A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice

Abstract

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Keywords: high phosphorus; lipidome; mice; skeletal muscle; transcriptome; β-oxidation.

Figure 1

Concentrations of individual species of phosphatidylethanolamine (PE) (A), phosphatidylglycerol (PG) (B), and lysophosphatidylcholine (LPC) (C) (including lysosphingomyelin (LSM)) in the M. gastrocnemius of adult male C57BL/6J mice. Mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. Data are mean values and standard deviations, n = 12 mice/group. * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) indicate statistically significant differences.

Figure 2

Principal component analysis (PCA) of the lipidome of the M. Gastrocnemius of adult male C57BL/6J mice. Shown are the scores plot (A) with a plotted 5% confidence interval and the corresponding loading plot (B) of the PCA. Mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. The data represent the principal components (PC 1 or PC 2) and their loadings that were calculated on the basis of the relative abundances of the individual lipid species within their respective lipid classes, n = 12 mice/group. Abbreviations: cardiolipin (CL), ceramide (Cer), hexosylceramide (HexCer), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), PC ether (PC O), phosphatidylethanolamine (PE), PE-based plasmalogens (PE P), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and triacylglycerols (TG).

Figure 3

Volcano plot of differentially regulated transcripts in the M. gastrocnemius of adult male C57BL/6J mice. Mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. Horizontal (p < 0.05) and vertical (fold change (FC): >log2(1.3) or <−log2(−1.3)) dashed lines indicate double filtering criteria (n = 6 mice/group). The transcripts in the upper left and the upper right corners represent the down-regulated and up-regulated transcripts, respectively. The ten most up- or down-regulated transcripts are labeled.

Figure 4

Enriched gene ontology (GO) terms by “biological process”, “cellular compartment”, “molecular function”, and KEGG pathways assigned to the transcripts that were up-regulated in the M. gastrocnemius by a high-phosphorus diet (P_1.2) compared with an adequate-phosphorus diet (P_0.3). Adult male C57BL/6J mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. GO terms and KEGG pathways are sorted using their enrichment p-values (EASE score) (top: lowest p-value; bottom: highest p-value). Only GO terms and KEGG pathways with p < 0.05 are shown. The number of linked up-regulated genes is indicated next to the bars. n = 6 mice/group.

Figure 5

Enriched gene ontology (GO) terms by “biological process”, “cellular compartment”, “molecular function”, and KEGG pathways assigned to the transcripts that were down-regulated in the M. gastrocnemius by a high-phosphorus diet (P_1.2) compared with an adequate-phosphorus diet (P_0.3). Adult male C57BL/6J mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. GO terms and KEGG pathways are sorted using their enrichment p-values (EASE score) (top: lowest p-value; bottom: highest p-value). Only GO terms and KEGG pathways with p < 0.05 are shown. The number of linked down-regulated genes is indicated next to the bars. n = 6 mice/group.

Figure 6

Normalized relative mRNA abundances of genes encoding myosin isoforms of muscle fiber types (A) and regulation of energy metabolism (B) in the M. gastrocnemius of adult male C57BL/6J mice determined using qPCR. Mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. Data are provided as single (circle) and mean values (horizontal line) (n = 12 mice/group) expressed as folds of relative mRNA abundance of the P_0.3 group. * (p < 0.05) and ** (p < 0.01) next to the horizontal line indicate statistically significant differences. Abbreviations: Myh7, myosin heavy chain 7 encoding for myosin heavy chain (MHC)-I; Myh2, myosin heavy chain 2 encoding for MHC-IIa; Myh1, myosin heavy chain 1 encoding for MHC-IIx; Myh4, myosin heavy chain 4 encoding for MHC-IIb; Ppargc1a, peroxisome proliferative-activated receptor, gamma, coactivator 1 alpha; Pdk4, pyruvate dehydrogenase kinase, isoenzyme 4; Lpl, lipoprotein lipase; Fgf21, fibroblast growth factor 21; and Foxo1, forkhead box O1.

Figure 7

Normalized relative protein level of phosphorylated protein kinase B (pAKT) in relation to relative AKT level (A) and level of phosphorylated forkhead box O 1 (FoxO1) in relation to the level of FoxO1 (B), and representative images of the Western blot analysis in the M. gastrocnemius of adult male C57BL/6J mice. Mice were fed a diet containing either 0.3% (P_0.3) or 1.2% (P_1.2) phosphorus for 6 weeks. Data are provided as single (circle) and mean values (horizontal line) (n = 12 mice/group) expressed as fold ratios of the P_0.3 group. * (p < 0.05) next to the horizontal line indicates statistically significant differences.