Hepatic steatosis and liver fat contents in liver transplant recipients are associated with serum adipokines and insulin resistance

Our data about pathogenesis of hepatic steatosis after liver transplantation is scarce. This study aimed to investigate the association between serum adipokines and insulin resistance with hepatic steatosis in liver transplant recipients. We investigated the association between insulin resistance, serum adiponectin, insulin, and leptin with hepatic steatosis in a cohort of liver transplant recipients. Homeostatic model assessment of insulin resistance 2 (HOMA 2-IR) was used for estimation of insulin resistance. Hepatic steatosis was determined using ultrasound and controlled attenuation parameter (CAP). A total of 178 patients were included. 79 patients (44.4%) had hepatic steatosis. Serum adiponectin (OR: 0.912; 95% CI 0.869–0.957; P < 0.001), serum leptin (OR: 1.060; 95% CI 1.017–1.102; P = 0.005), HOMA2-IR (OR: 1.671; 95% CI 1.049–2.662; P = 0.031), and post-transplant diabetes mellitus (PTDM) (OR: 5.988; 95% CI 1.680–21.276; P = 0.006) were independently associated with hepatic steatosis after liver transplantation. CAP values were negatively correlated with serum adiponectin (P = 0.011) and positively correlated with serum insulin (P = 0.001), leptin (P < 0.001) and HOMA2-IR (P < 0.001). Insulin resistance and alterations in adipokines might have central role in pathogenesis of hepatic steatosis after liver transplantation and can be targeted for diagnostic and therapeutic purposes.


Discussion
Our results demonstrate that hepatic steatosis diagnosed by ultrasound and TE in liver transplant recipients is associated with decreased serum adiponectin and increased serum leptin and insulin levels. Liver transplant recipients with hepatic steatosis had higher insulin resistance as assessed by HOMA2-IR. PTDM was also an independent predictor of hepatic steatosis after liver transplantation. We used CAP for non-invasive estimation of liver fat content. CAP values were different over different grades of hepatic steatosis in ultrasound and was correlated negatively with serum adiponectin and positively with serum leptin and HOMA2-IR. After adjustment for age, gender, BMI and other metabolic indices, serum adipokines and insulin resistance were independently associated with hepatic steatosis after liver transplantation highlighting their role in pathogenesis of hepatic steatosis after liver transplantation. In a subgroup of lean patients, decreased serum adiponectin level was an independent predictor of hepatic steatois after liver transplantation. Recurrent or de novo hepatic steatosis after liver transplantation has been reported to be associated with PTDM, higher BMI and other components of metabolic syndrome [16][17][18] . However, limited data is available about pathogenesis of hepatic steatosis after liver transplantation. Increased fasting hepatic gluconeogenesis and impairment of suppression of hepatic glucose production are two components of hepatic insulin resistance 19 . Diacylglycerol (DAG) and ceramide have been suggested to be the two major lipids involved in hepatic insulin resistance via activation of protein kinase and downstream impairment of hepatic insulin signaling 20 . Total hepatic DAG and ceramide have been correlated with HOMA-IR as a marker of insulin resistance 21,22 . It is well known that insulin resistance is strongly associated with hepatic steatosis 23 . In a population based cohort, HOMA-IR was closely associated with liver fat contents assessed by magnetic resonance spectropscopy 24 . However, the role of insulin resistance in the development of hepatic steatosis after liver transplantation is controversial.
In contrast to our findings, Andrade and colleagues reported that HOMA-IR, as a marker of insulin resistance, was not a predictor of NAFLD after liver transplantation 25 . In this study, we used HOMA2-IR (the updated HOMA model) that is a better indicator of hepatic insulin resistance as it accounts for variations in both peripheral and hepatic glucose output 26 . Insulin resistance is prevalent among patients undergoing liver transplantation for hepatitis C virus (HCV) cirrhosis 27 and other causes of liver cirrhosis 28 . A cross-sectional study showed that www.nature.com/scientificreports/ insulin resistance is highly prevalent and associated with new onset diabetes after liver transplantation 29 . None of these studies reported the impact of insulin resistance on the development of hepatic steatosis after liver transplantation.
Leptin and adiponectin are two major adipokines that are produced and secreted mainly from adipocytes and are involved in the pathogenesis of hepatic steatosis 15 . Leptin acts in liver cells via activation of the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT)-3 pathway resulting in increased expression of suppressor of cytokine signaling (SOCS)-3 which is a feedback inhibitor of leptin signaling 30 . It has been shown that inhibition of SOCS-3 improves leptin resistance and hepatic steatosis 31 . A meta-analysis of 33 studies has shown that serum leptin level was higher in patients with simple steatosis and NASH compared to the healthy controls 32 . Adiponectin is decreased when adipose tissue mass increases and has anti-steatotic properties on hepatocytes. Adiponectin promotes oxidation of free fatty acids, inhibits gluconeogenesis and prevents apoptosis of hepatocytes. It has also anti-fibrotic and anti-inflammatory effects by acting on Kupffer cells and hepatic satellite cells [33][34][35] .
Alterations of serum adipokines after liver transplantation have been reported previously in few studies, none of them focused on hepatic steatosis. Watt et al. reported that increased serum leptin and decreased serum adiponectin levels were seen in liver recipients with cardiovascular events 36 . A recent study reported that hypoadiponectinemia was an independent predictor of future cardiovascular events in liver transplant recipients 37 . However, a cross-sectional study among a small group of patients failed to demonstrate any correlation between serum adipokines and metabolic syndrome after liver transplantation 38 . In a previous study no association was demonstrated between hepatic steatosis and serum leptin after liver transplantation 39 . In the mentioned study, authors only included patients with HCV infection and did not evaluate the impact of serum adiponectin. It has been recently suggested that patients with NAFLD and adiponectin gene polymorphisms were susceptible to hepatic steatosis after liver transplantation 40 .
In this study, we showed, for the first time, that alterations in serum adiponectin and leptin as well as insulin resistance were correlated with hepatic steatosis and hepatic fat contents in liver transplant recipients independent of traditional risk factors of hepatic steatosis. We used ultrasound and CAP for non-invasive estimation of hepatic steatosis. CAP is a non-invasive, cheap and easy technique (compared to other methods) for quantitative measurement of liver fat content during transient elastography 41,42 . This parameter has been increasingly measured in patients with NAFLD 43 and suggested to have good efficacy for assessment of hepatic steatosis after liver transplantation 44 .
Finally, cumulative evidence suggests that hepatic steatosis is prevalent after liver transplantation, however, its long term impact on allograft and patient outcomes is less evident. Our study expands current knowledge about pathophysiology of hepatic steatosis after liver transplantation. Liver transplant recipients with insulin resistance and alterations in adiponectin and leptin are susceptible to hepatic steatosis and should be actively diagnosed and treated. Targeting insulin resistance and adipokines can be considered as therapeutic options for prevention and management of hepatic steatosis after liver transplantation.

Methods and materials
patients. In a cross-sectional study, all adult (> 18 years) liver transplant recipients who referred for their routine post-transplant follow-up between July 2017 and November 2017 were included in the study. All patients had undergone liver transplantation from deceased donors at Shiraz Organ Transplant Center, Shiraz, Iran. We included patients who have passed at least 6 months after transplant surgery. Tacrolimus based immunosuppressive regimen was used for the maintenance of immunosuppression after liver transplantation for all patients. Patients with acute rejection episodes and those with serum aminotransferase levels higher than 5 times upper limit of normal range were excluded from the study. Clinical characteristics of patients were recorded at the time of study inclusion. Body mass index (BMI) was calculated using this formula: weight (kg) divided by height (m 2 ) squared. Patients with BMI < 25 kg/m 2 were defined to be lean. Hepatic steatosis in transplant recipients was assessed by ultrasound and classified as grades 0, 1, 2, and 3 based on the severity of liver echogenicity as a marker of severity of hepatic steatosis.

Measurement of serum adiponectin, leptin and insulin.
Peripheral venous blood samples were collected from patients in the morning after an overnight fasting. Blood samples were centrifuged immediately at 4 °C and separated serum samples were stored at − 80 °C. Serum adiponectin levels were measured by enzyme linked immunoassay (ELISA) method using human adiponectin ELISA kit (Mediagnost, Reutlingen, Germany). Serum insulin levels were measured by ELISA method using human insulin kit (Monobind Inc., Lake Forest, CA (92630), USA). Serum leptin levels were measured using human leptin ELISA kit (Mediagnost, Reutlingen, Germany). Homeostatic model assessment of insulin resistance (HOMA-IR) is a method for assessment of insulin resistance and β-cell function based on the fasting plasma insulin and glucose concentrations. Insulin resistance was estimated using HOMA 2-IR software, as described before 45 . Blood sampling was performed on the day of clinical visit and assessment of hepatic steatosis by ultrasound and CAP measurement.

Measurement of controlled attenuation parameter (CAP).
Non-invasive estimation of hepatic fat content was measured for all patients using vibration-controlled transient elastography (VCTE) and expressed as CAP. Measurement of CAP was done on the day of clinical visit and blood sampling and after an overnight fasting. All CAP measurements was done by one person. CAP was obtained along the inter-axillary line and intercostal spaces on the liver while the patient was in supine position. CAP measurement was considered to be reliable if 10 valid successful acquisitions were made correctly. TE was performed using M probe in all patients and XL probe was used in obese patients in whom use of M probe was failed. CAP measurement was expressed in Scientific RepoRtS | (2020) 10:12701 | https://doi.org/10.1038/s41598-020-69571-1 www.nature.com/scientificreports/ decibel per meter (dB/m) 46 . The cutoff scores for CAP estimation of hepatic steatosis were as follows: ≥ 238 dB/m for S1 (corresponding to 11-32% liver fat), ≥ 259 dB/m for S2 (33-65% liver fat), and ≥ 292 dB/m for S3 (≥ 66% liver fat), as previously determined.
Statistical analysis. Continuous and categorical variables were compared using Student's t-test and Chisquare test, respectively. Data were presented using means ± standard deviation for numeric variables, and percents and counts for categorical variables. A one way analysis of variance (ANOVA) and post-hoc Tukey test were used to compare serum levels of adiponectin, leptin and insulin with different grades of hepatic steatosis. Logistic regression analysis was used to identify the independent variables and association of serum adipokines and insulin resistance with hepatic steatosis after liver transplantation. Pearson's and Spearman's correlation coefficients were used to assess correlation of serum adipokines and insulin resistance with CAP values after liver transplantation. Linear regression analysis was used to identify continuous variables independently associated with CAP values. Statistical analysis was performed with SPSS 20.0 (SPSS Inc.; Chicago, IL, USA). A Pvalue of < 0.05 was considered statistically significant.
ethics and consent. The study protocol was approved by the institutional review board of Organ Transplant Center, Shiraz University of Medical Sciences on 5/11/2016. The study protocol was performed in accordance with the Helsinki Declaration as revised in Seoul 2008. Written informed consents were obtained from each patient after explaining the study harms and benefits.

Data availability
Due to patient privacy the datasets in the current study are not publicly available but can be available by corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/.