Sirtuin 1 regulates SREBP-1c expression in a LXR-dependent manner in skeletal muscle

Abstract

Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has emerged as a main determinant of whole body homeostasis in mammals by regulating a large spectrum of transcriptional regulators in metabolically relevant tissue such as liver, adipose tissue and skeletal muscle. Sterol regulatory element binding protein (SREBP)-1c is a transcription factor that controls the expression of genes related to fatty acid and triglyceride synthesis in tissues with high lipid synthesis rates such as adipose tissue and liver. Previous studies indicate that SIRT1 can regulate the expression and function of SREBP-1c in liver. In the present study, we determined whether SIRT1 regulates SREBP-1c expression in skeletal muscle. SREBP-1c mRNA and protein levels were decreased in the gastrocnemius muscle of mice harboring deletion of the catalytic domain of SIRT1 (SIRT1(Δex4/Δex4) mice). By contrast, adenoviral expression of SIRT1 in human myotubes increased SREBP-1c mRNA and protein levels. Importantly, SREBP-1c promoter transactivation, which was significantly increased in response to SIRT1 overexpression by gene electrotransfer in skeletal muscle, was completely abolished when liver X receptor (LXR) response elements were deleted. Finally, our in vivo data from SIRT1(Δex4/Δex4) mice and in vitro data from human myotubes overexpressing SIRT1 show that SIRT1 regulates LXR acetylation in skeletal muscle cells. This suggests a possible mechanism by which the regulation of SREBP-1c gene expression by SIRT1 may require the deacetylation of LXR transcription factors.

Figure 1. Sterol regulatory element binding protein (SREBP)-1c expression.

(A) SREBP-1c mRNA level in the gastrocnemius muscle of SIRT1^+/Δex4 and SIRT1^Δex4/Δex4 mice. (B) SREBP-1c protein level in the gastrocnemius muscle of SIRT1^+/Δex4 and SIRT1^Δex4/Δex4 mice. (C) SREBP-1c mRNA level in human myotubes (7 days of differentiation) infected for 48 hours with recombinant adenovirus expressing either green fluorescent protein (AdGFP) or sirtuin 1 (AdSIRT1). (D) SREBP-1c protein level in human myotubes (7 days of differentiation) infected for 48 hours with AdGFP or AdSIRT1. For western blot analyses, equal protein loading was controlled by measuring total protein content and α-tubulin expression by western blot. Data are expressed as means ± SE (n = 4/group for animal study; n = 5/group for in vitro study). ** P<0.01 and * P<0.05: significantly different from the corresponding control condition.

Figure 2. Liver X receptor (LXR)-α and LXR-β expression.

(A) LXR-α and LXR-β mRNA level in the gastrocnemius muscle of SIRT1^+/Δex4 and SIRT1^Δex4/Δex4 mice. (B) LXR-α and LXR-β protein level in the gastrocnemius muscle of SIRT1^+/Δex4 and SIRT1^Δex4/Δex4 mice. (C) LXR-α and LXR-β mRNA level in human myotubes (7 days of differentiation) infected for 48 hours with recombinant adenovirus expressing either green fluorescent protein (AdGFP) or sirtuin 1 (AdSIRT1). For western blot analysis, equal protein loading was controlled by measuring total protein content and α-tubulin expression by western blot. Data are expressed as means ± SE (n = 4/group for animal study; n = 8/group for in vitro study). * P<0.05: significantly different from SIRT1^+/Δex4 mice.

Figure 3. Sirtuin 1 (SIRT1) regulates liver X receptor (LXR)-α and LXR-β acetylation.

(A) Immunoblot representing the acetylation profile of gastrocnemius muscle lysates of SIRT1^+/Δex4 and SIRT1^Δex4/Δex4 mice. Expected LXR band is indicated by a grey arrow. (B) Immunoblots showing LXR-α immunoprecipitates probed either with an acetyl-Lysine antibody (upper left) or a LXR-α antibody (lower left) and LXR-β immunoprecipitates probed either with an acetyl-Lysine antibody (upper right) or a LXR-β antibody (lower right). (C) Acetylation level of LXR-α and LXR-β normalized to the amount of LXR-α and LXR-β in the immunoprecipitate. (D) Acetylation level of LXR-β in human myotubes (7 days of differentiation) infected for 48 hours with recombinant adenovirus expressing either green fluorescent protein (AdGFP) or SIRT1 (AdSIRT1). Proteins were immunoprecipitated with a LXR-β antibody and then probed with an acetyl-lysine antibody. Data were normalized to the amount of LXR-β in the immunoprecipitate. Data are expressed as means ± SE (n = 4/group for animal study; n = 8/group for in vitro study). * P<0.05: significantly different from the corresponding control condition.

Figure 4. Sirtuin 1 (SIRT1) regulates sterol regulatory element binding protein (SREBP)-1c promoter transactivation.

(A) Human SREBP-1c promoter (−571/+90 bp) reporter construct (pSREBP-1c-Luc) and human SREBP-1c promoter reporter construct with two deleted mutations in LXR-response elements located at −311/−296 bp and −260/−245 bp (pSREBP-1c-Luc m1/2) . (B) SIRT1 protein level in mouse tibialis anterior muscle 7 days after electrotransfer of a control vector (pcDNA3.1-Zeo) or a SIRT1 expression vector (pBabe-SIRT1). Equal protein loading was controlled by measuring total protein content and α-tubulin expression by western blot. Data are means ± SE (n = 8/group). ** P<0.01: significantly different from pcDNA3.1-Zeo. (C) pSREBP-1c-Luc and pSREBP-1c-Luc m1/2 transactivation in absence and presence of a LXR-α expression vector (pCMV-LXR-α). Tibialis anterior muscles were removed 7 days after gene electrotransfer. Luciferase activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. Data are means ± SE (n = 8/group). ** P<0.01 and *** P<0.001: significantly different from pSREBP-1c-Luc alone. ††† P<0.001: significantly different from pSREBP-1c-Luc with pCMV-LXR-α. (D) pSREBP-1c-Luc and pSREBP-1c-Luc m1/2 transactivation in absence and presence of pBabe-SIRT1. Tibialis anterior muscles were removed 7 days after gene electrotransfer. Luciferase activity was normalized to β-galactosidase activity to correct for variations in transfection efficiency. Data are means ± SE (n = 8/group). * P<0.05 and ** P<0.01: significantly different from pSREBP-1c-Luc alone. ††† P<0.001: significantly different from pSREBP-1c-Luc with pBabeSIRT1.