Ji Yeon Kim^{a, 1}, Keon Jae Park^{a, d, 1}, Joo-Yeon Hwang^{b, c, 1}, Gyu Hee Kim^a, DaeYeon Lee^{a, e}, Yoo Jeong Lee^a, Eun Hyun Song^a, Min-Gyu Yoo^a,  Bong-Jo Kim^c, Young Ho Suh^f, Gu Seob Roh^g, Bin Gao^h, Won Kim^i,*, Won-Ho Kim<sup>a,*

a</sup> Division of Metabolic Disease, National Institute of Health, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk, 363-700, Korea
^b Division of Cardiovascular and Rare Disease, Center for Biomedical Science, National Institute of Health, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk, 363-700, Korea
^c Division of Structural and Functional Genomics, Center for Genomic Science, National Institute of Health, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk, 363-700, Korea
^d Department of Anatomy and Cardiology, Chungbuk University, Chungbuk, Korea
^e Department of Biotechnology, Korea University, Seoul, Korea
^f Department of Molecular Medicine, Seoul National University, Seoul, Korea
^g Department of Anatomy and Neurobiology, Gyeongsang National University, Jinju, Gyeongnam
^h Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA
ⁱ Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea

¹ JYK, KJP, and JYH contributed equally to this work.

^*Corresponding author. Division of Metabolic Disease, Center for Biomedical Science, National Institute of Health, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk, 363-700, Republic of Korea(W.-H. Kim), Division of Gastroenterology and Hepatology, Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center 20, Boramae-ro 5-gil, Dongjak-gu, Seoul 156-707,
Republic of Korea(W. Kim).

Abstract
Background & Aims
Nonalcoholic fatty liver disease (NAFLD) contributes to impaired glucose tolerance, leading to type 2 diabetes (T2D); however, the precise mechanisms and target molecules that are involved remain unclear. Activating transcription factor 3 (ATF3) is associated with β-cell dysfunction that is induced by severe stress signals in T2D. We aimed to explore the exact functional role of ATF3 as a mechanistic link between hepatic steatosis and T2D development.

Methods
Zucker diabetic fatty (ZDF) rats were utilized for animal experiments. An in vivo-jetPEI siRNA delivery system against ATF3 was used for loss-of-function experiments. We analyzed the baseline cross-sectional data derived from the biopsy-proven NAFLD registry (n=322). Human sera and liver tissues were obtained from 43 patients with biopsy-proven NAFLD and from seven healthy subjects.

Results
ATF3 was highly expressed in the livers of ZDF rats and in human subjects with NAFLD and/or T2D. Insulin resistance and hepatic steatosis were associated with increased ATF3 expression and decreased fatty acid oxidation via mitochondrial dysfunction and were attenuated by in vivo ATF3 silencing. Knockdown of ATF3 also ameliorated glucose intolerance, impaired insulin action, and inflammatory responses in ZDF rats. In patients with NAFLD and/or T2D, a significant positive correlation was observed between hepatic ATF3 expression and surrogate markers of T2D, mitochondrial dysfunction, and macrophage infiltration.

Conclusions
Increased hepatic ATF3 expression is closely associated with hepatic steatosis and incident T2D; therefore, ATF3 may serve as a potential therapeutic target for NAFLD and hepatic steatosis-induced T2D.

Lay summary
Hepatic activating transcription factor 3 (ATF3) may play an important role in oxidative stress-mediated hepatic steatosis and the development of type 2 diabetes (T2D) in a Zucker diabetic fatty (ZDF) rat model and in human patients with nonalcoholic fatty liver disease (NAFLD). Therefore, ATF3 may be a useful biomarker for predicting the progression of NAFLD and the development of T2D. Furthermore, given the significant association between hepatic ATF3 expression and both hepatic steatosis and impaired glucose homeostasis, in vivo ATF3 silencing may be a potential central strategy for preventing and managing NAFLD and T2D.