The Novel Hepatokine Ectodysplasin A Is Increased in Obesity and Reduced after Liraglutide Management

Background Ectodysplasin A (EDA), a new hepatokine, has been recently characterized to play a role in liver lipid metabolism and insulin resistance, but its physiological role remains scarcely acquainted in obesity. This study was the rst time to determine the level of serum EDA in obesity, and to assess change in the levels of EDA after weight loss in obese mice. Methods We analyzed the serum concentrations of EDA by enzyme-linked immunosorbent assay (ELISA) in 60 subjects including 30 normal weight and 30 obesity. Male C57BL/6J mice were fed with high-fat diet and injected by liraglutide to reduce weight. AML12 cells were induced by palmitic acid and treated with liraglutide. Quantitative real-time PCR and Western blot analysis were conducted to evaluate the expression of EDA. Results Serum EDA levels were signicantly higher in obesity than in normal weight subjects. It was positively correlated with body mass index (BMI). More importantly, the mRNA and protein expression of EDA reduced after liraglutide management in vivo and in vitro. Conclusions The level of EDA increased signicantly in obesity and decreased signicantly after weight loss. It is suggested that EDA may be a novel hepatokine associated with obesity-related metabolic diseases. hemoglobin 1c; HC, hip circumference, HDL-C, high density lipoprotein cholesterol; high-fat HOMA, HOMA-IR, β-cell ITS, transferrin selenium; test; LDL-C, lipoprotein cholesterol; steatohepatitis; liver disease; paraformaldehyde; mellitus;


Animals and experimental design
Twenty-four healthy male C57BL/6J mice, four weeks of age, were maintained in a room with controlled lighting (12 hours light/dark cycle) and regulated temperature (23±2 • C) and humidity (50-60%). All mice were fed with free access to food and water for 1 week to be adapted for the environment. After 1 week of acclimation, eight mice were randomly fed with regular chow as normal control group (NC group) and throughout the study, while the remaining mice were fed with HFD (HFD: 36% carbohydrate, 19% protein and 45% fat) for 20 weeks. Then, the HFD animals were randomly divided into two groups: HFD+saline group (HFD group), or liraglutide (LIRA, Victoza, Novo Nordisk)-treated group (HFD+LIRA group). They were received daily subcutaneous injections with either liraglutide (0.2 mg/kg daily) or the same volume of saline at about 12.30pm for 12 weeks and normal chow mice were given saline injections during the same period. The body weight of all mice measured every 2 weeks.
At the end of the experiment, the mice were fasted overnight and euthanized using 1% sodium pentobarbital (50 mg/kg). Eyeballs were removed and blood samples were collected and serum was obtained by centrifugation at 3,000 rpm at 4 • C for 20min. Tissues collected were either immediately fresh frozen in liquid nitrogen after dissection and stored at -80 °C until further processing or were xed in 4% paraformaldehyde (PFA) solution in PBS.
2.5 Biochemical analyses of animals' serum Serum blood glucose, TC, TG, LDL-C, as well as AST and ALT activities were examined by sop of Beckman Coulter Biochemical Analyzer (AU5800, USA).

Insulin tolerance test and glucose tolerance test
The insulin tolerance test (ITT) and glucose tolerance test (GTT) were performed as previously described [21,22]. Brie y, in ITT and GTT, mice were starved for 4 h and 16 h respectively, and then insulin (0.75 units/kg) or glucose (2.0 g/kg) were injected intraperitoneally. Accu-check glucometer (Sandhofer Strasse 116,68305 Mannheim, Germany) was used to measure the blood glucose levels in the tail vein. Blood glucose was measured before injection (time 0) and at 15, 30, 60, 90, 120 min after injection in ITT, and before injection (time 0) and at 30, 60, 90, 120 min after injection in GTT.

Tissue histology and liver triglyceride assay
The sections of liver tissues were xed in 4% PFA, embedded in para n and sliced (4μm). Hematoxylin and eosin (HE) staining was performed according to the standard procedure, and imaged under a light microscope (Nikon, Japan). Hepatic lipid accumulation was also determined using Oil Red O staining.
The imaging system was used to collect the images on the tissue staining section, and the analysis software was used to automatically read the tissue measurement area, calculate the positive area and tissue area in the measurement area, and calculate the proportion of the positive area (Servicebio, China).

Quantitative real-time PCR
Total RNA was isolated from cells or livers using TRIzol Reagent (Invitrogen) followed by the manufacturer's guidelines and cDNA was generated with PrimeScript RT-PCR Kit (Vazyme Biotech, Nanjing, China). PrimeScript RT-PCR Kit (Vazyme Biotech, Nanjing, China) was used for measuring relative mRNA expression by quantitative real-time PCR (Quant Studio 5). EDA gene expression levels were normalized to β-actin levels. The sequences of primers (Sangon Biotech, Shanghai, China) used in the study were listed as followed: EDA, forward TGAATAGCAGCCCATTAGTAGG and reverse CAGAGAATAAATGGCATTGGCA; -actin, forward TGGAATCCTGTGGCATCCATGAAAC and reverse TAAAACGCAGCTCAGTAACAGTCCG.

Western blot analysis
Total proteins were extracted from livers and cells by with ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and quanti ed with BCA kits (Beyotime, Shanghai, China). The denatured protein was loaded and separated on a 10% SDS-PAGE gel, transferred onto PVDF membranes, and then blocked with 5% non-fat milk in Tris-buffered saline for 1h. Subsequently, the membranes were incubated with the EDA (Abcam) and β-actin (CST) primary antibody overnight at 4°C. The following day, appropriate secondary antibodies conjugated to horseradish peroxidase were incubated with respective membranes for 1h at room temperature. The bands were detected by enhanced chemiluminescence (ECL) method using kit (Vazyme Biotech, Nanjing, China). The relative expression of target protein was normalized to that of β-actin.
Protein band densities were quanti ed using ImageJ.

Statistical analysis
All statistical analyses were performed using SPSS 25.0. Data are presented as mean ± standard deviation (x ±SD) for normally distributed variables and percentage (n%) for categorical variables. Data were tested for normality before the use of a parametric test. Independent student t test was used for comparison between the two groups. One-way ANOVA were used for multiple comparisons. Categorical variables were compared by χ2 test. Relationships between EDA and other parameters were examined by calculation of Pearson correlation coe cients and Spearman correlation coe cients. Multivariate regression models were t for EDA as a dependent variable to demonstrate the relative contribution of these parameters to the outcome ones after collinearity diagnostics. Drawing with software of GraphPad Prism 9.0 (GraphPad software, Inc., La Jolla, CA, USA). P<0.05 was considered signi cant.

Clinical and biochemical characteristics among groups
The basic clinical and biochemical parameters of subjects were summarized and presented in Table 1. There was no statistically signi cant difference in age, sex, smoking status, alcohol consumption, HbA1c, FPG, TC, LDL-C, AST and γ-GT between the two groups. Compared with the NW group, BMI, WC, HC, WHR, SBP, DBP, FINS, TG, ALT, HOMA-IR and HOMA-β in OB group were signi cantly increased (P<0.05 or P<0.01), while HDL-C were largely decreased (P<0.01). Serum EDA level was higher in OB than NW group (P<0.001).

Clinical and biochemical characteristics of the study objects according to the tertiles of EDA
Subjects in the upper and middle serum EDA tertile had higher BMI, WC, HC, WHR and SBP compared with subjects in the lower serum EDA tertile (P<0.05 or P<0.01). HDL-C was lower in the upper tertile group than in the lower tertile group (Table 2, P<0.05). Other metabolic parameter such as TG, FINS, HOMA-IR and HOMA-β were higher in the upper tertile group than in the middle one, although the differences did not reach statistical signi cance.

Relationships between serum EDA levels and metabolic parameters
Serum EDA concentrations were positively correlated with BMI, WC, HC, WHR and SBP but inversely correlated with AST ( Fig.1). Multivariable linear regression models revealed that BMI was independently related to the serum EDA levels (Table3).

Liraglutide induced body weight and liver weight loss
The body weight of mice throughout the study was shown in Figure 2. At the beginning of the study, there was no signi cant difference in body weight between NC group and HFD group. At the 20th week, the weight of mice in HFD group was increased obviously compared to that of NC group, and the difference was statistically signi cant ( Fig. 2A, P<0.05). It is suggested that the model of obesity mice fed with HFD was successfully established. After intervention with liraglutide, the body weight of mice in HFD+LIRA group was signi cantly lower than that in HFD group (Fig. 2B, P<0.01). We also found that the HFD group showed clear increase in liver weight compared with NC group, and liraglutide treatment markedly reduced liver weight compared with HFD group (Fig. 2C).

Liraglutide increased insulin sensitivity in HFD mice
After 12 weeks of treatment, GTT and ITT were performed. After administration of glucose, there was no signi cant difference in blood glucose level and the area under the curve (AUC) of GTT between HFD+LIRA group and HFD group (Fig. 3A, B). What's more, the blood glucose level and AUC of ITT in HFD+LIRA group were signi cantly lower than those in HFD group after insulin administration ( Fig. 3C, D, P<0.01). These results suggested that liraglutide treatment could promote insulin hypoglycemic effect and improved HFD-induced insulin resistance.

Liraglutide improved hepatic steatosis and reduced blood lipid levels
In order to determine whether liraglutide could affect blood lipid levels, we further analyzed the lipid pro le. The serum levels of TC, TG, LDL-C and ALT in HFD group were signi cantly higher than those in NC group (Fig. 4A, B, C, D, P<0.05), but the level of AST had no signi cant difference (Fig. 4E). After 12 weeks of liraglutide treatment, the results showed that the levels of TC, TG, LDL-C and ALT in HFD+LIRA group were signi cantly decreased compared to that HFD group (Fig. 4A, B, C, D, P<0.05). Then we investigated the effect of liraglutide on hepatic steatosis. By no accident, HE staining of liver tissue was consistent with lipid pro le and the liver appearance was shown in Figure 5A. The ratios of the Oil Red Ostained area to the total area in the liver were signi cantly lower in the HFD+LIRA group than in the HFD group (Fig. 5C, P<0.01). Although there was no difference between NC group and HFD group, the hepatic lipid accumulation in the HFD mice was more severe than that in the NC group in Figure 5B. In the HFD group, most of the hepatocytes showed vesicular degeneration and steatosis, and in ammatory cell in ltration was obvious. After liraglutide treatment, the hepatic lipid accumulation and liver steatosis were drastically improved. Also, mice fed 20 weeks of a HFD had signi cantly higher TG contents, liraglutide signi cantly attenuated the intrahepatic TG contents (Fig. 5D, P<0.01).

The expression of EDA decreased after liraglutide treatment in HFD mice
To explore whether the level of EDA has changed after liver lipid metabolism improvement, we analyzed the mRNA and protein expression of it in liraglutide treated obese mice. The relative mRNA expression level of EDA in HFD mice was increased signi cantly than that of NC group (Fig. 6A, P<0.05). In accordance with these observations, Western blot assessments revealed greater EDA protein expression in liver tissues from HFD-fed obese mice than their controls (Fig. 6B). Further studies showed that the mRNA and protein levels of EDA in HFD+LIRA group were signi cantly decreased than those in HFD group (Fig. 6A, B, P<0.05 or P<0.01). These results suggested that the expression of EDA decreased after liraglutide intervention in vivo.

The expression of EDA decreased after liraglutide treatment in AML12 cells
To investigate whether there existed similar results in vitro, we next analyzed the EDA expression in PAinduced AML12 cells. Compared with the control group, the mRNA and protein expression levels of EDA in PA group were signi cantly increased, while the mRNA and protein expression levels of EDA reduced signi cantly after liraglutide management in a dose-dependent manner (Fig. 7A, B, P<0.05 or P<0.01).

Discussion
In recent years, hepatokines have been identi ed and examined for their role in the development of obesity, NAFLD and insulin resistance [23]. The present study con rmed the association between a novel hepatokine EDA and obesity. We found that serum EDA level was higher in obesity subjects (BMI≥25kg/m 2 , n=30) than normal weight group (BMI<25 kg/m 2 , n=30). There was also a positive correlation between EDA and BMI and WHR. More importantly, our experiment showed that the expression of EDA was elevated in HFD-fed obese mice and was decreased after liraglutide reduced body weight and improved hepatic lipid homeostasis. Similar to our results, 23 obese patients who underwent a two-step bariatric surgery strategy had lower liver EDA expression 12 months after the rst surgery in parallel with reduction in body weight and improvement to insulin sensitivity [10].
In this study, we provide the rst clinical evidence showing that serum levels of EDA, which has been suggested as a potential candidate for the treatment of NAFLD, are increased in obesity. Our results have demonstrated several parameters of adiposity (BMI, WC, HC, WHC), insulin sensitivity and β-cell function (FINS, HOMA-IR and HOMA-β), lipid pro les (TG) and liver enzyme (ALT) were higher while HDL-C was decreased in OB subjects, indicating that obesity caused the glucose metabolism disorder and liver function abnormality. Furthermore, subjects in the upper EDA tertile had higher levels of BMI, WC, HC, WHR, and lower HDL than subjects in the lower terile. Other parameters of glucose metabolism and insulin function such as FINS, HOMA-IR and HOMA-β were higher in the upper tertile group than in the middle one, although there was no statistical signi cance. Additionally, our study revealed a positive association between circulating EDA and BMI, WC, HC, WHR and a reverse with AST, probably because a relatively small sample size, different populations and different disease states. More importantly, BMI was independently related to the serum EDA levels, which demonstrated that BMI was a potential independent predictor of EDA. Recently, Awazawa et al. also found that in obese men ranging in BMI from 23 to 46 kg/m 2 , hepatic EDA expression increased and correlated with visceral fat area, liver fat content, insulin resistance and NASH scores, however it reversed as weight loss [10]. Consistent with our results, others found signi cantly positive associations of EDA with a series of anthropometric parameters, e.g. age, BMI and WHR. However, EDA also correlated with other metabolic parameters such as FPG, HbA1c, HOMA-IR, but no parameter of liver enzymes [11], which was different with our results. These differences may be due to sample size and selected subjects. In our study, we enrolled obese patients but others selected NAFLD cases. Considering the correlation between EDA and BMI and WHR, we reasonably believe that EDA might be a serum biomarker and a potential therapeutic target for obesity.
Similarly, we have found that EDA was associated with obesity in experiments in vivo and in vitro. Obesity is characterized by adipocyte dysfunction including dyslipidemia [24], which is the main cause of insulin resistance and the increasing global obesity prevalence is closely coupled to the parallel increase in global rates of T2DM [25]. Many studies have shown that mice fed with HFD develop insulin resistance and obesity [23]. In the current study, HFD-fed mice also showed obesity, hyperlipidemia, insulin resistance and liver fat accumulation. Body weight and liver weight were elevated in mice after HFD treatment for 20 weeks. Moreover, HFD contributed to the increase in some lipid pro les such as TC, TG, LDL-C. The liver enzyme ALT was higher in HFD-fed mice and most of the hepatocytes showed vesicular degeneration and steatosis, suggesting that the liver function was impaired by HFD. Our research revealed that the EDA content was higher in mice after HFD treatment than that in control group. Notably, the relative mRNA and protein expression of EDA was also elevated in PA-induced AML12 cells with insulin resistance, which has not been discussed before, suggesting the potential relationship between EDA and insulin sensitivity in AML12 cells with lipid metabolism disorder. These results suggest that EDA may play a detrimental role in the occurrence and development of obesity, insulin resistance, and hepatic steatosis. Awazawa et al. overexpressed EDA in the livers of mice via adeno-associated virus (AAV)mediated gene transfer. In a glucose tolerance test, mice injected with EDA-AAV exhibited higher glucose concentrations and a decreasing tendency in energy expenditure than control GFP-AAV-injected mice [10]. In addition, another study showed that EDA knockdown attenuated hepatic lipogenesis via mediating lipolytic and lipogenic genes in vivo and in vitro. They found that expression of the key fatty acid oxidative enzyme carnitine palmitoyl transferase 1α (CPT1α) were signi cantly elevated in EDAknockdown HepG2 cell [11]. Previous studies and our results all indicated that EDA is associated with obesity, hyperlipidemia, abnormal liver function and insulin resistance.
It is known that GLP-1 RAs can improve glucose and cause weight loss, so they are receiving increasing attention for the treatment of diabetes-obesity. Recently, GLP-1 RAs have been used as a preferred treatment for patients with diabetes and obesity [26,27]. Previous studies have suggested that liraglutide can play a hypoglycemic role by increasing insulin secretion, improving islet cell function and reducing body weight in diabetic mice and obese rats [28][29][30]. Others also con rmed that GLP-1 RAs inhibited the formation of lipid droplets in different cells lines [31,32]. Our results also showed that liraglutide promoted weight loss and improved insulin sensitivity in HFD-fed mice. What's more, our data suggested that liraglutide can alter the lipid pro le by reducing serum TC and TG levels in HFD-fed mice. At the same time, it also signi cantly reduced the liver weight and hepatic steatosis of HFD-induced obese mice. Although the ratios of the Oil Red O-stained area to the total area had no statistical signi cance after HFD feeding, the hepatic lipid accumulation in images was more severe in HFD mice compared to NC group, probably because the large intra-group differences. More interestingly, we found that the expression of EDA was reduced signi cantly in the liver of HFD-fed mice and in AML-12 cells induced by PA after liraglutide treatment, suggesting that when obesity, insulin resistance and liver function were improved, the level of EDA was also decreased accordingly.
Based on our results, EDA may play a role in the development of obesity. However, there are several limitations in our study, including a relatively small sample size, limited population races and inadequate mechanism exploration. Our sample size is only 60 and limited to the Chinese population, which was not representative of the general population. In addition, our study design was cross-sectional and did not address the cause-effect relationship between serum EDA and obesity-related medical disorders. Also, in experiments in vivo and in vitro, there was no further study on the pathway mechanism of the relationship between EDA and obesity. Thus, additional studies are required to provide new insights into this subject.

Conclusions
In a whole, this study found that elevated circulating EDA levels were associated with obesity and mRNA and protein expression of it was progressively increased in liver tissue of obese mice and PA-induced hepatic cells, however it was decreased when liraglutide, a GLP-1 RA, reduced weight and improved lipid metabolism as well as insulin sensitivity in vivo and in vitro. This study for the rst time demonstrated that EDA might be a potential biomarker for obesity and obesity-related glycolipid metabolism abnormity, but the deep mechanisms and causality are still largely unknown and need further researches.

Declarations
Ethics approval and consent to participate Approval for the study was obtained from the Clinical Research Ethics Committee, A liated Hospital of Jiangsu University. All subjects signed informed consent forms. All animal experiments were in accordance with the guidelines and policies formulated by the Animal Care Committee of Jiangsu University experimental animal center. The study was carried out in compliance with the ARRIVE guidelines.

Consent for publication
Not Applicable.

Competing interest
The authors declare that they have no con icts of interest associated with this article. No bene ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this paper.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.     Figure 1 Correlations between serum EDA levels and metabolic parameters such as body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), systolic blood pressure (SBP) and aspartate aminotransferase (AST).