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. Author manuscript; available in PMC: 2012 Apr 6.
Published in final edited form as: Cell Metab. 2011 Apr 6;13(4):376–388. doi: 10.1016/j.cmet.2011.03.009

Fig. 7. SREBP-1c is a direct target of AMPK.

Fig. 7

A. Active AMPK phosphorylates human SREBP-1c at Ser372 in vitro. Purified recombinant GST-tagged nuclear forms of SREBP-1c, wild type (WT), the mutations of S372A or S336A, from transfected HEK293T cells, were incubated with purified rat AMPK in the presence of [32P]-ATP and 100 μM of ATP and AMP at 30°C for 30 min. Phosphorylation of SREBP-1c was visualized by 32P-autoradiography or by immunoblots with phosphor-specific Ser372 antibody in the in vitro kinase assay. B. AMPK activation by phenformin specifically stimulates Ser372 phosphorylation of SREBP-1c. HEK293T cells expressing GST-tagged human full-length SREBP-1c, wild type (WT) or S372A mutant, were treated with phenformin (5 mM) for 1 h. Total cell lysates were Immunoblotted with phospho-specific Ser372 and total SREBP-1 antibodies. C. AMPK is required for Ser372 phosphorylation in response to polyphenols and AICAR. AMPK+/+ or AMPKα1/α2 double knockout (AMPK−/−) MEFs were treated with AMPK activators for 1 h. D. AMPK deficient cells exhibit the inability of AMPK activators to repress autoregulation of nuclear SREBP-1c. AMPK+/+ and AMPK−/− MEFs were co-transfected with the plasmids encoding nuclear SREBP-1c and FAS promoter and treated with AMPK activators for 16 h. E. Ser372 phosphorylation of SREBP-1c is required for the inhibition of cleavage of SREBP-1c in response to S17834 in HEK293T cells. F. The mutation of full-length SREBP-1c S372A enhances the basal transcription of SREBP-1c promoter (−257/+90) and abrogates the suppression of SREBP-1c gene transcription in response to AMPK activators in HepG2 cells. *P<0.05, vs untreated group; #P<0.05, vs treatment group. G. DN-AMPK diminishes polyphenol-induced phosphorylation of SREBP-1c in primary mouse hepatocytes under high glucose conditions. H. AMPK activation by S17834 counteracts impaired Ser372 phosphorylation of SREBP-1c precursor in the liver of insulin resistant LDLR−/− mice. I. Proposed model of the phosphorylation regulation of SREBP-1c and −2 by AMPK in the liver: potential therapeutic implication in hepatic steatosis, insulin resistance and risk of atherosclerosis.