Fig. 2.

Net proteolytic activity is increased in male TIMP-2 KO hypothalamus. (A) qPCR revealed that, at least at the mRNA level, there is no overall compensation by other TIMPs in response to TIMP-2 absence. There was no effect of diet at the study termination, but when compared to the study start TIMP-1 mRNA expression was increased in chow-fed (p = 0.03) and HFD-fed (p = 0.002) TIMP-2 KO mice while no increase was observed in WT mice. (B) Using a caged substrate that emits fluorescence when cleaved by proteases, it was revealed that net proteolytic activity was increased in TIMP-2 KO hypothalamus, but not globally increased throughout the entire TIMP-2 KO brain. Interestingly, while WT mice decreased proteolytic activity on the HFD, proteolysis remained elevated in HFD-fed TIMP-2 KO mice, suggesting that the inability to effectively inhibit proteolysis may contribute to defective energy homeostasis and obesity. (C) In situ zymography confirmed the results of the gelatinase assay and specifically revealed increased proteolysis within the arcuate nucleus of TIMP-2 KO mice. Data are mean ± SEM. n = 4 – 5 per genotype and diet. *p < 0.05, **p ≤ 0.01, #p≤0.001 relative to WT mice on the same diet, unless otherwise indicated.