 hours. Triglycerides and cholesterol were measured in cell lysates and media alone. ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone at each corresponding time point. Pal, palmitate; CBB, Coomassie brilliant blue.</p

Aucubin and geniposide inhibit palmitate-induced ER stress response.

<p>Cells were treated with 300 μM palmitate in the absence or presence of 10 ug/mL aucubin or geniposide for 0, 3, 6, 9, 12, 18, or 24 hours. Immunoblotting was performed using antibodies against GRP78, PERK, p-PERK, CHOP, IRE1α, p-eIF2α, eIF2α, or β-actin. Quantification of immunoblot data is shown (lower panel). ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone at the corresponding time point. Pal, palmitate; EUE, <i>E. ulmoides</i> Oliver extracts.</p

<i>E. ulmoides</i> Oliver extract, aucubin, and geniposide enhance lysosomal activity.

<p>(A) Cells were treated with 100 μg/mL EUE, 10 μg/mL aucubin, or 10 µg/mL geniposide in the presence or absence of 300 µM palmitate for 12 hours, followed by exposure to 5 μM LysoTracker and image acquisition and quantification (lower panel). (B) Cells were fixed and immunostained with antibodies for cathepsin B or Lamp-1. Quantification of fluorescence is shown (lower). (C) The activities of α-mannosidase, β-galactosidase, and β-glucuronidase were analyzed from lysosomal extracts of cells treated for the indicated time periods. ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone (A, B); ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone at each corresponding time point (C). DIC, differential interference contrast microscopy; Pal, palmitate; EUE, <i>E. ulmoides</i> Oliver extract.</p

<i>E. ulmoides</i> Oliver extracts inhibit palmitate-induced ER stress response.

<p>(A) Cells were treated with 300 μM palmitate and 0, 25, 50, or 100 μg/mL extract for 12 hours. Immunoblotting was performed using antibodies against GRP78, PERK, p-PERK, CHOP, IRE1α, p-eIF2α, eIF2α, or β-actin. (B) Cells were treated with 300 μM palmitate in the presence of 100 μg/mL EUE for 0, 3, 6, 9, 12, 18, or 24 hours. Quantification of immunoblot data is shown (right panel). Immunoblotting was performed with antibodies against GRP78, PERK, p-PERK, CHOP, IRE1α, p-eIF2α, eIF2α, or β-actin. Data shown are the mean ± SE of three independent experiments, each performed with triplicate biological replicates. ^*<i>p</i><0.05, significantly different from cells treated with palmitate alone at the corresponding time point. Pal, palmitate; EUE, <i>E. ulmoides</i> Oliver extract.</p