MAFLD in Patients with Cushing's Disease Is Negatively Associated with Low Free Thyroxine Levels Rather than with Cortisol or TSH Levels

Purpose This study aims to analyze the clinical characteristic of metabolic associated fatty liver disease (MAFLD) in patients with active Cushing's disease (CD) and determine associations of thyroid hormones with MAFLD. Methods Patients with active CD were included in this cross-sectional study. All subjects were assessed for hepatic steatosis by abdominal ultrasonography and thyroid functions. Demographic and clinical characteristic parameters were collected for correlation analysis and logistic analysis. Results 290 individuals with active CD were included in Huashan hospital from January 2014 to February 2022. We found that the prevalence of CD with MAFLD was 33.79%. The MAFLD group had a lower level of FT4 and a higher level of FT3/FT4 but no difference in levels of cortisol, 24 h UFC, TSH, TT4, TT3, and FT3. Correlation analysis showed positive associations of TSH, TT4, TT3, FT3, and FT3/FT4 with BMI. In age-, BMI-, sex-, cortisol-, and 24 h UFC-adjusted analysis, FT4 was independently associated with MAFLD in patients with CD. This association remained similar even after adjusting for the presence of metabolic syndrome components. Conclusion Lower FT4 levels were associated with higher risk of MAFLD in patients with CD. FT4 may be used as a helpful indicator to predict MAFLD and provide novel ideas for the treatment of MAFLD in patients with CD in the future.


Introduction
Cushing's disease (CD), the most common cause of endogenous Cushing's syndrome (CS), is characterized by a lengthy and inappropriate exposure to excessive free glucocorticoids due to adrenocorticotropic hormone (ACTH) producing pituitary tumors [1]. Long-term excessive glucocorticoids infuence the metabolism of glucose, protein, and lipids and thus lead to a complex metabolic syndrome, which consists of hypertension, abnormal glucose metabolism, dyslipidaemia, and obesity [2]. However, liver steatosis, regarded as the hepatic manifestation of metabolic syndrome and is associated with an increased risk of cardiovascular disease, has not been fully investigated in CD [3].
In addition to metabolic complications, excessive cortisol also leads to alterations in functions of the remaining endocrine hormone axes, including hypothalamic-pituitarythyroid (HPT) axes. Usually, excessive cortisol inhibits the release of thyrotropin-releasing hormone (TRH) in the hypothalamus and thyroid stimulating hormone (TSH) in the pituitary, leading to a decrease in thyroxine (T4) and triiodothyronine (T3) in CS patients [4]. Tyroid functions would gradually normalize in most remitted or cured CD patients after transsphenoidal pituitary tumor resection [5]. In adrenal CS patients, serum TSH and free T4 levels were also decreased, and dysfunction of the hypothalamic-pituitary-thyroid axis was reversible after adrenalectomy [4].
It has been known that thyroid hormones are critical regulators for lipid metabolism by stimulating lipolysis in adipose tissues, regulating cholesterol metabolism, and increasing mitochondrial β-oxidation, autophagy, and the expression of genes involved in de novo lipogenesis in the liver [6]. It reports that patients in hypothyroidism had associated dyslipidemia and subsequently associated with nonalcoholic fatty liver disease (NAFLD) [7]. Mildly hypothyroid decreased insulin secretion and therefore suppressed the lipolysis response of adipose tissue to insulin and increased shuttling of fatty acids (FAs) to the liver, where they are esterifed and accumulated as triglycerides [7]. However, characteristics of thyroid hormones in MAFLD in patients with CD were also rarely investigated. Terefore, this cross-sectional study aims to evaluate the prevalence, biochemical characteristics, and risk/protective factors in CD patients with metabolic associated fatty liver disease (MAFLD) [8], especially exam associations of thyroid hormones with MAFLD in active CD.

Study Design.
Tis was a cross-sectional study performed at the department of Endocrinology and Metabolism in Huashan Hospital. According to ICD-10, all subjects diagnosed to Cushing's disease and admitted to hospital treatment were included from January 2014 to February 2022. To avoid subjective bias, patients received detailed clinical examination after enrollment by the same group of endocrinology specialists. Our study was approved by the Ethics Committee of Huashan Hospital.

Participants and Defnitions.
We included patients diagnosed with Cushing's disease at the department of Endocrinology and Metabolism in 2014-2022. Cushing's disease was defned as follows [9]: (1) exclude excessive exogenous glucocorticoid exposure and physiologic causes; (2) conditions and clinical features of Cushing's syndrome; (3) an elevated levels of 24 h UFC (at least two measurements); (4) failure of plasma cortisol decreased after an overnight 1 mg dexamethasone suppression test; and (5) pituitary lesion >6 mm showed by a magnetic resonance imaging (MRI) scan or positive results in an inferior petrosal sinus sampling in patients with negative MRI images or a lesion <6 mm.
Te defnition of MAFLD was according to a consensus proposed by a panel of experts from 22 countries, namely, hepatic fat content >5% (diagnosed by abdominal ultrasonography here) and accompany with any one of the following conditions: (1) overweight or obesity, (2) presence of diabetes mellitus, and (3) or at least two metabolic risk abnormalities (waist circumference ≥90/ 80 cm in men or women; blood pressure ≥130/85 mmHg or drug treatment; TG ≥ 1.70 mmol/L or drug treatment; HDL-c < 1.0 mmol/L for men and <1.3 mmol/L for women or drug treatment; prediabetes; HOMA-IR ≥ 2.5; and high-sensitivity C-reactive protein >2 mg/L) [8]. Patients with active CD were categorized into two groups for further analysis according to whether they sufered from MAFLD.
Additional exclusion criteria were as follows: (1) after transsphenoidal surgery, radiation therapy or pharmacological therapy for pituitary tumor, (2) use of anti-thyroid drugs or thyroid hormone or a history of thyroid surgery, and (3) missing thyroid function test results.

Data Collection and Calculations.
All data were obtained by reviewing the medical history in the electronic medical record system. Age, sex, weight, and height were asked or measured after registration. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gammaglutamyl transferase (GGT), albumin, platelets, total triglycerides (TG), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), glycated hemoglobin (HbA1c), blood glucose, insulin, C-peptide, and morning cortisol were detected under fasting condition between 8.00-10.00 am. Collecting 24-hour urine for detecting 24 h urine-free cortisol (24 h UFC) was also needed. All subjects were assessed for thyroid function, namely, thyroid stimulating hormone (TSH), total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3), and abdominal ultrasonography. After a systematic examination, comorbidities of each patient were judged and recorded by specialists. Te data about epidemiologic, clinical, and biochemical data were gathered by two researchers after carefully reviewing the patient fles. All the hormones were tested by the same assay.

Statistical Analysis.
All statistical analyses were performed by two researchers and were carried out by using Statistical Package for Social Sciences (version 26.0, SPSS, Inc., IBM, USA). A two-tailP value <0.05 was regarded as statistically signifcant. Continuous variables are given as the mean ± standard deviation when normally distributed and as the median with an interquartile range when skewedly distributed. In contrast, categorical variables are presented as absolute number (percentage). Te parametric Student's t-test was used to compare two groups for continuous variables, and the chi-squared test was used for categorical variables. 24 h UFC, TSH, AST, ALT, GGT, TG and HDL-c were log transformed before analysis. Te associations of thyroid functions with BMI were examined by Pearson's correlation and then graphed as line regression curves. Odds ratios (ORs) are given with 95% confdence intervals by using univariable logistic analysis and multivariable logistic analysis adjusted for confounders.

Results
We originally selected 479 patients with Cushing's disease (CD), 34.66% of which had ultrasonography diagnosed hepatic steatosis, from the Department of Endocrinology and Metabolism in Huashan Hospital from January 2014 to February 2022. After applying the exclusion criteria, we fnally included 290 patients with active CD in this study (as shown in Figure 1).

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International Journal of Endocrinology Among 290 enrolled patients with CD (mean age: 38.52 ± 12.84), more than thirty percent of patients had MAFLD (89/290, 33.79%). Females were more prone to sufer from CD when compared to males (78.28% versus 21.72%). 62.76% of patients with CD were overweight or obese (BMI > 24 kg/m 2 ), 58.62% of patients had diabetes or impaired glucose tolerance, 73.10% of patients had hypertension, 30.00% of patients had hyperlipidemia, and 15.86% of patients had central hypothyroidism (FT4 below the lower reference bound without elevated TSH). Compared with patients without MAFLD, CD patients with MAFLD had signifcantly higher BMI (P < 0.001), percentage of hypertension (P � 0.004), percentage of hyperlipidemia (P � 0.002), AST (P � 0.010), ALT (P < 0.001), GGT (P < 0.001), TG (P < 0.001), C-peptide (P � 0.013) levels, and a lower HDL level (P < 0.001). No diferences were found between two groups in the percentage of diabetes mellitus or impaired glucose tolerance, albumin, platelets, TC, LDL-c, fasting blood glucose (FBG), fasting insulin (FIN), 24 h free urine cortisol (24 h UFC), and morning cortisol levels as shown in Table 1 (All P > 0.05). Interestingly, serum FT4 levels were decreased and the FT3/ FT4 ratio was increased in patients with MAFLD (both P < 0.05), whereas the levels of TSH, TT3, and FT3 had not diference (all P > 0.05). Among all subjects with CD, the percentages of subjects with TSH, TT4, TT3, FT4, and FT3 levels below the lower limit of reference range were 24.48%, 18.28%, 49.66%, 16.2%, and 32.07%, respectively. Further analyses showed that the percentage of patients with FT4 below the reference range was lower in MAFLD than that in non-MAFLD (P � 0.040), but there were no diferences in the percentage of patients with TSH, TT4, TT3, and FT3 below the normal range between two groups (all P > 0.05).
We analyzed associations between hormones and MAFLD in patients with CD by logistic regression analyses ( Multivariate logistic regressions were performed to assess the associations between quartiles of thyroid hormones and MAFLD in patients with CD (Table 3). All patients were divided into 4 groups according to TSH, TT4, TT3, FT4, and FT3 quartiles, respectively. Te frst quartile was set to be the reference group. Te ORs for the third and fourth quartile groups of FT4 were 0.257 and 0.220 (95% CI: 0.089-0.742, P � 0.012; 95% CI: 0.059−0.824, P � 0.014) as shown in Table 3 (the P value for trend � 0.018). Nevertheless, no diferent association was observed between MAFLD and TSH, TT4, TT3, or FT3 (all P > 0.05).

Discussion
Tis study frstly described the characteristic of MAFLD in CD and explored the associations between thyroid parameters and MAFLD in patients with CD. Among all patients with CD, the prevalence of MAFLD was 33.79%. We found that subjects with MAFLD in patients with CD had lower FT4 levels than subjects without. However, the TSH, FT3, and cortisol levels were similar between two groups. Furthermore, there was an independent association between lower FT4 and higher risk of MAFLD in patients with active CD.
Hepatic steatosis, afected nearly 25.24% people around the world and regarded as the hepatic manifestation of metabolic syndrome [10], was not taken into consideration frequently when referred to the comorbidities of CS. A Chinese retrospective study enrolled 1652 patients with CS and found that 27.4% patients had hepatic steatosis [11]. Te prevalence of ultrasonography-diagnosed hepatic steatosis in all patients with CD (34.79%, 166/479) in this study was similar to the prevalence of hepatic steatosis in the normal population [10]. Although it is generally believed that glucocorticoids drive the development of NAFLD by acting on the liver and adipose tissue [12], no diferences in morning   International Journal of Endocrinology cortisol and 24 h UFC between MAFLD and non-MAFLD groups in patients with CD were found in this study. A large epidemiological study also showed that the plasma cortisol had no association with hepatic steatosis in the urban population [13]. However, no correlation between MAFLD and serum cortisol, whether in patients with CD and the normal population does not rule out that glucocorticoids play an important role in the development of hepatic steatosis. In vivo and in vitro studies showed that glucocorticoids drive hepatic steatosis by promoting De Novo Lipogenesis (DNL), free fatty acid utilization, and reducing VLDL secretion in the liver and increasing delivery of fatty acid from adipose tissues into the liver [12]. However, the exact course of active CD before diagnosed in this study was unclear, which would infuence the development of hepatic steatosis and the level of serum cortisol.
Hypothalamic-pituitary-thyroid axis is inhibited after long-term exposure to excess glucocorticoid to some extent [14]. We found that only a total of 94 (32.41%) had normal thyroid hormones. 15.86% patients with CD had secondary hypothyroidism, the percentage of which was lower than that in a Cushing's syndrome cohort (53%). Te etiology and course of disease may contribute to the diferent prevalence. Tere is a reciprocal causation relationship between obesity and thyroid disorders [15,16]. Te BMI is associated with dysfunction of thyroid function especially primary hypothyroidism [17]. Although the associations between thyroid hormones and body weight status vary slightly in diferent studies, most available data showed that lower levels of FT4 or higher levels of FT3 or TSH were associated with a higher BMI in euthyroid subjects [18][19][20][21]. In primary hypothyroidism patients, TSH had no signifcant relationship with International Journal of Endocrinology the BMI [22,23]. In patients with subclinical hypothyroidism, the BMI was positively correlated with TSH and negatively correlated with FT4 after adjusting for age [24]. However, central hypothyroidism has been much less frequently studied because of its rarity. It reported that FT4 was also negatively associated with the BMI and waist circumference in growth hormone-defcient induced hypothyroidism patients [25]. In patients with CD, we found that lower levels of FT3 and TSH, but not FT4, were associated with a higher BMI. Usually, the divergent associations between the BMI and FT3 or FT4 would refect an increased deiodinase activity [20]. However, patients with CD had an impaired deiodinase activity, which decreased the rate of conversion of T4 to T3 [26]. Moreover, muscle atrophy in CD patients also masks the relationship between the BMI and FT4 [27]. Numerous epidemiologists investigated the relationship between thyroid hormones and the incidence of the fatty liver. In a large population-based cohort study, a higher TSH level or lower FT4 was associated with a higher NAFLD risk [28]. A Lifeline cohort study considered 20289 euthyroid subjects and reported that NAFLD (FLI ≥ 60) was independently associated with a higher FT3 and a lower FT4 but not by TSH [29]. A meta-analysis reported that lower FT4 and higher TSH, but not FT3, had associations with the risk of NAFLD in primary hypothyroidism or subclinical hypothyroidism patients [30]. However, the association between MAFLD in patients with CD and thyroid hormones has not been investigated yet. Results in our study were partly similar to previous studies, namely an independent association of MAFLD and FT4 after adjusting confounders in patients with active CD, rather than TSH or FT3. After patients being divided into 4 groups according to TSH, TT4, TT3, FT4, and FT3 quartiles, respectively, the ORs for the third and fourth quartile groups of FT4 were negatively associated with MAFLD compared to the frst quartile. A dose-response relationship was observed on FT4 quartile and MAFLD among patients with active CD. Tese fndings were strengthened when similar associations were observed through various analytic strategies. Moreover, we found that cortisol-associated hypothyroidism had no association with MAFLD in patients with CD, which was distinct from the common view that primary hypothyroidism was highly associated with the fatty liver. Excessive glucocorticoid induced central hypothyroidism by reducing thyrotropin-releasing hormone expression and release in the hypothalamus, decreasing TSH secretion in pituitary, as well as inhibiting FT4 and FT3 levels. In Cushing's disease, the association between TSH and MAFLD might be blunted since there was not a relatively high TSH levels normally seen at a lower FT4 levels.
It is well known that thyroid hormones have prominent efects on hepatic fatty acid and cholesterol synthesis and metabolism [31]. As an active form of thyroid hormones, T3 has much greater thyroid hormone receptor afnity than T4 [32,33]. Although higher FT3 levels were associated with higher risk of the fatty liver in several studies that were conducted in euthyroid subjects [29,34,35], it was FT4, but not fT3, that was associated with MAFLD in patients with CD in this cohort. High cortisol concentrations not only could inhibit TSHstimulated T3 and T4 levels but also inhibit the conversion of T4 to T3, which made T3 levels in subjects more complex [36]. In addition, FT3 was susceptible to nutritional status or being sick. However, FT4 is more meaningful for the diagnosis of thyroid status, and the decreased FT4 concentration was used to mark the symptoms of hypothyroidism in patients with CD.

Limitation
Te present study had several limitations. First, similar to other cross-sectional studies, causal correlation cannot be established with certainty in this study. Te incidence or degree of hepatic steatosis was related to the duration of CD which was tough to evaluate in this study. Second, we did not analysis the relationship of thyroid hormones with MAFLD in active CD with euthyroid subjects or central hypothyroidism groups separately due to the limited statistical power. Tird, the BMI in isolation is an insufcient biomarker of abdominal obesity, and waist circumference was recommended to be included in the evaluation of overweight and the obesity group [37]. Terefore, it would be better to investigate the association between thyroid hormones and obesity or MAFLD by using waist circumference or the whole-body MRI, which was a limitation of this study.

Conclusion
In conclusion, CD patients with MAFLD were associated with a lower level of FT4 rather than FT3 or cortisol. Teoretically, glucocorticoids do have direct and indirect actions on the development of hepatic steatosis, but it seemed that serum cortisol had no independent value in the diagnosis of MAFLD in patients with CD. Moreover, our results found that lower FT4 increased the risk of MAFLD in patients with CD. More attention should be paid to optimize the replacement therapy in the future and improve awareness of thyroid status, especially the level of FT4, to decrease the risk of MAFLD in patients with CD.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Disclosure
Tis study was performed as part of the employment of Department of Endocrinology and Metabolism, Huashan Hospital.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
Kuangyang Chen designed the methodology of study, performed the data extraction, carried out all statistical analyses, and drafted the manuscript. Lijiao Chen collected the original clinical data, checked the data from fnal included patients, and revised the manuscript. Jiarong Dai reviewed the manuscript and provided guidance on statistical analyses. Hongying Ye conceptualized the study, revised the manuscript, and accepted responsibility for these data analyzed. All authors have read and approved the fnal version of the manuscript to be submitted and published. Te authors, Kuangyang Chen and Lijiao Chen, have contributed equally to this work. International Journal of Endocrinology 7