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Sensitivity to thyroid hormones is associated with advanced fibrosis in euthyroid patients with non-alcoholic fatty liver disease: A cross-sectional study

Open AccessPublished:July 16, 2022DOI:https://doi.org/10.1016/j.dld.2022.06.021

      Abstract

      Background and aims

      The relationship between thyroid hormone sensitivity and the occurrence of advanced hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) remains unclear. We aimed to explore the association between sensitivity to thyroid hormones and advanced fibrosis (F3-F4) of NAFLD in patients with biopsy-proven euthyroid NAFLD.

      Methods

      In this study, 129 participants with biopsy-proven euthyroid NAFLD were enrolled, all of whom underwent thyroid function tests and liver biopsy. Indicators reflecting the sensitivity to thyroid hormones were also calculated. A logistic regression model was used to evaluate the association between thyroid hormone sensitivity and risk of advanced liver fibrosis.

      Results

      Among the 129 participants, 40 (31.0%) had advanced fibrosis. Advanced fibrosis was independently associated with TSH, FT3, FT3/FT4, thyrotroph T4 resistance index (TT4RI), TSH index (TSHI), and thyroid feedback quantile-based index (TFQI) (P<0.05), even after adjusting for sex, age, and metabolic factors. The combination of TFQI with age, waist circumference (WC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) performed best for advanced fibrosis diagnosis.

      Conclusion

      In euthyroid NAFLD patients, higher FT3/FT4, TFQI, TT4RI, and TSHI values were strongly associated with an increased incidence of advanced liver fibrosis. The combination of TFQI with age, WC, TGs, and LDL-C can be used as a predictor for advanced fibrosis in patients with NAFLD.

      Keywords

      1. Introduction

      Non-alcoholic fatty liver disease (NAFLD) is a predominant chronic liver disease characterized by increased abnormal triglycerides (TGs) accumulation in hepatocytes [
      • Younossi Z.
      • et al.
      Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.
      ,
      • Zheng K.I.
      • et al.
      Validation of Baveno VI and expanded Baveno VI criteria to identify high-risk varices in patients with MAFLD-related compensated cirrhosis.
      . NAFLD is a wide-spectrum clinical pathological syndrome involving simple steatosis and non-alcoholic steatohepatitis (NASH), where the latter may gradually progress to liver fibrosis or cirrhosis without effective intervention [
      • Younossi Z.M.
      • et al.
      Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes.
      ]. In addition, an increasing number of studies have shown that metabolic disorders are involved in the occurrence and development of NAFLD [
      • Anstee Q.M.
      • Targher G.
      • Day C.P.
      Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.
      ].
      Dysregulation of hepatic lipid metabolism may contribute to intrahepatic fat deposition and the subsequent progression to NAFLD. Thyroid hormones (THs) play an important role in liver lipid metabolism by regulating de novo lipogenesis (DNL), fatty acid oxidation, and cholesterol and carbohydrate metabolism [
      • Sinha R.A.
      • Singh B.K.
      • Yen P.M.
      Direct effects of thyroid hormones on hepatic lipid metabolism.
      ,
      • Ritter M.J.
      • Amano I.
      • Hollenberg A.N.
      Thyroid hormone signaling and the liver.
      . Candidate genes participating in these processes are mostly upregulated by THs via nuclear TH Receptor-β (THR-β) [
      • Braun D.
      • Schweizer U.
      Thyroid hormone transport and transporters.
      ,
      • Lazar M.A.
      Thyroid hormone receptors: multiple forms, multiple possibilities.
      . Results from clinical studies have indicated that patients with hypothyroidism are more likely to develop NAFLD [
      • Wang B.
      • et al.
      Thyroid function and non-alcoholic fatty liver disease in hyperthyroidism patients.
      ,
      • Xu C.
      • et al.
      Association between thyroid function and nonalcoholic fatty liver disease in euthyroid elderly Chinese.
      . Researchers also found that higher serum TSH and lower FT4 concentrations were significantly associated with steatosis and fibrosis in NAFLD [
      • Kim D.
      • et al.
      Low-normal thyroid function is associated with advanced fibrosis among adults in the United States.
      ,
      • Kim D.
      • et al.
      Subclinical hypothyroidism and low-normal thyroid function are associated with nonalcoholic steatohepatitis and fibrosis.
      . Hypothyroidism-induced NAFLD has been attributed to decreased lipid utilization, including reduced β-oxidation of free fatty acids (FFAs) and the clearance of TGs, with a consequent increase in the accumulation of TGs and low-density lipoprotein within hepatocytes [
      • Mavromati M.
      • Jornayvaz F.R.
      Hypothyroidism-associated dyslipidemia: potential molecular mechanisms leading to NAFLD.
      ]. Moreover, hypothyroidism is correlated with hepatic insulin resistance, which fails to suppress the production of endogenous glucose and stimulates DNL [
      • Ferrandino G.
      • et al.
      Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms.
      ].
      Nevertheless, the relationship between NAFLD with thyroid function remains disputable in clinical studies [
      • Kim D.
      • et al.
      Low-normal thyroid function is associated with advanced fibrosis among adults in the United States.
      ,
      • Kim D.
      • et al.
      Subclinical hypothyroidism and low-normal thyroid function are associated with nonalcoholic steatohepatitis and fibrosis.
      ,
      • Liu Y.
      • et al.
      Thyroid function and risk of non-alcoholic fatty liver disease in euthyroid subjects.
      ,
      • Manka P.
      • et al.
      Low free triiodothyronine is associated with advanced fibrosis in patients at high risk for nonalcoholic steatohepatitis.
      . In particular, only a few studies have investigated the potential correlation between thyroid hormones and histological characteristics related to NAFLD progression, including steatosis and fibrosis. To our knowledge, no study has focused on the relationship between sensitivity to THs and the occurrence of advanced liver fibrosis in patients with NAFLD. Thyroid feedback quantile-based index (TFQI) is a novel metabolism-related indicator that reflects the sensitivity of the hypothalamus–pituitary–thyroid axis to THs [
      • Laclaustra M.
      • et al.
      Impaired sensitivity to thyroid hormones is associated with diabetes and metabolic syndrome.
      ]. Therefore, we analyzed thyroid function in a biopsy-proven NAFLD cohort with euthyroidism, and investigated the potential relationship between THs sensitivity and NAFLD-related fibrosis.

      2. Materials and methods

      2.1 Study population

      We designed a cross-sectional study using a biopsy-proven NAFLD cohort from the Department of Hepatology, Tianjin Second People's Hospital, China from January 2017 to January 2021. The inclusion criteria were biopsy-proven NAFLD patients between the age of 18–75 years. The exclusion criteria were as follows:1) excessive alcohol intake (> 140 g/week for women or > 210 g/week for men, patients with alcohol dependence or alcoholism); 2) presence of other forms of liver disease, including Wilson disease, viral hepatitis, autoimmune liver disease, primary biliary cholangitis or other cholangitis, drug-induced liver injury, Budd-Chiari syndrome, and human immunodeficiency virus infection; and 3) presence of over-hypothyroidism or hyperthyroidism. Finally, 129 patients with biopsy-proven NAFLD and euthyroidism were enrolled in this study. The protocols and informed consent were approved by the Ethics Committee of Tianjin Second People's Hospital. The study complied with the principles of the Declaration of Helsinki. All participants provided informed consent before the study.

      2.2 Clinical data and demographic information

      Clinical data, including sex, age, medical history (hypertension, diabetes, or impaired fasting glucose [IFG]), self-reported alcohol consumption, and medication history, were obtained from medical records. Anthropometric information included blood pressure (BP), WC, height, and body weight. Body mass index (BMI) was calculated by dividing the weight by the square of height. The anthropometric characteristics of the participants were measured according to standard methods.

      2.3 Serological data

      All participants underwent blood tests at the time of liver biopsy. Blood samples were collected at 6:00 a.m. after overnight fasting. The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TIBL), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), albumin (ALB), prothrombin time (PT), platelets (PLT), fasting blood glucose (FBG), high-density lipoprotein cholesterol (HDL-C), LDL-C, total cholesterol (TC), and TGs were biochemically analyzed using a Hitachi-7180 (Hitachi Co., Tokyo, Japan) analyzer.
      Serum levels of FT3, FT4, and TSH were obtained using electrochemical luminescent immunoassays (Cobas e 411 analyzer, Roche Diagnostics, Switzerland). Normal reference value ranges of thyroid function in adults were 0.27–4.2 mIU/L for TSH, 3.1–6.8 pmol/L for FT3, and 12.0–22.0 pmol/L for FT4. Serum levels of TSH, FT3, and FT4 within the normal reference range were defined as the euthyroid state [
      • Amouzegar A.
      • et al.
      Reference limit of thyrotropin (TSH) and free thyroxine (FT4) in thyroperoxidase positive and negative subjects: a population based study.
      ]. Three indices (TFQI, thyrotroph T4 resistance index [TT4RI], and TSH index [TSHI]) were used to evaluate the central sensitivity to thyroid hormones. The calculation formulas for TSHI and TT4RI were obtained from a previous study [
      • Mehran L.
      • et al.
      Reduced sensitivity to thyroid hormone is associated with diabetes and hypertension.
      ]. TFQI was calculated according to the method proposed by Laclaustra et al. [
      • Laclaustra M.
      • et al.
      Impaired sensitivity to thyroid hormones is associated with diabetes and metabolic syndrome.
      ]. Lower TT4RI, TSHI, and TFQI values indicate higher central sensitivity to thyroid hormones.

      2.4 Histological assessments

      Each liver tissue section measured at least 10 mm and contained more than 11 portal tracks. Liver sections were stained with hematoxylin-eosin and Masson trichrome after formalin fixation and paraffin embedding. Liver sections were reviewed and described by experienced pathologists, who were unable to obtain any clinical information. The diagnosis of NAFLD was based on the Brunt criteria [
      • Musso G.
      • et al.
      Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity.
      ] and the degree of hepatic steatosis and inflammation was evaluated using the NAFLD activity score (NAS) system [
      • Kleiner D.E.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ]. Liver fibrosis stages were graded from F0 to F4 according to the Kleiner score (stage F0: Absence of fibrosis; stage F1: perisinusoidal or periportal fibrosis without septa; stage F2: combined periportal and perisinusoidal fibrosis with tiny septa; stage F3: septal or bridging fibrosis, without cirrhosis; and stage F4: cirrhosis) [
      • Kleiner D.E.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ]. Fibrosis stages–F3-F4 are regarded as advanced fibrosis [
      • Staufer K.
      • et al.
      Evaluation and comparison of six noninvasive tests for prediction of significant or advanced fibrosis in nonalcoholic fatty liver disease.
      ].

      2.5 Statistical analysis

      Categorical variables are shown as numbers (n) with proportions (%) and were compared using the chi-square test. Continuous variables with or without normal distribution are shown as mean ± standard deviation (SD) or median (25th quartile and 75th quartile), respectively. The Student's t-test or Mann–Whitney U test was used to compare the variables in different groups. The association between thyroid parameters and the clinical and pathological characteristics of the patients was evaluated using Spearman's coefficient correlation analysis (r). Logistic regression analysis was performed to evaluate the association between thyroid parameters and advanced fibrosis. Confidence intervals (CIs) and odds ratios (ORs) were calculated. Receiver operating characteristic (ROC) was also analyzed to evaluate the effectiveness of the indicators in predicting advanced fibrosis. The optimal cut-off points, sensitivity, and specificity of the indicators were determined using the DeLong method. P values < 0.05 (two-sided) were considered statistically significant. Statistical analysis was performed using IBM SPSS software (version 23 for Mac).

      3. Results

      3.1 General characteristics of the cohort

      Table 1 presents the baseline characteristics of the study cohort. In total, 129 patients with a median age of 47.0 years were enrolled in this study. In this study population, 65 (50.4%) patients were women, and the occurrence of advanced liver fibrosis was 31.0%. The prevalence of diabetes and IFG was 46.5%. The mean level of BMI and the median level of WC were 27.7 kg/m2 and 95.0 cm, respectively. The number and proportion of patients based on fibrosis stage were as follows: F0, 28 (21.7%); F1, 31 (24.0%); F2, 30 (23.3%); F3, 21 (16.3%); and F4, 19 (14.7%). The patients were divided into two groups according to sex. Female patients were more likely to have higher serum TC and HDL-C levels than male patients. Additional information regarding the participants is provided in Table 1.
      Table 1General characteristics of euthyroid NAFLD patients.
      IndexTotal (N = 129)Male (n = 64)Female (n = 65)p
      Age47.0 (32.0, 57.0)47.0 (30.5, 55.8)49.0 (33.0, 59.5)0.287
      Hypertension, n (%)44 (34.1)22 (34.4)22 (33.8)0.949
      Diabetes/IFG, n (%)60 (46.5)26 (40.6)34 (52.3)0.183
      BMI(Kg/m2)27.7 ± 3.727.5 ± 3.627.8 ± 3.60.572
      WC (cm)95.0 (89.0, 101.5)95.5 (90.0, 102.5)95.0 (87.0, 101.5)0.424
      ALT (U/L)32.0 (20.7, 49.0)30.0 (21.3, 48.2)33.0 (20.0, 50.0)0.779
      AST (U/L)26.0 (19.5, 35.0)26.5 (19.3, 34.0)26.0 (19.5, 39.5)0.458
      ALP (U/L)74.0 (58.0, 95.5)78.0 (64.0, 97.1)71.0 (55.6, 95.5)0.208
      GGT (U/L)66.0 (43.0, 107.0)64.5 (44.0, 104.0)71.0 (42.0, 110.7)0.903
      TBIL (umol/L)14.0 (10.3, 19.0)14.1 (10.7, 17.9)13.8 (9.9, 19.3)0.989
      ALB (g/L)45.1 (41.0, 47.8)46.1 (41.1, 48.2)44.4 (40.5, 47.0)0.219
      PLT (109/L)221.2 ± 83.6220.1 ± 81.9222.3 ± 85.90.88
      PT (s)12.2 (10.9, 12.9)12.2 (10.8, 13.0)12.0 (11.0, 12.8)0.576
      TC (mmol/L)5.43 (4.60, 5.97)5.15 (4.30, 5.77)5.48 (4.88, 6.05)0.03
      TGs (mmol/L)1.69 (1.31, 2.06)1.77 (1.33, 2.06)1.63 (1.30, 2.12)0.262
      HDL-C (mmol/L)1.32±0.361.24±0.331.39±0.370.016
      LDL-C (mmol/L)2.99±0.793.00±0.752.99±0.840.974
      FBG (mmol/L)5.86 (5.27, 6.63)5.75 (5.08, 6.51)6.01 (5.37, 6.70)0.166
      FT3 (pmol/L)4.86±0.724.91±0.664.80±0.770.392
      FT4 (pmol/L)15.72±2.2115.73±2.2115.72±2.220.986
      TSH (uIU/ml)2.29 (1.60, 3.40)2.22 (1.55, 3.54)2.32 (1.65, 3.33)0.929
      FT3/FT40.31±0.050.32±0.050.31±0.050.357
      TT4RI36.2 (26.3, 48.3)35.7 (25.3, 49.6)38.6 (26.7, 48.1)0.966
      TSHI3.01 (2.71, 3.20)3.06 (2.66, 3.20)2.99 (2.72, 3.20)0.845
      TFQI0.01±0.310.01±0.320.01±0.310.951
      NAFLD activity score (NAS), n (%)
      0–444 (34.1)25 (39.1)19 (29.2)0.239
      5–885 (65.9)39 (60.9)46 (70.8)0.239
      Fibrosis stage, n (%)
      028 (21.7)12 (18.8)16 (24.6)0.419
      131 (24.0)16 (25.0)15 (23.1)0.798
      230 (23.3)14 (21.9)16 (24.6)0.713
      321 (16.3)14 (21.9)7 (10.8)0.088
      419 (14.7)8 (12.5)11 (16.9)0.478
      Advanced fibrosis, n (%)40 (31.0)22 (34.4)18 (27.7)0.412
      Data are expressed as mean (± standard deviation), median (quartile 25, quartile 75).
      BMI, body mass index; WC, waist circumference; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; TBIL, total bilirubin; ALB, albumin; PLT, Platelets; PT, prothrombin time; TC, Total cholesterol; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; FT3, free triiodothyronine; FT4, free thyroxine; TSH, thyroid stimulating hormone; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 resistance index; TSHI, TSH Index; TFQI, Thyroid Feedback Quantile-based Index.
      Patients were further divided into advanced (stages F3-F4) and non-advanced (stages F0-F2) fibrosis groups. Table 2 shows the general characteristics of the patients with different liver fibrosis stages. The incidence of advanced fibrosis in this cohort was 31.0%. There was no statistically significant difference in sex between the two groups (P = 0.412). Compared with patients without advanced fibrosis, patients with advanced fibrosis were older, and the serum concentrations of TGs, AST, ALP, PT, TSH, FT3, and values of FT3/FT4, TT4RI, TSHI, and TFQI were statistically higher (P<0.05), whereas the PLT count and ALB were significantly lower (P<0.05).
      Table 2General characteristics of euthyroid NAFLD patients according to fibrosis stage.
      IndexF (0–2) n = 89F (3–4) n = 40p
      Female sex, n (%)47 (52.8)18 (45.0)0.412
      Age43.0 (28.0,56.0)51.5 (42.0,61.0)0.031
      Diabetes/IFG, n (%)39 (43.8)21 (52.5)0.361
      BMI(Kg/m2)27.4 ± 3.828.1 ± 3.50.539
      WC (cm)95.0 (87.0, 99.5)96.0 (91.3, 103.0)0.106
      TC (mmol/L)5.43 (4.51, 5.87)5.45 (4.88, 6.07)0.378
      TGs(mmol/L)1.55 (1.21, 1.99)1.81 (1.58, 2.26)0.006
      HDL-C (mmol/L)1.33±0.381.30±0.280.676
      LDL-C (mmol/L)2.94 (2.43, 3.45)3.31 (2.57, 3.55)0.268
      FBG (mmol/L)5.82 (5.22, 6.65)6.28 (5.30, 6.61)0.521
      ALT (U/L)35.6 ± 23.742.3 ± 21.80.131
      AST (U/L)27.4 ± 15.038.1 ± 18.70.001
      ALP (U/L)79.4 ± 30.494.7 ± 54.10.042
      GGT (U/L)65.0 (42.5, 103.1)68.0 (45.3, 115.3)0.656
      TBIL (umol/L)15.1 ± 6.815.8 ± 7.50.613
      ALB (g/L)46.0 (42.6, 48.1)41.7 (39.5, 47.0)0.003
      PLT (109/L)238.0 (186.0, 282.5)173.5 (136.5, 242.3)<0.001
      PT (s)11.5 (10.6, 12.6)12.9 (12.1, 13.7)<0.001
      FT3 (pmol/L)4.74±0.785.11±0.490.002
      FT4 (pmol/L)15.75±2.2315.67±2.180.838
      TSH (uIU/ml)2.11 (1.45, 3.02)2.87 (2.08, 3.79)0.001
      FT3/FT40.30±0.050.33±0.050.004
      TT4RI34.8 ± 13.544.4 ± 13.4<0.001
      TSHI2.93 (2.57, 3.14)3.12 (2.88, 3.34)0.001
      TFQI0.01 (−0.24, 0.16)0.16 (−0.14, 0.33)0.023
      Data are expressed as mean (±standard deviation), median (quartile 25, quartile 75).
      IFG, impaired fasting glucose; BMI, body mass index; WC, waist circumference; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; TBIL, total bilirubin; ALB, albumin; PLT, Platelets; PT, prothrombin time; TC, Total cholesterol; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; FT3, free triiodothyronine; FT4, free thyroxine; TSH, thyroid stimulating hormone; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 resistance index; TSHI, TSH Index; TFQI, Thyroid Feedback Quantile-based Index.

      3.2 Correlation between thyroid parameters with clinical and pathological characteristics of NAFLD patients

      Table 3 shows the correlations between thyroid parameters and the clinical and pathological characteristics of the participants. Serum FT3 levels were positively correlated with WC, BMI, TGs, LDL-C, AST, and PT, but negatively correlated with HDL-C and PLT (P<0.05). Serum TSH levels were positively correlated with age, AST, and PT but negatively correlated with ALB and PLT (P<0.05). FT3/FT4 ratio was positively correlated with WC and PT and negatively correlated with PLT (P<0.05). TT4RI was positively correlated with age, AST, and PT and negatively correlated with PLT and ALB (P<0.05). TSHI was positively correlated with age and TGs, AST, and PT levels and negatively correlated with ALB (P<0.05). The TFQI was positively correlated with age, TGs, AST, and PT (P<0.05). All variables except FT4 were positively correlated with fibrosis stage (P<0.05).
      Table 3Association of thyroid parameters with clinical characteristics of patients.
      IndexFT3FT4TSHFT3/FT4TT4RITSHITFQI
      rprprprprprprp
      Age0.0670.448−0.010.9080.210.0170.0660.460.2280.0090.260.0030.230.009
      BMI (Kg/m2)0.1820.0390.0370.6790.0430.6260.1570.0760.070.4280.1040.240.1140.198
      WC (cm)0.3020.0010.0380.670.030.7380.2620.0030.050.5770.0770.3830.10.26
      TC (mmol/L)0.050.574−0.0350.6950.1610.0690.0780.3790.1650.0610.1340.130.0920.298
      TGs (mmol/L)0.2180.0130.0610.4890.0650.4660.1240.160.1330.2010.1770.0450.1780.044
      HDL-C (mmol/L)−0.1930.029−0.1040.240.1630.065−0.0580.5140.1410.1110.1050.2360.0650.463
      LDL-C (mmol/L)0.1930.0280.0090.9160.1260.1540.160.070.1490.0920.1270.1510.1030.248
      FBG (mmol/L)0.0310.724−0.080.370.0970.2730.0820.3580.0860.330.0670.4490.0280.753
      ALT (U/L)0.1280.1490.0220.8030.0870.3250.0710.4250.1220.1680.1480.0940.0990.264
      AST (U/L)0.210.0170.0470.5970.2550.0040.1410.1120.2930.0010.3010.0010.250.004
      ALB (g/L)−0.0070.9380.0410.643−0.2060.019−0.0710.421−0.2030.021−0.1760.046−0.1580.074
      PLT (109/L)−0.1850.0360.1070.227−0.2290.009−0.2810.001−0.2010.023−0.150.09−0.0880.322
      PT (s)0.306<0.0010.0570.5230.180.0420.2410.0050.2180.0130.2380.0070.2160.014
      Fibrosis stage0.2090.017−0.0750.40.352<0.0010.2890.0010.348<0.0010.315<0.0010.2420.006
      BMI, body mass index; WC, waist circumference; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; ALB, albumin; PLT, Platelets; PT, prothrombin time; TC, Total cholesterol; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; FT3, free triiodothyronine; FT4, free thyroxine; TSH, thyroid stimulating hormone; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 resistance index; TSHI, TSH Index; TFQI, Thyroid Feedback Quantile-based Index.

      3.3 Relationship between thyroid parameters with advanced fibrosis

      Table 4 shows the association between thyroid parameters and advanced fibrosis. In the unadjusted model (Model 1), increased levels of TSH (OR=1.868, 95% CI 1.263–2.765), FT3 (OR=2.135, 95% CI 1.205–3.785), FT3/FT4 (OR=1.755, 95% CI 1.178–2.615), TT4RI (OR=2.078, 95% CI 1.367–3.16), TSHI (OR=2.152, 95% CI 1.339–3.459), and TFQI (OR=1.725, 95% CI 1.133–2.627) were associated with a higher risk of advanced fibrosis. After adjusting for age and sex (Model 2), increased TSH (OR=1.777, 95% CI 1.189–2.656), FT3 (OR=2.102, 95% CI 1.178–3.75), FT3/FT4 (OR=1.697, 95% CI 1.133–2.541), TT4RI (OR=1.933, 95% CI 1.302–3.05), TSHI (OR=2.047, 95% CI 1.261–3.322), and TFQI (OR=1.65, 95% CI 1.072–2.539) were positively associated with advanced fibrosis. After further adjustments for age, sex, diabetes or IFG, BMI, WC, TC, TGs, and FBG (Model 3), elevated TSH (OR=1.831, 95% CI 1.2–2.795), FT3 (OR=2.128, 95% CI 1.122–4.037), FT3/FT4 (OR=1.726, 95% CI 1.116–2.67), TT4RI (OR=2.026, 95% CI 1.296–3.167), TSHI (OR=2.046, 95% CI 1.242–3.369), and TFQI (OR=1.628, 95% CI 1.047–2.53) remained significantly positively associated with advanced fibrosis (Table 4).
      Table 4Association of thyroid parameters with advanced fibrosis.
      Thyroid parametersModel 1Model 2Model 3
      OR (95% CI)pOR (95% CI)pOR (95% CI)p
      FT32.135 (1.205, 3.785)0.0092.102 (1.178, 3.75)0.0122.128 (1.122, 4.037)0.021
      FT40.982 (0.829, 1.164)0.8360.982 (0.826, 1.166)0.8330.969 (0.809, 1.161)0.734
      TSH1.868 (1.263, 2.765)0.0021.777 (1.189, 2.656)0.0051.831 (1.2, 2.795)0.008
      FT3/FT4 (+1SD)1.755 (1.178, 2.615)0.0061.697 (1.133, 2.541)0.011.726 (1.116, 2.67)0.014
      TT4RI (+1SD)2.078 (1.367, 3.16)0.0011.993 (1.302, 3.05)0.0012.026 (1.296, 3.167)0.002
      TSHI (+1SD)2.152 (1.339, 3.459)0.0022.047 (1.261, 3.322)0.0042.046 (1.242, 3.369)0.005
      TFQI (+1SD)1.725 (1.133, 2.627)0.0111.65 (1.072, 2.539)0.0231.628 (1.047, 2.53)0.03
      Model 1: unadjusted.
      Model 2: adjusted for age and sex.
      Model 3: Model 2 + further adjusted for diabetes/IFG, WC, BMI, TC, TGs and FBG.
      IFG, impaired fasting glucose; WC, waist circumference; BMI, body mass index; TC, Total cholesterol; TGs, triglycerides; FBG, fasting blood glucose; OR, odds ratio; CI, confidence interval; FT3, free triiodothyronine; FT4, free thyroxine; TSH, thyroid stimulating hormone; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 Resistance Index; TSHI, TSH Index; TFQI, Thyroid Feedback Quantile-based Index.

      3.4 ROC analyses for advanced fibrosis prediction

      The TFQI, FT3/FT4, TT4RI, and TSHI were selected for predicting advanced fibrosis in patients with NAFLD. As shown in Fig. 1, the area under the ROC curve (AUC), optimal cut-off points, sensitivity, and specificity for advanced fibrosis prediction were as follows: TFQI, 0.625, 0.14, 0.525, and 0.73; FT3/FT4, 0.658, 0.30, 0.775, and 0.584; TT4RI, 0.698, 43.0, 0.55, and 0.764; and TSHI, 0.678, 3.07, 0.65, and 0.64, respectively. Multifactor models, including age, WC, TGs, and indicators reflecting sensitivity to THs, were further applied to better predict advanced fibrosis (Fig. 2). The combination of TFQI with age, WC, TGs, and LDL-C performed best for advanced fibrosis diagnosis, with 0.725 sensitivity, 0.64 specificity, and an AUC of 0.7.
      Fig 1
      Fig. 1ROC curves for prediction of advanced liver fibrosis in NAFLD patients. (A) ROC curve for prediction of advanced liver fibrosis from TFQI; (B) ROC curve for prediction of advanced liver fibrosis from FT3/FT4. (C) ROC curve for prediction of advanced fibrosis from TT4RI. (D) ROC curve for prediction of advanced fibrosis from TSHI. ROC, receiver operating characteristic; FT3, free triiodothyronine; FT4, free thyroxine; TFQI, Thyroid Feedback Quantile-based Index; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 resistance Index; TSHI, TSH Index.
      Fig 2
      Fig. 2Multifactor models for prediction of advanced liver fibrosis in NAFLD patients. (A) ROC curve for prediction of advanced liver fibrosis from TFQI + age + WC + TGs + LDL-C; (B) ROC curve for prediction of advanced liver fibrosis from FT3/FT4 + age + WC + TGs + LDL-C. (C) ROC curve for prediction of advanced fibrosis from TT4RI+ age + WC + TGs + LDL-C. (D) ROC curve for prediction of advanced fibrosis from TSHI+ age + WC + TGs + LDL-C.  ROC, receiver operating characteristic; FT3, free triiodothyronine; FT4, free thyroxine; TFQI, Thyroid Feedback Quantile-based Index; FT3/FT4, FT3 to FT4 ratio; TT4RI, TSH T4 resistance Index; TSHI, TSH Index; WC, waist circumference; TGs, triglycerides.

      4. Discussion

      To the best of our knowledge, the association between sensitivity to THs and NAFLD-related advanced liver fibrosis has not been studied, especially in euthyroid individuals. The present study demonstrated that increased central resistance to thyroid hormones was positively associated with an increased incidence of advanced hepatic fibrosis. The innovation of this study was the inclusion of sensitivity to THs, in addition to thyroid function, as a predictor of advanced fibrosis.
      THs are critical regulators of hepatic lipid metabolism, including the synthesis and metabolism of fatty acids and cholesterol [
      • Jung K.Y.
      • et al.
      Association between thyroid function and lipid profiles, apolipoproteins, and high-density lipoprotein function.
      ]. THs have profound effects on stimulating lipolysis to generate free fatty acids (FFAs) and regulate FFA uptake in the liver [
      • Klieverik L.P.
      • et al.
      Thyroid hormone effects on whole-body energy homeostasis and tissue-specific fatty acid uptake in vivo.
      ]. Additionally, THs can stimulate DNL in the liver by upregulating the expression of the main genes involved in lipogenesis [
      • Mendoza A.
      • et al.
      Thyroid hormone signaling promotes hepatic lipogenesis through the transcription factor.
      ]. Moreover, THs can help regulate serum concentrations of cholesterol by increasing the biosynthesis, export, reverse transport, and conversion of cholesterol into bile acids [
      • Ness G.C.
      Thyroid hormone. Basis for its hypocholesterolemic effect.
      ]. Furthermore, THs can regulate hepatic mitochondrial activity and fatty acid oxidation (FAO) via the electron transport chain and tricarboxylic acid (TCA) cycle [
      • Sinha R.A.
      • et al.
      Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.
      ,
      • Harper M.E.
      • Seifert E.L.
      Thyroid hormone effects on mitochondrial energetics.
      . Another effect of THs is increased peripheral glucose uptake and insulin sensitivity via action on skeletal muscles [
      • Dimitriadis G.D.
      • Raptis S.A.
      Thyroid hormone excess and glucose intolerance.
      ,
      • Kalra S.
      • Aggarwal S.
      • Khandelwal D.
      Thyroid dysfunction and type 2 diabetes mellitus: screening strategies and implications for management.
      .
      NAFLD is a metabolic syndrome that manifests in the liver. The development of NAFLD is closely associated with metabolic abnormalities, including diabetes mellitus, hyperlipidemia, and cardiovascular disease [
      • Adams L.A.
      • et al.
      Non-alcoholic fatty liver disease and its relationship with cardiovascular disease and other extrahepatic diseases.
      ]. Studies focusing on the link between thyroid function and the incidence and development of NAFLD are increasing [
      • Efstathiadou Z.A.
      • Kita M.D.
      • Polyzos S.A.
      Thyroid dysfunction and non-alcoholic fatty liver disease.
      ]. As noted, THs exhibit multiple effects on hepatic lipid metabolism; therefore, thyroid dysfunction is positively correlated with obesity and metabolic disorders [
      • Liu F.H.
      • et al.
      Subclinical hypothyroidism and metabolic risk factors association: a health examination-based study in northern Taiwan.
      ,
      • Oh H.S.
      • et al.
      Association between thyroid dysfunction and lipid profiles differs according to age and sex: results from the Korean national health and nutrition examination survey.
      . Recently, several clinical studies conducted in euthyroid individuals have investigated the link between thyroid function and the occurrence of NAFLD; however, the results failed to reach a consensus. Previous studies have shown that lower serum concentrations of FT4 are strongly correlated with the occurrence of NAFLD [
      • Tao Y.
      • et al.
      Thyroid function is associated with non-alcoholic fatty liver disease in euthyroid subjects.
      ,
      • Zhang X.
      • et al.
      Serum thyroid hormones levels are significantly associated with nonalcoholic fatty liver disease in euthyroid chinese population.
      ,
      • Shao C.
      • et al.
      Serum level of free thyroxine is an independent risk factor for non-alcoholic fatty liver disease in euthyroid people.
      ], while the results from two cross-sectional studies conducted in the Chinese population failed to support this [
      • Liu Y.
      • et al.
      Thyroid function and risk of non-alcoholic fatty liver disease in euthyroid subjects.
      ,
      • Lai S.
      • et al.
      Sensitivity to thyroid hormone indices are closely associated with NAFLD.
      . The reported results were inconsistent regarding the association between FT3 and NAFLD-related fibrosis [
      • Manka P.
      • et al.
      Low free triiodothyronine is associated with advanced fibrosis in patients at high risk for nonalcoholic steatohepatitis.
      ,
      • Du J.
      • et al.
      Association between thyroid hormone levels and advanced liver fibrosis in patients with type 2 diabetes mellitus and non-alcoholic fatty liver disease.
      ,
      • Guo W.
      • et al.
      Free triiodothyronine is associated with hepatic steatosis and liver stiffness in euthyroid Chinese adults with non-alcoholic fatty liver disease.
      . Some studies have indicated that higher TSH levels may be an essential risk factor accelerating the progression of NAFLD [
      • Martínez-Escudé A.
      • et al.
      TSH levels as an independent risk factor for NAFLD and liver fibrosis in the general population.
      ,
      • Tahara K.
      • et al.
      Thyroid-stimulating hormone is an independent risk factor of non-alcoholic fatty liver disease.
      , while another study reported that the occurrence of NAFLD was strongly correlated with increased concentration of FT3 and decreased concentration of FT4, but not TSH [
      • van den Berg E.H.
      • et al.
      Higher free triiodothyronine is associated with non-alcoholic fatty liver disease in euthyroid subjects: the lifelines cohort study.
      ]. In addition, a large meta-analysis conducted by Mantovani et al. indicated that hypothyroidism is closely related to the prevalence and severity of NAFLD [
      • Mantovani A.
      • et al.
      Association between primary hypothyroidism and nonalcoholic fatty liver disease: a systematic review and meta-analysis.
      ]. All of the above studies failed to explore the link between thyroid parameters and NAFLD-related advanced fibrosis, especially in a biopsy-proven euthyroid NAFLD population.
      In the present study, our results indicated that both TSH and FT3 levels were independently correlated with advanced fibrosis, even after adjusting for metabolic factors. However, we failed to find an association between advanced fibrosis and serum FT4 level. Higher levels of FT3-induced advanced fibrosis may be related to the expression of iodothyronine deiodinase (DIO), which is increased in obese people and animals, and accelerates the conversion from FT4 to FT3 [
      • Ortega F.J.
      • et al.
      Type I iodothyronine 5′-deiodinase mRNA and activity is increased in adipose tissue of obese subjects.
      ]. In addition, higher FT3 levels also increase lipolysis and stimulated DNL within hepatocytes [
      • Cachefo A.
      • et al.
      Hepatic lipogenesis and cholesterol synthesis in hyperthyroid patients.
      ], which may accelerate the progression of NAFLD. Studies have also confirmed that higher FT3 levels are strongly linked to metabolic risk factors that participate in NAFLD, such as TGs, blood glucose, and insulin [
      • Kim D.
      • et al.
      Low-normal thyroid function is associated with advanced fibrosis among adults in the United States.
      ,
      • Bano A.
      • et al.
      Thyroid function and the risk of nonalcoholic fatty liver disease: the Rotterdam study.
      . We also found that FT3 levels were correlated with BMI, WC, TC, and LDL-C levels in the present study. However, the causal relationship between FT3 level and advanced fibrosis remains unclear.
      We further explored the role and mechanism of the hypothalamus–pituitary–thyroid (HPT) axis in regulating circulating THs. Disorders caused by metabolic abnormalities are often accompanied by the dysregulation of the HPT axis [
      • Chaker L.
      • et al.
      Clinical aspects of thyroid function during ageing.
      ]. In Jostel et al. first defined the TSH index to estimate pituitary thyrotroph function [
      • Jostel A.
      • Ryder W.D.
      • Shalet S.M.
      The use of thyroid function tests in the diagnosis of hypopituitarism: definition and evaluation of the TSH Index.
      ]. Reduced sensitivity to THs has been confirmed to be associated with metabolic syndromes and its components, including hypertension and diabetes [
      • Mehran L.
      • et al.
      Reduced sensitivity to thyroid hormone is associated with diabetes and hypertension.
      ]. In Laclaustra et al. first reported that the TFQI was better in assessing sensitivity to THs than the TSHI and TT4RI. This study also indicated that higher TFQI values were significantly related to metabolic syndrome, diabetes, and diabetes-related mortality [
      • Laclaustra M.
      • et al.
      Impaired sensitivity to thyroid hormones is associated with diabetes and metabolic syndrome.
      ]. Recently, a cross-sectional study conducted by Lai et al. also reported that higher TFQI and FT3/FT4 values were positively correlated with dyslipidemia and NAFLD [
      • Lai S.
      • et al.
      Sensitivity to thyroid hormone indices are closely associated with NAFLD.
      ]. These studies provide an explanation for the link between thyroid dysfunction and metabolic disorders. However, to the best of our knowledge, the present study is the first to evaluate the relationship between sensitivity to THs and the risk of advanced fibrosis among patients with NAFLD.
      In addition to the HPT axis, abnormal peripheral DIO activity is associated with the prevalence of metabolic disorders. As mentioned earlier, the upregulation of peripheral DIO activity in obese subjects leads to increased conversion of FT4 to FT3, accelerating the progression of NAFLD [
      • Ortega F.J.
      • et al.
      Type I iodothyronine 5′-deiodinase mRNA and activity is increased in adipose tissue of obese subjects.
      ,
      • Cachefo A.
      • et al.
      Hepatic lipogenesis and cholesterol synthesis in hyperthyroid patients.
      . Many studies have shown that the FT3/FT4 ratio is associated with liver fibrosis and FT3 imbalance [
      • Guo W.
      • et al.
      Free triiodothyronine is associated with hepatic steatosis and liver stiffness in euthyroid Chinese adults with non-alcoholic fatty liver disease.
      ,
      • Chen P.
      • et al.
      Free triiodothyronine is associated with the occurrence and remission of nonalcoholic fatty liver disease in euthyroid women.
      . In this study, our results also showed that the incidence of advanced fibrosis increased in NAFLD patients with an increased FT3/FT4 ratio. Previous studies have confirmed that higher FT3/FT4 levels are positively correlated with metabolic disorders, such as obesity, elevated BP, and FBG levels [
      • Nie X.
      • et al.
      Characteristics of serum thyroid hormones in different metabolic phenotypes of obesity.
      ,
      • Roef G.L.
      • et al.
      Triiodothyronine and free thyroxine levels are differentially associated with metabolic profile and adiposity-related cardiovascular risk markers in euthyroid middle-aged subjects.
      ,
      • Urrunaga-Pastor D.
      • et al.
      High free triiodothyronine and free-triiodothyronine-to-free-thyroxine ratio levels are associated with metabolic syndrome in a euthyroid population.
      ]. Consistent with our results, a Lifelines Cohort Study found that the prevalence of NAFLD significantly increased with elevated FT3/FT4 in a population with euthyroidism [
      • van den Berg E.H.
      • et al.
      Higher free triiodothyronine is associated with non-alcoholic fatty liver disease in euthyroid subjects: the lifelines cohort study.
      ].
      Since various metabolic risk factors accelerate the occurrence and progression of NAFLD, we speculate that sensitivity to THs may be a potential predictor of advanced liver fibrosis. In this study, TFQI, TSHI, TT4RI, and FT3/FT4 were used, and all of these variables were found to be significantly positively associated with higher risks of advanced fibrosis. Few studies have focused on the relationship between TFQI and NAFLD-related fibrosis, and the exact mechanisms remain unclear.
      The strengths of the present study are that all participants were diagnosed with NAFLD by liver biopsy, and thyroid parameters were measured under the same facilities and conditions. However, this study has several shortcomings. First, this cross-sectional study was insufficient to assess the causal relationship between the thyroid parameters and advanced fibrosis. Second, the participants in this cohort were all Chinese individuals from a single medical center; therefore, the conclusions of this study may not be applicable to other populations. Thirdly, the total number of cases was small, because only patients with biopsy-proven euthyroid NAFLD were included to ensure the accuracy of the study. Finally, individuals without NAFLD were not included and analyzed in this study. Therefore, further multicenter prospective studies are required to confirm our results.

      5. Conclusions

      In conclusion, this study showed that FT3/FT4, TT4RI, TSHI, and TFQI were positively associated with advanced fibrosis in euthyroid NAFLD patients. This study provides evidence that TFQI combined with age, WC, TGs, and LDL-C levels may be a novel predictor of advanced liver fibrosis. Therefore, older patients with NAFLD with lower central sensitivity to THs and dyslipidemia should be treated to prevent the progression of liver fibrosis. However, extensive studies are necessary to confirm our results and reveal their potential mechanisms.

      Funding

      This work was funded by the Natural Science Foundation of Tianjin City (No. 20JCYBJC01150) and Tianjin Health Science and Technology Project (No. TJWJ2021QN063).

      Declaration of Competing Interest

      None declared.

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