Fig 2: MMP2 cleavage site is localized in an extracellular domain of the GLUT4 receptor.(A) AlphaFold model of interaction between murine MMP2 and murine GLUT4 extracellular loop (residues 64–77). Pink represents contacts between MMP2 and the extracellular loop. (B) MMP fluorescent cleavage competition assay: MMP substrate, which becomes fluorescent after cleavage, was incubated at 37 °C with active MMP2, MMP2 with EDTA, MMP2 with GLUT4 loop peptide, MMP2 with negative control peptide, and MMP2 with positive control peptide. After 30 minutes, fluorescent intensity of the reactions was determined using a plate reader. (C) Sequence alignment of the extracellular loop between the first and second helix of human GLUT4 and murine GLUT4; murine GLUT1, murine GLUT3, and murine GLUT4; and human GLUT1, human GLUT3, and human GLUT4. Red box indicates MMP2 cleavage site. Values are expressed as means + SEM. *, p<0.05; **, p<0.01; ***, p>0.001 by nonparametric t-test (U-test). n=3 (B)

Fig 3: MMP2 decreases glucose uptake and glycolysis in 3T3L1 adipocytes.(A) Aerobic glycolysis of adipocytes after treatment with 200 ng/mL, 400 ng/mL, or 800 ng/mL of active MMP2. ECAR, extracellular acidification rate. (B) Aerobic glycolysis of adipocytes after treatment with 400 ng/mL of active MMP2, MMP2 inhibitor, or MMP2 with MMP2 inhibitor. (C) Glucose uptake of 3T3-L1 adipocytes after treatment with 200 ng/mL, 400 ng/mL, or 800 ng/mL of active MMP2. Glucose uptake was assessed by mean fluorescent intensity of 2-NBDG in live adipocytes. (D) Glucose uptake of 3T3-L1 adipocytes after treatment with 400 ng/mL of active MMP2, MMP2 inhibitor, or MMP2 with MMP2 inhibitor. Glucose uptake was assessed by mean fluorescent intensity of 2-NBDG in live adipocytes. Values are expressed as means + SEM. *, p<0.05; **, p<0.01; ***, p>0.001, ****, p<0.0001 by parametric unpaired t-test. n=5 (A & B) n=6 (C & D)

Fig 4: A putative MMP2 cleavage site is localized in an extracellular domain of the GLUT4 receptor. (A) AlphaFold model of interaction between murine MMP2 and murine GLUT4 extracellular loop (residues 64–77). Pink represents contacts between MMP2 and the extracellular loop. (B) MMP FRET Substrate cleavage competition assay: MMP FRET Substrate was incubated at 37 °C with 5 nM MMP2 alone or MMP2 with 50 mM EDTA, 500 µM GLUT4 loop peptide, 500 µM negative control peptide (GLUT4 LoopRev), or 500 µM positive control peptide. After 30 min, fluorescent intensity of the reactions was determined using a plate reader. (C) Size exclusion chromatography of peptide and MMP2 mixtures: an MMP substrate peptide (MMPsub), GLUT4 loop peptide (GLUT4 Loop), or the reverse peptide (GLUT4 LoopRev) at 20 µM was incubated with 10 nM MMP2 for 18 h at 37 °C and analyzed using a size exclusion chromatography column. The amide bonds in the peptides were detected using UV absorbance 214 nM. ‘#’ indicates the broad peak for MMP2-peptide complex. (D) Sequence alignment of the extracellular loop between the first and second helix of human GLUT4 and murine GLUT4; murine GLUT1, murine GLUT3, and murine GLUT4; and human GLUT1, human GLUT3, and human GLUT4. Red box indicates MMP2 cleavage site. Values are expressed as means + SEM. ****, p > 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. n = 6 (B).

Fig 5: MMP2 decreases insulin-stimulated glucose uptake in 3T3-L1 adipocytes. (A) Flow cytometry gating for adipocytes in glucose uptake assay. Live adipocytes were used to determine glucose uptake as shown in (B,C). (B) Left panel shows the histograms of 2-NBDG fluorescence of live adipocytes after treatment with 200 ng/mL, 400 ng/mL, or 800 ng/mL of MMP2 and right panel shows the quantification of mean fluorescence intensity (MFI). As a negative control of glucose update, some cells were pretreated with Cytochalasin D (10 µM, CytoD) for 1 h. (C) Left panel shows the histograms of 2-NBDG of live adipocytes after treatment with 400 ng/mL (5 nM) of MMP2, MMP2 inhibitor I (1 µM), or MMP2 with MMP2 inhibitor I and right panel shows the quantification of MFI. Values are expressed as means + SEM. ***, p > 0.001, ****, p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. n = 6 (A–C)