Mouse skeletal muscle cell lines (C2C12 myoblasts) were obtained from Shanghai FuHeng Biology (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Co., Ltd., Grand Island, NY, United States) containing 10% (v/v) fetal bovine serum (Gibco) and 1% (w/v) penicillin–streptomycin (P/S; NCM Biotech, Suzhou, China) in a humidified incubator under 5% (v/v) CO₂ at 37°C. To initiate differentiation, cells were grown to 70%–80% confluence, and then incubated with DMEM with 2% (v/v) heat-inactivated horse serum (Gibco) and 1% (w/v) P/S for 4–6 days. The medium was changed daily. The fully differentiated myotubes were used in the following experiments.

C2C12 myoblasts were seeded in 96-well plates and grown for differentiation. After the formation of myotubes, 0, .25, .5, .75 or 1 mM PA was added for 24 h, and then cell viability was measured using cell counting kit-8 (Beyotime) according to the manufacturer’s protocol. Next, the absorbance was measured at a wavelength of 450 nm using Microplate Reader (Thermo Fisher Scientific). Cell viability was expressed as a percentage of the optical density of each treatment group relative to the control group.

Firstly, palmitate (33.40mg, Sigma, St. Louis, MO, United States) was dissolved in 3 mL double distilled water (v/v) with heating at 75°C. Next, preparation 40% free fatty acid-free bovine serum albumin solution (BSA), fatty acid free BSA powder (1.2 g, Beyotime, Shanghai, China) dissolved in phosphate-buffered saline (PBS), centrifugated at room temperature 8,000 rpm for 15 min until BSA completely dissolved, and then fixed to 3 mL with PBS. Thirdly, mixing the two liquids together to make a 20 mM solution and then sterilized by passing through a .22-μm-pore-sized filter, and this solution was stored at −20°C and used within 2 weeks. Fully differentiated myotubes were treated with .25 mM PA for 24 h (Petersen and Shulman, 2018; Shen et al., 2019). Control cells were treated with the same volume of PBS–BSA DMEM with or without insulin. PA-treated C2C12 myotubes were serum-depleted for 4.5 h and then incubated with or without 10 ng/mL NRG-1β (PeproTech, Rocky Hill, NJ, United States) for 1.5 h (Suárez et al., 2001; Canto et al., 2004; Heim et al., 2020). After 1 h treatment of NRG-1β, cells were then incubated with or without insulin (100 nM) for 30 min.

Glucose uptake by differentiated C2C12 myotubes was measured using the 2-NBDG assay that employs a fluorescent D-glucose analogue (Zou et al., 2005; Bala et al., 2021). After incubation with or without NRG-1β, cells were incubated with 80 µM 2-NBDG (APExBIO, Houston, TX, United States) at 37°C for 30 min 2-NBDG uptake was stopped by removing the incubation medium and washing the cells with 1×PBS followed to remove free 2-NBDG. And then discarding the PBS, the cells were subsequently resuspended in pre-cool RIPA (Beyotime) and transferred to 96-well culture plates (Xu et al., 2018). Next, 2-NBDG levels were determined using a fluorescence microplate (excitation 485 nm, emission 535 nm; Thermo Fisher Scientific, Massachusetts, MA, United States). And we were very careful to avoid light during the experiment.

The animal protocol was approved (Protocol number: 2019-A005-1, Shanghai, China) by the Shanghai General Hospital Clinical Center Laboratory Animal Welfare and Ethics Committee. Thirty pathogen-free 3–4-week-old male C57BL/6J wild-type mice (SLAC, Shanghai, China) (18–20 g) were used. The animals were housed in a temperature-controlled (25°C ± 2°C) room under a 12 h light/dark cycle with a relative humidity of 50%–70%. T2DM was induced as described previously (Hamza et al., 2011; Kleinert et al., 2018; Yang et al., 2020). The mice were randomly divided into a control group (CON, n = 10), a T2DM + PBS group (DM, n = 10) and a T2DM + NRG-1β group (DM + NRG-1β, n = 10). All mice were acclimatized for 1 week prior to experimentation. The CON group was fed normal chow; the DM and the DM + NRG-1β groups were fed a high-fat diet [high fat (60FDC) Purified Rodent Diet; D12492; Research Diets, New Brunswick, NJ, United States) for five consecutive weeks. Then, after 12 h of fasting, the DM and DM + NRG-1β groups were intraperitoneally injected with 50 mg/kg streptozocin (STZ) (S0130; Sigma, St. Louis, MO, United States) daily for 3 days. The CON group was intraperitoneally injected with isometric citric acid buffer. The blood glucose levels were measured 7 days after the final injection. When that level was >16.7 mmol/L, T2DM was considered established (Kleinert et al., 2018; Yang et al., 2020). DM + NRG-1β group and DM group received 30 μg/kg NRG-1β (via intraperitoneal injection) or the same volume of PBS weekly for 4 weeks, respectively (Ennequin et al., 2015; Wu et al., 2019; Liu et al., 2020). After a week final treatment of NRG-1β, mice were anesthetized with 1% (w/v) pentobarbital sodium and euthanized by decapitation. The gastrocnemius tissues were dissected, weighed, and soaked overnight at 4°C in 4% (v/v) paraformaldehyde (PFA) and then in 30% (w/v) sucrose at 4°C for 48 h to immunofluorescence analysis or frozen in liquid nitrogen and stored at −80°C for later biochemical analysis.

After washing with ice-cold PBS, cells or gastrocnemius tissues were homogenized. Total protein was extracted into pre-cooled RIPA lysis buffer (Beyotime) with 1% (w/v) of a protease/phosphatase inhibitor cocktail (NCM Biotech) at 4°C for 15 min, and the protein concentration was measured using a BCA protein assay kit (Beyotime). Protein loading buffer (5X) (NCM Biotech) was added to the lysates followed by protein denaturation by placing the tubes in boiling water for 10 min.

Membrane proteins (including GLUT4) were obtained using Membrane and Cytosol Protein Extraction Kit (Beyotime), according to the manufacturer’s instructions. Western blotting was employed to determine GLUT4 expression levels on plasma membranes. The membrane marker Na⁺-K⁺-ATPase served as a control.

Proteins (20–30 μg/lane) were subjected to 10% (v/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (Epizyme, Suzhou, China) and transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore, Billerica, MA, United States) and the blots saturated with blocking buffer (NCM Biotech) for 15 min at room temperature and then incubated overnight at 4°C with antibodies against β-actin (1:5,000; Proteintech, Wuhan, China), myosin heavy chain (MHC; 1:1,000; Proteintech), Phosphor-Akt (ser 473) (1:1,000; Cell Signaling Technology, Massachusetts, MA, United States), AKT (1:1,000; Proteintech), GLUT4 (1:1,000; Cell Signaling Technology) and Na⁺-K⁺-ATPase (1:1,000; Cell Signaling Technology). The gray intensity of protein was measured using ImageJ software (United States National Institutes of Health). Three independent experiments were performed and the data averaged.

C2C12 myoblasts were cultured on 35-mm-diameter confocal dishes and allowed to differentiate for 4–5 days. After treatment with NRG-1β, the myotubes were fixed in 4% (v/v) PFA for 15 min, permeabilized by adding a buffer containing saponin (Beyotime) for 10 min at room temperature, and then incubated in PBS with 3% (w/v) BSA for 1 h at room temperature, followed by incubation with anti-GLUT4 (1:300; Affinity, Suzhou, China) overnight at 4°C. The cells were washed three times with PBS (10 min each time) and then incubated with Alexa Fluor 594-conjugated goat anti-mouse secondary antibody for 2 h at room temperature. Detection was performed using the confocal microscope (TCS SP8 X; Leica Heidelberg, Germany).

Mice were anesthetized with 1% (w/v) pentobarbital sodium and perfused with saline for a few minutes, followed by perfusion with 4% (v/v) PFA in PBS. The gastrocnemius tissues were dissected and soaked overnight at 4°C in 4% (v/v) PFA and then in 30% (w/v) sucrose at 4°C for 48 h. Gastrocnemius slices (20 μm) were prepared and immunofluorescence detected as described above.

All experiments were repeated at least three times. Data are presented as means ± standard error of the mean (SEM) and were compared via one-way analysis of variance. p-values <.05 were considered statistically significant.

C2C12 myoblasts differentiated into myotubes, as confirmed by the development of the myotube-like multi-nuclear structure (Supplementary Figures S1A, B) and a structure protein marker MHC (Supplementary Figures S1C, D). To explore the effects of NRG-1β on glucose uptake and GLUT4 translocation in IR environment, we reproduced the classic cellular model in the C2C12 myotubes (Nieuwoudt et al., 2017; Petersen and Shulman, 2018). Glucose uptake was measured with the aid of the 2-NBDG assay (Zou et al., 2005; Bala et al., 2021). PA at .25 mM did not affect C2C12 myotube cell viability (Figure 1A). And then, C2C12 myotubes were incubated with various concentrations of NRG-1β for 1.5 h (Suárez et al., 2001; Canto et al., 2004). NRG-1β at 10 ng/mL optimally increased glucose uptake (Figure 1B). Glucose uptake was decreased in PA-treated (plus insulin) C2C12 myotubes compared to BSA (plus insulin) group, but NRG-1β rescued the uptake (Figure 1C). Thus, NRG-1β increased glucose uptake in PA-treated C2C12 myotubes.

To explore the effects of NRG-1β on GLUT4 translocation in PA-treated C2C12 myotubes, we used immunofluorescence staining to detect GLUT4. The GLUT4 levels were significantly decreased in PA-treated (plus insulin) C2C12 myotubes compared BSA (plus insulin) group; however, NRG-1β rescued the decrease (Figures 2A, B). Next, we examined cell membrane GLUT4 levels in the presence or absence of NRG-1β. GLUT4 membrane translocation decreased in PA-treated C2C12 myotubes, but NRG-1β rescued the fall (Figures 2C, D). Therefore, NRG-1β promoted GLUT4 translocation in PA-treated C2C12 myotubes.

The PI3K/AKT pathway triggers the GLUT4 translocation that is essential for glucose uptake and metabolism (Zhu et al., 2013; Li et al., 2017; Sharma and Dey, 2021). To explore the molecular mechanisms in play, we measured the AKT and phosphorylation of AKT protein levels. Phosphor-Akt (ser 473) expression was significantly decreased in PA-treated (plus insulin) C2C12 myotubes compared to the BSA (plus insulin) group, but NRG-1β increased the level significantly (Figures 3A, B), indicating that NRG-1β activated PI3K/AKT signaling in PA-treated C2C12 myotubes. Next, we determined the effects of pretreatment with MK2206 (an AKT inhibitor) on glucose uptake and translocation of GLUT4 to the plasma membrane of PA-treated C2C12 myotubes. Pretreatment with MK2206 significantly reduced the effects of NRG-1β on glucose uptake (Figure 3C) and GLUT4 translocation to the plasma membrane (Figure 4). These data suggested that NRG-1β increased glucose uptake and GLUT4 translocation in PA-treated C2C12 myotubes by activating PI3K/AKT signaling.

We established a T2DM animal model by combing high-fat diet with a low dose of STZ to evaluate the effects of NRG-1β. Seven days after the final intraperitoneal injection of STZ, establishment was successful (the blood glucose level was >16.7 mmol/L) (Hamza et al., 2011; Kleinert et al., 2018; Yang et al., 2020). The body weight of the DM group began to decrease after STZ injection, in contrast to the constant weight gain of the CON group, but NRG-1β improved diabetes-induced weight loss (Figure 5A). The blood glucose level remained high after STZ injection in the DM group; NRG-1β attenuated diabetes-induced hyperglycemia significantly (Figure 5B).

T2DM induced a remarkable change in the distribution of GLUT4 in skeletal muscle tissue (Fang et al., 2017; Fujiwara et al., 2017). After treatment with 30 μg/kg NRG-1β once weekly for 4 weeks, we examined the translocation of GLUT4 in gastrocnemius tissues in C57BL/6J and T2DM mice. The GLUT4 fluorescence intensity of T2DM mice were significantly lower than those of C57BL/6J mice of the same age; however, NRG-1β inhibited the reduction (Figures 6A, B). These observations were confirmed by Western blotting (Figures 6C, D).