School of Pharmacy and Institute of New Drug Development, Jeonbuk National University, Jeonju, Republic of Korea.
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.
College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul, Goyang-si, Gyeonggi-do, Republic of Korea.
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, USA.
College of Pharmacy, Graduate School of Pharmaceutical Sciences, and.
Graduate Program in Innovative Biomaterials Convergence, Ewha Womans University, Seoul, Republic of Korea.
Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Department of Biochemistry and Molecular Biology, Jeonbuk National University Medical School, Jeonju, Republic of Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul Metropolitan Government Boramae Medical Center, Seoul, Republic of Korea.
Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea.
Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Department of Internal Medicine, Gil Medical Center, Gachon University College of Medicine, Incheon, Republic of Korea.
MRC-OSTRC, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Chosun University, Gwangju, Republic of Korea.
Abstract
Iron overload has emerged as a significant risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), a growing global health concern. Despite this association, the precise mechanisms by which hepatic iron and its regulatory genes connect liver pathology to systemic metabolic dysfunction remain elusive. Here, we demonstrate that humoral signals originating from iron-overloaded hepatocytes acted as critical mediators driving systemic metabolic dysfunction in MASLD. Ferroportin (FPN, SLC40A1), the sole cellular iron exporter, exhibited markedly reduced expression in hepatocytes of both patients with MASLD and mouse models of the disease, concomitant with hepatic iron accumulation. Functionally, hepatocyte-specific FPN deletion significantly exacerbated diet-induced obesity and insulin resistance, with these metabolic perturbations accompanied by decreased energy expenditure and impaired thermogenic capacity. Mechanistically, we establish that hepatic iron accumulation resulting from FPN deficiency enhanced the production of 2 specific hepatokines, fetuin-A and LECT2, through activation of the transcription factor FoxO1. Notably, therapeutic interventions - including genetic silencing of these hepatokines, hepatocyte-specific FPN overexpression, or oral iron chelation - effectively reversed the metabolic dysfunction phenotypes. These findings provide critical insights into the pathophysiological mechanisms linking MASLD to systemic metabolic disorders and highlight promising therapeutic strategies to combat these diseases.
Keywords
DiabetesHepatologyHomeostasisMetabolismObesity
Figures
Figure 1 Serum ferritin levels correlate with metabolic parameters in patients with MASLD. (A) Overview of correlation analyses between serum ferritin and metabolic parameters in patients with MASLD. (B–P) Serum ferritin levels were analyzed across patient subgroups stratified by sex and clinical parameters: NAS (B–D), HOMA-IR (E–G), adipo-IR (H–J), fat mass (K–M), and BMI (N–P). Subgroups in panels E–G represent normoglycemia (NG) (HOMA-IR <3), impaired glucose tolerance (IGT) (3~10), and overt T2D (>10). (Q) Correlation plot summarizing relationships between serum ferritin and metabolic parameters. Data are presented as the median ± 95% CI. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparison test.Figure 2 Hepatic FPN expression is downregulated in human and murine MASLD. (A–C) Immunohistochemical staining for FPN and DAB-enhanced Perls’ iron staining in liver tissues from the MASLD cohort (A), with corresponding quantifications (B and C). Normal controls (n = 3); MASL, simple steatosis with or without IR (n = 7 per group); eMASH with no or mild fibrosis (F0–1) (n = 8); and aMASH with F ≥2 (n = 3). (D) Inverse correlation between FPN expression and hepatic iron accumulation. Pearson correlation coefficients (r) and corresponding P values are shown. (E) Serum ferritin levels across MASLD subgroups. (F–H) FPN immunostaining and Perls’ iron staining in mice fed a CDAHFD (F), a HFHFrHC diet (G), or a standard HFD (H). (I) IF staining for FPN in primary hepatocytes isolated from HFD-fed mice. Scale bars: 20 μm. (J) Live-cell fluorescence imaging of intracellular labile iron levels in primary hepatocytes, as shown in I. (K) Schematic illustrating the proposed role of hepatic FPN deficiency in MASLD-associated systemic metabolic dysfunction. Data are presented as the mean ± SEM. Scale bars: 50 μm (upper images) and 20 μm (lower images) (A and F–H). *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Tukey’s multiple-comparison test.Figure 3 Hepatocyte-specific FPN deletion exacerbates obesity and IR under HFD conditions. (A) Study design for metabolic phenotyping of hepatocyte-specific Fpn-KO mice fed a HFD. (B) Body weight curves of LT control and Fpn-LKO mice fed a ND or a HFD for 19 weeks (n = 7–8 per group). Arrowheads indicate the timing of GTT and ITT analyses. (C and D) Body composition analysis measuring lean and fat mass (n = 7–8 per group). (E–H) Representative images of eWAT, iWAT, and BAT from HFD-fed mice and tissue weights of eWAT, iWAT, and BAT (n = 7–8 per group). (I–K) H&E staining of eWAT, iWAT, and BAT. Scale bars: 50 mm. Adipocyte size in H&E-stained sections of eWAT and iWAT (n = 3 per group). (L and M) GTT and ITT analyses in HFD-fed mice (n = 6–7 per group). (N–Q) Western blot analysis of p-Akt (S473) in eWAT, iWAT, and GAS tissues from HFD-fed mice. Relative band intensities were quantified. (R and S) Body weight (n = 12–13 per group) and fasting blood glucose levels (n = 6 per group) in female LT and Fpn-LKO mice fed a HFD for 9 weeks. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 4 Hepatic FPN deficiency reduces energy expenditure and impairs thermogenic capacity. (A–D) Comprehensive metabolic cage analyses measuring energy expenditure (EE) (A and B) and oxygen consumption rates (VO2) (C and D) in LT control versus Fpn-LKO mice (n = 7 per group) fed a HFD during light/dark cycles. (E) Rectal temperature (temp.) measurements in LT and Fpn-LKO (n = 5 per group) after cold exposure at 6°C for the indicated times. T.N., thermoneutral condition. (F) Surface body temperature measurements and representative thermal imaging after cold exposure at 6°C for 48 hours. (G and H) Western blotting and immunohistochemical staining for UCP1 in iWAT from the mice shown in F following cold exposure. The UCP1 band is indicated by a pound sign, and molecular weight markers are shown. Scale bars: 50 μm. Rel., relative. (I–L) Endurance exercise performance tests assessing running distance, duration, and final speed achieved by LT control versus Fpn-LKO mice (n = 10 per group) on treadmill exercise protocols. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 5 Hepatic FPN deficiency increases the expression of hepatokines, fetuin-A, and LECT2. (A and B) Overview of bulk RNA-seq analysis strategy used to identify hepatokines associated with hepatic iron overload in MASLD patient liver samples. Sequential filtering criteria are illustrated schematically in B. (C) Western blot analysis of secreted fetuin-A and LECT2 proteins from CM derived from primary hepatocytes isolated from HFD-fed LT mice versus Fpn-LKO mice. (D and E) Hepatic mRNA expression and serum levels of fetuin-A and LECT2 in ND- versus HFD-fed LT and Fpn-LKO mice (n = 6–8 per group). (F) snRNA-seq analyses demonstrating cell-type–specific expression patterns of fetuin-A and LECT2 across human liver cells. (G) snRNA-seq analyses depicting fetuin-A and LECT2 expression levels in hepatocytes from patients with MASLD according to MASLD severity. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 6 Hepatic iron overload induces fetuin-A and LECT2 expression via FoxO1 activation. (A and B) qPCR analyses showing increased Fetua and Lect2 mRNA levels induced by the iron treatments FAC (100 μM, n = 3 per group) or FeCl3 (100 μM, n = 4 per group). (C) Identification of conserved FoxO1-binding motifs (forkhead response element [FHRE]) within FETUA and LECT2 promoters. TF, transcription factor. (D and E) Western blot analysis of FoxO1 in primary hepatocytes from LT and Fpn-LKO mice (D) or in primary mouse hepatocytes treated with FeCl3 (E). (F) IF staining demonstrating FoxO1 nuclear translocation in primary mouse hepatocytes treated with FAC or FeCl3. Scale bars: 20 μm. (G) Luciferase reporter assays confirming FoxO1-mediated transcriptional activation of FETUA and LECT2 in HepG2 cells overexpressing FoxO1 (n = 3 per group). (H) ChIP assays validating direct FoxO1 binding to promoter regions of target genes (n = 3 per group). (I and J) Western blot analysis of FoxO1 and its quantification in the livers of ND- or HFD-fed LT and Fpn-LKO mice (n = 6–8 per group). (K) Positive correlations between hepatic FoxO1 expression and transcript levels of Fetua and Lect2. Pearson correlation coefficients (r) and corresponding P values are shown. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 7 Silencing of fetuin-A and LECT2 ameliorates systemic metabolic dysfunction. (A) Experimental design illustrating hepatocyte-specific silencing of fetuin-A or LECT2 via AAV-mediated shRNA delivery under the control of the TBG promoter during HFD feeding of Fpn-LKO mice. (B and C) qPCR analysis validating effective knockdown of Fetua and Lect2 mRNA levels (n = 8–9 per group). (D and E) GTT analysis (n = 5–6 per group). (F and G) ITT analysis (n = 5 per group). (H–K) Western blot analysis of p-Akt (S473) status across peripheral tissues including eWAT, iWAT, and GAS muscle tissues following knockdown intervention, with quantification of band intensities (n = 5 per group). Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 8 Exogenous fetuin-A and LECT2 impair insulin sensitivity in adipocytes and myotubes. (A–D) Inhibitory effects of exogenous fetuin-A (A and B) and LECT2 (C and D) on insulin signaling in differentiated 3T3-L1 adipocytes and C2C12 myotubes (n = 3 per group). (E–J) Serum levels of fetuin-A (E) and LECT2 (H), along with their positive correlations with serum ferritin (F and I) and NAS (G and J) levels. Box-and-whisker plots (median, IQR, minimum–maximum) with all individual data points are shown. Pearson correlation coefficients (r) and corresponding P values are indicated. Data are presented as the mean ± SEM. **P < 0.01, by unpaired, 2-tailed Student’s t test (A–D) or 1-way ANOVA followed by Tukey’s multiple-comparison test (E and H).Figure 9 Hepatocyte-specific FPN overexpression improves systemic metabolic dysfunction. (A) Experimental design illustrating hepatocyte-specific overexpression of FPN via AAV-mediated delivery under the control of the TBG promoter during HFD feeding in LT and Fpn-LKO mice. (B and C) qPCR analysis confirming hepatic FPN overexpression in LT mice (B) (n = 4–8 per group) and Fpn-LKO mice (C) (n = 3–4 per group). (D and E) DAB-enhanced Perls’ staining demonstrating reduced hepatic iron levels following FPN overexpression. Scale bars: 50 μm. (F–I) Western blot analysis showing decreased FoxO1 expression following FPN overexpression in LT mice (F and H) (n = 4 per group) and Fpn-LKO mice (G and I) (n = 3–4 per group). (J and K) qPCR analysis demonstrating downregulation of Fetua and Lect2 following FPN overexpression in LT mice (J) (n = 4–8 per group) and Fpn-LKO mice (K) (n = 3–4 per group). (L and M) GTT and ITT analyses in LT mice (n = 3–8 per group). (N and O) GTT and ITT analyses in Fpn-LKO mice (n = 3–4 per group). Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.Figure 10 Oral iron chelation ameliorates systemic metabolic dysfunction. (A) Therapeutic administration protocol of an oral iron chelator, DFP, in HFD-fed Fpn-LKO mice. (B) Quantification of hepatic iron content (n = 7–8 per group). (C) Western blot analysis of hepatic FoxO1 expression (n = 3–4 per group). (D and E) Western blot analysis of serum fetuin-A and corresponding quantification (n = 4 per group). (F) qPCR analysis of hepatic Lect2 mRNA levels (n = 7–8 per group). (G and H) GTT analysis (n = 7 per group). (I and J) ITT analysis (n = 6 per group). Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.
그림 출처: PubMed Central 공개 논문 (PMC13262725)
Conflict of interest statement
The authors have declared that no conflict of interest exists.
그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
The Journal of clinical investigation · eISSN 1558-8238
