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

Additional file 1: Figure S1. of Turmeric extract and its active compound, curcumin, protect against chronic CCl4-induced liver damage by enhancing antioxidation

Chromatograms of blank (A), standard (B), turmeric extract (C). (PDF 164 kb

TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

Lysosomal Ca2+ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca2+ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca2+ levels. The Ca2+ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca2+ level, in which ITPR-independent regulation of Ca2+ status was observed. Further, TMBIM6 regulated the local release of Ca2+ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca2+ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca2+ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved Tmbim6+/+ mice compared with that in the counter tmbim6−/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca2+ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca2+ modulator that coordinates with autophagy to alleviate metabolism stress.Abbreviations: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6</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