Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SLpm) is integral to skeletal muscle function and health. Previous studies assessing SLpm PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SLpm PL composition. Mechanically skinned SLpm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SLpm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SLpm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SLpm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.
Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration which outpaces regeneration, yielding fragile membranes with phospholipids high in 18:1 and low in 18:2n6. Central to the regenerative process are sphingolipids, specifically sphingosine‐1‐phosphate (S1P), which promote satellite cell proliferation and muscle regeneration. In mdx mice, a model of DMD, S1P is deficient. Thus the purpose of this study was to examine the impact of a dietary source of sphingolipids on muscle membrane phospholipid composition in mdx mouse. C57BL10 (WT) and mdx mice were fed either an AIN76A (CON) or AIN76A with 0.1% sphingomyelin (SM) diet for 7 wks starting at age 4 wks. Phospholipids were extracted and analyzed using thin layer‐gas chromatography. Diaphragm muscle from mdx demonstrated significantly higher percent mole fraction of monoenes (specifically 18:1) and lower polyenes (specifically 18:2n6) in the three major membrane phospholipid species (phosphatidylcholine, PC; phosphatidylethanolamine, PE; cardiolipin, CL) compared to WT. SM significantly increased both n3 (specifically PE 22:6n3) and n6 (specifically CL 18:2n6) polyenes and decreased monoenes (specifically CL 18:1) compared to CON. These results suggest that dietary sphingolipids influence membrane phospholipid composition. The project was funded by NSERC (PJL) and VBI/Fralin (VT) Seed Research Fund (RWG & EMS).
High saturated fat (HSFA) diets in rodent models can have adverse effects on healthy bone development. Past studies have focused on bone development of the primary consumer (parent) and did not examine indirect (in utero programming) or direct (fat consumption through milk) diet‐mediated effects in offspring. Thus, the objective of this study was to determine if maternal consumption of HSFA diet influences bone lipid content in female rat offspring at weaning (19 days) and young adulthood (3 months). Female Wistar rats (28 days old) were fed control (CON; AIN93G, 7% soybean oil) or HSFA (HF; AIN93G, 20% lard) diet for 10 weeks, bred, and remained on the same diet throughout gestation and lactation. After weaning, female offspring from both treatments were fed CON. Femur lipids of mothers and their 19 day and 3 month old offspring were analyzed. After 16 weeks on HSFA, maternal femurs had 12 and 34% more saturates (SFA) and monoenes (MUFA), respectively, and 45% less polyenes (PUFA) compared to CON. Similar effects were seen with 19 day olds (2 and 35% less SFA and PUFA, respectively, and 51% higher MUFA). However, after 9 weeks of CON, 3 month olds from mothers fed HF became more similar to CON (3 and 11% lower MUFA and PUFA, respectively, and 14% higher SFA). These results suggest that maternal diet can influence offspring bone lipids and the effects are somewhat reversible by early adulthood. The project was funded by NSERC (PJL & WEW).
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