Role Of Metalloproteases In Shedding Of Dipeptidyl-peptidase Iv From Skeletal Muscle Cells

Abstract

Dipeptidyl- Peptidase IV (DPP-IV) is a membrane bound myokine that is involved in converting the powerful vasoconstrictor, neuropeptide Y, into the non-vasoconstricting form. Previous work in this lab suggested that DPP-IV is released from myocyte cell cultures, when a whey protein solution is applied, but not when leucine is applied. A possible mechanism is a process called shedding that occurs via the metalloproteases MMP2 and MMP9. These MMP’s are known to mediate the shedding of DPP-IV from adipocytes and smooth muscle cells and exist in commercially available whey protein, but not in leucine products. PURPOSE: To determine if the shedding of DPP-IV from skeletal muscle cells is mediated by metalloproteases. We hypothesized that when MMP2 and MMP9 were inhibited (i), the amount DPP-IV released in the presence of whey protein would be decreased. METHODS: C2C12 cells underwent 4–5 days of differentiation. They were treated for 6 hours with one of the following: leucine (Leu) or whey protein (WP), LEU+MMP2i, Leu+MMP9i, Leu+complete protease inhibitor (CP), WP+MMP2i, WP+MMP9i, or WP+CP. Control plates were also run with one of the following: MMP2i, MMP9i, CP, or dimethyl sulfoxide (DMSO-vehicle for inhibitors). The media was collected for DPP-IV activity measurements, which were measured using a fluorometric assay. RESULTS: The DPP-IV activity of the control cells treated with only the inhibitors and vehicle, as well as Leu+MMP2i and Leu+MMP9i were not significantly different from Leu. Only Leu+CP had a significant decrease in DPP-IV activity from Leu (22.5% decrease; p=0.01). DPP-IV activity of the DMSO group was significantly increased from WP (35.4% increase; p<0.001); however when the inhibitors were added, this increase was no longer significant. When whey protein was given with the inhibitors, DPP-IV activity significantly decreased for all 3 groups from WP (WP+MMP2i: 32.3% decrease; WP+MMP9i: 40.6% decrease, and WP+CP: 51.0% decrease; all p<0.0001) CONCLUSION: The shedding of DPP-IV from skeletal muscle cells is mediated by metalloproteases 2 and 9, which are present in whey protein. This is a possible mechanism by which milk products may affect neuropeptide Y-mediated blood flow.