Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: (A) RT-PCR analysis of GIPR and GLP-1R expression in differentiated primary human myotubes from six osteoarthritis patients (three technical replicates per donor). GIPR was consistently detected across all donors; GLP-1R was undetectable in all samples. (B) CT and ΔCT values for GIPR and GLP-1R across all six donors (ΔCT values: 35.00 ± 0.81, 33.07 ± 0.71, 35.01 ± 0.73, 34.85 ± 1.21, 35.80 ± 0.54, 33.36 ± 0.23; mean ± SD). (C) Mean fluorescence intensity (MFI) fold change relative to unstained controls in human primary myoblasts live-stained with LUXendin645 (500 nM, 15 minutes). Data from three donors shown with individual patient values connected by lines; bar represents the group mean. *p = 0.0383, paired t-test. (D) Representative gating strategy for LUXendin645 flow cytometry on the BD Accuri C6 Plus. Upper row: unstained human primary myoblasts. Lower row: LUXendin645-stained human primary myoblasts. Sequential gating: FSC/SSC scatter to exclude debris, (2) singlet discrimination, (3 & 4) fluorescent signal as histogram and dot plot to identify LUXendin645-positive cells. (E) MFI values and percentage LUXendin645-positive cells for each of the three donors.

Article Snippet: Differentiated human skeletal muscle myotubes (n = 6 donors) were treated with semaglutide (10 nM; MedChemExpress HY-114118; DMSO vehicle) or GIP peptide (5.02 μM; MedChemExpress HY-P0276; H 2 O vehicle).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: Data from n = 6 donors unless otherwise stated. All PCR comparisons by Wilcoxon signed-rank test; ns = not significant. (A) mRNA expression (fold change to vehicle control) of atrophy- and inflammation-related genes in human primary myotubes 4 hours after treatment with semaglutide vs DMSO (left) or GIP vs H ₂ O (right). Genes assessed: GIPR, MAFBX, MURF1, FOXO3, IL6, and TGFB. GLP-1R was also assessed but was undetectable in all samples, consistent with . (B) Principal component analysis (PCA) of Olink Reveal normalised protein expression (NPX) data from primary human myotubes treated with GIP (H ₂ O vehicle control), semaglutide (DMSO vehicle control), or their respective vehicle controls. Points represent individual samples; 95% confidence ellipses are shown for each treatment group. (C) Volcano plots showing differential protein expression between GIP vs H ₂ O vehicle control (left) and semaglutide vs DMSO vehicle control (right). The x-axis shows log ₂ fold change (NPX) and the y-axis shows −log ₁₀ (nominal p-value) from Welch’s two-sample t- test. Dashed lines indicate thresholds of |log ₂ FC| > 0.58 and p < 0.05 (unadjusted). No proteins reached statistical significance after Benjamini–Hochberg correction across 1,034 proteins tested (minimum BH-adjusted p = 1.0, both comparisons). At nominal thresholds, three proteins were upregulated in GIP-treated myotubes (UBE2B, S100A12, CRNN), and one following semaglutide treatment (CD79B). (D) Heatmaps showing z-scored NPX values for the 100 proteins with the lowest nominal p- values from GIP vs H ₂ O comparison (left) and semaglutide vs DMSO comparison (right). Rows are hierarchically clustered; columns ordered by treatment group. Colour scale: red = high NPX, blue = low NPX. (E) Individual delta NPX values (NPX − mean NPX of vehicle control) for six proteins of functional relevance to muscle biology: FOXO1, MSTN, IGF1R, TGFB1, FOXO3, and IL6. Data shown for semaglutide vs DMSO vehicle control and GIP vs H ₂ O vehicle control. Individual donor values are plotted with mean ± SEM.

Article Snippet: Differentiated human skeletal muscle myotubes (n = 6 donors) were treated with semaglutide (10 nM; MedChemExpress HY-114118; DMSO vehicle) or GIP peptide (5.02 μM; MedChemExpress HY-P0276; H 2 O vehicle).

Techniques: Expressing, Control, Comparison, Functional Assay

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: (A) Representative desmin/DAPI immunofluorescence images of human primary myoblasts before and after differentiation. Undifferentiated myoblasts appear as long, thin, mononucleated cells; differentiated myotubes are thicker and multinucleated. (B) Representative desmin/DAPI images of differentiated myotubes treated with or without TNFα for 24 hours. TNFα treatment induced visible myotube thinning while preserving multinucleation, confirming TNFα as a positive control for atrophy induction. (C) Representative desmin/DAPI images of control myotubes and myotubes following 24 hours of incretin treatment. (D) Myotube thickness (µm) following 24 hours of treatment with semaglutide vs vehicle (DMSO; left) or GIP vs vehicle (H ₂ O; right).

Article Snippet: Differentiated human skeletal muscle myotubes (n = 6 donors) were treated with semaglutide (10 nM; MedChemExpress HY-114118; DMSO vehicle) or GIP peptide (5.02 μM; MedChemExpress HY-P0276; H 2 O vehicle).

Techniques: Immunofluorescence, Preserving, Positive Control, Control

Journal: bioRxiv

Article Title: GLP-1 and GIP receptor agonism does not directly drive skeletal muscle atrophy or impair myogenesis in primary human myotubes

doi: 10.64898/2026.07.20.739515

Figure Lengend Snippet: Data shown are from n = 3 biological replicates (PCR) or n = 6 biological replicates (all other assays). Myotubes were treated with semaglutide or GIP (with appropriate vehicle controls) every 2 days during the 8-day differentiation protocol. All comparisons by Wilcoxon signed-rank test; ns = not significant. (A) MyoG, MyoD, and Myf5 mRNA expression (fold change to vehicle control) at Day 2 and Day 4 of differentiation, for semaglutide vs vehicle control (left, blue) and GIP vs vehicle control (right, red). (B) Nuclear fusion index (NFI, %) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Representative desmin/DAPI immunofluorescence images are shown for each condition (DMSO, semaglutide, H ₂ O, GIP). (C) Myotube coverage (%) following 8 days of differentiation. (D) Representative images used for lactate dehydrogenase (LDH) quantification, following 8 days of differentiation.| (E) LDH release (absorbance 450–650 nm) following 8 days of differentiation, shown for all four conditions. LDH release was consistent between semaglutide, DMSO, and GIP, but significantly elevated in the H ₂ O vehicle control condition relative to all three (vs DMSO, p = 0.0014; vs semaglutide, p = 0.0004; vs GIP, p = 0.0003), consistent with a hypotonic vehicle effect rather than a treatment effect. (F) Myotube thickness (µm) following 8 days of differentiation with semaglutide vs DMSO (left) and GIP vs H ₂ O (right). Semaglutide p = 0.7708; GIP p = 0.0205.

Article Snippet: Differentiated human skeletal muscle myotubes (n = 6 donors) were treated with semaglutide (10 nM; MedChemExpress HY-114118; DMSO vehicle) or GIP peptide (5.02 μM; MedChemExpress HY-P0276; H 2 O vehicle).

Techniques: Expressing, Control, Immunofluorescence