Metformin Administration Protects Against Deltoid Tendon Damage Through Activation of Notch Signaling

Abstract

Type 2 Diabetes Mellitus (T2DM) is a growing global health concern that is associated with severe complications including diabetic tendinopathy. In this study, we found that T2DM patients had a significantly higher prevalence of tendon surgery compared to non-T2DM patients, which were alongside impaired ECM and cell adhesion. Notably, metformin-treated T2DM patients had a lower prevalence of tendon surgery compared to other medications, along with improved tendon fiber structure, downregulation of tendon damage marker MMP3, and upregulation of HES1, a Notch signaling effector gene. Metformin also activates Notch signaling in cultured tenocytes, and tendons from diabetic mice and aged monkey. These findings highlight metformin's potential to protect tendons by activating Notch signaling, offering novel insights into its therapeutic benefits beyond glucose regulation.

FIGURE 1

Patients with type 2 diabetes undergo deltoid tendon damage. (A) Statistical analysis of patients with shoulder pain. (B) Percentage of non‐type 2 diabetes mellitus (T2DM) and T2DM patients with conservative treatment or undergo tendon surgery. (C) Relative image of H&E staining using deltoid tendons from non‐T2DM and T2DM patients. (D) Volcano plot of the RNA‐seq results from Control and DB groups, n = 32 and 30, respectively. (E) KEGG analysis of deltoid tendon differentially expressed genes (DEGs). Heatmap of DEGs from Control versus DB that enriched in cell adhesion molecules (F), ECM–receptor interaction (G), and PPAR signaling (H) pathways. (I) Statistical analysis of T2DM patients with shoulder pain taking metformin or other medications. (J) Relative image of H&E staining using deltoid tendons from T2DM patients and T2DM patients with metformin. (K) Heatmap of the expression of genes in ECM–receptor interaction pathway. (L) Relative mRNA levels of MMP3 from DB and DB+Met groups, n = 13 and 8, respectively. Data represent mean ± SEM (t‐test: ***p < 0.001).

FIGURE 2

Metformin administration ameliorates deltoid tendon damage and activates Notch signaling. (A) Relative mRNA levels of HES1 expression in type 2 diabetes mellitus (T2DM) patients (n = 13) versus Met‐treated T2DM patients (n = 8). (B) HES1 protein levels in deltoid tendons of Control, T2DM, and Met‐treated T2DM groups. (C) Body weight of WT and DB/DB mice after 3‐month of metformin treatment from WT and DB/DB groups. (D) Serum glucose levels of DB/DB mice with or without metformin, n = 5 and 6, respectively. (E) Volcano plot analysis of tendon transcriptomes in DB/DB mice versus Met‐treated DB/DB mice, n = 4. (F) Heatmap of Notch1 and Hes1 expressions in mouse deltoid tendon from WT, WT+Met, DB and DB+Met groups, n = 4. (G) Western blot analysis of HES1 protein levels in deltoid tendon from WT, DB and DB+Met groups. (H, I) Gene set enrichment analysis (GSEA) of DEGs from Control and Met‐treated DB/DB mice. (J) Fasting blood glucose in streptozotocin (STZ)‐induced T2DM mice, n = 13. (K) Glucose tolerance tests (GTT) in STZ‐T2DM mice, n = 5. (L) Insulin tolerance tests (ITT) in STZ‐T2DM mice, n = 5. (M) HES1 protein levels in tendon of Control and STZ‐induced T2DM mice, n = 4. (N) Metformin intervened bone marrow stem cells (BMSCs) were collected 24 h post‐intervention. Western‐blot analysis (O) of NICD and HES1 and quantification (P) after 10 μM metformin treatment, n = 3. Western‐blot analysis (Q) of NICD and HES1 and quantification (R) after 10 nM metformin treatment, n = 3. (S) A scheme showing metformin treatment on BMSCs after induction of tenogenic differentiation. Differentiated BMSCs were collected after sequential treatment: 24‐h nutrient deprivation, followed by 12‐h BMP‐12 exposure, culture in normal media until Day 14.5, 24‐h Met administration, and final sample collection. Western‐blot analysis (T) of NICD and HES1 and quantification (U) after 10 nM metformin treatment in tenogenic differentiated BMSCs, n = 3. Western‐blot analysis (V) of NICD and HES1 and quantification (W) after 10 nM metformin treatment in tenogenic differentiated BMSCs with or without notch inhibitor RO4929097, n = 3. (X) Relative mRNA expressions of SCX, SOX9, TNMD, COL1A1, COL3A1, RUNX2, HES1, HEY1, HEYL, DLL1, DLL4, JAG1 in BMSCs from DMSO or Met groups, n = 3. (Y) Relative mRNA expressions of SCX, SOX9, TNMD, COL1A1, COL3A1, RUNX2, HES1, HEY1, HEYL, DLL1, DLL4, JAG1 in BMSCs from Met or RO4929097 groups, n = 3. Data represent mean ± SEM (t‐test, two‐way ANOVA, and ANCOVA: *p < 0.05, **p < 0.01, ***p < 0.001).