Role of S6K1 in regulation of SREBP1c expression in the liver

doi:10.1016/j.bbrc.2011.07.038

Biochemical and Biophysical Research Communications

Volume 412, Issue 2, 26 August 2011, Pages 197-202

https://doi.org/10.1016/j.bbrc.2011.07.038 Get rights and content

Abstract

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays an important role in the control of fatty acid metabolism in the liver. Evidence suggests that mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contributes to the regulation of SREBP1c expression, but signaling downstream of mTORC1 remains unclear. We have now shown that medium rich in branched-chain amino acids stimulates expression of the SREBP1c gene in cultured hepatocytes in a manner sensitive both to rapamycin, a pharmacological inhibitor of mTORC1, and to a short hairpin RNA (shRNA) specific for S6 kinase 1 (S6K1), a downstream effector of mTORC1. The phosphorylation of S6K1 was increased in the liver of obese db/db mice. Furthermore, depletion of hepatic S6K1 in db/db mice with the use of an adenovirus vector encoding S6K1 shRNA resulted in down-regulation of SREBP1c gene expression in the liver as well as a reduced hepatic triglyceride content and serum triglyceride concentration. These results thus suggest that S6K1 regulates SREBP1c expression both in cultured hepatocytes and in mouse liver, and that increased hepatic activity of S6K1 contributes at least in part to the pathogenesis of obesity-induced hepatic steatosis and hypertriglyceridemia.

Highlights

► S6K1 regulates nutrient signal-induced expression of the SREBP1c in cultured hepatocytes. ► The phosphorylation of S6K1 was increased in the liver of obese db/db mice. ► Depletion of hepatic S6K1 in db/db mice decreased the expression of SREBP1c. ► Depletion of hepatic S6K1 in db/db mice ameliorated hepatic steatosis and hypertriglyceridemia.

Introduction

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays a key role in lipid metabolism by regulating the expression of various genes that contribute to fatty acid synthesis [1]. The observations that the abundance of SREBP1c in the liver is increased in animal models of obesity [2] and in humans with hepatic steatosis [3], [4] and that transgenic overexpression of SREBP1c in the liver triggers hypertriglyceridemia and hepatic steatosis in mice [5] suggest that this transcription factor contributes to the pathogenesis of such obesity-related disorders. The mechanism underlying the exaggerated expression of SREBP1c in the liver of obese animals has remained unclear, however [6]. Although insulin is an important physiological regulator of SREBP1c expression [7], [8], [9], it appears not to contribute to the up-regulation of SREBP1c in the liver associated with obesity, given that the liver of obese animals is resistant to insulin.

Mammalian target of rapamycin (mTOR) is a protein kinase that is implicated in a variety of biological processes [10]. It contributes to two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2, which transmit distinct signals in cells [10]. Signaling by mTORC1 is inhibited by the drug rapamycin, whereas the activity of mTORC2 is insensitive to this agent [10]. Rapamycin was recently shown to inhibit expression of the SREBP1c gene induced by a constitutively active form of the protein kinase Akt in retinal pigment epithelial cells [11]. Moreover, the expression of SREBP1c induced by serum in fibroblasts [12] as well as that induced by insulin in cultured hepatocytes [13] were subsequently shown to be sensitive to rapamycin. Although these results suggest that mTORC1 contributes to regulation of the SREBP1c gene, the mechanism by which it does so has remained unclear. S6 kinase 1 (S6K1) is a protein kinase that acts as a downstream effector of mTORC1 [14]. Regulation of SREBP1c expression has been reported to be independent of [13] or dependent on [12] S6K1, however. Moreover, the role of the mTORC1–S6K1 pathway in the regulation of SREBP1c expression in the liver of living animals has not been determined.

We have now investigated the role of S6K1 in the regulation of SREBP1c gene expression. We found that nutrient signaling induced by medium rich in branched-chain amino acids (BCAAs) stimulated the expression of SREBP1c in cultured hepatocytes in a manner dependent on mTORC1 and S6K1. Furthermore, ablation of S6K1 in the liver of obese mice reduced the hepatic expression of SREBP1c as well as ameliorated both hypertriglyceridemia and hepatic steatosis. Our results thus suggest that S6K1 plays an important role in the exaggerated expression of SREBP1c in the liver of obese animals.

Section snippets

Cell culture, mice, and adenoviral vectors

AML12 mouse hepatocytes [15] were obtained from American Type Culture Collection. For the induction of SREBP1c gene expression, AML12 cells that had been cultured in Dulbecco’s modified Eagle medium/Ham’s Nutrient Mixture F-12 (DMEM/F-12, Sigma) supplemented with 10% fetal bovine serum, Insulin–Transferrin–Selenium liquid media supplement (Sigma) and 100 nM dexamethasone were incubated with serum-free DMEM/F-12 for 16 h and then with phosphate-buffered saline (PBS) for 3 h to deprive them of

Effect of nutrient signaling on SREBP1c gene expression in cultured hepatocytes

Evidence suggests that mTORC1 participates in the regulation of SREBP1c gene expression [11], [12], [13]. Given that medium rich in BCAAs, such as DMEM, induces robust activation of mTORC1 signaling [20], we investigated whether such nutrient signaling might stimulate expression of the SREBP1c gene. AML12 mouse hepatocytes were deprived of serum as well as of nutrients by incubation with PBS and were then exposed to insulin or DMEM. Insulin induced an ∼2.2-fold increase in the amount of SREBP1c

Discussion

We have shown that nutrient signaling induced by medium rich in BCAAs (DMEM) stimulated expression of the SREBP1c gene in cultured hepatocytes. This effect was inhibited by rapamycin and by shRNA-mediated depletion of S6K1, implicating S6K1 as a mediator of DMEM-induced expression of the SREBP1c gene. Whereas mTORC1 has previously been suggested to contribute to the regulation of SREBP1c expression [11], [12], [13], the role of S6K1, a downstream effector of mTORC1, in such regulation has been

Acknowledgments

This study was supported by Grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to M.K (Grant-in-Aid for Creative Scientific Research) and to W.O. (Grant-in-Aid for Scientific Research on Innovative Areas), and Tianjin Municipal Science and Technology Commission (No. 11JCYBJC11200) to S.L.

References (30)

H. Shimano
Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes
Prog. Lipid Res.
(2001)
I. Shimomura et al.
Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice
Mol. Cell
(2000)
M.S. Brown et al.
Selective versus total insulin resistance: a pathogenic paradox
Cell Metab.
(2008)
S.G. Dann et al.
MTOR complex1–S6K1 signaling: at the crossroads of obesity, diabetes and cancer
Trends Mol. Med.
(2007)
T. Porstmann et al.
SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth
Cell Metab.
(2008)
K. Duvel et al.
Activation of a metabolic gene regulatory network downstream of mTOR complex 1
Mol. Cell
(2010)
T.R. Fenton et al.
Functions and regulation of the 70 kDa ribosomal S6 kinases
Int. J. Biochem. Cell Biol.
(2011)
K. Hara et al.
Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism
J. Biol. Chem.
(1998)
I. Gout et al.
Molecular cloning and characterization of a novel p70 S6 kinase, p70 S6 kinase beta containing a proline-rich region
J. Biol. Chem.
(1998)
B. Caballero et al.
Plasma amino acids and insulin levels in obesity: response to carbohydrate intake and tryptophan supplements
Metabolism
(1988)

N. Higuchi et al.

Liver X receptor in cooperation with SREBP-1c is a major lipid synthesis regulator in nonalcoholic fatty liver disease

Hepatol. Res.

(2008)

M. Kohjima et al.

Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease

Int. J. Mol. Med.

(2007)

H. Shimano et al.

Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells

J. Clin. Invest.

(1997)

I. Shimomura et al.

Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes

Proc. Natl. Acad. Sci. USA

(1999)

M. Matsumoto et al.

Role of the insulin receptor substrate 1 and phosphatidylinositol 3-kinase signaling pathway in insulin-induced expression of sterol regulatory element binding protein 1c and glucokinase genes in rat hepatocytes

Diabetes

(2002)

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