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

Journal: Bioactive Materials

Article Title: Muscle-fiber-inspired nanofibrillar microbundles induce myogenic differentiation in human adipose-derived stem cells

doi: 10.1016/j.bioactmat.2026.03.020

Figure Lengend Snippet: RNA-seq profiling of human adipose-derived stem cells (hASCs) after 21 days of culture in myogenic differentiation medium. (a) Principal component analysis (PCA) based on transcriptome expression values (FPKM), showing clustering of biological replicates for Monolayer (2D), PCL, and Fibril conditions. (b) Venn diagram showing the overlap of detected genes among Monolayer, PCL, and Fibril groups (numbers indicate gene counts in each intersection). (c) Gene Ontology (GO) Biological Process (BP) over-representation analysis (ORA) for differentially expressed genes in 2D vs nFMBs (left) and PCL-mFiBs vs nFMBs (right); bars are plotted as −log10 (adjusted p value), with terms enriched among genes upregulated in the first condition shown to the right (red) and terms enriched among genes downregulated in nFMBs shown to the left (blue). (d) KEGG pathway enrichment analysis for differentially expressed genes between PCL-mFiBs and nFMBs groups; dot size represents the number of genes mapped to each pathway (Count), dot color indicates adjusted p value, and the x-axis denotes Gene Ratio. (e) Category network plot (CNP; category–gene network plot) for the PCL-mFiBs vs nFMBs comparison, visualizing representative enriched GO BP terms and their associated genes; gene nodes are colored by fold change and term nodes reflect enrichment significance. (f–i) Heatmaps of selected genes associated with representative GO terms: GO:0000280 (nuclear division), GO:0030198 (extracellular matrix organization), GO:0003012 (muscle system process), and GO:0007519 (skeletal muscle tissue development), respectively; expression patterns are shown across 2D, PCL-mFiBs, and nFMBs, with gene symbols listed alongside each heatmap.

Article Snippet: For HSkMCs, myogenic differentiation was induced using Skeletal Muscle Differentiation Medium (ATCC, Manassas, USA).

Techniques: RNA Sequencing, Derivative Assay, Cell Characterization, Expressing, Comparison

Journal: Human Genetics and Genomics Advances

Article Title: Using a modular massively parallel reporter assay to discover context-dependent regulatory activity in type 2 diabetes-linked noncoding regions

doi: 10.1016/j.xhgg.2026.100606

Figure Lengend Snippet: HNF1 transcription factor motifs contribute to enhancer activity near selected T2D-associated variants (A) For 47 HNF1-motif-overlapping fragments with significant INS promoter-bias effects on activity, we designed three versions: original (motif intact), deleted (motif removed and sequence adjusted), and shuffled (dinucleotide-shuffled motif). When the tested variant was adjacent to the motif, we synthesized both reference and alternative alleles for each version. For variants directly overlapping the motif, we generated only one deletion and one shuffled fragment. (B) We synthesized four fragments corresponding to the variant rs1635852, which overlaps an HNF1 motif at a high-information-content position. The T2D risk allele (T) disrupts this motif, while the non-risk allele (C) matches the consensus. (C) Shuffling the motif significantly decreased enhancer activity compared to intact fragments with either the risk T (Wilcoxon rank-sum test p = 0.016) or non-risk C ( p = 0.008) allele. Motif deletion also significantly decreased enhancer activity compared to the non-risk C allele ( p = 0.016). (D) For the variant rs11819995, located 11 bp upstream of an HNF1 motif, we synthesized six fragments. (E) Deletion of the motif significantly decreased enhancer activity for both the reference (C, non-risk) and alternative (T, risk) alleles ( p = 0.008 for both alleles). Shuffling the motif likewise reduced activity for both alleles ( p = 0.008 for the reference allele and p = 0.056 for the alternative allele). (F) To assess context-specific effects, we cloned these fragments into MPRA vectors with the SCP1 or skeletal-muscle-specific MYBPC2 promoter and delivered all three to LHCN-M2 human skeletal muscle myotubes ( n = 6). (G) When paired with the INS promoter, the shuffled rs11819995-containing fragment showed increased activity relative to the original fragment ( p = 0.015); however, none of the fragments containing rs11819995 functioned as enhancers in LHCN-M2 myotubes, regardless of promoter context. Overall, their activity is highest when paired with the skeletal-muscle-specific promoter.

Article Snippet: We obtained INS-1 832/13 rat insulinoma cells from Dr. Christopher Newgard (Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC) and LHCN-M2 human skeletal muscle myoblasts from Evercyte.

Techniques: Activity Assay, Sequencing, Variant Assay, Synthesized, Generated, Clone Assay