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ACS Omega
2024 Oct 12;945:45564-45571. doi: 10.1021/acsomega.4c07865.
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Identification of the Active EPA/AA-Binding Ether-Type Phosphatidylcholine Derived from the Starfish Patiria pectinifera for C2C12 Myotube Growth.
Fukushima A
,
Imamura K
,
Takatani N
,
Hosokawa M
,
Beppu F
.
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Concerns about nutritional approaches for promoting skeletal muscle mass and function have increased. This study assessed the effects of starfish-derived glycerophospholipids (PLs) (SPL), characterized by unique ether-linked subclasses, alkylacyl (Alk)- and alkenylacyl (Pls)-PL, on skeletal muscle function, focusing on myotube formation in C2C12 myoblasts. SPL was prepared via chloroform/methanol extraction from Patiria pectinifera, followed by silica gel chromatography fractionation. Myoblasts were induced to differentiate with or without SPL treatment. On day 7 of differentiation, 50 μg/mL of SPL treatment increased myotube diameter. The phosphatidylcholine (PC) fraction (SPC) also enhanced myotube growth at 30 μg/mL. LC-MS/MS analysis indicated the most abundant PC molecular species in SPC were Alk- and Pls-PC with eicosapentaenoic acid and arachidonic acid. Treatment with 1-O-hexadecyl-2-arachidonoyl-PC, 1-1(Z)-hexadecenyl-2-arachidonoyl-PC or 1-O-hexadecyl-2-eicosapentaenoyl-PC increased myotube diameter and myokine Il-15 mRNA expression. These results demonstrate a novel functionality of SPC and highlight the role of ether-type PC molecules in muscle function.
Figure 1. Effect of SPL on myotube formation in C2C12 cells. C2C12
myoblasts
were induced to differentiate with starfish phospholipids (SPL) (0–50
μg/mL) and stained with HE on day 7 of differentiation. (a)
Representative images of HE-stained myotubes. (b) Myotube diameter
on day 7 of differentiation. (c) mRNA expression levels of MyoD, myogenin,
and IL-15, measured using RT-PCR. Values are presented as mean ±
SD (n = 3). Statistical significance was determined
using Dunnett’s method, * p < 0.05.
Figure 2. Immunostaining
of myotubes with myosin heavy chain (MyHC) and analysis
of myotube diameter and myoblast fusion index. C2C12 cells were induced
to differentiate with or without SPC (30 μg/mL). (a) Representative
immunofluorescence images of myotubes stained with MyHC (green) and
DAPI (blue). (b) Myotube diameters relative to the control. (c) Myoblast
fusion index, expressed as the percentage of fused myoblasts over
total nuclei. Values are presented as mean ± SD (n = 3). Statistical significance was determined using the Student’s t-test, * p < 0.05.
Figure 3. Comparison between SPC
and soy-PC on C2C12 myotube formation. C2C12
cells were induced to differentiate in the presence or absence of
SPC or soy-PC at 30 μg/mL. (a) Microscopic images of HE-stained
myotubes. (b) RT-PCR analysis of MyHC mRNA expression. Values are
presented as mean ± SD (n = 3). Statistical
significance was determined using the Student’s t-test, * p < 0.05, ** p <
0.01.
Figure 4. Analysis of the subclass composition and molecular species in starfish
PC. (a) Structures and composition of PC subclasses in SPC measured
using the Rouser method and HPLC-ELSD. (b) ESI+ MS spectrum of SPC,
with “(O)” indicating alkylacyl and “(P)”
indicating alkenylacyl. (c) Chromatogram showing the precursor ion
([M + H]+) to product ion transitions m/z 794.8 > 184, 792.8 > 184, and 796.8 >
184, and
chromatogram displaying the acetate-adducted parent ion ([M + CH3COO]−) transitioning to product ions with m/z 301.5 and 303.5 (ion intensity multiplied
by 20). Retention times are indicated on the x-axis,
and ion intensity is represented on the y-axis.
Figure 5. Effects of EPA/AA-binding ether-linked
PC on C2C12 myotube formation.
C2C12 cells were treated with (O)16:0/EPA-,(O)16:0/AA-, 16:0/AA-,
or (P)16:0/AA-PC during differentiation at a concentration of 5, 10,
or 20 μM. (a) Microscopic images of HE-stained myotubes. (b)
Myotube diameter and (c) IL-15 mRNA expression on day 7 of differentiation.
Values are presented as mean ± SD (n = 3). Statistical
significance was determined using Dunnett’s method, * p < 0.05, ** p < 0.01
