Fig 1: SIRT6 functions in lipolysis mediated by TNFR2. (A–B) Tumour necrosis factor receptor (TNFR1 and TNFR2) protein levels in the eWAT from WT + PBS, WT + LLC, SIRT6 TG + PBS and SIRT6 TG + LLC mice were determined by western blot (n = 6 per group). (C) Serum TNFR2 concentrations in mice were measured by ELISA (PBS, n = 7 per group; LLC, n = 11 per group). (D) cAMP levels in WT + DMEM, WT + LLC‐CM, TG + DMEM, TG + LLC‐CM adipocytes (n = 3 per group) or WT + DMEM, WT + LLC‐CM, KO + DMEM, KO + LLC‐CM adipocytes (n = 4 per group) were measured. WT + DMEM (WT adipocytes were treated with DMEM medium), WT + LLC‐CM (WT adipocytes were treated with LLC cell‐conditioned medium), TG + DMEM (SIRT6 TG adipocytes were treated with DMEM medium), TG + LLC‐CM (SIRT6 TG adipocytes were treated with LLC cell‐conditioned medium), KO + DMEM (SIRT6 KO adipocytes were treated with DMEM medium) and KO + LLC‐CM (SIRT6 KO adipocytes were treated with LLC cell‐conditioned medium). (E) MEFs isolated from KO and WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 12.5 μg/mL TNFR2 antagonist. Representative images of oil red O staining and the quantification of lipid content were shown (n = 4 per group). (F) Serum TNFα and TNFR2 concentrations of cachectic cancer patients (n = 12) and non‐cachectic cancer patients (n = 10) were compared. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: MDL800 reverses LLC‐induced adipocytes lipolysis. (A) Western blot analysis of SIRT6 and its substrates H3K9ac and H3K56ac in WT adipocytes with or without 20‐μM MDL‐800 treatment for 24 h (n = 3 per group). (B) MEFs isolated from WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 20‐μM MDL800. Representative images of oil red O staining and the quantification of lipid content were shown (n = 3 per group). (C) Glycerol levels in medium supernatant were measured (n = 6 per group). (D) The expression and phosphorylation levels of lipolysis associated proteins in the WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20‐μM MDL800 were determined by western blot (n = 4–6 per group). (E) cAMP levels in WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20 μM MDL800 adipocytes were measured (n = 3 per group). (F) Role of SIRT6 in cancer cachexia. TNFα, secreted from tumour cell, interacts with membrane TNFR2 and then induces the activation of lipolysis signalling pathway, mainly indicated by increased expression of ATGL and over‐phosphorylation of HSL and Perilipin 1. Without SIRT6, lipolysis is enhanced due to increased expression of TNFR2 and interaction of TNFα and TNFR2, which led to adipose wasting in tumour‐bearing mice. When SIRT6 is overexpressed or pharmaceutically activated, decreased expression of TNFR2 attenuates the activation of lipolysis signalling, which led to adipose homeostasis and then metabolic balance in mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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