Advertisement

Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis

  • Author Footnotes
    † These authors contributed equally as joint first authors.
    Ji Yeon Kim
    Footnotes
    † These authors contributed equally as joint first authors.
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally as joint first authors.
    Keon Jae Park
    Footnotes
    † These authors contributed equally as joint first authors.
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea

    Department of Anatomy and Cardiology, Chungbuk University, Chungbuk, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally as joint first authors.
    Joo-Yeon Hwang
    Footnotes
    † These authors contributed equally as joint first authors.
    Affiliations
    Division of Cardiovascular and Rare Disease, Center for Biomedical Science, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea

    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, Republic of Korea
    Search for articles by this author
  • Gyu Hee Kim
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • DaeYeon Lee
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea

    Department of Biotechnology, Korea University, Seoul, Republic of Korea
    Search for articles by this author
  • Yoo Jeong Lee
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • Eun Hyun Song
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • Min-Gyu Yoo
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • Bong-Jo Kim
    Affiliations
    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, Republic of Korea
    Search for articles by this author
  • Young Ho Suh
    Affiliations
    Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
    Search for articles by this author
  • Gu Seob Roh
    Affiliations
    Department of Anatomy and Neurobiology, Gyeongsang National University, Jinju, Gyeongnam, Republic of Korea
    Search for articles by this author
  • Bin Gao
    Affiliations
    Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA
    Search for articles by this author
  • Won Kim
    Correspondence
    Corresponding authors. Addresses: 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); 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).
    Affiliations
    Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Republic of Korea
    Search for articles by this author
  • Won-Ho Kim
    Correspondence
    Corresponding authors. Addresses: 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); 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).
    Affiliations
    Division of Metabolic Disease, #187 Osong Saengmyeong2-ro, Osong-eup, Heungdeok-gu, Cheongju, Chungbuk 363-700, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally as joint first authors.
Open AccessPublished:March 29, 2017DOI:https://doi.org/10.1016/j.jhep.2017.03.023

      Background & Aims

      Non-alcoholic 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 participants.

      Results

      ATF3 was highly expressed in the livers of ZDF rats and in human participants 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 non-alcoholic 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.

      Graphical abstract

      Keywords

      Introduction

      Obesity is a major underlying risk factor for type 2 diabetes (T2D).
      • Finelli C.
      • Tarantino G.
      Is there any consensus as to what diet or lifestyle approach is the right one for NAFLD patients?.
      A common pathogenic event in both animals and humans with obesity and T2D is hepatic lipid accumulation, which is the earliest phenotype of non-alcoholic fatty liver disease (NAFLD).
      • Anstee Q.M.
      • Daly A.K.
      • Day C.P.
      Genetic modifiers of non-alcoholic fatty liver disease progression.
      NAFLD has reached epidemic levels worldwide because its incidence has gradually increased in the non-obese Asian population as well as the obese populations of the United States and Europe.
      • Bellentani S.
      • Saccoccio G.
      • Masutti F.
      • Crocè L.S.
      • Brandi G.
      • Sasso F.
      • et al.
      Prevalence of and risk factors for hepatic steatosis in Northern Italy.
      Non-alcoholic steatohepatitis (NASH) is the histologic form of NAFLD that is associated with increased morbidity and mortality, and it is closely linked to hepatic insulin resistance and abnormal glucose metabolism.
      • Samuel V.T.
      • Shulman G.I.
      Mechanisms for insulin resistance: common threads and missing links.
      However, the exact mechanism by which NAFLD induces T2D is poorly understood, and effective pharmacotherapies for NAFLD-induced T2D are unsatisfactory.
      Hepatic oxidative stress, including endoplasmic reticulum (ER) and mitochondrial stress, is closely associated with the progression from simple steatosis to NASH and with the development of insulin resistance and T2D.
      • Yamamoto S.
      • Matsushita Y.
      • Nakagawa T.
      • Honda T.
      • Hayashi T.
      • Noda M.
      • et al.
      Visceral fat accumulation, insulin resistance, and elevated depressive symptoms in middle-aged Japanese men.
      In the liver and adipose tissue of both diet-induced and ob/ob mice, lipid-induced ER and oxidative stress exacerbated hepatic steatosis via impaired hepatic lipid, glucose, and insulin metabolism.
      • Basseri S.
      • Austin R.C.
      Endoplasmic reticulum stress and lipid metabolism: mechanisms and therapeutic potential.
      • Bravo R.
      • Parra V.
      • Gatica D.
      • Rodriguez A.E.
      • Torrealba N.
      • Paredes F.
      • et al.
      Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration.
      Hepatic steatosis also contributes to the development of metabolic dysfunction associated with obesity, such as macrophage infiltration in adipose tissues, lipid accumulation and insulin resistance in skeletal muscle and liver, hyperglycemia, and hyperinsulinemia.
      • Apostolova N.
      • Blas-Garcia A.
      • Esplugues J.V.
      Mitochondria sentencing about cellular life and death: a matter of oxidative stress.
      • Brown G.T.
      • Kleiner D.E.
      Histopathology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      Moreover, several liver-derived endocrine factors that affect the peripheral metabolism have been identified
      • Meex R.C.
      • Hoy A.J.
      • Morris A.
      • Brown R.D.
      • Lo J.C.
      • Burke M.
      • et al.
      Fetuin B Is a Secreted Hepatocyte Factor Linking Steatosis to Impaired Glucose Metabolism.
      • Kharitonenkov A.
      • Shiyanova T.L.
      • Koester A.
      • Ford A.M.
      • Micanovic R.
      • Galbreath E.J.
      • et al.
      FGF-21 as a novel metabolic regulator.
      • Misu H.
      • Takamura T.
      • Takayama H.
      • Hayashi H.
      • Matsuzawa-Nagata N.
      • Kurita S.
      • et al.
      A liver-derived secretory protein, selenoprotein P, causes insulin resistance.
      as key hepatokines associated with insulin resistance and NAFLD. However, the specific target molecule underlying metabolic dysfunction and linking hepatic steatosis to T2D remains obscure.
      Stress-inducible activating transcription factor 3 (ATF3) can function as either a transcriptional activator or repressor.
      • Hai T.
      • Wolford C.C.
      • Chang Y.S.
      ATF3, a hub of the cellular adaptive-response network, in the pathogenesis of diseases: is modulation of inflammation a unifying component?.
      Although several studies using ATF3 transgenic or knockout (KO) mice have been performed,
      • Hartman M.G.
      • Lu D.
      • Kim M.L.
      • Kociba G.J.
      • Shukri T.
      • Buteau J.
      • et al.
      Role for activating transcription factor 3 in stress-induced beta-cell apoptosis.
      • Rynes J.
      • Donohoe C.D.
      • Frommolt P.
      • Brodesser S.
      • Jindra M.
      • Uhlirova M.
      Activating transcription factor 3 regulates immune and metabolic homeostasis.
      • Lee Y.S.
      • Sasaki T.
      • Kobayashi M.
      • Kikuchi O.
      • Kim H.J.
      • Yokota-Hashimoto H.
      • et al.
      Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.
      it is still unclear whether the physiological role of ATF3 is beneficial or detrimental in the development of metabolic dysfunction. ATF3 is induced by various stresses and promotes a compensatory or adaptive homeostatic response, alleviating cellular stress.
      • Gilchrist M.
      • Thorsson V.
      • Li B.
      • Rust A.G.
      • Korb M.
      • Roach J.C.
      • et al.
      Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4.
      However, recent studies have shown that ATF3 is also associated with β-cell dysfunction induced by severe stress signals related to T2D.
      • Zmuda E.J.
      • Qi L.
      • Zhu M.X.
      • Mirmira R.G.
      • Montminy M.R.
      • Hai T.
      The roles of ATF3, an adaptive-response gene, in HFD-induced diabetes and pancreatic β-cell dysfunction.
      • Kim J.Y.
      • Park K.J.
      • Kim G.H.
      • Jeong E.A.
      • Lee D.Y.
      • Lee S.S.
      • et al.
      In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes.
      Therefore, persistent ATF3 expression induced by excessive reactive oxygen species (ROS) or ER stress likely has detrimental effects, overwhelming its initial compensatory role. Nonetheless, the exact functional role of ATF3 as a target molecule responsible for oxidative stress-mediated hepatic steatosis and impaired glucose metabolism is largely unknown. Hence, the aim of this study was to investigate whether ATF3 has a beneficial or detrimental effect on hepatic steatosis and incident T2D in Zucker diabetic fatty (ZDF) rats and human participants with NAFLD.

      Patients and methods

      Human participants

      We prospectively enrolled this cross-sectional cohort derived from the ongoing NAFLD registry (NCT02206841; n = 322) of the Seoul Metropolitan Government Seoul National University Boramae Medical Center. The eligibility criteria and liver biopsy indications are presented in the Supplementary information and described elsewhere.
      • Koo B.K.
      • Kim D.
      • Joo S.K.
      • Kim J.H.
      • Chang M.S.
      • Kim B.G.
      • et al.
      Sarcopenia is an independent risk factor for non-alcoholic steatohepatitis and significant fibrosis.
      Informed consent was obtained from all participants under a protocol approved by the institutional review board (#20130320/16-2013-45/041). The study was conducted in accordance with the Declaration of Helsinki.

      Statistical analysis

      In human studies, data are expressed as a percentage, median (interquartile range) or mean ± standard deviation. Data are reported as the mean ± standard error of the mean in animal studies and in vitro experiments. Group comparisons were performed using an unpaired t test and one- or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc tests, where p <0.05 indicates statistical significance. In the human studies, continuous variables are described as the mean (95% confidence intervals [CI]). Categorical variables are shown as counts and percentages. Comparisons between groups were performed using Student’s t test or the Mann-Whitney U test and Kruskal-Wallis test when appropriate. Differences between categorical variables were assessed with the chi-squared test or Fisher’s exact test. Correlation analyses were performed using Spearman’s analysis. The SPSS statistical package (SPSS Inc., version 18.0, Chicago, IL, USA) was used for all analyses.
      Additional procedures and detailed methods are described in the Supplementary material and CTAT form.

      Results

      Hepatic steatosis is correlated with impaired insulin action and glucose intolerance

      To clarify the relationship between NAFLD and T2D, we used ZDF rats, which phenotypically mimic human participants with obesity and T2D. Impaired glucose tolerance (IGT) and insulin resistance observed during the progression of T2D in ZDF rats significantly exacerbated hepatic steatosis, as shown by the serum alanine aminotransferase (ALT), hepatic triglyceride (TG), and cholesterol levels as well as the liver/body weight ratios (Fig. S1A, B). In parallel, a reduction in the insulin action due to hepatic insulin resistance was also observed in the livers of ZDF rats (Fig. S1C, D). Insulin receptor substrate 1 (IRS-1) phosphorylation at inhibitory Ser-307 was increased in the livers of 6-week-old ZDF rats compared to those of Zucker lean rats and was enhanced in 19-week-old rats with hyperglycemia, hypoinsulinemia, and pancreatic β-cell dysfunction. In contrast, active IRS-1 (Tyr-941) phosphorylation was increased in 6-week-old ZDF rats and was significantly decreased in 19-week-old rats. This trend was consistent with the changes in phosphorylation of Akt, a downstream signaling molecule of IRS-1. Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, the rate-limiting enzymes associated with hepatic gluconeogenesis, were increased in the livers of 6-week-old rats and were significantly enhanced in 19-week-old rats.

      Hepatic expression of ATF3 is enhanced in ZDF rats and NAFLD participants

      Stress-inducible ATF3 levels were significantly increased in the livers of 6-week-old ZDF rats with mild steatosis and were further significantly increased in 19-week-old ZDF rats with severe steatosis and T2D (Fig. 1A). Hepatic expression of 4-hydroxy-2-nonenal (4-HNE), a marker of oxidative stress, showed a similar trend to that of ATF3 expression in the liver tissues of ZDF rats (Fig. 1B). In particular, ATF3 expression was positively correlated with hepatic TG accumulation and liver weight (Fig. S1E, F), suggesting an explicit role of ATF3 in the progression of hepatic steatosis. For all functional elements of ATF3, we conducted literature-based functional connectivity analyses using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database resource. ATF3-centered regulatory networks were significantly associated with NAFLD pathway-associated terms, including apoptosis-related gene networks in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps (Fig. 1C). Next, to confirm the biological significance of ATF3 in the progression of hepatic steatosis and the development of T2D in participants with NAFLD, we conducted gene expression profiling analyses using human liver samples from different phases in the accessible public domain (GSE48452). Hepatic ATF3 mRNA expression levels were significantly higher in participants with an NAFLD activity score (NAS) >5 compared to that of those without NAFLD (NAS = 0) or with mild NAFLD (NAS 1–5) (Fig. 1D). A lipogenesis-related gene, fatty acid synthase (FAS), which is a target molecule of sterol regulatory element binding protein 1c (SREBP-1c), was also significantly upregulated in NAFLD participants with a NAS >5. A strong positive correlation was observed between the FAS and ATF3 mRNA expression levels in the livers of morbidly obese participants with NAS >5. ATF3 expression in the livers of NAFLD participants was also positively correlated with JUN expression, which is related to ER and oxidative stress (Fig. 1E). Intriguingly, the serum adiponectin levels, a predictive marker of T2D, were significantly downregulated in participants with NAFLD compared to those of healthy individuals, and a negative correlation was observed between the circulating adiponectin levels and hepatic ATF3 mRNA expression (Fig. 1F).
      Figure thumbnail gr1
      Fig. 1ATF3 overexpression in the livers of ZDF rats and human participants with NAFLD. (A) Imaging and quantification of ATF3 expression in the livers of ZDF rats (n = 8). (B) Immunostaining analyses of ATF3 (ICC) and 4-HNE (IHC) in liver tissues of 19-week-old rats (100×; n = 8, each group). (C) An integrative functional significance of ATF3 in NAFLD-related gene network. Red and blue colors indicate the genes that were closely linked to ATF3, which are upregulated and downregulated, respectively, in human livers with NAFLD. (D) Hepatic expression of ATF3 (left) and FAS mRNA levels (middle) in participants with no NAFLD (NAS = 0), mild NAFLD (NAS 1–5), and morbidly obese NAFLD (NAS >5) from a public database (GSE48452) and their correlation analyses (right). (E and F) Correlation between ATF3 and JUN mRNA levels (E; p = 0.421 between NAS = 0 vs. NAS 1–5) or serum adiponectin levels (F; p = 0.513 between NAS = 0 vs. NAS 1–5). (A and B) All data are given as the mean ± SEM with *p <0.01 and **p <0.05 from the t test. (D, E, and F) Data are presented as box plots (median, IQR). The top and bottom of each box indicate the 1st and 3rd quartiles (interquartile range, IQR), and the band inside the box indicates the median. The dot indicates an outlier more than 1.5 times the IQR below the 25th percentile or more than 1.5 times the IQR above the 75th percentile of all data. p values in D–F from post hoc analysis for the ANOVA; p <0.001 form the ANOVA. ZL, Zucker lean; ZDF, Zucker diabetic fatty; ATF3, activating transcription factor 3; 4-HNE, 4-hydroxy-2-nonenal; ICC, immunocytochemistry; IHC, immunohistochemistry; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; FAS, fatty acid synthase; SEM, standard error of the mean; ANOVA, analysis of variance. (This figure appears in colour on the web.)

      In vivo ATF3 silencing ameliorates glucose intolerance and hepatic steatosis

      Next, we hypothesized that ATF3 may act as a potential target molecule for hepatic steatosis and IGT in the development of obese T2D. To test this hypothesis, we performed an in vivo loss-of-function study by intravenously administering ATF3 siRNA to 15-week-old rats (Fig. S2A–F). ATF3 silencing in ZDF rats significantly improved impaired glucose homeostasis, as assessed by an oral glucose tolerance test (Fig. 2A). ATF3 silencing also markedly reduced the ALT levels, intrahepatic TGs, and liver weights, which were significantly increased in ZDF rats (Fig. S2G). A significant positive correlation was observed between ATF3 protein expression and both the hepatic TG accumulation and liver weights in ZDF rats (Fig. 2B). Moreover, ATF3 knockdown significantly restored the reduction in signaling capacity of the insulin receptor, which was observed in ZDF rats (Fig. 2C). The hepatic expression levels of SREBP-1c, FAS, and stearoyl-coenzyme A (CoA) desaturase 1 (SCD-1) increased in ZDF rats, and these changes were reversed by ATF3 silencing (Fig. 2D), as confirmed by immunohistochemical analysis (Fig. 2E).
      Figure thumbnail gr2
      Fig. 2In vivo ATF3 silencing ameliorates impaired glucose metabolism and hepatic steatosis in ZDF rats. Either in vivo-jetPEI ATF3 siRNA (A3 si) or scrambled siRNA (Scr.) was intravenously administered to 15-week-old ZL or ZDF rats (n = 8, each group). (A) Oral glucose tolerance test (OGTT). (B) Correlation between ATF3 protein expression and liver TG levels or liver/body weight ratios. (C) Representative Western blots and densitometric quantification relative to IRS-1. (D) Hepatic expression of lipogenesis-related proteins. (E) Hematoxylin and eosin (H&E) staining and IHC analysis (100×) of SREBP-1c and FAS proteins and staining hepatocytes were scored. All data are given as the mean ± SEM with *p <0.01 and **p <0.05 from the t test. ZL, Zucker lean; ZDF, Zucker diabetic fatty; ATF3; activating transcription factor 3; TG, triglyceride; IRS-1, insulin receptor substrate 1; pY (941), active IRS-1 (Tyr-941) phosphorylation; pS (307), inhibitory IRS-1 (Ser-307) phosphorylation; IHC, immunohistochemistry; SREBP-1c, sterol regulatory element binding protein 1c; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1. (This figure appears in colour on the web.)

      ATF3 silencing ameliorates hepatic steatosis by restoring reduced fatty acid oxidation

      Next, we examined the role of ATF3 in fatty acid oxidation in the livers of ZDF rats. Following an increase in the lipogenic proteins, the hepatic protein levels of peroxisome proliferator-activated receptor-α (PPAR-α) and PPAR-γ coactivator 1α (PGC-1α) were significantly decreased in ZDF rats, and they were restored by ATF3 knockdown (Fig. 3A, Fig. S2H). Consistent with these findings, the hepatic expression and enzymatic activity of carnitine palmitoyltransferase 1 (CPT-1) were suppressed in ZDF rats, which were reversed by ATF3 depletion (Fig. 3A, B). Conversely, the PPAR-γ and fat-specific protein 27 (FSP27) expression levels were increased in ZDF rats and were markedly suppressed by ATF3 silencing. The mitochondrial and peroxisomal fatty acid oxidation rates, including total oxidation, decreased in ZDF rats; however, this was also restored by ATF3 silencing (Fig. 3C, Fig. S2I). ATF3 induction in ZDF rats was negatively correlated with the rate of β-oxidation (Fig. 3D).
      Figure thumbnail gr3
      Fig. 3ATF3 silencing counteracts impaired fatty acid oxidation and metabolic rates in ZDF rats. (A) Hepatic expression of proteins related to fatty acid oxidation and lipid accumulation. (B) CPT-1 activity in the mitochondria of primary hepatocytes isolated from rats. (C) Total liver and mitochondrial β-oxidation rates. (D) Correlation between the ATF3 protein expression and β-oxidation rates. (E) Real-time PCR analyses for hepatic mRNA expression of fatty acid uptake (left), synthesis/lipogenesis (middle), and oxidation (right) markers. (F) The lactate/pyruvate ratio in the livers of ZDF rats and the NADH/NAD+ ratio were determined. All data are given as the mean ± SEM with *p <0.01 and **p <0.05 from the t test (n = 8, each group). ZL, Zucker lean; ZDF, Zucker diabetic fatty; PGC-1α, PPAR-γ coactivator 1α; PPAR-α, peroxisome proliferator-activated receptor α; CPT-1, carnitine palmitoyltransferase 1; PPAR-γ, peroxisome proliferator-activated receptor γ; FSP27, fat-specific protein 27; CD36, fatty acid translocases; FATP2, fatty acyl transport protein 2; FABP1, fatty acid binding protein 1; ACSL1, acyl-CoA synthetase long-chain family member 1; ACC, acetyl CoA carboxylase; FAS, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1; DGAT1, diglyceride acyltransferase 1; SREBP1, sterol regulatory element binding protein 1; SPT1, serine palmitoyltransferase 1; ACOX1, acyl-coenzyme A oxidase 1; LCAD, long-chain fatty acyl-CoA dehydrogenase; SEM, standard error of the mean.

      Hepatic lipogenesis and metabolic dysfunction depend on ATF3 induction

      Next, the effects of ATF3 silencing on the expression of hepatic genes encoding rate-limiting enzymes in lipid homeostasis were examined (Fig. 3E). The mRNA levels of fatty acid translocases (CD36), fatty acyl transport protein 2 (FATP2), fatty acid binding protein 1 (FABP1), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in fatty acid uptake or intracellular fatty acid binding, were consistently elevated in ZDF rats. Among these, the hepatic expression levels of CD36, FATP2, and ACSL1, but not FABP1, were markedly attenuated by ATF3 silencing. Moreover, ATF3 knockdown significantly inhibited the expression of hepatic genes related to fatty acid synthesis (ACC, FAS, and SCD-1) and lipogenesis (DGAT1 and SREBP-1c), which were upregulated in ZDF rats. Among the genes related to fatty acid oxidation, SPT-1 was upregulated, but mitochondrial CPT-1, peroxisomal acyl-coenzyme A oxidase 1 (ACOX1), and long-chain fatty acyl-CoA dehydrogenase (LCAD) were downregulated in ZDF rats. These effects were offset by ATF3 siRNA. We also demonstrated that hepatic steatosis in ZDF rats was associated with a reduction in the metabolic rate. The increased lactate/pyruvate ratio in ZDF rats, which is considered an alteration in cellular redox homeostasis, was strongly counterbalanced by ATF3 silencing (Fig. 3F left, Fig. S3A). Accordingly, the ratio of NADH/NAD+, which represents the hepatic levels of lactate and pyruvate, increased approximately twofold in ZDF rats and was diminished by ATF3 siRNA (Fig. 3F, right). Hepatic oxidative stress and lipid peroxidation are implicated in the progression of simple steatosis to NASH, and they are accompanied by a reduction in the metabolic rate. Similarly, ATF3 silencing substantially reduced the levels of malondialdehyde, a stable indicator of lipid peroxidation and oxidative stress that is increased in the livers of ZDF rats, and inhibited oxidative stress- or apoptosis-related protein expression levels (Fig. S3B, C). Then, to clarify the effect of oxidative stress-mediated ATF3 on the mitochondrial function in hepatocytes, we first isolated the mitochondrial fraction from the livers of ZDF rats and measured the mitochondrial adenosine triphosphate (ATP) and ROS production. ATF3 impaired mitochondrial function in the livers of ZDF rats, as demonstrated by their restoration with ATF3 silencing (Fig. S3D). To further confirm the effect of ATF3 silencing on oxidative stress-induced mitochondrial damage, we transfected primary hepatocytes isolated from 6-week-old, male Sprague Dawley (SD) rats with ATF3 siRNA. The cells were then treated with palmitic acid at final concentrations of 10–400 μM to mimic oxidative conditions, inducing overexpression of ATF3 as in the ZDF rats. Palmitic acid strongly reduced the intracellular ATP levels and increased the ROS generation in a dose-dependent manner, which was markedly attenuated by ATF3 silencing (Fig. S3E). Accordingly, the critical role of ATF3 in metabolic alterations regarding ER stress, lipogenesis, fatty acid oxidation, insulin action, and apoptosis was also verified by examining the effect of endogenous ATF3 silencing on palmitic acid-treated primary hepatocytes (Fig. S3F).

      Reducing ATF3 expression inhibits hepatic inflammatory responses in ZDF rats

      We investigated whether the changes in the metabolism and oxidative stress responses observed after ATF3 silencing in the livers of ZDF rats resulted in changes in hepatic inflammation. As expected, the loss of ATF3 led to a significant reduction in the serum pro-inflammatory cytokine (interleuking [IL]-6, interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and methyl-accepting chemotaxis protein [MCP]-1) and leptin levels, which were significantly elevated in ZDF rats (Fig. 4A). Similarly, ATF3 silencing diminished the increased hepatic mRNA levels of pro-inflammatory cytokines and adhesion- or fibrosis-related molecules (ICAM1 and COL4) (Fig. 4B). The hepatic inflammatory markers, including suppressor of cytokine signaling (SOCS) 1 and 3, chemokine (C-C motif) receptor 2 for monocytes, chemokine (C-X-C motif) ligand 2 (CXCL2/MIP2) for macrophages, and TNF receptor 1 (TNFR1), but not TNFR2, increased in ZDF rats, and they were markedly reduced by ATF3 siRNA (Fig. 4C). Additionally, ATF3 knockdown reduced the infiltration of inflammatory CD68-positive macrophages into the livers of ZDF rats (Fig. 4D). The inhibitory effects of ATF3 siRNA were similar at both 100 and 150 μg of ATF3 siRNA. The in vitro chemotaxis assay revealed that ATF3 silencing led to a striking reduction in macrophage migration in response to TNF-α (Fig. 4E). NF-κB plays a critical role in various inflammatory diseases, and its activation is essential for cytokine production. The increased IκBα phosphorylation and decreased IκBα protein expression in ZDF rats were reversed by ATF3 silencing (Fig. 4F). Phosphorylated NF-κB-p65 proteins were abundant in the liver nuclear fraction from ZDF rats, and their expression levels correlated with an increase in CCL5/regulated on activation, normal T cell expressed and secreted (RANTES), a chemokine released from activated T-cells. The levels were markedly diminished by ATF3 siRNA, suggesting that ATF3 acts as a chemoattractant in NF-κB-mediated inflammatory responses.
      Figure thumbnail gr4
      Fig. 4In vivo ATF3 silencing inhibits the hepatic inflammatory responses in ZDF rats. Either 100 or 150 μg of ATF3 siRNA was administered to 15-week-old ZL or ZDF rats (n = 8, each group). (A) Serum pro-inflammatory cytokines. (B and C) Real-time PCR analyses of hepatic mRNA expression of pro-inflammatory cytokines (B) and chemokines (C). (D) IHC analysis for CD68 (100x) and the quantification of CD68-positive cells. (E) Chemotaxis analysis for macrophage migration. (F) ATF3 silencing reduces NF-κB activation in ZDF rats. All data are given as the mean ± SEM with *p <0.01 and **p <0.05 from the t test. ATF3, activating transcription factor 3; Scr. si, scrambled siRNA; ATF3 si, ATF3 siRNA; ZL, Zucker lean; ZDF, Zucker diabetic fatty; IL-6, interleukin 6; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein 1; IL-1, interleukin 1; MIP-1, macrophage inflammatory protein 1; ICAM1, intercellular adhesion molecule 1; Col4, collagen type IV; SOCS, suppressor of cytokine signaling; CCR2, chemokine (C-C motif) receptor 2; CXCL2, chemokine (C-X-C motif) ligand 2; TNFR, tumor necrosis factor receptor; CTL, control; RANTES, regulated on activation, normal T cell expressed and secreted; IHC, immunohistochemistry; SEM, standard error of the mean. (This figure appears in colour on the web.)

      NAFLD is closely linked to incident T2D

      Next, we investigated the relationship between NAFLD and T2D development in 322 human participants who were included in the prospective NAFLD registry cohort. The clinical characteristics of the study participants are presented in Table S3. When they were grouped according to histologic NAFLD status, patients with hepatic steatosis showed more metabolic dysfunction than those without steatosis. NAFLD was closely associated with obesity-related (fat mass and body mass index), lipid-related (high-density lipoprotein cholesterol [HDL-cholesterol] and TG), liver injury-related (aspartate aminotransferase [AST], ALT) (Fig. S4A), and T2D-related (insulin, the homeostasis model assessment of insulin resistance [HOMA-IR], c-peptide, HbA1c, and fasting blood glucose) traits (Fig. 5A). Histologic grades of steatosis were determined by liver biopsy (Fig. 5B), and they were positively associated with body mass index (BMI); total fat mass; and serum ALT, AST, and TG levels, but were negatively associated with HDL-cholesterol levels (Fig. S4B). We also confirmed the reproducible associations between phenotypic variables, including anthropometric or metabolic traits and liver injury markers or NAS (Table S3, Fig. S4C). HOMA-IR (r = 0.401, p = 0.003), insulin (r = 0.449, p <0.001), BMI (r = 0.399, p <0.001), and total fat mass (r = 0.398, p <0.001) were positively correlated with the serum ALT levels in participants with NAFLD; however, a negative correlation was observed between serum HDL-cholesterol (r = −0.329, p = 0.009) and ALT levels (data not shown). Notably, there was a marked difference in the overall prevalence of T2D between participants with and without NAFLD (Fig. 5C). Next, we found that the prevalence of T2D was significantly increased according to the histological severity of NAFLD, suggesting that liver fat accumulation per se or NASH (with inflammation and fibrosis) may be prerequisites for T2D development (data not shown). Moreover, major T2D surrogate markers, such as fasting blood glucose, HbA1c, and HOMA-IR, could effectively discriminate between NAFLD participants with and without T2D (Fig. 5D). However, this discrimination was not consistent with BMI; fat mass; or serum AST, TG, insulin, and c-peptide levels, suggesting that participants with NAFLD with non-alcoholic fatty liver (NAFL) and/or NASH might be at a higher risk of developing T2D.
      Figure thumbnail gr5
      Fig. 5NAFLD is closely associated with T2D. (A) Comparison of diabetes-related, biochemical parameters in participants with and without NAFLD (n = 322). (B) The histologic grades of steatosis by H&E staining in liver biopsy tissues. (C) Comparison of the prevalence (%) of diabetes in participants with or without NAFLD (p value was determined by the chi-squared test). (D) Comparison of diabetes-related, biochemical parameters between participants with NAFLD alone and those with NAFLD plus diabetes. (A and D) Data are presented as box plots (median, IQR) and p value was determined by the t test. FBG, fasting blood glucose; HOMA-IR, the homeostasis model assessment of insulin resistance; NAFLD, non-alcoholic fatty liver disease; DM, diabetes mellitus. (This figure appears in colour on the web.)

      ATF3 induction is responsible for hepatic steatosis in participants with NAFLD

      Next, 50 liver biopsy samples selected from a prospective NAFLD registry were examined for changes in ATF3 expression. The clinical characteristics of the 50 participants are presented and were similar to those of an entire cohort (n = 305), as summarized in Table S4. Notably, hepatic ATF3 mRNA expression dramatically increased in participants with steatosis, and its expression was highest in the livers of participants with grade 2, which positively correlated with lipogenesis (SREBP-1c, FAS and PPAR-γ)-, ER stress (CHOP)-, and macrophage infiltration (MCP-1)-related gene expression levels (Fig. 6A–C). Unexpectedly, β-oxidation-related PGC-1α mRNA expression was significantly increased in the livers of participants with steatosis, whereas no significant changes in PPAR-α expression were found (Fig. 6B). Additionally, there was a positive correlation between the ATF3 protein staining score and the histologic grade of steatosis (Fig. 6D). The positive correlation between ATF3 staining intensity (Fig. 6E) and ER/oxidative stress- and lipogenesis-related protein expression (Fig. S5A) was confirmed by immunohistochemical analysis. Similar to the mRNA expression, the majority of human liver tissues with a low level of ATF3 also displayed low expression levels of CHOP, 4-HNE, and SREBP-1c; conversely, the majority of liver tissues with a high level of ATF3 showed high protein expression levels.
      Figure thumbnail gr6
      Fig. 6ATF3 induction correlates with hepatic steatosis in NAFLD participants. Fifty liver biopsy samples with or without steatosis were randomly selected. (A) Real-time PCR for ATF3 mRNA in the livers of controls (n = 7) and participants with steatosis grades 1 (n = 13), 2 (n = 17), and 3 (n = 13). (B) Real-time PCR for the hepatic mRNA levels in the livers of NAFLD participants. All data are given as the mean ± SEM with *p <0.05, **p <0.005, and ***p <0.0005 from the t test. (C) Correlation analysis based on real-time PCR data. Correlation coefficients (r) and p values are given. (D) IHC analysis and quantification of ATF3 in liver tissues across steatosis grades. Data are presented as box plots (median, IQR) (*p <0.05 and **p <0.005 vs. steatosis grade 0 from the t test). (E) IHC analysis of CHOP and SREBP-1c in liver tissues with low (n = 18) and high (n = 32) expression levels of ATF3. The expression scores were assessed based on their intensity and distribution, and they were divided into three groups: low (0–1.49), middle (1.50–2.99), and high (≥3). ATF3, activating transcription factor 3; NAFLD, non-alcoholic fatty liver disease; SREBP1c, sterol regulatory element binding protein 1c; FAS, fatty acid synthase; PPAR-γ, peroxisome proliferator-activated receptor γ; PPAR-α, peroxisome proliferator-activated receptor α; PGC-1α, PPAR-γ coactivator 1α; CHOP, CCAAT-enhancer-binding protein homologous protein; MCP-1, monocyte chemoattractant protein 1; IHC, immunohistochemistry; SEM, standard error of the mean. (This figure appears in colour on the web.)

      ATF3 expression is correlated with macrophage infiltration, mitochondrial dysfunction, and insulin resistance in participants with NAFLD

      CD68-positive macrophage infiltration was more prominent in NAFLD participants with higher grades of steatosis, and the level positively correlated with ATF3 expression (Fig. 7A, B). In participants with NAFLD, we also found a positive correlation between hepatic ATF3 protein expression and the prognostic markers of T2D as well as the TG levels (r = 0.295, p = 0.037), whereas no correlation was found between ATF3 expression and BMI (r = 0.094, p = 0.518) or fat mass (r = 0.198, p = 0.178) (Fig. S5B, C), highlighting the specific role of ATF3 as a key driver of development of hepatic steatosis and T2D independent of obesity. Indeed, the ATF3 mRNA and protein levels initially increased in NAFLD participants and were more significantly increased during the progression from NAFLD to T2D (Fig. 7C). Moreover, the ATF3 expression levels displayed a statistically significant correlation with both mitochondrial ROS production (r = 0.721, p <0.001) and ATP depletion (r = −0.645, p <0.001) in the livers of participants with NAFLD and/or T2D (Fig. S5D). We also found that in HepG2 cells, ATF3 expression significantly increased after exposure to 10% sera obtained from participants with NAFLD and/or T2D for 48 h, which correlated with lipid accumulation (Fig. S6A, B). This prompted us to examine the direct role of ATF3 stimulation in the sera of participants with NAFLD and/or T2D in mitochondrial dysfunction. ROS generation and intracellular ATP formation were significantly elevated and reduced, respectively, in HepG2 cells exposed to the sera of participants with NAFLD and/or T2D. However, their impaired mitochondrial responses were significantly ameliorated by endogenous ATF3 silencing (Fig. S6C). The critical roles of ATF3 in mitochondrial dysfunction and impaired insulin action were also corroborated by ATF3 cDNA-overexpressing HepG2 cells (Fig. S6D, E). Consistent with these findings, 48 h of incubation with the sera of participants with NAFLD and/or T2D strongly inhibited the insulin-stimulated activation of IRS-1 (Y941) and Akt (S473) and conversely increased the insulin-suppressed IRS-1 (S307) phosphorylation in HepG2 cells, which was completely restored or attenuated by endogenous ATF3 silencing (Fig. S6F). Finally, to further clarify the relative impact of insulin resistance on hepatic ATF3 mRNA expression, we compared the hepatic ATF3 expression level between participants with (HOMA-IR ≥2) and without (HOMA-IR <2) insulin resistance. Hepatic ATF3 expression was significantly higher in NAFLD participants with insulin resistance (n = 33; median, 3.24) than in those without (n = 16; median, 1.54) (Fig. 7D). Furthermore, there was a significant increase in the prevalence of T2D in participants with insulin resistance among both the selected 50 participants and the entire cohort of 322 participants with NAFLD (Fig. 7E, p <0.05).
      Figure thumbnail gr7
      Fig. 7ATF3 induction precedes macrophage infiltration and diabetes in NAFLD participants. (A) IHC analysis and quantification of CD68-positive cells in human liver tissues (*p <0.01 vs. steatosis grade 0 from the t test). (B) Correlation of an ATF3 staining score with CD68-positive cells in NAFLD participants. (C) Comparison of ATF3 mRNA and protein expression among controls, participants with NAFLD alone, and those with NAFLD plus diabetes (p <0.001 and p <0.005, respectively from post hoc analysis for the ANOVA; p <0.001 from the ANOVA). (D) Comparison of hepatic ATF3 mRNA expression in NAFLD participants with (HOMA-IR ≥2) and without (HOMA-IR <2) insulin resistance (p <0.05 from the t test). (E) The prevalence (%) of diabetes with or without insulin resistance among the participants with NAFLD (p value was determined by the chi-squared test). (A, C, and D) Data are presented as box plots (median, IQR). ATF3, activating transcription factor 3; NAFLD, non-alcoholic fatty liver disease; DM, diabetes mellitus; HOMA-IR, the homeostasis model assessment of insulin resistance. (This figure appears in colour on the web.)

      Discussion

      In the current study, we identified ATF3 as a target protein that was highly elevated in the livers of ZDF rats and human participants with NAFLD and/or T2D. Loss-of-function experiments using an in vivo-jetPEI siRNA delivery system against ATF3 ameliorated the oxidative stress-mediated hepatic steatosis and glucose intolerance. To the best of our knowledge, this is the first study demonstrating a novel functional role of ATF3 as a potent regulatory factor in induction of hepatic steatosis and T2D. Moreover, our findings suggest that ATF3 promotes the development of T2D among human participants with NAFLD and that it may be a useful biomarker for predicting hepatic steatosis-induced T2D at an earlier stage.
      We demonstrated that ATF3 may be a detrimental regulator of the oxidative stress-mediated hepatic steatosis and insulin insensitivity during the progressive development of T2D in ZDF rats and human participants with NAFLD. Specifically, ATF3 expression was positively associated with the severity of hepatic steatosis in the liver tissues obtained from participants with NAFLD, and its expression level was higher in participants with NAFLD and T2D than that of participants with NAFLD alone. We also found a positive correlation between hepatic ATF3 expression and the prognosticators of T2D in participants with NAFLD. Despite previous extensive studies on the relationship between NAFLD and T2D, their inter-regulated pathogenesis remains obscure, and no target molecules have been reported to prevent NAFLD-induced T2D. Therefore, to further elucidate the molecular basis underlying the progression of NAFLD to T2D, we used ZDF rats to mimic human T2D participants
      • Katsuda Y.
      • Ohta T.
      • Miyajima K.
      • Kemmochi Y.
      • Tong B.
      • Shinohara M.
      • et al.
      Diabetic complications inobese type 2 diabetes rat models.
      and implemented integrative therapeutic approaches to prevent obesity-mediated T2D using an in vivo-siRNA delivery system for exploring target pathways and molecules.
      Oxidative or ER stress is closely associated with obesity development via lipid accumulation and insulin resistance in insulin-responsive tissues, leading to T2D.
      • Yamamoto S.
      • Matsushita Y.
      • Nakagawa T.
      • Honda T.
      • Hayashi T.
      • Noda M.
      • et al.
      Visceral fat accumulation, insulin resistance, and elevated depressive symptoms in middle-aged Japanese men.
      Our recent study demonstrated that ROS production and ER stress are increased during the progression of T2D in ZDF rats, triggering hyperglycemia-mediated pancreatic β-cell dysfunction and apoptosis.
      • Kim J.Y.
      • Park K.J.
      • Kim G.H.
      • Jeong E.A.
      • Lee D.Y.
      • Lee S.S.
      • et al.
      In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes.
      However, the exact molecular mechanism and key regulatory proteins involved in the progressive development of T2D with hepatic steatosis remain elusive. In this study, several lines of evidence showed that oxidative stress-mediated ATF3 may be an effective target molecule for the development of hepatic steatosis and/or T2D in obese ZDF rats. The potent roles of ATF3 in the pathogenesis of NAFLD and progressive development of T2D are also supported by a loss-of-function study through in vivo delivery of ATF3-specific siRNA to 15-week-old ZDF rats. In vivo ATF3 silencing ameliorated impaired insulin action and reduced metabolic rates, preventing hepatic steatosis and T2D development. Based on the aforementioned results, we hypothesized that ATF3 may function as a feedback regulator in the acceleration of oxidative stress or as an executive downstream target molecule in severe liver injury. However, the in vivo-jetPEI siRNA delivery system used in this study did not specifically affect the liver because ATF3 silencing was observed in adipose tissue and muscle as well as in the liver. These data suggest that the therapeutic effects of ATF3 siRNA on hepatic steatosis and impaired glucose homeostasis may be indirectly mediated via metabolic control in adipose tissue, muscle, and liver. ATF3, as such, may play an important role in the maintenance of glucose and lipid homeostasis via interregulation between liver and other metabolic organs including adipose tissue and muscle which are closely associated with glucose and lipid metabolism. Therefore, the optimal regulation of ATF3 in each organ exposed to various stresses may be necessary to maintain body homeostasis. Accordingly, synchronous inhibition of ATF3 expression in each organ using an in vivo delivery system during the progressive development of obese T2D may be a more effective therapeutic strategy to ameliorate impaired glucose and lipid metabolism. Previous studies demonstrated that the ATF3 is initially induced by stresses and engaged in a compensatory or adaptive homeostatic response to alleviate cellular stress during the development of T2D.
      • Hai T.
      • Wolford C.C.
      • Chang Y.S.
      ATF3, a hub of the cellular adaptive-response network, in the pathogenesis of diseases: is modulation of inflammation a unifying component?.
      However, in a high fat diet (HFD)-induced animal model for T2D, ATF3 deficiency in knockout mice exacerbated HFD-induced glucose intolerance and metabolic disorder,
      • Zmuda E.J.
      • Qi L.
      • Zhu M.X.
      • Mirmira R.G.
      • Montminy M.R.
      • Hai T.
      The roles of ATF3, an adaptive-response gene, in HFD-induced diabetes and pancreatic β-cell dysfunction.
      suggesting a protective rather than detrimental role of ATF3. At the early stage of disease development, stress-induced ATF3 is believed to help each organ cope with high metabolic demands. However, because ATF3 is a double-edged sword, this adaptation or coping mechanism in each organ comes with a cost, and after prolonged overwork in the cells of each organ, its detrimental and proapoptotic function comes into play. Therefore, to prevent or alleviate impaired glucose homeostasis and hepatic steatosis observed in ZDF rats, we hypothesized that synchronous inhibition of ATF3 expression increased in each organ under obese stress may be absolutely necessary. Nonetheless, to dissect the potential roles of ATF3 in each organ, especially the liver, during the progression of T2D in ZDF rat models, we need to perform additional experiments using organ-specific knockout or silencing animal models.
      In the current study, the literature-based functional network and gene expression profiling analyses based on an open public database domain (GSE48452) also supported the hypothesis that ATF3 is a potential target molecule that regulates the progressive development of T2D in participants with NAFLD. GSE48452 included morbidly obese Caucasian participants at all stages of NAFLD. Accordingly, the ATF3 mRNA and protein expression levels were significantly increased in our Asian NAFLD cohort with or without T2D. In multiple linear regression analyses, the severity of steatosis was positively associated with the serum AST, ALT, and TG levels; BMI; and total fat mass, and it was negatively associated with the HDL-cholesterol levels. In particular, the upregulation of hepatic ATF3 mRNA was positively associated with the severity of hepatic steatosis and correlated with the lipogenesis-, ER stress-, and macrophage infiltration-related gene expression levels. Indeed, ATF3 immunoreactivity in human liver tissues was predominantly observed in hepatocytes, and the staining patterns of ATF3 changed depending on the histologic grade of steatosis. In contrast to participants with mild or moderate steatosis, which was principally restricted to the cytoplasm of hepatocytes, ATF3 expression in participants with severe steatosis was high in the nucleus. The reduced cytoplasmic ATF3 expression in participants with severe steatosis may be due to the accumulation of lipid droplets that push ATF3 to the side of hepatocytes; thereafter, the staining for ATF3 was detectable at the border of lipid droplets in the cytoplasm of hepatocytes.
      Intriguingly, we also found a significant correlation between hepatic ATF3 protein expression and prognostic markers of T2D. The elevation of ATF3 mRNA and protein levels was further enhanced in NAFLD participants with T2D compared to those with NAFLD alone. The prevalence of diabetes among both the selected 50 participants and the entire NAFLD cohort significantly increased in participants with insulin resistance compared to that of those without. ATF3 protein expression was also associated with mitochondrial dysfunction in the liver tissues of participants with NAFLD and/or T2D, suggesting the potential role of ATF3 in the pathogenesis of NAFLD and progressive development of T2D.
      Despite the positive correlation of ATF3 expression with lipid accumulation and T2D prognostic markers, no significant correlation was found between ATF3 expression and obesity-related traits, such as the BMI and fat mass (Fig. S6C). Therefore, hepatic overexpression of ATF3 in participants with NAFLD might account for the mechanistic basis underlying the development of hepatic steatosis and T2D and might not simply depend on obesity.
      Herein, an in vivo ATF3 silencing system was used as a prominent therapeutic strategy to prevent oxidative stress-mediated hepatic steatosis or T2D. Recently, ATF3 has been considered an adaptive response gene with a dual mode of action to activate or repress target gene expression.
      • Hai T.
      • Wolford C.C.
      • Chang Y.S.
      ATF3, a hub of the cellular adaptive-response network, in the pathogenesis of diseases: is modulation of inflammation a unifying component?.
      Stress-inducible ATF3 signaling may initially elicit a compensatory or adaptive response for the acute ER or oxidative stress.
      • Lee Y.S.
      • Sasaki T.
      • Kobayashi M.
      • Kikuchi O.
      • Kim H.J.
      • Yokota-Hashimoto H.
      • et al.
      Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.
      Based on the previous experiments,
      • Zmuda E.J.
      • Qi L.
      • Zhu M.X.
      • Mirmira R.G.
      • Montminy M.R.
      • Hai T.
      The roles of ATF3, an adaptive-response gene, in HFD-induced diabetes and pancreatic β-cell dysfunction.
      ATF3 KO mice with complete depletion of the ATF3 gene may not induce an adequate compensatory or adaptive response for acute oxidative or ER stress; therefore, they are likely to suffer from various stress-mediated metabolic diseases. Despite the efforts for moderate induction of ATF3 to cope with metabolic stresses at the early stage, the increased ATF3 and excessive ROS or ER stress in the obese, diabetic stage may be associated with severe metabolic diseases. Accordingly, moderate inhibition via in vivo ATF3 silencing during the progressive development of obese T2D may be a more effective therapeutic strategy than the complete depletion of ATF3 expression as observed in KO mice. Although hepatic ATF3 expression was positively correlated with the histologic grade of steatosis, the exact functional role of ATF3 at different steatosis grades in participants with NAFLD and/or T2D remains obscure. In this context, the debate about whether the transient or persistent expression of ATF3 in hepatocytes determines the severity of liver injury or T2D merits further scrutiny.
      HepG2 cells exposed to the sera of participants with NAFLD and/or T2D exhibited significant mitochondrial dysfunction and impaired insulin action, which was accompanied by increased ATF3 expression and lipid accumulation. These results indicate that dysregulated hepatic or secretory proteins might induce endogenous ATF3 production and mediate the development of steatosis and T2D. Although data were not shown here, we have found that ATF3 expression or its activation may be also closely associated with cAMP response element-binding protein (CREB) activation in our model because CREB phosphorylation was highly increased during the progressive development of hepatic steatosis and/or T2D in ZDF rats. However, CREB activation was significantly decreased in ZDF rats injected with ATF3 siRNA. These are correlated with the previous results because hepatic CREB activation is constitutively upregulated in diabetes.
      • Dentin R.
      • Liu Y.
      • Koo S.H.
      • Hedrick S.
      • Vargas T.
      • Heredia J.
      • et al.
      Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2.
      • Dentin R.
      • Hedrick S.
      • Xie J.
      • Yates J.
      • Montminy M.
      Hepatic glucose sensing via the CREB coactivator CRTC2.
      Therefore, we need to perform further studies on the relationship between ATF3 expression and CREB activation during the progression of hepatic steatosis and diabetes. However, additional studies using combined omics platforms are needed to identify novel endocrine factors that are secreted by hepatocytes, such as hepatokines, proteins, cytokines, or chemokines that affect metabolic disorders and, especially, ATF3 induction in the peripheral tissues of participants with NAFLD and/or T2D. Furthermore, abundant ATF3 levels in hepatocytes with excessive oxidative stress might result from diverse secretory processes.
      • Zhang M.
      • Schekman R.
      Cell Biology. Unconventional secretion, unconventional solutions.
      Collectively, our findings suggest that hepatic ATF3 protein induction plays an important role in oxidative stress-mediated hepatic steatosis and the development of T2D in both ZDF rats and human participants with NAFLD. Therefore, ATF3 proteins 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 metabolism, in vivo ATF3 silencing may be a potential central strategy for preventing and managing NAFLD and T2D.

      Financial support

      This work was supported by research grants from the Korean National Institute of Health (2011-NG64001-00).

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      Conceptualization, J.Y.K., K.J.P., and W.H.K.; investigation and data analysis, J.Y.K., K.J.P., J.Y.H., G.H.K., D.Y.L., Y.J.L., E.H.S., M.G.Y., Y.H.S., B.G., W.K., and W.H.K.; discussion, J.Y.K., K.J.P., J.Y.H., BJK, G.S.R., S.I.P., B.G., W.K., and W.H.K; and writing, review & editing, J.Y.K., K.J.P., J.Y.H., B.G., W.K., and W.H.K.

      Acknowledgments

      We acknowledge the technical assistance of Do-Hyung Kim (Korea Biomedical Animal Institute, Korea) and Jong-ho Park (Cosmo Gene Tec., Korea) in the animal experiments (administration of drug and in vivo-jetPEI reagent with ATF3 siRNA).

      Supplementary data

      References

      Author names in bold designate shared co-first authorship

        • Finelli C.
        • Tarantino G.
        Is there any consensus as to what diet or lifestyle approach is the right one for NAFLD patients?.
        J Gastrointestin Liver Dis. 2012; 21: 293-302
        • Anstee Q.M.
        • Daly A.K.
        • Day C.P.
        Genetic modifiers of non-alcoholic fatty liver disease progression.
        Biochim Biophys Acta. 2011; 1812: 1557-1566
        • Bellentani S.
        • Saccoccio G.
        • Masutti F.
        • Crocè L.S.
        • Brandi G.
        • Sasso F.
        • et al.
        Prevalence of and risk factors for hepatic steatosis in Northern Italy.
        Ann Intern Med. 2000; 132: 112-117
        • Samuel V.T.
        • Shulman G.I.
        Mechanisms for insulin resistance: common threads and missing links.
        Cell. 2012; 148: 852-871
        • Yamamoto S.
        • Matsushita Y.
        • Nakagawa T.
        • Honda T.
        • Hayashi T.
        • Noda M.
        • et al.
        Visceral fat accumulation, insulin resistance, and elevated depressive symptoms in middle-aged Japanese men.
        PLoS One. 2016; 11: e0149436
        • Basseri S.
        • Austin R.C.
        Endoplasmic reticulum stress and lipid metabolism: mechanisms and therapeutic potential.
        Biochem Res Int. 2012; : 841362
        • Bravo R.
        • Parra V.
        • Gatica D.
        • Rodriguez A.E.
        • Torrealba N.
        • Paredes F.
        • et al.
        Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration.
        Int Rev Cell Mol Biol. 2013; 301: 215-290
        • Apostolova N.
        • Blas-Garcia A.
        • Esplugues J.V.
        Mitochondria sentencing about cellular life and death: a matter of oxidative stress.
        Curr Pharm Des. 2011; 17: 4047-4060
        • Brown G.T.
        • Kleiner D.E.
        Histopathology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
        Metabolism. 2015; https://doi.org/10.1016/j.metabol.2015.11.008
        • Meex R.C.
        • Hoy A.J.
        • Morris A.
        • Brown R.D.
        • Lo J.C.
        • Burke M.
        • et al.
        Fetuin B Is a Secreted Hepatocyte Factor Linking Steatosis to Impaired Glucose Metabolism.
        Cell Metab. 2015; 22: 1078-1089
        • Kharitonenkov A.
        • Shiyanova T.L.
        • Koester A.
        • Ford A.M.
        • Micanovic R.
        • Galbreath E.J.
        • et al.
        FGF-21 as a novel metabolic regulator.
        J Clin Invest. 2005; 115: 1627-1635
        • Misu H.
        • Takamura T.
        • Takayama H.
        • Hayashi H.
        • Matsuzawa-Nagata N.
        • Kurita S.
        • et al.
        A liver-derived secretory protein, selenoprotein P, causes insulin resistance.
        Cell Metab. 2010; 12: 483-495
        • Hai T.
        • Wolford C.C.
        • Chang Y.S.
        ATF3, a hub of the cellular adaptive-response network, in the pathogenesis of diseases: is modulation of inflammation a unifying component?.
        Gene Expr. 2010; 15: 1-11
        • Hartman M.G.
        • Lu D.
        • Kim M.L.
        • Kociba G.J.
        • Shukri T.
        • Buteau J.
        • et al.
        Role for activating transcription factor 3 in stress-induced beta-cell apoptosis.
        Mol Cell Biol. 2004; 24: 5721-5732
        • Rynes J.
        • Donohoe C.D.
        • Frommolt P.
        • Brodesser S.
        • Jindra M.
        • Uhlirova M.
        Activating transcription factor 3 regulates immune and metabolic homeostasis.
        Mol Cell Biol. 2012; 32: 3949-3962
        • Lee Y.S.
        • Sasaki T.
        • Kobayashi M.
        • Kikuchi O.
        • Kim H.J.
        • Yokota-Hashimoto H.
        • et al.
        Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.
        Diabetologia. 2013; 56: 1383-1393
        • Gilchrist M.
        • Thorsson V.
        • Li B.
        • Rust A.G.
        • Korb M.
        • Roach J.C.
        • et al.
        Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4.
        Nature. 2006; 441: 173-178
        • Zmuda E.J.
        • Qi L.
        • Zhu M.X.
        • Mirmira R.G.
        • Montminy M.R.
        • Hai T.
        The roles of ATF3, an adaptive-response gene, in HFD-induced diabetes and pancreatic β-cell dysfunction.
        Mol Endocrinol. 2010; 24: 1423-1433
        • Kim J.Y.
        • Park K.J.
        • Kim G.H.
        • Jeong E.A.
        • Lee D.Y.
        • Lee S.S.
        • et al.
        In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes.
        Cell Signal. 2013; 25: 2348-2361
        • Koo B.K.
        • Kim D.
        • Joo S.K.
        • Kim J.H.
        • Chang M.S.
        • Kim B.G.
        • et al.
        Sarcopenia is an independent risk factor for non-alcoholic steatohepatitis and significant fibrosis.
        J Hepatol. 2017; 66: 123-131
        • Katsuda Y.
        • Ohta T.
        • Miyajima K.
        • Kemmochi Y.
        • Tong B.
        • Shinohara M.
        • et al.
        Diabetic complications inobese type 2 diabetes rat models.
        Exp Anim. 2014; 63: 121-132
        • Dentin R.
        • Liu Y.
        • Koo S.H.
        • Hedrick S.
        • Vargas T.
        • Heredia J.
        • et al.
        Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2.
        Nature. 2007; 449: 366-369
        • Dentin R.
        • Hedrick S.
        • Xie J.
        • Yates J.
        • Montminy M.
        Hepatic glucose sensing via the CREB coactivator CRTC2.
        Science. 2008; 319: 1402-1405
        • Zhang M.
        • Schekman R.
        Cell Biology. Unconventional secretion, unconventional solutions.
        Science. 2013; 340: 559-561