Levels of circulating adipokines and their relation with glycemic control and insulin resistance in Saudi patients with non-alcoholic fatty liver disease

Background: Non-alcoholic fatty liver disease (NAFLD) is a prevalent condition associated with obesity and insulin resistance (IR). Adipokines include fat-secreted proteins such as leptin or adiponectin and fator liver-derived cytokines such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) are considered to play an important role in the pathogenesis of the metabolic syndrome, including NAFLD. Objective: The present study aimed to explore the role of adipokines in the pathogenesis of NAFLD and correlate them with glucose control. Material and Methods: One hundred Saudi patients with NAFLD (45 males and 55 females) with NAFLD diagnosed by ultrasonographic findings, our group includes one gender, age and body mass index (BMI) matched hundred healthy volunteers. Adiopkines and parameters of glucose control of all participants were detected. Results: Serum glucose, insulin, aspartate aminotransferase(AST), alanine aminotransferase (ALT), aspartate aminotransferase /alanine aminotransferase ratio (AST/ALT), serum levels of total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-C), serum TNF-α and L-6 levels were significantly higher in patients with NAFLD when compared to control group. However, serum high density lipoprotein cholesterol (HDL-C) and adiponectin levels were significantly lower in patients with NAFLD when compared to control group. Moreover, serum levels of adipokines showed an association with insulin resistance. Conclusion: Within the limit of this study non-alcoholic fatty liver disease is associated with adipokines alteration that is correlated with abnormal glucose control and


INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease, affecting 20-40% of the general population in the Western countries (1).The increasing prevalence of NAFLD parallels the rise of obesity and its complications as NAFLD occurs in 60%~95% of people with obesity (2).However, obesity, insulin resistance (IR) and metabolic syndrome are the major risk factors associated with NAFLD pathophysiology (3).IR appears plays the key role in the genesis of NAFLD, suggesting a possible interplay among IR, atherosclerosis and NAFLD (4).
Adiponectin is an adipose tissue derived cytokine playing an important role in the regulation of glucose homeostasis and insulin sensitivity (5).Previous studies have shown that adiponectin improves insulin sensitivity and possesses antiinflammatory and anti-atherosclerotic properties (6).Lower levels of adiponectin might represent an independent risk factor for NAFLD and has been associated with IR and NAFLD (7).
Obesity is associated with high circulating serum leptin levels which adversely affects glucose homeostasis (8).Initial studies demonstrated an association of leptin levels with NASH and the degree of liver steatosis and inflammation (9).Therefore, the role of leptin in NAFLD is still unclear Resistin is primarily secreted by monocytes or macrophages in humans (10).The link between resistin and NAFLD remains under debate in humans.It is accepted that obesity, particularly central obesity is a risk factor strongly associated with NAFLD (11).Moreover, accumulating evidence indicates that resistin has pro-inflammatory properties.Resistin enhances interleukin 6 (IL-6) and TNF-α production, both are increased in NAFLD patients (12).
The aim of the present study is to explore the role of adipokines in the pathogenesis of NAFLD and correlate them with glucose metabolism.

Subjects
One hundred patients with NAFLD with body mass index (BMI) ranged from 30 to 35 Kg/m 2 , their age ranged from 35 to 55 years were identified from a large number of patients attending the Liver Clinic in King Abdulaziz University Teaching Hospital.Control group included one hundred healthy volunteers, who were gender, age and BMI matched with study group, were enrolled.NAFLD diagnosis was based according to the standard criteria accepted by the American Gastroenterology Association (13).The diagnosis was based on ultrasonograhpic finding of bright liver (the diagnosis of bright liver was based on abnormally intense, high level echoes arising from the hepatic parenchyma, with amplitude similar to that of echoes arising from the diaphragm); In all patients, liver biopsy could not be done because the stage and grade of the NAFLD was not of importance in this study.Otherwise, according to Saverymuttu et al., ultrasound examinations can accurately identify steatosis with a sensitivity of 94% and a specificity of 84% (14).
We excluded patients with; hepatitis B and hepatitis C infection and other known liver diseases like cirrhosis.We also excluded patients with diabetes, hypertension, malignancy, hypohyperthyroid disease, coronary artery disease, pregnancy, alcohol consumption, cigarette smoking, use of amiodarone, corticosteroids, tamoxifen, methotrexate, or oral contraceptives.
This study was approved by the Scientific Research Ethical Committee, Faculty of Applied Sciences, King Abdulaziz University.
Informed consent was obtained from all participants.All participants were free to withdraw from the study at any time.
All persons included in the study were subjected to detailed history taking, complete clinical examination, anthropometric evaluation (height, weight, and body mass index "BMI" were recorded.Overweight and obesity were defined as BMI between 25 and 30 kg/m 2 (<30) and ≥ 30 kg/m 2 , respectively (15).

Statistical Analysis
Independent t-test was used to compare mean differences between both groups.Statistical analysis of data was performed using SPSS (Chicago, IL, USA) version 17.The degree of correlation between adiopkines and parameters of glucose control in NAFLD patients was detected by Pearson's product moment correlation coefficients (r).All data were expressed as the mean ± SD.P<0.05 indicated statistical significance.

RESULTS
One hundred NAFLD Saudi subjects were enrolled including 55 women and 45 men, had age ranged from 30 to 56 years and one hundred healthy subjects had age ranged from 31 to 58 years, there was no significant differences in demographic and anthropometric characteristics between both groups (Table 1).NAFLD patients were more insulin resistant as indicated by significantly higher values of fasting glucose, insulin and HOMA-IR and lower values of QUICKI, with no significant differences in leptin concentration between patients and controls.Also, NAFLD patients showed significantly higher resistin level, aspartate aminotransferase(AST), alanine aminotransferase (ALT), aspartate aminotransferase /alanine aminotransferase ratio (AST/ALT), serum levels of total cholesterol, triglycerides, serum high density lipoprotein cholesterol (HDL-C) and adiponectin levels in comparison to controls (Table 3).
Table 3 summarizes the relationship between adipokines and parameters of glucose control in NAFLD patients.Serum levels of leptin, resistin, TNF-α and IL-6 showed an inverse relationship with QUICKI and a direct relationship with serum insulin, HOM-IR.However, levels of adiponectin showed a direct relationship with QUICKI and an inverse relationship with serum insulin, HOM-IR (Table 3)

DISCUSSION
It is believed that IR is related to adipose tissue plays a key role in the development of NAFLD (18).Our study underscores that NAFLD is associated with IR and adipokines alterations.Though NAFLD patients had raised ALT suggestive of some hepatocellular injury.In our study, NAFLD patients showed significantly higher serum glucose, insulin, HOMA-IR, resistin level, AST, ALT, AST/ALT ratio, lipid profile, serum TNF-α levels, serum IL-6 levels and significantly lower values of QUICKI, serum HDL-C and adiponectin levels in comparison to controls.
A recent study conducted by Sanches and colleagues proved that patients with IR have 65% greater risk of developing NAFLD.Because they found that obese patients with NAFLD presented greater baseline HOMA-IR values and insulin concentration than their peers without NAFLD (19).Willner et al. reported a strong relation between NAFLD and IR, showing that about 80% of patients with NAFLD have IR (20).
The present study revealed that serum levels of adiponectin were significantly lower in NAFLD patients in comparison to   those of the controls.Also, our study showed that adiponectin levels were also associated with increased IR in NAFLD patients.
A number of studies have demonstrated that reduced systemic levels of adiponectin have clinically significant effects and associate closely with obesity-related diseases, including NAFLD (21,22).Also, in the study by Hu et al., these authors found that adiponectin expression decreases both in obese patients and genetically obese mice (23).
Our study confirmed a previous study found that leptin higher in NAFLD children as well as in obese control subjects (24) and an earlier clinical trial reported significantly higher serum leptin levels in patients with NASH as compared to controls (25).The increases in leptin levels in NASH is not explained by obesity alone but is also due to peripheral leptin resistance.In NASH leptin receptors become resistant to its effect leading to hyperleptinemia which alters insulin signaling and promotes accumulation of intracellular fatty acids in hepatocytes thereby increasing hepatic steatosis and steatohepatitis (26).
Our results revealed higher levels of resistin among the NAFLD patient than control, previous studies have demonstrated that obese individuals display higher serum resistin values than lean subjects and a positive correlation may exist between BMI and resistin (27).Shen et al. showed that resistin was increased in liver of NASH patients, and there was a positive correlation between resistin and inflammatory severity in the liver of NASH patients (28).

CONCLUSION
Within the limit of this study non-alcoholic fatty liver disease is associated with adipokines alteration that is correlated with abnormal glucose control and insulin resistance.

Table 1 :
Demographic and anthropometric characteristics of NAFLD patients and control subjects

Table 2 :
Mean value and significance of biochemical parameters of NAFLD and control subjects

Table 3 :
Correlation coefficient (r) of adiopkines and parameters of glucose control in NAFLD patients