Additional file 1: Figure S1. of Methyl group donors abrogate adaptive responses to dietary restriction in C. elegans

Similar changes of the triglyceride-to-phospholipid ratio and the fatty acid composition of phosphatidylcholine (PC) in dietary restricted L4 larvae (P0 generation) compared to embryos (F1 generation) obtained from dietary restricted worms. (PPTX 106 kb

Effect of lipid extracts from LDL and OxLDL on CerS activity.

<p>(A) Oxidation of LDL was performed as described under “Experimental Procedure”. RAW 264.7 cells were stimulated with lipid extracts from native LDL and OxLDL (50 µg protein/mL respectively) for 24 h. CerS activity in cell homogenates was measured using C₁₆-CoA and C₂₂-CoA substrates. Results are means ± S.D., *p < 0.05, of a typical experiment repeated four times with similar results. (B) Cells were treated with intact LDL and OxLDL (50 µg protein/mL respectively) as described above for 24 h. CerS activity in cell homogenates was measured using C₁₆-CoA and C₂₂-CoA substrates. Results are means ± S.D. *p < 0.05, of a typical experiment repeated four times with similar results. (C) Cells were treated as described above and lipids were extracted and analyzed for ceramide content as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (D) Ceramide species were analyzed after treatment with lipid extracts from native LDL and OxLDL as described earlier. The data are means ± S.E., *p < 0.05, n = 4.</p

Influence of POVPC and PGPC on ceramide levels and CerS activation in RAW 264.7 cells.

<p>(A) Both POVPC and PGPC elevate ceramide generation in RAW 264.7 cells. Cells were stimulated for 24 h with respective OxPLs (50 µM) in parallel to ethanol treated control cells. Lipids were extracted and analyzed for ceramide levels by LC/MS-MS as described under “Experimental Procedure”. No probability. values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide speciation was performed after OxPL treatment as above. The data are means ± S.E., *p<u><</u>0.05, **p < 0.01 compared with control, n = 4. (C) After OxPL treatment cells were harvested and CerS activity in cell homogenates was measured as described earlier. Results are means ± S.E., *p < 0.01, **p < 0.05, of a typical experiment repeated four times with similar results. (D) CerS mRNA levels were measured by RT-qPCR after 24 h incubation with 50 µM POVPC or PGPC as described under “Experimental Procedures”. The data are normalized to GAPDH mRNA expression and data are means ± S.E. for four independent experiments performed in triplicate. (E) nSMase activity was measured in cell homogenates after exposure to OxPL as described under “Experimental Procedures”. The data are represented as means ± S.E., n = 4.</p

Effect of FB1 on OxPL induced CerS activation and ceramide levels.

<p>(A) Raw 264.7 cells were pre-incubated with FB1 (20 µM) for 2 h prior to the 24 h OxPL treatment. Lipids were extracted and ceramide levels were analyzed as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide species were analyzed as earlier. The data are means ± S.E., *p < 0.05, **p < 0.01 compared with control, n = 4.</p

The oxidized phospholipids POVPC and PGPC are cytotoxic and induce apoptosis.

<p>(A) Chemical structures of POVPC and PGPC. Dotted circle represents the functional group at <i>sn-2</i> position. (B) RAW 264.7 cells were incubated with 50 µM POVPC and PGPC for indicated periods. Control cells were treated with 1% ethanol. Cell viability was determined by Vybrant® MTT assay kit. Results are expressed as a percentage of viable cell number in treated cells compared with that of untreated control cells. Data are means ± S.D., n = 8 in each group. (C) Cells were incubated with the stated concentrations of POVPC and PGPC for 4 h. The cells were analyzed for Alexa Fluor₄₈₈-annexin V and propidium iodide fluorescence staining by Flow cytometry as described under “Experimental Procedures”. Results are represented as means ± S.D. Probabilities compared to control were determined by Student’s t-test (two-tailed, unpaired); ***p < 0.001,(n = 8 in each group).</p

Alternate display format of TAG/DAG-species and FA-species.

<p><b>A</b> fructose-feeding vs control in rat liver; <b>B</b> FF treatment vs Fructose-feeding in rat liver; <b>C</b> fructose-feeding vs control in rat serum; <b>D</b> FF treatment vs Fructose-feeding in rat serum; An algorithm was developed to generate the alternate display form of DAG/TAG-species. In the diagrams the species shown in red have increased levels between the two respective groups, while a blue color indicates a decreased level of the respective species between the two groups. A black coloring indicates no change of the species between the two groups. All species have been colored if the difference of levels between the two groups were >5% of the total amount of the higher level, regardless of the significance of the change. For clarity, only the sections of the diagram containing the combinations of DAG C36 With FA C18 to yield TAG C54 are shown. If there is a fatty acid-DAG combination present with the same direction of change as the corresponding TAG-species, then this combination seems at least to contribute to the composition of the TAG-species. Significant changes are indicated using *: P<0.05; **: P<0.01; ***: P<0,001.</p

TAG-species in rat liver.

<p>Values given are means±s.d. of the sums of the different chain lengths (C46–C60) on the left and degree of desaturation (sat – deca) on the right. Serum data is shown for day 14 and day 20; the control group is shown as black bar, the fructose-fed group is shown as red bar and the FF treated group is shown as a blue bar; significant changes are indicated using *: P<0.05; **: P<0.01; ***: P<0,001.</p

Tissue weights and overall TAG- and DAG-levels.

<p><b>A</b> Organ weight of the animals after the end of treatment; <b>B</b> TAG-levels in rat liver, jejunum and serum and DAG-levels in rat liver and rat jejunum; <b>C</b> Sums of TAG- and DAG-species in rat serum. Tissue samples were taken during study 1, serum samples were taken during study 2; the control group is shown as black bar, the fructose-fed group is shown as red bar and the FF treated group is shown as a blue bar; values given are means±s.d.; significant changes are indicated using *: P<0.05; **: P<0.01; ***: P<0,001.</p

Clinical chemistry data, organ weight, food and water consumption.

<p>Values determined in study 1. Values given are mean values of the 6 animals/group with standard deviation included. Significant changes are indicated using *: P<0.05; **: P<0.01; ***: P<0,001. ASAT: aspartate aminotransferase, ALAT: alanine aminotransferase, AP: alkaline phosphatase (AP).</p><p>Clinical chemistry data, organ weight, food and water consumption.</p

DAG-species in rat liver and serum.

<p>DAG-species in <b>A</b> rat liver and <b>B</b> rat serum. Values given are means±s.d. of the sums of the different chain lengths (C32 – C38) on the left and degree of desaturation (sat – hexa) on the right. Serum data is shown for day 14 and day 20; the control group is shown as black bar, the fructose-fed group is shown as red bar and the FF treated group is shown as a blue bar; significant changes are indicated using *: P<0.05; **: P<0.01; ***: P<0,001.</p