Mouse tissue extraction
Reagents:
- Protease inhibitor cocktail from Roche, #11697498001.
This method is used to make extracts from mouse tissues except adipose tissues. The extracts are for regular SDS-PAGE and WB only. To measure phosphorylation, you need phosphatase inhibitors.
1. Harvest mouse tissues and snap freeze in liquid nitrogen.
2. Add 1 ml TNET (Tris, 50 mM, NaCl, 150 mM, EDTA, 1 mM, Triton X-100, 1%, pH 8.0) with protease inhibitors and homogenize until clear. Keep on ice.
3. Spin down at 14K RPM for 15 min at 4C.
4. Transfer the supernatant for further experiments.
Mouse adipose tissue extraction
1. Harvest adipose tissues and snap freeze in liquid nitrogen.
2. Add 1 ml TNE (No Triton X-100) with protease inhibitor and homogenize until clear. Keep on ice.
3. Spin down at 5000 RPM for 5 min.
4. Carefully remove the fat cake and resuspend the loose pellet. Alternatively, use the pipette tip to penetrate the fat cake and transfer the solution and the pellet to a new tube.
5. Add 10% Triton X-100 to a final concentration of 1-2%.
6. Spin down at 14K RPM for 15 min at 4C.
7. Transfer the supernatant for further experiments.
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SIRT6 links glucose and lipid metabolism
SIRT1, a histone deacetylase, has drawn considerable attention due to its important roles in metabolic regulation and longevity. SIRT1 is induced by fasting and suppressed by feeding. SIRT1 increases glucose production and beta oxidation in liver to meet energy needs during food deprivation. Another member of SIRT family, SIRT6 follows similar expression patterns as SIRT1. The function of SIRT6 in metabolic regulation remains unknown.
Kim and colleagues found SIRT1 can regulate SIRT6 expression in liver. Specifically, SIRT1 functions together with FOXO3a and stimulates SIRT6 transcription during fasting. The activation of SIRT6 directly suppresses gene expression of metabolic enzymes involved in triglyceride synthesis and glycolysis in liver. Deficiency of SIRT6 causes increases of glucose utilization, reduced beta oxidation and as a result, fatty liver. More importantly, Kim et al found the expression of SIRT6 is decreased in human fatty liver samples which indicates SIRT6 may play critical roles in liver steatosis in clinical settings. This study landed in Cell Metabolism of this month.
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