Journal: Theranostics

Article Title: Silybin A from Silybum marianum reprograms lipid metabolism to induce a cell fate-dependent class switch from triglycerides to phospholipids

doi: 10.7150/thno.99562

Figure Lengend Snippet: Shift from TGs to phospholipids in human monocytes, hepatocytes, and mouse liver. (A) Main components of silymarin. (B-D) Total amounts of lipid classes were determined by UPLC-MS/MS. (B, C) Human primary monocytes (B) and HepG2 cells (C) were treated with silymarin (50 µg/ml for monocytes and 10 µg/ml for HepG2 cells), silybin (20 µM), or vehicle (ethanol for silymarin, DMSO for silybin) for 24 h. Individual values and mean + SEM; n = 3 (B: except TG, C: silymarin except TG), n = 4 (C: TG silymarin), n = 6 (B: TG silymarin and silybin, C: TG silybin), n = 7 (C: silybin except TG). (D, E) Mice received silybin hemisuccinate ('silybin'; 200 mg/kg, i.p.) or vehicle (0.9% NaCl) trice at 0, 12, and 24 h and were sacrificed after 37 h. (E) Body temperature, body weight and organ weight of mice upon administration of silybin. Temperature and body weight were measured after 37 h before animals were sacrificed and organs collected. The box-and-whisker plot shows fold-changes upon silybin gavage. The median fold change belonging to each group is shown as bold line. The boxes extend from the 25 th to 75 th percentiles, and whiskers extend to minimal and maximal values. Vehicle control; body temperature: 37.2 ± 0.2 [°C]; body weight: 21.9 ± 0.2 [g]; liver: 1.31 ± 0.03 [g]; spleen: 0.070 ± 0.002 [g]; kidney: 0.1445 ± 0.004 [g]; heart: 0.126 ± 0.0023 [g]; brain: 0.439 ± 0.006 [g]; thymus: 0.049 ± 0.002 [g]; adrenal: 0.0105 ± 0.001 [g]; Lipid contents are given as nmol / 1×10 6 cells for PC and units / 1×10 6 cells for PE, PS, PI, PG, SM and TG. Individual values and mean + SEM (D) or box plots and individual values (E) from n = 5 (D: CE and TG), n = 7 (D: PE; ctrl, E: body temperature), n = 8 (D: except CE and TG, E: body and organ weights) mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control. Two-tailed paired (B, C) or unpaired (D, E) Student's t -test.

Article Snippet: Cultured cell lines: Human HepG2 liver carcinoma cells (1×10 5 cells/cm 2 , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS, GE Healthcare, Freiburg, Germany or Merck) at 37°C and 5% CO 2 .

Techniques: Tandem Mass Spectroscopy, Whisker Assay, Control, Two Tailed Test

Journal: Theranostics

Article Title: Silybin A from Silybum marianum reprograms lipid metabolism to induce a cell fate-dependent class switch from triglycerides to phospholipids

doi: 10.7150/thno.99562

Figure Lengend Snippet: Silymarin/silybin induces global changes in phospholipid, TG and sterol metabolism. (A-F) Comparative analysis of transcriptome data from silymarin-treated HepG2 (A) and Huh7.5.1 hepatocarcinoma cells (B), silymarin- and silybin-treated Caco-2 colon carcinoma cells (C), and hepatocytes derived from HCV-infected mice receiving silybin (D, E). Volcano plots compare the expression of lipid metabolic genes upon silymarin (A-C) or silybin (C-E) treatment vs. vehicle control. Differentially expressed genes are defined as those that show consistent regulation in the same direction in at least two independent model systems at a significance level of P < 0.05 (without adjustment for multiple comparisons) and are annotated in the corresponding plots. The dashed line indicates a P -value of 0.05; multiple two-tailed unpaired Student's t -tests. (F) Radar plots indicating the fold change in PLA2G1 , DGAT1 , and INSIG1 expression by silymarin (HepG2, Huh7.5.1, Caco-2) or silybin (hepatocytes, Caco-2) relative to vehicle control. Non-adjusted P values given vs. vehicle control; multiple two-tailed unpaired Student's t -tests (G-J). HepG2 cells were incubated with silymarin (10 µg/ml), silybin (20 µM) or vehicle (ethanol for silymarin, DMSO for silybin) for 24 h. (G) mRNA levels of GPAT2-4 normalized to β-actin. Individual values and mean + SEM as fold-change of control; n = 4 (GPAT2 and GPAT4), n = 5 ( GPAT3 ). (H) Effects of silymarin and silybin on the cellular ratio of short-chain acyl-CoAs, normalized to the internal standard [ 13 C 3 ]-malonyl-CoA. Individual values and mean + SEM; n = 5 (silybin) and n = 6 (silymarin). * P < 0.05, *** P < 0.001 vs. vehicle controls; two-tailed paired Student's t -tests. (I, J) Incorporation of isotopically labeled sodium acetate- 13 C 2 , d 3 in PE (I) and TG (J) by HepG2 cells treated with silymarin (10 µg/ml), silybin (20 µM), or vehicle (ethanol for silymarin, DMSO for silybin) for 24 h. The total amount of the isotopically labeled PE and TG species analyzed is shown. Individual values and means + SEM; n = 3. * P < 0.05 vs. vehicle controls; two-tailed paired Student's t -tests.

Article Snippet: Cultured cell lines: Human HepG2 liver carcinoma cells (1×10 5 cells/cm 2 , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS, GE Healthcare, Freiburg, Germany or Merck) at 37°C and 5% CO 2 .

Techniques: Derivative Assay, Infection, Expressing, Control, Two Tailed Test, Incubation, Labeling

Journal: Theranostics

Article Title: Silybin A from Silybum marianum reprograms lipid metabolism to induce a cell fate-dependent class switch from triglycerides to phospholipids

doi: 10.7150/thno.99562

Figure Lengend Snippet: Proposed mechanisms of silymarin and its bioactive constituent silybin A in hepatocytes. Acetyl-CoA-carboxylase (ACC/ACACA) converts acetyl-CoA to malonyl-CoA, which is elongated to long-chain fatty acids by fatty acid synthase (FASN). Acyl-CoA esters are formed from free fatty acids (FAs) by acyl-CoA synthetases (ACSLs), which also activate exogenous fatty acids for further metabolism. Saturated acyl-CoAs are converted into monounsaturated acyl-CoAs (MUFA-CoA) preferentially by Δ9-desaturases, such as the stearoyl-CoA desaturase (SCD) isoenzyme 5. Acyl-CoA thioesterases (ACOTs) catalyze the opposite reaction, hydrolyzing acyl-CoAs to free fatty acids (FAs). Acyl-CoAs are used by glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid (LPL) acyltransferases/lysophosphatidic acid acyltransferases (LPLATs/LPAATs) to introduce fatty acyl-chains into the sn -1 and sn -2 positions of glycerol-3-phosphate and lysophosphatidic acid (LPA), respectively. The resulting PA is either converted to CDP-DAG for PI, PG, and PS biosynthesis or dephosphorylated to DAG for TG, PC, and PE biosynthesis by lipins (LPINs) and other PA phosphatases. LPIN2 also plays an important role in the regulation of fatty acid metabolism as nuclear transcriptional coactivator. Acylation of DAG by DGATs yields TGs, which are stored in lipid droplets and mobilized by ATGL/PNPLA2 and other triglyceride lipases, providing DAG and FAs. Phospholipid degradation is driven by a large number of phospholipases with different specificities. PLA2G6 releases saturated and unsaturated long-chain fatty acids from the sn -1 or sn -2 position of phospholipids, such as PC, PE and PA, whereas PLD1 specifically cleaves PC to PA and choline. By targeting multiple nodes, silymarin/silybin triggers a switch from TGs to phospholipids, thereby enriching intracellular membranes with phospholipids that have a balanced fatty acid composition. The increase in intracellular membranes is associated with enhanced membrane-associated biotransformation capacities. Mechanistically, silymarin/silybin inhibits phospholipid degradation, while moderately activating de novo phospholipid biosynthesis and stimulating TG catabolism in lipid droplets (LD), which in combination results in an effective channeling of TG-derived DAG and FAs into membrane biogenesis. In addition, silymarin induces the expression of genes involved in peroxisomal fatty acid degradation (HAO2, ACOX3), upregulates ACOT7, which hydrolyzes acyl-CoAs into FAs and CoA, and decreases the expression of ACSLs, that activate long-chain fatty acids. The color scale in the pathway diagram indicates the percentage changes in metabolite levels, lipid droplet counts, and enzyme expression by silybin relative to vehicle control in HepG2 cells (black bordered boxes) or by silymarin relative to vehicle control in HepG2 cells (orange bordered boxes). GPAM, glycerol-3-phosphate acyltransferase, mitochondrial.

Article Snippet: Cultured cell lines: Human HepG2 liver carcinoma cells (1×10 5 cells/cm 2 , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS, GE Healthcare, Freiburg, Germany or Merck) at 37°C and 5% CO 2 .

Techniques: Introduce, Membrane, Derivative Assay, Expressing, Control

Journal: Theranostics

Article Title: Silybin A from Silybum marianum reprograms lipid metabolism to induce a cell fate-dependent class switch from triglycerides to phospholipids

doi: 10.7150/thno.99562

Figure Lengend Snippet: Phospholipid profiling indicates an upregulation of diverse species. Human primary monocytes and HepG2 cells were treated with silymarin (50 µg/ml for monocytes and 10 µg/ml for HepG2 cells), silybin (20 µM) or vehicle (ethanol for silymarin, DMSO for silybin) for 24 h. Mice received silybin hemisuccinate ('silybin'; 200 mg/kg) or vehicle (0.9% NaCl) trice at 0, 12, and 24 h and were sacrificed after 37 h. (A) Volcano plots showing the cellular proportion of phospholipid species that increase (yellow background) or decrease (grey background) upon treatment with silymarin or silybin. Adjusted P values given vs. vehicle control. The dashed line indicates a P -value of 0.05. (B) Forest plots depicting phospholipid species that are up- (positive values) or down-regulated (negative values) by silymarin (squares) or silybin (circles). Values, calculated as percentage of control, show the difference to 100%, with the dashed line at 0% indicating no difference to control. The dot size describes the mean relative abundance of phospholipid species within the phospholipid subclass (relative intensities). Data and the number of experiments are identical to Figure .

Article Snippet: Cultured cell lines: Human HepG2 liver carcinoma cells (1×10 5 cells/cm 2 , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS, GE Healthcare, Freiburg, Germany or Merck) at 37°C and 5% CO 2 .

Techniques: Control

Journal: Theranostics

Article Title: Silybin A from Silybum marianum reprograms lipid metabolism to induce a cell fate-dependent class switch from triglycerides to phospholipids

doi: 10.7150/thno.99562

Figure Lengend Snippet: Silybin A is the active isomer that causes the switch from TGs to phospholipids. HepG2 cells were treated with the indicated compounds (20 µM) or vehicle (DMSO) for 24 h. Total amounts of lipid classes were determined by UPLC-MS/MS and are given as nmol / 1×10 6 cells for PC and units / 1×10 6 cells for PE, PS, PI, PG, SM and TG. Individual values and mean + SEM; n = 3 (PE silybin A, PS dehydrosilybin, TGs, free fatty acids (FFA) 2,3-dehydrosilybin A and B) n = 4 (except PE silybin A, PS dehydrosilybin, TGs, free fatty acids FFA 2,3-dehydrosilybin A and B). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control (DMSO). Two-tailed paired Student's t -test of log-transformed data.

Article Snippet: Cultured cell lines: Human HepG2 liver carcinoma cells (1×10 5 cells/cm 2 , Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS, GE Healthcare, Freiburg, Germany or Merck) at 37°C and 5% CO 2 .

Techniques: Tandem Mass Spectroscopy, Control, Two Tailed Test, Transformation Assay