Impact of Vitamin D3 Deficiency on Phosphatidylcholine-/Ethanolamine, Plasmalogen-, Lyso-Phosphatidylcholine-/Ethanolamine, Carnitine- and Triacyl Glyceride-Homeostasis in Neuroblastoma Cells and Murine Brain

Abstract

Vitamin D₃ hypovitaminosis is associated with several neurological diseases such as Alzheimer's disease, Parkinson's disease or multiple sclerosis but also with other diseases such as cancer, diabetes or diseases linked to inflammatory processes. Importantly, in all of these diseases lipids have at least a disease modifying effect. Besides its well-known property to modulate gene-expression via the VDR-receptor, less is known if vitamin D hypovitaminosis influences lipid homeostasis and if these potential changes contribute to the pathology of the diseases themselves. Therefore, we analyzed mouse brain with a mild vitamin D hypovitaminosis via a targeted shotgun lipidomic approach, including phosphatidylcholine, plasmalogens, lyso-phosphatidylcholine, (acyl-/acetyl-) carnitines and triglycerides. Alterations were compared with neuroblastoma cells cultivated in the presence and with decreased levels of vitamin D. Both in cell culture and in vivo, decreased vitamin D level resulted in changed lipid levels. While triglycerides were decreased, carnitines were increased under vitamin D hypovitaminosis suggesting an impact of vitamin D on energy metabolism. Additionally, lyso-phosphatidylcholines in particular saturated phosphatidylcholine (e.g., PC aa 48:0) and plasmalogen species (e.g., PC ae 42:0) tended to be increased. Our results suggest that vitamin D hypovitaminosis not only may affect gene expression but also may directly influence cellular lipid homeostasis and affect lipid turnover in disease states that are known for vitamin D hypovitaminosis.

Keywords: calcitriol; carnitine; lyso-phosphatidylcholine; neurodegenerative diseases; phosphatidylcholine; phosphatidylcholine plasmalogen; shotgun lipidomics; triacyl glyceride; vitamin D hypovitaminosis.

Figure 1

Effect of vitamin D₃ deficiency on phosphatidylcholine (PCaa) species. (A) The levels of PCaa in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 43 analyzed PCaa species (dots) was plotted against the corresponding p-value (y-axis). The two vertical lines represent the mean SEM. The horizontal line marks the p value of 0.05, which was set as statistical significance. Filled black dots symbolize PCaa species with a fold change greater than the mean SEM without reaching significance and empty dots represent species with a fold change within the mean SEM. The bar chart on the right shows the relative fold change of all measured PCaa species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. Below the volcano plot, ratios indicating the saturation state of the fatty acids within the PCaa species and the distribution of saturation and chain length in mol% are shown. (B) Analyzed PCaa species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured PCaa species in a bar chart. Error bars represent the standard error of the mean (SEM). Statistical significance was set as * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001; n.s. not significant.

Figure 2

Effect of vitamin D₃ deficiency on phosphatidylcholine plasmalogens (PCae) species. (A) The levels of PCae in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 39 analyzed PCae species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured PCae species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. Below the volcano plot, ratios dealing with the saturation state of the fatty acids within the PCae species and the distribution of chain length in mol% are shown. (B) Analyzed PCae species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured PCae species in a bar chart. Error bars represent the standard error of the mean (SEM). Statistical significance was set as * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001; n.s. not significant.

Figure 3

Effect of vitamin D₃ deficiency on lyso phosphatidylcholine (lyso-PC) species. (A) The levels of lyso-PC in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot the fold change (x-axis) of each of the 22 analyzed lyso-PC species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured lyso-PC species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. Below the volcano plot, ratios dealing with the saturation state of the fatty acids within the lyso-PC species and the distribution of chain length in mol% are shown. (B) Analyzed lyso-PC species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured lyso-PC species in a bar chart. Error bars represent the standard error of the mean (SEM). Statistical significance was set as * p ≤ 0.05 and ** p ≤ 0.01; n.s. not significant.

Figure 4

Effect of vitamin D₃ deficiency on phosphatidylethanolamine (PE) species. (A) The levels of PEaa in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 35 analyzed PEaa species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured PEaa species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. (B) Analyzed PEaa species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured PEaa species in a bar chart. Besides PEaa, also levels of PEae (C,D) and Lyso-PE species (E,F) under hypovitaminosis D3 conditions were examined. Error bars represent the standard error of the mean (SEM). Statistical significance was set as *** p ≤ 0.001; n.s. not significant.

Figure 4

Effect of vitamin D₃ deficiency on phosphatidylethanolamine (PE) species. (A) The levels of PEaa in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 35 analyzed PEaa species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured PEaa species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. (B) Analyzed PEaa species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured PEaa species in a bar chart. Besides PEaa, also levels of PEae (C,D) and Lyso-PE species (E,F) under hypovitaminosis D3 conditions were examined. Error bars represent the standard error of the mean (SEM). Statistical significance was set as *** p ≤ 0.001; n.s. not significant.

Figure 5

Effect of vitamin D₃ deficiency on carnitine species. (A) The levels of carnitines in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 41 analyzed carnitine species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured carnitine species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. The bar chart below the volcano plot shows the changes in C0, C2, CX (with X > 3), C18/C2- and (C16 + C18)/C2 ratio in SH-SY5Y cells deficient in vitamin D₃ compared to calcitriol-treated cells. (B) Analyzed carnitine species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured carnitine species in a bar chart. Error bars represent the standard error of the mean (SEM). Statistical significance was set as *** p ≤ 0.001; n.s. not significant.

Figure 6

Effect of vitamin D₃ deficiency on triacyl glycerides (TAG) species. (A) The levels of TAG in SH-SY5Y wt cells incubated with the solvent control (ethanol) were compared to cells treated with 1,25-dihydroxy vitamin D₃. In the volcano plot, the fold change (x-axis) of each of the 14 analyzed TAG species (dots) was plotted against the corresponding p-value (y-axis). The volcano plots are structured as described in detail in the legend of Figure 1. The bar chart on the right shows the relative fold change of all measured TAG species comparing calcitriol-treated with solvent-control-treated SH-SY5Y cells. (B) Analyzed TAG species in brain samples of vitamin D deficient mice compared to control-fed mice in a volcano plot and relative fold changes of all measured TAG species in a bar chart. Error bars represent the standard error of the mean (SEM). Statistical significance was set as *** p ≤ 0.001; n.s. not significant.

Figure 7

Overview of the effects of vitamin D₃ hypovitaminosis on lipid metabolism in neuroblastoma cells and brain samples of mice with mild to moderate vitamin D deficiency. (A) Effects observed influencing the metabolism of phospholipids (phosphatidylcholine, PCaa; phosphatidylcholine plasmalogens, PCae; lyso-phosphatidylcholine, lyso-PC) in a Venn diagram. (B) The effect of vitamin D3 deficit on lipids involved in cellular energy metabolism and β-oxidation (carnitine and triacyl glycerides, TAG) in a Venn diagram. (C) Summary of the influence of vitamin D₃ deficit on different lipid classes known to be affected in disorders such as neurodegenerative Alzheimer’s disease (AD).