2.9. SIRT1 Activity Assay

The SIRT1 activity in liver, calf skeletal muscles and eWAT was determined using a SIRT1 direct fluorescent screening assay kit (Cayman, Ann Arbor, MI, USA) as previously described [31]. Briefly, a total of 25 μL of assay buffer (50 mM Tris-HCl, pH 8.0, containing 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂), 5 μL of tissue extract (1.5 mg/mL), and 15 μL of substrate (Arg-His-Lys-Lys(ε-acetyl)--7-amino-4-methylcoumarin) solution were added to all wells. The fluorescence intensity was monitored every 2 min for 1 h using a BertholdTech TriStar2S fluorescence plate reader (Berthold Technologies, Bad Wildbad, Germany) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The results were expressed as the rate of reaction for the first 30 min, when there was a linear relationship between fluorescence and time.