We evaluated syncytin-1 expression in muscle biopsy samples from humans with BMI > 30 kg/m² and control humans with BMI < 25 kg/m². Muscle samples analyzed were collected in a previous study where we found that humans with BMI > 30 kg/m² have lower muscle protein synthesis compared to that of control subjects with BMI < 25 kg/m² (Tran et al., 2018). Study participants were recruited through flyer advertisements from the greater Phoenix Metro area, and the campuses of Arizona State University and Mayo Clinic in Arizona. Study exclusion criteria included medication or supplements known to affect protein metabolism (i.e. amino acids, protein, fish oil), presence of acute illness, liver disease, renal disease, heart disease, clinically abnormal hemoglobin or hematocrit values, Diabetes, current participation in a weight-loss program, extreme dietary practices, smoking, and use of anabolic steroids or corticosteroids within the last 3 months. Limited evidence describing syncytin-1 gene expression in skeletal muscle of healthy humans (Frese et al., 2015) suggests an effect size of approximately 1.5 in association with a change in syncytin-1 gene expression in muscle. Statistical power calculations (Faul et al., 2007) indicated that for a standard power of 80% and α error of 0.05, approximately eight subjects per group can detect a difference corresponding to a comparable effect size (i.e., 1.5) between subjects with increased BMI and controls subjects. The study procedures were approved by the Institutional Review Board at Mayo Clinic. Research took place in the Clinical Studies Infusion Unit (CSIU) at Mayo Clinic in Arizona, Scottsdale campus.

Subjects arrived in the CSIU in the morning after an overnight fasting period (∼10 h). Subjects had a blood draw to determine fasting blood chemistry parameters and then underwent a 2 h oral glucose tolerance test (OGTT). Subjects with evidence of diabetes (i.e., fasting plasma glucose ≥ 126 mg/dl or 2 h plasma glucose during the OGTT ≥ 200 mg/dl) were excluded from the study. Body composition was determined using bioelectrical impedance analysis (BIA; BIA 310e, Biodynamics Corp., Shoreline, Washington). All subjects were asked to follow specific instructions prior to the BIA to improve measurements of body composition (i.e., no caffeine, food, or alcohol for > 10–12 h; arrive well-hydrated) (Utter et al., 1999; Chittawatanarat et al., 2011). Waist and hip circumference measurements were performed using procedures previously described (WHO, 2011). Table 1 displays the characteristics of the two subject populations. Participants in the group of subjects with increased BMI identified as White (n = 10) (2 Hispanic) and those in the control group identified as White (n = 7) and Asian (n = 1) (1 Hispanic).

A catheter was placed into an antecubital arm vein for infusion (15 μmol kg FFM⁻¹ min⁻¹; priming dose, 6.4 μmol kg FFM⁻¹) of d₁₀-leucine (L-[2,3,3,4,5,5,5,6,6,6-²H₁₀]leucine) to measure rate of muscle protein synthesis, while another catheter was placed in a retrograde fashion in a dorsal hand vein for blood sampling. A percutaneous muscle biopsy (∼100 mg) of the vastus lateralis was obtained with a Bergström biopsy needle under local anesthesia (lidocaine, 2%) at 120 and 300 min after the start of the d₁₀-leucine infusion, and while subjects remained resting in bed. The muscle sample was rinsed with cold saline to remove blood, blotted dry, and cleaned of any visible fat and connective tissue before placing in liquid nitrogen for later analysis. Blood samples were collected at 110, 115, 140, 260, 280, and 300 for determining blood d₉-leucine enrichment.

Leucine enrichments in the muscle and blood samples were used to quantify protein fractional synthesis rate in skeletal muscle, and by following procedures we have previously described (Tran et al., 2015). Briefly, blood samples were collected in tubes containing 15% sulfosalicylic acid (SSA), and mixed well prior to centrifugation to collect the supernatant. The supernatant was passed through cation-exchange columns (AG 50W-8x 100–200-mesh; Bio-Rad Laboratories, Inc.) to isolate the blood amino acids, and which were eluted using 8 ml of 2N NH4OH. For muscle, ∼15 mg of muscle was homogenized in the presence of 0.5 ml of 5% sulfosalicylic acid to precipitate the muscle proteins. The proteins were hydrolyzed with 6 N HCl at 110°C for 24 h. This sample was passed through cation-exchange column (AG 50W-8x 200–400-mesh; Bio-Rad Laboratories, Inc.) to isolate/purify the amino acids, and which were eluted from the columns with of 2N NH4OH. Isotopic enrichment of amino acids with d₉-leucine was measured using liquid chromatography tandem mass spectrometry (LC/MS/MS), and by following procedures we have also previously described (Tran et al., 2015). Fractional synthesis rate (FSR; %/hour) of muscle protein was calculated as: FSR = Δ Em Eb  x  60  x  100

Where ΔEm is the increment in muscle protein leucine enrichment between the two biopsies, Eb is the average leucine enrichment in blood between the biopsies, and T is the time interval (i.e., in mins) between the biopsies (60 and 100 are used as factors to express the FSR values in %/hour).

Plasma glucose concentrations were measured using an automated glucose analyzer (YSI 2300, Yellow Springs, OH). Plasma insulin concentrations were measured using a commercially available ELISA kit (80-INSHU-E01.1; ALPCO Diagnostics, Windham, NH). Plasma glucose and insulin concentrations during the OGTT were used to calculate the Matsuda insulin-sensitivity index (Matsuda and DeFronzo, 1999). The rest of the blood chemistry parameters reported in Table 1 were measured by the Mayo Clinic Clinical Laboratory. The concentration of major species of free fatty acids in plasma (arachidonic acid, elaidic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid) was measured by LC/MS (Persson et al., 2010).

Using a C2C12 cell culture model we sought to evaluate how biological signals altered within the metabolic environment of obesity affect syncytin-A expression in muscle. This experimental model allows isolating specific effects on syncytin-A expression in muscle, and in the absence of other concurrent biological signals present in vivo in humans. Insulin is well-established as a muscle anabolic hormone. Current evidence indicates specific roles of palmitate and oleate in regulating muscle growth and protein metabolism (da Paixão et al., 2021; Tardif et al., 2011; Lee et al., 2017; Kwon and Querfurth, 2015). Also, we found differential contribution of these fatty acid species to the total plasma free fatty acid concentrations in our human subjects with increased BMI (see Results section). Therefore, we chose to specifically test the effects of palmitate and oleate in our cell culture experiments.

C2C12 myoblasts (RRID:CVCL_0188) obtained from American Type Culture Collection (ATCC; Cat#: CRL-1772) were used for the cell culture experiments. C2C12 myoblasts were grown in growth media containing Dulbecco’s Modified Eagle’s Medium (DMEM) with 20% fetal bovine serum and 1% antibiotic-antimycotic at 37°C. The cells were grown in 6-well Collagen I-coated plates (Thermo Fisher Scientific; Cat #: A1142801). After 95% confluency, cells were differentiated in differentiation media containing DMEM with 2% horse serum and 1% antibiotic-antimycotic for 5 days, and until the cells were spindle-shaped (Kwon and Querfurth, 2015).

A 200 mM stock solution of palmitate and oleate were prepared using sodium oleate and palmitate dissolved in 50% ethanol for 30 min at 70°C. A 10% solution of fatty acid-free bovine serum albumin (BSA) was prepared in phosphate-buffered saline for conjugation purposes. Then, 5 mM stock solution of BSA conjugated fatty acid solutions (i.e., palmitate and oleate) were prepared by adding 200 mM stock solution of oleate and palmitate in 10% fatty acid-free BSA. Conjugation of the fatty acids and BSA was performed at 37°C for 1 h before cell treatment. C2C12 cells were incubated in serum free DMEM media for 2 h before treatment. The conjugated fatty acid-BSA serum was filtered and added to serum free DMEM media. Cells were treated with either 300 μm palmitate, 300 μm oleate, their combination, and with or without 20 nM insulin for 24 h. Before harvesting, cells were stimulated with 100 nM of insulin for 15 min. Myotubes were harvested as described by Kwon and Querfurth (Kwon and Querfurth, 2015).

Lysates of human muscle and C2C12 myotubes were prepared following procedures we have previously used in our laboratory (Tran et al., 2016). Protein concentration in the lysates was measured using Pierce™ Coomassie Plus (Bradford) protein assay reagent kit (ThermoFisher Scientific; Cat# 23200), and by following the manufacturer’s protocol. Briefly, BSA standards were used in the concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg for standard curve measurements. Lysates were diluted in a ratio of 1:40 (Lysate: Mili Q water). A 10 μl of the standard or the lysate was loaded into a 96 well plate. 190 μl of the Coomassie/Bradford reagent was added and the plate was incubated at room temperature for 10 min before measuring the absorbance at 595 nm in a spectrophotometer. Approximately 40 ug of protein from the human muscle lysate and 15 ug of protein from the myotube lysate was diluted (1:1) in a 2X Laemmli sample buffer and boiled for 5 min at 95°C, and the proteins were separated by SDS-PAGE on Any kD™ precast polyacrylamide gels (Mini-PROTEAN TGX, Bio-Rad Laboratories, Inc.). Proteins were transferred to nitrocellulose membrane in the case of human muscle homogenates and polyvinylidene difluoride membrane in case of C2C12 cell lysates for 1 h at 90 V, followed by incubation of the membrane with primary antibodies overnight at 4°C. Prior to incubation, the membranes were blocked at room temperature with TBST +5% non-fat dry milk (Syncytin-1/A) or TBST +5% BSA (4E-BP1, p-4E-BP1) for 60 min.

Syncytin-1 expression was quantified using an anti-syncytin-1 primary antibody (Biorbyt; Cat# orb100573; RRID:AB_2857960). The same antibody was used also in the C2C12 myotube experiments. Although the immunogen sequence was designed to detect human syncytin-1, it allows for mouse cross-reactivity and detection of syncytin-A in a mouse cell line (Ethiraj et al., 2018). Other primary antibodies used were: anti-GAPDH (Rockland Immunochemicals; Cat# 600-401-A33; RRID:AB_2107593), anti-4E-BP1 (Cell Signaling Technology; Cat# 9452; RRID:AB_331692), anti-phospho-4E-BP1 (Thr^37/46) (Cell Signaling Technology; Cat# 9459; RRID:AB_330985), and anti-myosin (Santa Cruz Biotechnology; Cat# sc-32732; RRID:AB_670118). A dilution of 1:1000 was used for all primary antibodies in Tris-buffered saline with 0.1% Tween®-20 (TBST) + 5% BSA, except for that for syncytin-1, which was diluted at 1:250 in TBST. We chose myosin (instead of GAPDH) as the housekeeping protein for the cell culture experiments because GAPDH showed considerable variation across the cell culture experimental conditions (see Results section).

The following day, the membrane was washed with TBST and incubated for 60 min at room temperature with secondary antibody. Secondary antibodies used were anti-rabbit (Cat. # 205718; Abcam) and anti-mouse (Cat. # sc-516102; Santa Cruz Biotechnology) IgG HRP-linked antibodies at a dilution of 1:2000 (anti-mouse) or 1:5000 (anti-rabbit) in TBST. Excess secondary antibody was washed with TBST.

Protein bands were visualized using the Clarity™ Western ECL Blotting Substrate (Bio-Rad, Hershey, PA) and imaged using the ImageQuant LAS 4000 (GE Healthcare, Wauwatosa, WI). Images of the membranes were captured in the increasing exposure intervals of 30 s except for GAPDH which was imaged at 5 s. Density of bands was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD). The band intensity of the protein of interest was normalized against the respective band intensity of the housekeeping protein present in the same lane.

Unpaired t-test or one-way ANOVA with Dunnett’s post-hoc tests were employed to compare data from two or more than two experimental groups, respectively. A two-way ANOVA with Bonferroni correction for multiple comparisons was used for two-factor analysis (i.e., BMI x sex). Correlations were evaluated using the Pearson product-moment correlation coefficient (r). Data are presented as means ± SD. p value of <0.05 was considered statistically significant. All statistical tests were two-sided. Statistical analyses were performed using GraphPad Prism version 8.4 (GraphPad Software, La Jolla, CA).

Subject characteristics shown in Table 1 indicate that the two subject groups represent distinct populations in terms of body composition and metabolic characteristics. In addition to having greater percent body fat, subjects with increased BMI were also characterized by significantly higher waist-to-hip ratio, and increased plasma insulin, glucose and triglyceride concentrations, as well as insulin resistance, all typical observations in humans with obesity.

Although the total concentration of the measured plasma free fatty acids did not differ between subjects with increased BMI and controls, plasma oleate concentrations were lower in the group with increased BMI (Supplementary Table S1). Plasma oleate and palmitate concentrations constituted ∼62% of the total plasma free fatty acids measured, with palmitic acid contributing significantly more (27.6 ± 1.7 versus 25.9 ± 1.1%; p = 0.02) and oleic acid contributing significantly less (33.4 ± 2.0 versus 37.2 ± 2.4%; p < 0.01) to the total plasma free fatty acids in the subjects with increased BMI compared to controls.

When compared to that in control subjects, skeletal muscle syncytin-1 expression was higher in the group of subjects with increased BMI and concomitant reduction in muscle protein synthesis (protein synthesis: 0.059 ± 0.014 versus 0.084 ± 0.019%/hour; p = 0.01) (Figure 1A). ANOVA analysis indicated no overall effect for sex (p = 0.54). Interestingly, although it did not reach statistical significance (probably because of low sample size), female (0.134 ± 0.038 versus 0.078 ± 0.027; p = 0.07), but not male (0.109 ± 0.031 versus 0.080 ± 0.052; p = 0.32), subjects with increased BMI displayed increased expression of syncytin-1 in skeletal muscle.

Expression of syncytin-1 in muscle across study subjects correlated significantly and inversely with muscle protein synthesis (Figure 1B). Moreover, this correlation was significant for female (r = −0.70; p = 0.03), but not male (r = −0.29; p = 0.45), subjects. Among the subject characteristics evaluated (Table 1), syncytin-1 expression displayed positive correlation with HbA1c (r = 0.58; p = 0.01) and negative correlation with HDL-Cholesterol:Total Cholesterol (r = −0.47; p = 0.05).

Expression of GAPDH in skeletal muscle did not differ between subjects with increased BMI and control subjects (p > 0.05). However, ANOVA analysis showed significant effect of cell culture treatments on GAPDH in the myotube experiments (p = 0.02). On the other hand, there was no significant effect of cell culture treatments on myosin expression in the myotube epxeriments (p = 0.66). Therefore, myosin was used as the housekeeping gene to express responses of syncytin-A and 4E-BP1 in the myotube experiments.

Insulin treatment alone consistently increased the expression of syncytin-A, but this effect was not significant in the presence of fatty acids (Figure 2). Neither insulin or any of the fatty acid treatments affected the response of p-4E-BP1/t-4E-BP1 (for all p > 0.05) (Figure 3A). On the other hand, p-4E-BP1 expression increased by insulin alone, but this effect was not evident when either palmitate or oleate was present along with the insulin (Figure 3B). However, there was a significant effect of combined palmitate and oleate (i.e., without the presence of insulin) on increasing p-4E-BP1 expression (Figure 3B). The effects of insulin and fatty acid treatments on t-4E-BP1 were identical to those for p-4E-BP1 (Figure 3C).