SREBP-1c Ablation Protects Against ER Stress-induced Hepatic Steatosis by Preventing Impaired Fatty Acid Oxidation

Young-Seung Lee(이영승),Timothy F. Osborne(티모씨 에프 오스본),Young-Kyo Seo(서영교),Tae-Il Jeon(전태일) 한국생명과학회 2021 생명과학회지 Vol.31 No.9

간 소포체(ER) 스트레스는 비알콜성지방간과 인슐린 저항성의 발달에 기여하고, unfolded protein response(UPR)의 구성요소는 지질 대사를 조절한다. 최근 연구에 따르면 ER 스트레스와 비정상적인 세포 지질 대사 사이의 연관성이 보고되었으며, 이 과정에서 지질 대사의 중심 조절자인 sterol regulatory element binding proteins(SREBPs)의 관련성이 확인되었다. 그러나 ER 스트레스 동안 지질 대사를 조절하는 SREBP의 정확한 역할과 비알콜성지방간에 대한 기여는 아직 밝혀지지 않았다. 본 연구에서 SREBP-1c 결핍은 UPR, 염증 및 지방산 산화 조절을 통해 ER 스트레스에 의해 유도된 비알콜성지방간으로부터 생쥐를 보호한다는 것을 보여준다. SREBP-1c는 inositol requiring kinase 1α (IRE1α) 발현을 직접적으로 조절하고 ER 스트레스에 의해 유도된 tumor necrosis factor-α의 활성화를 매개하여 peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α)의 감소와 그에 따른 지방산 산화의 장애를 유발한다. 그러나, SREBP-1c의 유전적 결핍은 이러한 현상을 보호하여 간 염증과 지방축적을 완화시킨다. SREBP-1c 결핍이 ER 스트레스에 의해 유도된 염증 신호를 방지하는 메커니즘은 아직 밝혀지지 않았지만, SREBP-1c가 결핍된 Kupffer 세포에서 IRE1α 신호의 변화가 염증 신호에 관여할 수 있을 것으로 생각된다. 본 연구결과는 SREBP-1c가 ER 스트레스에 의해 유도된 비알콜성지방간에서 UPR 및 염증의 조절에 중요한 역할을 함을 시사한다. Hepatic endoplasmic reticulum (ER) stress contributes to the development of steatosis and insulin resistance. The components of unfolded protein response (UPR) regulate lipid metabolism. Recent studies have reported an association between ER stress and aberrant cellular lipid control; moreover, research has confirmed the involvement of sterol regulatory element-binding proteins (SREBPs)-the central regulators of lipid metabolism-in the process. However, the exact role of SREBPs in controlling lipid metabolism during ER stress and its contribution to fatty liver disease remain unknown. Here, we show that SREBP-1c deficiency protects against ER stress-induced hepatic steatosis in mice by regulating UPR, inflammation, and fatty acid oxidation. SREBP-1c directly regulated inositol-requiring kinase 1α (IRE1α) expression and mediated ER stress-induced tumor necrosis factor-α activation, leading to a reduction in expression of peroxisome proliferator-activated receptor γ coactivator 1-α and subsequent impairment of fatty acid oxidation. However, the genetic ablation of SREBP-1c prevented these events, alleviating hepatic inflammation and steatosis. Although the mechanism by which SREBP-1c deficiency prevents ER stress-induced inflammatory signaling remains to be elucidated, alteration of the IRE1α signal in SREBP-1c-depleted Kupffer cells might be involved in the signaling. Overall, the results suggest that SREBP-1c plays a crucial role in the regulation of UPR and inflammation in ER stress-induced hepatic steatosis.

Estrogen-related receptor γ controls sterol regulatory element-binding protein-1c expression and alcoholic fatty liver

Kim, Don-Kyu,Kim, Yong-Hoon,Lee, Jae-Ho,Jung, Yoon Seok,Kim, Jina,Feng, Rilu,Jeon, Tae-Il,Lee, In-Kyu,Cho, Sung Jin,Im, Seung-Soon,Dooley, Steven,Osborne, Timothy F.,Lee, Chul-Ho,Choi, Hueng-Sik Elsevier 2019 Biochimica et biophysica acta, Molecular and cell Vol.1864 No.12

<P><B>Abstract</B></P> <P>Although SREBP-1c regulates key enzymes required for hepatic <I>de novo</I> lipogenesis, the mechanisms underlying transcriptional regulation of SREBP-1c in pathogenesis of alcoholic fatty liver is still incompletely understood. In this study, we investigated the role of ERRγ in alcohol-mediated hepatic lipogenesis and examined the possibility to ameliorate alcoholic fatty liver through its inverse agonist. Hepatic ERRγ and SREBP-1c expression was increased by alcohol-mediated activation of CB<SUB>1</SUB> receptor signaling. Deletion and mutation analyses of the <I>Srebp-1c</I> gene promoter showed that ERRγ directly regulates <I>Srebp-1c</I> gene transcription <I>via</I> binding to an ERR-response element. Overexpression of ERRγ significantly induced SREBP-1c expression and fat accumulation in liver of mice, which were blocked in <I>Srebp-1c</I>-knockout hepatocytes. Conversely, liver-specific ablation of <I>ERRγ</I> gene expression attenuated alcohol-mediated induction of SREBP-1c expression. Finally, an ERRγ inverse agonist, GSK5182, significantly ameliorates fatty liver disease in chronically alcohol-fed mice through inhibition of SREBP-1c-mediated fat accumulation. ERRγ mediates alcohol-induced hepatic lipogenesis by upregulating SREBP-1c expression, which can be blunted by the inverse agonist for ERRγ, which may be an attractive therapeutic strategy for the treatment of alcoholic fatty liver disease in human.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ERRγ is induced by alcohol-mediated activation of CB<SUB>1</SUB> receptor signaling. </LI> <LI> ERRγ increases hepatic SREBP-1c expression and alcohol-mediated hepatic lipogenesis </LI> <LI> An ERRγ inverse agonist inhibits SREBP-1c-induced hepatic <I>de novo</I> lipogenesis. </LI> <LI> An ERRγ inverse agonist ameliorates alcohol fatty liver disease. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

알파-리포산이 HepG2 간세포에서 SREBP-1c 발현에 미치는 효과

윤태승,민애경,김남경,김미경,조호찬,김혜순,황재석,류성열,박근규,이인규 대한내분비학회 2008 Endocrinology and metabolism Vol.23 No.1

Background: Non-alcoholic fatty liver disease is common in patients with insulin resistance. Sterol regulatory element binding protein-1c (SREBP-1c) is a member of a family of transcription factors that have been recognized as key regulators for lipid accumulation in the liver that activate enzymes involved in the fatty acid biosynthetic pathway. This study was designed to evaluate whether alpha-lipoic acid (ALA) inhibits insulin-stimulated SREBP-1c expression.Methods: We investigated the effects of ALA on insulin-stimulated SREBP-1c expression in a human hepatoma cell line (HepG2 cells) using Northern and Western blot analysis. We also examined the effect of ALA on the promoter activity of the SREBP-1c gene to examine whether ALA can affect SREBP-1c expression at the transcriptional level. To discern the mechanism by which ALA inhibits SREBP-1c expression, we examined the role of AMP-activated protein kinase (AMPK). Results: Insulin increased the expression of SREBP-1c mRNA and protein in HepG2 cells in a dose depended manner. Co-treatment with ALA inhibited the insulin increased SREBP-1c expression in a dose-dependent manner. ALA also inhibited insulin-stimulated activation of the SREBP-1c promoter activity, indicating that ALA inhibited SREBP-1c expression at the transcriptional level. ALA increased phosphorylation of AMPK in HepG2 cells. Inhibition of the AMPK activity by compound C markedly reversed the inhibitory effects of ALA for insulin-stimulated SREBP-1c expression. These results suggest that ALA-induced suppression of SREBP-1c expression is at least in part mediated via AMPK activation.Conclusion: The present study suggests that ALA has an inhibitory effect on insulin-stimulated SREBP-1c expression. Therefore, further studies on the effects of ALA on hepatic steatosis in an animal model need to be performed. (J Kor Endocr Soc 23:27~34, 2008)

Cilostazol inhibits insulin-stimulated expression of sterol regulatory binding protein-1c via inhibition of LXR and Sp1

Jung, Yun-A,Kim, Hee Kyoung,Bae, Kwi-Hyun,Seo, Hye-Young,Kim, Hye-Soon,Jang, Byoung Kuk,Jung, Gwon-Soo,Lee, In-Kyu,Kim, Mi-Kyung,Park, Keun-Gyu Nature Publishing Group 2014 Experimental and molecular medicine Vol.46 No.1

<P>Hepatic steatosis is common in obese individuals with hyperinsulinemia and is an important hepatic manifestation of metabolic syndrome. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces transcription of SREBP-1c via activation of liver X receptor (LXR) and specificity protein 1 (Sp1). Cilostazol is an antiplatelet agent that prevents atherosclerosis and decreases serum triglyceride levels. However, little is known about the effects of cilostazol on hepatic lipogenesis. Here, we examined the role of cilostazol in the regulation of SREBP-1c transcription in the liver. The effects of cilostazol on the expression of SREBP-1c and its target genes in response to insulin or an LXR agonist (T0901317) were examined using real-time RT-PCR and western blot analysis on cultured hepatocytes. To investigate the effect of cilostazol on SREBP-1c at the transcriptional level, transient transfection reporter assays and electrophoretic mobility shift assays (EMSAs) were performed. Cilostazol inhibited insulin-induced and LXR-agonist-induced expression of SREBP-1c and its downstream targets, acetyl-CoA carboxylase and fatty acid synthase, in cultured hepatocytes. Cilostazol also inhibited activation of the SREBP-1c promoter by insulin, T0901317 and Sp1 in a luciferase reporter assay. EMSA analysis showed that cilostazol inhibits SREBP-1c expression by repressing the binding of LXR and Sp1 to the promoter region. These results indicate that cilostazol inhibits insulin-induced hepatic SREBP-1c expression via the inhibition of LXR and Sp1 activity and that cilostazol is a negative regulator of hepatic lipogenesis.</P>

Cilostazol inhibits insulin-stimulated expression of sterol regulatory binding protein-1c via inhibition of LXR and Sp1

정윤아,김희경,배기현,서혜영,김혜순,장병국,정권수,이인규,박근규,김미경 생화학분자생물학회 2014 Experimental and molecular medicine Vol.46 No.-

Hepatic steatosis is common in obese individuals with hyperinsulinemia and is an important hepatic manifestation of metabolicsyndrome. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces transcription of SREBP-1c via activation of liver X receptor (LXR) and specificity protein 1 (Sp1). Cilostazol is an antiplatelet agent that prevents atherosclerosis and decreases serum triglyceride levels. However, little is knownabout the effects of cilostazol on hepatic lipogenesis. Here, we examined the role of cilostazol in the regulation of SREBP-1ctranscription in the liver. The effects of cilostazol on the expression of SREBP-1c and its target genes in response to insulin oran LXR agonist (T0901317) were examined using real-time RT-PCR and western blot analysis on cultured hepatocytes. Toinvestigate the effect of cilostazol on SREBP-1c at the transcriptional level, transient transfection reporter assays andelectrophoretic mobility shift assays (EMSAs) were performed. Cilostazol inhibited insulin-induced and LXR-agonist-inducedexpression of SREBP-1c and its downstream targets, acetyl-CoA carboxylase and fatty acid synthase, in cultured hepatocytes. Cilostazol also inhibited activation of the SREBP-1c promoter by insulin, T0901317 and Sp1 in a luciferase reporter assay. EMSA analysis showed that cilostazol inhibits SREBP-1c expression by repressing the binding of LXR and Sp1 to the promoterregion. These results indicate that cilostazol inhibits insulin-induced hepatic SREBP-1c expression via the inhibition of LXRand Sp1 activity and that cilostazol is a negative regulator of hepatic lipogenesis.

Protective Effects of Korean Red Ginseng against Alcohol-induced Hepatosteatosis

Sun Ju Kim(김선주),Sung Hwan Ki(기성환),Sangkyu Lee(이상규) 한국생명과학회 2015 생명과학회지 Vol.25 No.3

알코올에 의한 지방간(지방변성증)은 에탄올 대사에 의해 감소되는 당량의 과도한 발생에 의해 유발된다. 일반적으로 만성적인 에탄올 투여는 간 지질의 합성을 증가시키는 sterol regulatory element-binding protein 1c (SREBP-1c)를 조절함으로써 지방변성증을 유발시킨다. SPEBP-1c에서 에탄올의 영향은 간에서의 지방대사를 조절하는 NAD<SUP>+</SUP> 의존적 단백질 탈아세틸화효소인 mammalian sirtuin-1 (SIRT-1)에 의해 조정된다. 홍삼은 항당뇨와 항비만 효과를 위해 아시아에서 광범위하게 사용되는 한약재이다. 홍삼의 약리학적 치료학적인 효과는 진세노사이드와 같은 생물활성 성분에 의해 주로 일어난다. 따라서 우리는 마우스 간세포주인 AML-12 세포에서 SREBP-1과 SIRT-1에대한 한국홍삼 추출물의 조절효과를 평가하였다. 알코올과 홍삼추출물(0-1,000 μg/ml)을 AML-12 세포주에 처리하고, 지방소립을 Oil red O 염색법으로 확인하고, western blots을 사용해 SIRT-1과 SREBP-1의 발현을 확인하였다. 에탄올을 처리한 세포에서 홍삼추출물은 SIRT-1과 SREBP-1c의 발현을 회복시켰다. 또한 에탄올이 처리된 세포에서 홍삼추출물과 진세노사이드 Rb2와 Rd가 SREBP-1을 유의적으로 감소시키는 것으로 확인 되었다. 결과적으로 홍삼과 활성 진세노사이드 성분인 Rb2와 Rd가 SIRT-1과 간 지질대사를 변화시키는 SREBP-1c의 아세틸화의 조절을 통해 알코올에 의한 간지방변성을 억제하는 것을 확인하였다. Alcohol-induced fatty liver (steatosis) results from excessive generation of reducing equivalents by ethanol metabolism. Generally, chronic ethanol treatment causes hepatosteatosis by regulating sterol regulatory element-binding protein 1c (SREBP-1c), which increases the synthesis of hepatic lipids. The effect of ethanol on SREBP-1c is mediated through mammalian sirtuin-1 (SIRT-1), a NAD<SUP>+</SUP>-dependent protein deacetylase that regulates hepatic lipid metabolism. Ginseng is a widely used herbal medicine that is used in Asia for its anti-diabetes and anti-obesity effects. The pharmacological and therapeutic effects of ginseng are primarily produced by bioactive constituents known as ginsenosides. Here, we examined the regulatory effects of Korean red ginseng (KRG) extracts on SREBP-1c and SIRT-1 on lipid homeostasis in AML-12 mouse hepatocytes. AML-12 cells were treated with ethanol and/or KRG extracts (0 - 1,000 μg/ml). Lipid droplets were assayed using Oil red O staining, and western blotting was used to measure SIRT-1 and SREBP-1 expression. Treatment with KRG extracts restored SIRT-1 expression and reduced SREBP-1c expression in ethanol-treated cells. We also showed that KRG extract and ginsenosides Rb2 and Rd significantly decreased SREBP-1 acetylation in ethanol-treated cells. These results show that treatment with KRG extract and its active ginsenoside constituents Rb2 and Rd protected against alcohol-related hepatosteatosis via regulation of SIRT-1 and downstream acetylation of SREBP-1c, which altered hepatic lipid metabolism.

Identification of Ku70/Ku80 as ADD1/SREBP1c Interacting Proteins

Lee, Yun Sok,Koh, Hae-Young,Park, Sang Dai,Kim, Jae Bum The Korean Society for Integrative Biology 2004 Korean journal of biological sciences Vol.8 No.1

In vertebrates, multisubunit cofactors regulate gene expression through interacting with cell-type- and gene-specific DNA-binding proteins in a chromatin-selective manner. ADD1/SREBP1c regulates fatty acid metabolism and insulin-dependent gene expression through binding to SRE and E-box motif with dual DNA binding specificity. Although its transcriptional and post-translational regulation has been extensively studied, its regulation by interacting proteins is not well understood. To identify cellular proteins that associate with nuclear form of ADD1/SEBP1c, we employed the GST pull-down system with Hela cell nuclei extract. In this study, we demonstrated that Ku proteins interact specifically with ADD1/SREP1c protein. GST pull-down combined with peptide sequencing analysis revealed that Ku80 binds to ADD1/SREBP1c in vitro. Additionally, western blot analysis showed that Ku70, a heterodimerizing partner of Ku80, also associates with ADD1/SREBP1c. Furthermore, co-transfection of Ku70/Ku80 with ADD1/SREBP1c enhanced the transcriptional activity of ADD1/SREBP1c. Taken together, these results suggest that the Ku proteins might be involved in the lipogenic and/or adipogenic gene expression through interacting with ADD1/SREBP1c.

Deoxypodophyllotoxin in <i>Anthriscus sylvestris</i> alleviates fat accumulation in the liver via AMP-activated protein kinase, impeding SREBP-1c signal

Kim, Kwang-Youn,Park, Kwang-Il,Lee, Seul Gi,Baek, Su Youn,Lee, Eun Hye,Kim, Sang Chan,Kim, Sang-Hun,Park, Sul-Gi,Yu, Sun-Nyoung,Oh, Tae Woo,Kim, Joung-Hee,Kim, Keuk-Jun,Ahn, Soon-Cheol,Kim, Young Woo Elsevier 2018 Chemico-biological interactions Vol.294 No.-

<P><B>Abstract</B></P> <P>Deoxypodophyllotoxin (DPT) is a naturally occurring flavolignan in <I>Anthriscus sylvestris</I> known as cow parsley or wild chervil, and has been reported to have inhibitory effects against several pathological processes including cancer, inflammation and infection. Here, we report the effects of DPT in the fatty liver induced by high fat diet <I>in vivo</I> as well as its regulatory mechanism related with the transcription factor for lipogenic genes such as sterol regulatory element binding protein-1c (SREBP-1c) <I>in vitro</I>. C57BL/6 mice were fed high fat diet for 10 weeks and also orally administrated with DPT for additional 4 weeks. 5 and 10 mg/kg of DPT decreased lipid accumulation in the liver induced by high fat diet, as indicated by histological parameters such as Oil Red O staining and hematoxylin & eosin as well as the contents of hepatic triglyceride and cholesterol. In hepatocytes, DPT inhibited the liver X receptor α-mediated SREBP-1c induction and expression of the lipogenic genes, including fatty acid synthase, acetyl-CoA carboxylase and stearoyl-CoA desaturase-1. Moreover, DPT induced AMP-activated protein kinase (AMPK) activation, which has been known to inhibit the expression of SREBP-1c in hepatocyte. Also this compound restored the dysregulation of AMPK and SREBP-1c induced by high fat diet in mice. In conclusion, we demonstrated that DPT significantly inhibited fatty liver by adjusting lipid metabolism coordinated with AMPK activation and SREBP-1c inhibition.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Deoxypodophyllotoxin (DPT), a chemopreventive flavolignan, inhibited diet-induced fatty liver. </LI> <LI> DPT inhibited the LXR-α-mediated sterol regulatory element binding protein (SREBP)-1c. </LI> <LI> DPT blocked the expression of <I>de novo</I> lipogenic genes including FAS and ACC. </LI> <LI> DPT activated AMPK related with SREBP-1c inhibition. </LI> <LI> DPT might be a pharmaceutical candidate for hepatic steatosis. </LI> </UL> </P>

Clenbuterol Inhibits SREBP-1c Expression by Activating CREB1

Zhou, Lei,Li, Yixing,Nie, Tao,Feng, Shengqiu,Yuan, Jihong,Chen, Huaping,Yang, Zaiqing Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.4

As a $\beta_2$-adrenergic agonist, clenbuterol decreases body fat, but the molecular mechanism underlying this process is unclear. In the present study, we treated 293T and L-02 cells with clenbuterol and found that clenbuterol downregulates SREBP-1c expression and upregulates CREB1 expression. Considering SREBP-1c has the function of regulating the transcription of several lipogenic enzymes, we considered that the downregulation of SREBP-1c is responsible for body fat reduction by clenbuterol. Many previous studies have found that clenbuterol markedly increases intracellular cAMP levels, therefore, we also investigated whether CREB1 is involved in this process. The data from our experiments indicate that CREB1 overexpression inhibits SREBP-1c transcription, and that this action is antagonized by CREB2, a competitive inhibitor of CREB1. Furthermore, since PPARs are able to repress SREBP-1c transcription, we investigated whether clenbuterol and CREB1 function via a pathway involving PPAR activation. However, our results showed that clenbuterol or CREB1 overexpression suppressed PPARs transcription in 293T and L-02 cells, which suggested that they impair SREBP-1c expression in other ways.

Glucose and Insulin Stimulate Lipogenesis in Porcine Adipocytes: Dissimilar and Identical Regulation Pathway for Key Transcription Factors

Zhang, Guo Hua,Lu, Jian Xiong,Chen, Yan,Dai, Hong Wei,ZhaXi, YingPai,Zhao, Yong Qing,Qiao, Zi Lin,Feng, Ruo Fei,Wang, Ya Ling,Ma, Zhong Ren Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.11

Lipogenesis is under the concerted action of ChREBP, SREBP-1c and other transcription factors in response to glucose and insulin. The isolated porcine preadipocytes were differentiated into mature adipocytes to investigate the roles and interrelation of these transcription factors in the context of glucose- and insulin-induced lipogenesis in pigs. In ChREBP-silenced adipocytes, glucose-induced lipogenesis decreased by ~70%, however insulin-induced lipogenesis was unaffected. Moreover, insulin had no effect on ChREBP expression of unperturbed adipocytes irrespective of glucose concentration, suggesting ChREBP mediate glucose-induced lipogenesis. Insulin stimulated SREBP-1c expression and when SREBP-1c activation was blocked, and the insulin-induced lipogenesis decreased by ~55%, suggesting SREBP-1c is a key transcription factor mediating insulin-induced lipogenesis. $LXR{\alpha}$ activation promoted lipogenesis and lipogenic genes expression. In ChREBP-silenced or SREBP-1c activation blocked adipocytes, $LXR{\alpha}$ activation facilitated lipogenesis and SREBP-1c expression, but had no effect on ChREBP expression. Therefore, $LXR{\alpha}$ might mediate lipogenesis via SREBP-1c rather than ChREBP. When ChREBP expression was silenced and SREBP-1c activation blocked simultaneously, glucose and insulin were still able to stimulated lipogenesis and lipogenic genes expression, and $LXR{\alpha}$ activation enhanced these effects, suggesting $LXR{\alpha}$ mediated directly glucose- and insulin-induced lipogenesis. In summary, glucose and insulin stimulated lipogenesis through both dissimilar and identical regulation pathway in porcine adipocytes.