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adiponectin ratio correlates with hepatic steatosis but not arterial stiffness in nonalcoholic fatty liver disease in Japanese population
Cytokine 126 (2020) 154927
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Leptin/adiponectin ratio correlates with hepatic steatosis but not arterial stiffness in nonalcoholic fatty liver disease in Japanese population
T
⁎
Kenichiro Mikamia,b, , Tetsu Endob, Naoya Sawadab, Go Igarashib, Masayo Kimurab, Takuma Hasegawab, Chikara Iinob, Hirofumi Tomitac, Kaori Sawadad, Shigeyuki Nakajid, Masashi Matsuzakae, Natalie J. Torokf, Shinsaku Fukudab a
Department of Internal Medicine, Owani Hospital, Aomori, Japan Department of Gastroenterology, Hirosaki University Graduate School of Medicine, Aomori, Japan c Department of Cardiology, Hirosaki University Graduate School of Medicine, Aomori, Japan d Department of Social Medicine, Hirosaki University Graduate School of Medicine, Aomori, Japan e Clinical Research Support Center, Hirosaki University Hospital, Aomori, Japan f Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Leptin Adiponectin Nonalcoholic fatty liver disease Arterial stiffness Interleukin-6 Brief-type self-administered diet history questionnaire
Background and aims: Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD. Methods: The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV). Results: The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol. Conclusion: L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.
1. Introduction Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), are becoming the most common liver disorders worldwide. The prevalence of NAFLD has risen in all regions, with an overall prevalence worldwide of approximately 25% [1]. Fatigue is a
common symptom, but 48–100% of patients are asymptomatic [2]. Patients with advanced NASH, including cirrhosis, may have normal liver function tests, resulting in part from diminished inflammation as fibrosis progresses [3]. NAFLD is associated with components of metabolic syndrome, such as obesity, diabetes, hypertension, and dyslipidemia, and most notably the leading cause of death overall is
Abbreviations: NAFLD, nonalcoholic fatty liver disease; CVD, cardiovascular disease; BMI, body mass index; BFP, body fat percentage; BaPWV, brachial-ankle pulse wave velocity; L/A, leptin-to-adiponectin; BDHQ, brief-type self-administered diet history questionnaire; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GGT, gamma-glutamyl-transpeptidase; IL-6, interleukin-6; IL-17, interleukin-17 ⁎ Corresponding author at: Department of Gastroenterology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki City, Aomori 036-8562, Japan. E-mail address: [email protected] (K. Mikami). https://doi.org/10.1016/j.cyto.2019.154927 Received 10 August 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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food item, the subjects indicated their mean frequency of consumption of the food over the past month. The mean daily consumption of nutrients was calculated using an ad hoc computer program for BDHQ, which was based on the Standard Tables of Food Composition in Japan [39].
coronary artery disease among NAFLD patients [4,5]. However, the pathological association between NAFLD and cardiovascular disease (CVD) is still unclear, due to limitations of classical epidemiological studies and the absence of interventional studies for NAFLD treatment [6]. A major cause of CVD is atherosclerosis, which progresses to narrowing arteries leading to myocardial and cerebral ischemia [7]. Increased arterial stiffness is one of the earliest markers of functional and structural changes in arterial walls as atherosclerosis [8]. The measurement of pulse wave velocity (PWV) is widely used to assess arterial stiffness as a noninvasive, precise, and reproducible method [9,10]. Adipokines including leptin and adiponectin are polypeptides secreted by adipocytes and capable of acting at both local and systemic levels, providing a critical link between obesity, insulin resistance, and inflammation [11–13]. Leptin and adiponectin are two major adipokines with opposite effects in the pathogenesis of NAFLD [14]. Leptin promotes lipotoxicity and insulin resistance, and up-regulation of proinflammatory cytokines [15,16]. Leptin also plays a key role in hepatic fibrogenesis leading to cirrhosis via activation of hepatic stellate cells [17]. On the contrary, adiponectin has a protective role against obesity-related disorders including type 2 diabetes, metabolic syndrome, and cardiovascular disease, by reduction of inflammatory cytokines and oxidative stress leading to an improvement of insulin resistance [18–21]. Several recent studies have shown that the leptinto-adiponectin (L/A) ratio or adiponectin-to-leptin (A/L) ratio is a useful parameter to assess insulin resistance in patients with and without diabetes [22–26]. In NAFLD, adipokine imbalance is involved in increased leptin levels and decreased adiponectin levels [14,27]. However, these studies evaluated only patients with NAFLD, and there have been few general community population-based studies. Since NAFLD is generally asymptomatic, screening for NAFLD is very important to prevent disease progression. Therefore, we conducted a general population-based cross-sectional study to assess the relationship between L/A ratio and CVD risk in NAFLD in a Japanese community.
2.3. Clinical and laboratory assessment Hypertension was defined as blood pressure ≥140/90 mm Hg or use of antihypertensive medication. Diabetes was defined as fasting serum glucose ≥126 mg/dL, HbA1c ≥6.5%, use of diabetes medication, or a prior known diabetes diagnosis. Hyperlipidemia was defined as total cholesterol ≥220 mg/dL, triglycerides ≥150 mg/dL, or use of antihyperlipidemic medication. The following clinical characteristics were measured: height, body weight, and body composition. Body mass index (BMI) was calculated as body weight divided by height squared and expressed in kg/m2. Body fat percentage (BFP) was measured by a body composition analyzer MC-190 (Tanita Corp., Tokyo, Japan). Alcohol intake was determined from a questionnaire. Fasting whole blood samples were obtained after overnight fast, and laboratory tests included aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, glycated hemoglobin, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride, gamma-glutamyl-transpeptidase (GGT), interleukin6 (IL-6), leptin, and adiponectin. Serum levels of IL-6, leptin, and adiponectin were determined using chemiluminescent enzyme immunoassay, radioimmunoassay, and latex agglutination turbidimetric immunoassay, respectively, in a commercial laboratory (LSI Medience Corp., Tokyo, Japan). 2.4. Assessment of severity of fatty liver NAFLD was diagnosed based on abdominal ultrasound findings using a Prosound F37 (Hitachi Aloka Medical Ltd.). Ultrasound examinations were performed by one of five well-trained hepatology specialists, each with more than 5 years of experience, without detailed knowledge of the participant’s data. Images were stored and reevaluated by a single hepatologist with more than 20 years of experience. The severity of echogenicity was graded as follows: normal, normal echogenicity; mild, slight diffuse increase in fine echoes in liver parenchyma with normal visualization of diaphragm and intrahepatic vessel borders; moderate, moderate diffuse increase in fine echoes with slightly impaired visualization of hepatic vessel border and diaphragm; severe, marked increase in fine echoes with poor or non-visualization of the intrahepatic vessel borders, diaphragm, and posterior right lobe of the liver [40].
2. Materials and methods 2.1. Study population The Iwaki Health Promotion Project is an ongoing community-based health promotion study of Japanese people ≥20 years of age designed to prevent lifestyle-related diseases and prolong their lifespan. This program has been carried out annually since 2005 with about 1000 participants in the Iwaki region of Hirosaki City in Aomori Prefecture located in northern Japan [28–31]. All study subjects participated voluntarily in response to a public announcement, and approximately 600 data points were collected from each participant, including their demographics, medical history, lifestyle data, and microbiota and blood chemical analysis data. Our research on the association between fatty liver and adipokines is one part of this project. In 2016, 1148 individuals were enrolled in this project. Of these, all individuals who did not have complete clinical data, who were positive for hepatitis B surface antigen or anti-hepatitis C virus antibody, and/or had excessive alcohol consumption (daily alcohol intake greater than 30 g/day for men and 20 g/day for women), were excluded. Ultimately, 871 subjects (273 men and 598 women) were included in the current study. This study was approved by the Ethics Committee of the Hirosaki University School of Medicine, and written informed consent was obtained from all participants.
2.5. Assessment of arterial stiffness Arterial stiffness was diagnosed using brachial-ankle pulse wave velocity using a blood pressure pulse wave monitor (BP-203RPE; Omron Healthcare Co., Ltd., Kyoto, Japan). This device provides automated measurements of baPWV on both the right and left sides, and the average of the two sides was used in the current study [10]. 2.6. Statistical analysis All statistical analyses of collected data were performed using the Excel statistical software package for Macintosh (Ekuseru-Toukei 2016; Esumi Co., Ltd., Tokyo, Japan). Categorical variables were compared using the chi-square test. Ratios of serum leptin to adiponectin (L/A ratio) were compared using the Kruskal-Wallis test, followed by a SteelDwass test or Mann-Whitney U test. Spearman’s correlation coefficient was used to calculate the correlation between the L/A ratio and BMI or body fat percentage. Differences were considered to be significant with P-values of less than 0.05.
2.2. Dietary assessment Dietary habits were assessed by using a brief-type self-administered diet history questionnaire (BDHQ) that included questions on the consumption frequency of 56 foods and beverages and nine dishes commonly consumed in the general Japanese population [32–38]. For each 2
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Table 1 Baseline characteristics of study participants. Characteristic Gender Male, n (%) Female, n (%) Age (years), mean (SD) Degree of hepatic steatosis, n (%) Normal Mild Moderate Severe Alcohol consumption (g/day), mean (SD) Daily nutrition intake Energy (kcal), mean (SD) Protein (g), mean (SD) Total fat (g), mean (SD) Saturated fatty acid (g), mean (SD) Monounsaturated fatty acid (g), mean (SD) Polyunsaturated fatty acid (g), mean (SD) n-3 polyunsaturated (g), mean (SD) n-6 polyunsaturated (g), mean (SD) Cholesterol (mg), mean (SD) Carbohydrates (g), mean (SD) Body mass index (kg/m2), mean (SD) Body fat percentage (%), mean (SD) Hypertension, n (%) Medication for hypertension, n (%) Diabetes, n (%) Medication for diabetes, n (%) Hyperlipidemia, n (%) Medication for hyperlipidemia, n (%) Steroid exposure by medication, n (%) Glucose level (mg/dL), mean (SD) Glycated hemoglobin A1c level (%), mean (SD) Total cholesterol level (mg/dL), mean (SD) HDL-cholesterol level (mg/dL), mean (SD) LDL-cholesterol level (mg/dL), mean (SD) Triglyceride level (mg/dL), mean (SD) AST level (U/L), mean (SD) ALT level (U/L), mean (SD) GGT level (U/L), mean (SD) Leptin (ng/mL), mean (SD) Adiponectin (μg/mL), mean (SD) Leptin/Adiponectin ratio, mean (SD) IL-6 (pg/mL), mean (SD) BaPWV (cm/sec), mean (SD)
Total (n = 871)
Normal (n = 671)
NAFLD (n = 200)
p Value
273 (31.3) 598 (68.7) 54.0 (16.1)
190 (28.3) 481 (71.7) 53.6 (16.9)
83 (41.5) 117 (58.5) 55.3 (13.3)
< 0.001 0.246
671 (77.0) 111 (12.8) 74 (8.5) 15 (1.7) 4.0 (7.0)
671 (1 0 0)
3.9 (6.8)
111 (55.5) 74 (37.0) 15 (7.5) 4.6 (7.9)
0.795
1797.7 (557.5) 69.3 (26.9) 52.6 (19.9) 14.1 (5.8) 18.5 (7.2) 13.1 (4.8) 2.7 (1.3) 10.3 (3.7) 359.9 (178.3) 247.6 (80.0) 22.8 (3.5) 27.2 (8.1) 325 (37.3) 218 (25.0) 94 (10.8) 41 (4.70) 384 (44.1) 96 (11.0) 5 (0.57) 91.8 (20.9) 5.8 (0.6) 204.9 (35.9) 63.9 (16.5) 118.4 (30.3) 92.6 (60.5) 22.4 (8.6) 21.3 (14.8) 26.8 (23.9) 9.1 (6.6) 11.0 (6.1) 1.0 (1.0) 1.91 (6.44) 1467.1 (377.7)
1782.4 (564.7) 69.1 (27.6) 52.4 (20.5) 14.0 (6.0) 18.4 (7.4) 13.1 (4.9) 2.7 (1.3) 10.3 (3.8) 360.3 (183.9) 244.7 (80.0) 21.9 (2.9) 26.0 (7.6) 221 (32.9) 148 (22.0) 54 (8.0) 26 (3.87) 266 (39.6) 59 (8.7) 4 (0.59) 90.2 (21.3) 5.8 (0.6) 202.8 (36.3) 66.3 (15.6) 116.0 (30.4) 81.2 (43.6) 21.2 (7.0) 18.1 (10.8) 23.6 (20.8) 8.8 (5.5) 12.1 (6.3) 0.8 (0.7) 2.00 (7.27) 1452.9 (389.1)
1849.3 (530.6) 70.2 (24.4) 53.2 (17.7) 14.4 (5.0) 18.6 (6.3) 13.2 (4.4) 2.7 (1.1) 10.4 (3.3) 358.6 (158.2) 257.2 (79.5) 25.8 (3.6) 31.3 (8.1) 104 (52.0) 70 (35.0) 40 (20.0) 15 (7.50) 118 (59.0) 37 (18.5) 1 (0.50) 97.3 (18.9) 6.1 (0.7) 211.8 (33.7) 55.8 (16.9) 126.4 (28.6) 131.0 (87.5) 26.3 (11.7) 32.3 (20.3) 37.7 (29.9) 12.8 (8.4) 7.2 (2.8) 1.9 (1.4) 1.64 (1.80) 1514.8 (333.0)
0.029 0.206 0.276 0.120 0.297 0.425 0.766 0.342 0.694 0.017 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.034 < 0.001 < 0.001 0.875 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.003
Abbreviations: BaPWV, brachial-ankle pulse wave velocity; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl-transpeptidase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Interleukin-6, IL-6. Boldface type indicates a significant p value (p < 0.05). Hypertension was defined as blood pressure ≥140/90 mmHg or use of antihypertensive medication. Diabetes was defined as fasting serum glucose ≥126 mg/dL, HbA1c ≥6.5%, use of diabetes medication, or a prior known diabetes diagnosis. Hyperlipidemia was defined as total cholesterol ≥220 mg/dL, triglycerides ≥150 mg/dL, or use of antihyperlipidemic medication.
3. Results
consumption between subjects with and without NAFLD. Mean daily energy and carbohydrate intake determined by the BDHQ were significantly higher in the subjects with NAFLD compared the subjects without NAFLD (p = 0.029 and p = 0.017, respectively) (Table 1). On the other hand, intake of protein and total fat containing saturated fatty acid, monounsaturated fatty acid, polyunsaturated fatty acid including n-3 polyunsaturated fatty acid and n-6 polyunsaturated fatty acid, and cholesterol did not differ between subjects with or without NAFLD (Table 1). With regard to medications that subjects were taking, the subjects with NAFLD had higher medication rates compared with the subject without NAFLD for treatment of hypertension, diabetes, and hyperlipidemia (p < 0.001, p = 0.034, and p < 0.001, respectively). Furthermore, one subject with NAFLD and four subjects without NAFLD (0.50% and 0.59%, respectively) received steroid therapy for inflammatory disease such as arthritis. In addition, there were no subjects with conditions associated with steatosis such as genetic disease, parenteral nutrition, and malnutrition.
3.1. Characteristics of the participants with and without NAFLD The characteristics of the study subjects with and without NAFLD are shown in Table 1. Of the 871 subjects included, 200 subjects (22.9%) had NAFLD. Compared with the subjects without NAFLD, the subjects with NAFLD were predominantly male; had a greater BMI and body fat percentage (BFP); showed a higher prevalence of hypertension, diabetes, and hyperlipidemia; and presented higher levels of glucose, glycated hemoglobin A1c, total cholesterol, LDL-cholesterol, triglyceride, AST, ALT, and GGT, and lower level of HDL-cholesterol. A significantly higher level of leptin, lower level of adiponectin, and higher L/A ratio were observed in the subjects with NAFLD as compared with the subjects without NAFLD. IL-6 level was lower in the subjects with NAFLD than in the subjects without NAFLD (p < 0.001). In addition, the subjects with NAFLD had higher baPWV compared with the subjects without NAFLD. There were no differences in age and alcohol 3
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Fig. 1. Correlation between L/A ratio and severity of hepatic steatosis in NAFLD. (A, B) Serum leptin level correlated positively and serum adiponectin level correlated negatively with the severity of hepatic steatosis. (C) Consequently, L/A ratio correlated positively with the severity of hepatic steatosis. (D–F) The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. The correlation coefficient was calculated using Spearman’s correlation test.
3.2. Relationship between L/A ratio and NAFLD
3.3. Relationship between baPWV and severity of steatosis in NAFLD
Serum leptin level increased with increasing severity of steatosis (r = 0.302, p < 0.001), while serum adiponectin level decreased with increasing severity of steatosis (r = −0.412, p < 0.001) (Fig. 1A and B). Consequently, L/A ratio increased with increasing severity of steatosis (r = 0.482, p < 0.001) (Fig. 1C). The L/A ratio showed positive correlations with BMI (r = 0.588, p < 0.001) and BFP (r = 0.603, p < 0.001), and a negative correlation with age (r = −0.131, p < 0.001) (Fig. 1D–F). We examined the gender differences in the L/A ratio in an exploratory analysis. The L/A ratio was higher in women than men regardless of the presence (1.4 ± 0.8 vs. 2.3 ± 2.4, p < 0.001) or absence (0.6 ± 0.3 vs. 0.9 ± 0.6, p < 0.001) of NAFLD (Fig. 2A). Although BMI in subjects with NAFLD was higher in both men and women compared with subjects without NAFLD (p < 0.001, respectively), men had higher BMI than women without NAFLD (22.8 ± 2.6 kg/m2 vs. 21.6 ± 3.0 kg/m2, p < 0.001), and there was no difference in BMI between men and women with NAFLD (Fig. 2B). On the other hand, BFP was higher in women than men regardless of the presence (24.6 ± 5.0% vs. 36.1 ± 6.3%, p < 0.001) or absence (19.2 ± 5.7% vs. 28.7 ± 6.6%, p < 0.001) of NAFLD in the same manner as L/A ratio (Fig. 2C).
There was a weak positive correlation between baPWV and severity of steatosis (r = 0.100, p = 0.003) (Fig. 3A). Only in women, baPWV was higher in subjects with NAFLD compared with subjects without NAFLD (1435.5 ± 378.9 cm/sec vs. 1509.9 ± 330.7 cm/sec, p < 0.05), and there was no difference in baPWV between men and women regardless of the presence or absence of NAFLD (1521.5 ± 338.2 cm/sec vs. 1509.9 ± 330.7 cm/sec, p = 0.914 and 1496.9 ± 411.5 cm/sec vs. 1435.5 ± 378.9 cm/sec, p = 0.097, respectively) (Fig. 3B). BaPWV was strongly correlated with age (r = 0.803, p < 0.001), while no relation was found between baPWV and L/A ratio (r = −0.058, p = 0.086) (Fig. 3C and D).
3.4. Correlations between IL and 6 and L/A ratio or other measured factors Although IL-6 level was lower in the subjects with NAFLD than in the subjects without NAFLD (Table 1), Il-6 level was very weakly correlated with leptin level and L/A ratio (r = 0.133, p < 0.001 and r = 0.130, p < 0.001, respectively) (Fig. 4A, C), whereas there was no correlation between IL and 6 level and adiponectin level (Fig. 4B). On the other hand, IL-6 level was also correlated with BMI, BFP, and age, and baPWV in particular (r = 0.351, p < 0.001) (Fig. 4D–G).
Fig. 2. Gender difference in the L/A ratio. (A) Women had higher a L/A ratio compared with men regardless of the presence or absence of NAFLD. (B) Both men and women had a higher BMI in subjects with NAFLD than subjects without NAFLD. However, men had a higher BMI than women in subjects without NAFLD. BMIs in subjects with NAFLD in both men and women were not significantly different. (C) On the contrary, women had a higher BFP compared with men regardless of the presence or absence of NAFLD in the same manner as L/A ratio. ***P < 0.001. 4
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Fig. 3. Relationship between baPWV and NAFLD. (A) BaPWV correlated weakly with the severity of hepatic steatosis. (B) BaPWVs between men and women were not significantly different in subjects both with or without NAFLD. Only in women, baPWV was higher in subjects with NAFLD than subjects without NAFLD. (C) BaPWV was strongly correlated with age. (D) On the contrary, baPWV was not correlated with L/A ratio. The correlation coefficient was calculated using Spearman’s correlation test. *P < 0.05.
Fig. 4. Correlations between IL and 6 and L/A ratio or other measured factors. The correlation coefficient between IL and 6 and measured factors were calculated using Spearman’s correlation test (A-G). Il-6 level was very weakly correlated with leptin level (A) and L/A ratio (C), whereas there was no correlation between IL and 6 level and adiponectin level (B). On the other hand, IL-6 was also correlated with BMI (E), BFP (F), and age (G), and baPWV (D) in particular. 5
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Fig. 5. Correlations between L/A ratio and serum lipid profiles or other biochemistry. The correlation coefficients between L/A ratio and biochemical components were calculated using Spearman’s correlation test (A-I). L/A ratio was correlated best with triglyceride level (D). Furthermore, L/A ratio was negatively correlated with HDL-cholesterol level (B). On the other hand, L/A ratio was very weakly correlated with LDL-cholesterol level (C), and there was no significant correlation between L/A ratio and total cholesterol level (A). Overall, L/A ratio was also correlated with the ratio of total cholesterol to HDL-cholesterol (E). In relation to other parameters, L/A ratio was weakly correlated with glycated hemoglobin A1c, ALT, and GGT (F, H, I), and there was no correlation between L/A ratio and AST (G).
steatosis. However, arterial stiffness was strongly correlated with age, and there was no relationship between arterial stiffness and L/A ratio. Leptin and adiponectin might be associated with NAFLD development and progression [14,27]. In fact, patients with NAFLD or NASH have shown a higher L/A ratio or lower adiponectin-to-leptin (A/L) ratio, correlated with lobular necrosis and fibrosis of the liver [44–46]. Moreover, L/A ratio correlates with obesity [47], metabolic syndrome [22,48–51], insulin resistance [23,24,52], and atherosclerosis [25,53], which are often accompanied by NAFLD. Indeed, in our study, subjects with NAFLD showed a higher L/A ratio and arterial stiffness compared with subjects without NAFLD in the general population. Previous studies have shown that the L/A ratio in women is higher than that in men in the general population, and in patients with metabolic syndrome or obesity [47,51]. These gender-related differences of the L/A ratio are considered to be due to the direct effect of sex hormones on adipokine synthesis [54]. Our data also showed a higher L/A ratio in women than in men with or without NAFLD. Furthermore, although the L/A ratio was associated with both BMI and BFP, the L/A ratio was more correlated with BFP than BMI. Therefore, the gender difference of L/A ratio is presumed to be partly due to the body fat composition. Epidemiological studies have suggested that NAFLD is associated with higher risk of atherogenic CVD development [4,5]. However, the causal relationship between NAFLD and CVD risk has not been clarified, because there are many relative factors such as heterogeneity of NAFLD, genetic variants, metabolic factors, and lifestyle difference [6]. In regard to CVD risk, arterial stiffness is associated with age, and is an independent predictor of cardiovascular morbidity and mortality [55–57]. In our study, arterial stiffness evaluated by baPWV was correlated with the severity of hepatic steatosis; however, baPWV was actually more strongly correlated with age. Moreover, baPWV was not
3.5. Correlations between L/A ratio and serum lipid profiles or other biochemistry Regarding the correlation between L/A ratio and lipids, the L/A ratio was correlated best with triglyceride level (r = 0.342, p < 0.001) (Fig. 5D). Moreover, the L/A ratio was negatively correlated with HDLcholesterol level (r = −0.314, p < 0.001) (Fig. 5B). On the other hand, the L/A ratio very weakly correlated with LDL-cholesterol level (r = 0.117, p < 0.001) (Fig. 5C), and there was no significant correlation between the L/A ratio and total cholesterol level (r = 0.066, p = 0.053) (Fig. 5A). Overall, the L/A ratio was also correlated with the ratio of total cholesterol to HDL-cholesterol (Fig. 5E). With respect to the relation with other parameters, the L/A ratio was weakly correlated with glycated hemoglobin A1c, ALT, and GGT (Fig. 5F, H, I), and there was no correlation between L/A ratio and AST (Fig. 5G). 4. Discussion The present study investigated the relationship between severity of steatosis in NAFLD and L/A ratio and arterial stiffness in an unselected community-dwelling population using a cross-sectional study design. In this study, the prevalence of NAFLD diagnosed by ultrasonography was 22.9%. The prevalence in our study was within the range of previous results [41–43]. The participants with NAFLD had a higher L/A ratio than those without NAFLD, and the L/A ratio increased with increased severity of steatosis. In addition, the L/A ratio was higher in women than men regardless of the presence or absence of NAFLD. Furthermore, L/A ratio was positively correlated with BMI and BFP, i.e., obesity, and L/A ratio was negatively correlated with age. On the other hand, arterial stiffness evaluated by baPWV was also associated with severity of 6
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suitable as a marker of atherosclerosis in NAFLD than L/A ratio. On the other hand, dyslipidemia is one of the risk factors of atherosclerosis and cardiovascular disease. Regarding the relationship between L/A ratio and lipid profiles in our study, the L/A ratio was positively correlated with triglyceride level and the ratio of total cholesterol to HDL-cholesterol, and negatively correlated with HDL-cholesterol. Thus, from these results of correlations between L/A ratio and lipid profiles, we consider that L/A ratio might be related to CVD risk in NAFLD. Furthermore, L/A ratio is associated with the risk of obesity-related cancers such as breast and endometrium cancer [76–80]. On the other hand, the association between L/A ratio and hepatocarcinogenesis has not been reported. Therefore, prospective studies are needed to investigate the relationship between L/A ratio and hepatocellular carcinoma in NAFLD patients. There are several limitations to our study. First, this study was limited by the cross-sectional design. Thus, longitudinal studies must be considered in the future to investigate these causal associations among the measured items. Second, the diagnosis of fatty liver was made based on ultrasonography examination without liver biopsy. While liver biopsy remains the standard for confirming the diagnosis, staging fibrosis, grading activity, and judging response to treatment, liver biopsy is an invasive procedure. In our study based on mass surveillance, it was impractical to perform liver biopsy in the general population. Instead, a common ultrasonographic definition of fatty liver was established and used as a noninvasive modality [40,81]. Third, our survey was limited to Japanese subjects, and hence possible ethnic differences were not considered.
associated with L/A ratio in our study. With regard to this point, a previous report showed that baPWV was not correlated with visceral fat area, while arterial elasticity had a strong correlation with visceral fat area [58]. Thus, it is considered that baPWV, which is not correlated with visceral fat area, was not correlated with L/A ratio in our study. In addition, another report indicated that, like our findings, there was no association between aortic stiffness and liver steatosis in obese patients [59]. On the other hand, a few reports showed the relationship between L/A ratio and carotid intima-media thickness [25,26] or CVD risk in men [53]. These differences in results between our study and other reports might be due to the differences in age and ethnicity, and in evaluation endpoints such as baPWV, arterial elasticity, and carotid intima-media thickness. Therefore, further studies are needed to clarify the L/A ratio and CVD risk, especially in NAFLD. Dietary intake and medications have been reported as factors affecting the expression of adipokines [60,61]. With regard to dietary intake, Mediterranean diets, low-carbohydrate diets, and low-fat diets induce body weight loss and improve cardiometabolic risk parameters such as blood pressure, serum lipids, leptin, and adipokine levels [62,63]. Mediterranean diets are rich in vegetables, nuts, and olive oil, and low in red meat, providing increased monounsaturated fatty acids and decreased saturated fatty acids [64]. Therefore, when evaluating leptin and adiponectin levels, it is recommended that meals for the participants be standardized. However, due to the nature of the community health survey with over 1000 participants in our study, it was impossible to standardize their daily diet. Alternatively, according to the evaluation of daily nutrition intake using BDHQ, the subjects with NAFLD had a significantly higher intake of daily energy and carbohydrates compared with the subjects without NAFLD, but daily intake of protein and total fat did not differ between the subjects with or without NAFLD (Table 1). Therefore, we consider that daily fat intake had the least influence on the evaluation of L/A ratio in our study. On the other hand, certain kinds of medications for hyperlipidemia (e.g., ezetimibe) [65,66], diabetes (e.g., sodium-glucose cotransporter type-2 inhibitors), [67,68], and hypertension (e.g., renin-angiotensin system inhibitors) [69] influence the expression levels of leptin and adiponectin. In our study, although we asked participants if they were taking any medications for hyperlipidemia, diabetes, and hypertension, we could not analyze each type of medication in detail. In addition, it has been reported that antipsychotics [70] and oral contraceptives [71] influence the expression levels of leptin and adiponectin. Unfortunately, we could not analyze these medications in this study due to the lack of interview information. Thus, in our study, the influence of medications was not completely eliminated from the evaluation of L/A ratio. On the other hand, although steroid therapy influences hepatic steatosis, very few participants had taken steroid medication (0.57% of all participants), and there was no difference in the number of participants who had taken steroid therapy between subjects with or without NAFLD (0.50% and 0.59%, respectively). Therefore, inclusion of the participants with steroid therapy might have had little impact on the evaluation of L/A ratio in our study. Leptin has a proinflammatory and atherogenic function, whereas adiponectin has an anti-inflammatory and protective function to regulate vascular homeostasis in the pathophysiological condition leading to atherosclerosis [72]. Indeed, L/A ratio has been suggested as a biomarker of obesity- and metabolic syndrome-associated CVD risk [73]. Leptin is also involved in immune response and exerts proinflammatory activity linked with proinflammatory cytokines such as IL-6, tumor necrosis factor-alpha, and interleukin-17 (IL-17) [60,74]. Moreover, IL6 is associated with the prevalence and severity of atherosclerosis in NAFLD [75]. In our study, although IL-6 level was lower in the participants with NAFLD compared with the participants without NAFLD (Table 1), IL-6 level was very weakly correlated with L/A ratio (Fig. 4C). Moreover, IL-6 level was correlated with baPWV, which is closely related to atherosclerosis (Fig. 4D). Thus, according to our findings and those in a previous report, IL-6 level might be more
5. Conclusion We found that L/A ratio and arterial stiffness were associated with the severity of steatosis in NAFLD in a Japanese community population. L/A ratio was correlated with body fat composition with gender difference, and arterial stiffness was correlated with age, respectively, whereas there was no correlation between L/A ratio and arterial stiffness. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the underlying mechanisms for increased atherosclerosis in NAFLD. Further studies with longitudinal observations are needed to investigate the involvement of L/A ratio in the progression of NAFLD. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We are extremely grateful to all the participants in the Iwaki Health Promotion Project and the entire staff of the project, who conducted the interview and collected the data. We appreciate Mr. Jeffery G. Stocker’s contribution to proofreading. Author contributions KM, TE, NS, SN, and SF contributed to study conception and design. KM, TE, NS, GI, MK, TH, CI, KS, and SN contributed to data acquisition. KM, TE, HT, MM, and NT contributed to data interpretation. KM analyzed the data and drafted the manuscript. SN is the guarantor of this work and takes responsibility for the integrity and accuracy of the data. Funding This work was supported by JST COI Grant Number JPMJCE1302. 7
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Ethical approval [32]
This study was approved by the Ethics Committee of the Hirosaki University School of Medicine, and written informed consent was obtained from all participants.
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Leptin/adiponectin ratio correlates with hepatic steatosis but not arterial stiffness in nonalcoholic fatty liver disease in Japanese population
T
⁎
Kenichiro Mikamia,b, , Tetsu Endob, Naoya Sawadab, Go Igarashib, Masayo Kimurab, Takuma Hasegawab, Chikara Iinob, Hirofumi Tomitac, Kaori Sawadad, Shigeyuki Nakajid, Masashi Matsuzakae, Natalie J. Torokf, Shinsaku Fukudab a
Department of Internal Medicine, Owani Hospital, Aomori, Japan Department of Gastroenterology, Hirosaki University Graduate School of Medicine, Aomori, Japan c Department of Cardiology, Hirosaki University Graduate School of Medicine, Aomori, Japan d Department of Social Medicine, Hirosaki University Graduate School of Medicine, Aomori, Japan e Clinical Research Support Center, Hirosaki University Hospital, Aomori, Japan f Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Leptin Adiponectin Nonalcoholic fatty liver disease Arterial stiffness Interleukin-6 Brief-type self-administered diet history questionnaire
Background and aims: Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD. Methods: The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV). Results: The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol. Conclusion: L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.
1. Introduction Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), are becoming the most common liver disorders worldwide. The prevalence of NAFLD has risen in all regions, with an overall prevalence worldwide of approximately 25% [1]. Fatigue is a
common symptom, but 48–100% of patients are asymptomatic [2]. Patients with advanced NASH, including cirrhosis, may have normal liver function tests, resulting in part from diminished inflammation as fibrosis progresses [3]. NAFLD is associated with components of metabolic syndrome, such as obesity, diabetes, hypertension, and dyslipidemia, and most notably the leading cause of death overall is
Abbreviations: NAFLD, nonalcoholic fatty liver disease; CVD, cardiovascular disease; BMI, body mass index; BFP, body fat percentage; BaPWV, brachial-ankle pulse wave velocity; L/A, leptin-to-adiponectin; BDHQ, brief-type self-administered diet history questionnaire; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GGT, gamma-glutamyl-transpeptidase; IL-6, interleukin-6; IL-17, interleukin-17 ⁎ Corresponding author at: Department of Gastroenterology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki City, Aomori 036-8562, Japan. E-mail address: [email protected] (K. Mikami). https://doi.org/10.1016/j.cyto.2019.154927 Received 10 August 2019; Received in revised form 4 November 2019; Accepted 11 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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food item, the subjects indicated their mean frequency of consumption of the food over the past month. The mean daily consumption of nutrients was calculated using an ad hoc computer program for BDHQ, which was based on the Standard Tables of Food Composition in Japan [39].
coronary artery disease among NAFLD patients [4,5]. However, the pathological association between NAFLD and cardiovascular disease (CVD) is still unclear, due to limitations of classical epidemiological studies and the absence of interventional studies for NAFLD treatment [6]. A major cause of CVD is atherosclerosis, which progresses to narrowing arteries leading to myocardial and cerebral ischemia [7]. Increased arterial stiffness is one of the earliest markers of functional and structural changes in arterial walls as atherosclerosis [8]. The measurement of pulse wave velocity (PWV) is widely used to assess arterial stiffness as a noninvasive, precise, and reproducible method [9,10]. Adipokines including leptin and adiponectin are polypeptides secreted by adipocytes and capable of acting at both local and systemic levels, providing a critical link between obesity, insulin resistance, and inflammation [11–13]. Leptin and adiponectin are two major adipokines with opposite effects in the pathogenesis of NAFLD [14]. Leptin promotes lipotoxicity and insulin resistance, and up-regulation of proinflammatory cytokines [15,16]. Leptin also plays a key role in hepatic fibrogenesis leading to cirrhosis via activation of hepatic stellate cells [17]. On the contrary, adiponectin has a protective role against obesity-related disorders including type 2 diabetes, metabolic syndrome, and cardiovascular disease, by reduction of inflammatory cytokines and oxidative stress leading to an improvement of insulin resistance [18–21]. Several recent studies have shown that the leptinto-adiponectin (L/A) ratio or adiponectin-to-leptin (A/L) ratio is a useful parameter to assess insulin resistance in patients with and without diabetes [22–26]. In NAFLD, adipokine imbalance is involved in increased leptin levels and decreased adiponectin levels [14,27]. However, these studies evaluated only patients with NAFLD, and there have been few general community population-based studies. Since NAFLD is generally asymptomatic, screening for NAFLD is very important to prevent disease progression. Therefore, we conducted a general population-based cross-sectional study to assess the relationship between L/A ratio and CVD risk in NAFLD in a Japanese community.
2.3. Clinical and laboratory assessment Hypertension was defined as blood pressure ≥140/90 mm Hg or use of antihypertensive medication. Diabetes was defined as fasting serum glucose ≥126 mg/dL, HbA1c ≥6.5%, use of diabetes medication, or a prior known diabetes diagnosis. Hyperlipidemia was defined as total cholesterol ≥220 mg/dL, triglycerides ≥150 mg/dL, or use of antihyperlipidemic medication. The following clinical characteristics were measured: height, body weight, and body composition. Body mass index (BMI) was calculated as body weight divided by height squared and expressed in kg/m2. Body fat percentage (BFP) was measured by a body composition analyzer MC-190 (Tanita Corp., Tokyo, Japan). Alcohol intake was determined from a questionnaire. Fasting whole blood samples were obtained after overnight fast, and laboratory tests included aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, glycated hemoglobin, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride, gamma-glutamyl-transpeptidase (GGT), interleukin6 (IL-6), leptin, and adiponectin. Serum levels of IL-6, leptin, and adiponectin were determined using chemiluminescent enzyme immunoassay, radioimmunoassay, and latex agglutination turbidimetric immunoassay, respectively, in a commercial laboratory (LSI Medience Corp., Tokyo, Japan). 2.4. Assessment of severity of fatty liver NAFLD was diagnosed based on abdominal ultrasound findings using a Prosound F37 (Hitachi Aloka Medical Ltd.). Ultrasound examinations were performed by one of five well-trained hepatology specialists, each with more than 5 years of experience, without detailed knowledge of the participant’s data. Images were stored and reevaluated by a single hepatologist with more than 20 years of experience. The severity of echogenicity was graded as follows: normal, normal echogenicity; mild, slight diffuse increase in fine echoes in liver parenchyma with normal visualization of diaphragm and intrahepatic vessel borders; moderate, moderate diffuse increase in fine echoes with slightly impaired visualization of hepatic vessel border and diaphragm; severe, marked increase in fine echoes with poor or non-visualization of the intrahepatic vessel borders, diaphragm, and posterior right lobe of the liver [40].
2. Materials and methods 2.1. Study population The Iwaki Health Promotion Project is an ongoing community-based health promotion study of Japanese people ≥20 years of age designed to prevent lifestyle-related diseases and prolong their lifespan. This program has been carried out annually since 2005 with about 1000 participants in the Iwaki region of Hirosaki City in Aomori Prefecture located in northern Japan [28–31]. All study subjects participated voluntarily in response to a public announcement, and approximately 600 data points were collected from each participant, including their demographics, medical history, lifestyle data, and microbiota and blood chemical analysis data. Our research on the association between fatty liver and adipokines is one part of this project. In 2016, 1148 individuals were enrolled in this project. Of these, all individuals who did not have complete clinical data, who were positive for hepatitis B surface antigen or anti-hepatitis C virus antibody, and/or had excessive alcohol consumption (daily alcohol intake greater than 30 g/day for men and 20 g/day for women), were excluded. Ultimately, 871 subjects (273 men and 598 women) were included in the current study. This study was approved by the Ethics Committee of the Hirosaki University School of Medicine, and written informed consent was obtained from all participants.
2.5. Assessment of arterial stiffness Arterial stiffness was diagnosed using brachial-ankle pulse wave velocity using a blood pressure pulse wave monitor (BP-203RPE; Omron Healthcare Co., Ltd., Kyoto, Japan). This device provides automated measurements of baPWV on both the right and left sides, and the average of the two sides was used in the current study [10]. 2.6. Statistical analysis All statistical analyses of collected data were performed using the Excel statistical software package for Macintosh (Ekuseru-Toukei 2016; Esumi Co., Ltd., Tokyo, Japan). Categorical variables were compared using the chi-square test. Ratios of serum leptin to adiponectin (L/A ratio) were compared using the Kruskal-Wallis test, followed by a SteelDwass test or Mann-Whitney U test. Spearman’s correlation coefficient was used to calculate the correlation between the L/A ratio and BMI or body fat percentage. Differences were considered to be significant with P-values of less than 0.05.
2.2. Dietary assessment Dietary habits were assessed by using a brief-type self-administered diet history questionnaire (BDHQ) that included questions on the consumption frequency of 56 foods and beverages and nine dishes commonly consumed in the general Japanese population [32–38]. For each 2
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Table 1 Baseline characteristics of study participants. Characteristic Gender Male, n (%) Female, n (%) Age (years), mean (SD) Degree of hepatic steatosis, n (%) Normal Mild Moderate Severe Alcohol consumption (g/day), mean (SD) Daily nutrition intake Energy (kcal), mean (SD) Protein (g), mean (SD) Total fat (g), mean (SD) Saturated fatty acid (g), mean (SD) Monounsaturated fatty acid (g), mean (SD) Polyunsaturated fatty acid (g), mean (SD) n-3 polyunsaturated (g), mean (SD) n-6 polyunsaturated (g), mean (SD) Cholesterol (mg), mean (SD) Carbohydrates (g), mean (SD) Body mass index (kg/m2), mean (SD) Body fat percentage (%), mean (SD) Hypertension, n (%) Medication for hypertension, n (%) Diabetes, n (%) Medication for diabetes, n (%) Hyperlipidemia, n (%) Medication for hyperlipidemia, n (%) Steroid exposure by medication, n (%) Glucose level (mg/dL), mean (SD) Glycated hemoglobin A1c level (%), mean (SD) Total cholesterol level (mg/dL), mean (SD) HDL-cholesterol level (mg/dL), mean (SD) LDL-cholesterol level (mg/dL), mean (SD) Triglyceride level (mg/dL), mean (SD) AST level (U/L), mean (SD) ALT level (U/L), mean (SD) GGT level (U/L), mean (SD) Leptin (ng/mL), mean (SD) Adiponectin (μg/mL), mean (SD) Leptin/Adiponectin ratio, mean (SD) IL-6 (pg/mL), mean (SD) BaPWV (cm/sec), mean (SD)
Total (n = 871)
Normal (n = 671)
NAFLD (n = 200)
p Value
273 (31.3) 598 (68.7) 54.0 (16.1)
190 (28.3) 481 (71.7) 53.6 (16.9)
83 (41.5) 117 (58.5) 55.3 (13.3)
< 0.001 0.246
671 (77.0) 111 (12.8) 74 (8.5) 15 (1.7) 4.0 (7.0)
671 (1 0 0)
3.9 (6.8)
111 (55.5) 74 (37.0) 15 (7.5) 4.6 (7.9)
0.795
1797.7 (557.5) 69.3 (26.9) 52.6 (19.9) 14.1 (5.8) 18.5 (7.2) 13.1 (4.8) 2.7 (1.3) 10.3 (3.7) 359.9 (178.3) 247.6 (80.0) 22.8 (3.5) 27.2 (8.1) 325 (37.3) 218 (25.0) 94 (10.8) 41 (4.70) 384 (44.1) 96 (11.0) 5 (0.57) 91.8 (20.9) 5.8 (0.6) 204.9 (35.9) 63.9 (16.5) 118.4 (30.3) 92.6 (60.5) 22.4 (8.6) 21.3 (14.8) 26.8 (23.9) 9.1 (6.6) 11.0 (6.1) 1.0 (1.0) 1.91 (6.44) 1467.1 (377.7)
1782.4 (564.7) 69.1 (27.6) 52.4 (20.5) 14.0 (6.0) 18.4 (7.4) 13.1 (4.9) 2.7 (1.3) 10.3 (3.8) 360.3 (183.9) 244.7 (80.0) 21.9 (2.9) 26.0 (7.6) 221 (32.9) 148 (22.0) 54 (8.0) 26 (3.87) 266 (39.6) 59 (8.7) 4 (0.59) 90.2 (21.3) 5.8 (0.6) 202.8 (36.3) 66.3 (15.6) 116.0 (30.4) 81.2 (43.6) 21.2 (7.0) 18.1 (10.8) 23.6 (20.8) 8.8 (5.5) 12.1 (6.3) 0.8 (0.7) 2.00 (7.27) 1452.9 (389.1)
1849.3 (530.6) 70.2 (24.4) 53.2 (17.7) 14.4 (5.0) 18.6 (6.3) 13.2 (4.4) 2.7 (1.1) 10.4 (3.3) 358.6 (158.2) 257.2 (79.5) 25.8 (3.6) 31.3 (8.1) 104 (52.0) 70 (35.0) 40 (20.0) 15 (7.50) 118 (59.0) 37 (18.5) 1 (0.50) 97.3 (18.9) 6.1 (0.7) 211.8 (33.7) 55.8 (16.9) 126.4 (28.6) 131.0 (87.5) 26.3 (11.7) 32.3 (20.3) 37.7 (29.9) 12.8 (8.4) 7.2 (2.8) 1.9 (1.4) 1.64 (1.80) 1514.8 (333.0)
0.029 0.206 0.276 0.120 0.297 0.425 0.766 0.342 0.694 0.017 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.034 < 0.001 < 0.001 0.875 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.003
Abbreviations: BaPWV, brachial-ankle pulse wave velocity; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl-transpeptidase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Interleukin-6, IL-6. Boldface type indicates a significant p value (p < 0.05). Hypertension was defined as blood pressure ≥140/90 mmHg or use of antihypertensive medication. Diabetes was defined as fasting serum glucose ≥126 mg/dL, HbA1c ≥6.5%, use of diabetes medication, or a prior known diabetes diagnosis. Hyperlipidemia was defined as total cholesterol ≥220 mg/dL, triglycerides ≥150 mg/dL, or use of antihyperlipidemic medication.
3. Results
consumption between subjects with and without NAFLD. Mean daily energy and carbohydrate intake determined by the BDHQ were significantly higher in the subjects with NAFLD compared the subjects without NAFLD (p = 0.029 and p = 0.017, respectively) (Table 1). On the other hand, intake of protein and total fat containing saturated fatty acid, monounsaturated fatty acid, polyunsaturated fatty acid including n-3 polyunsaturated fatty acid and n-6 polyunsaturated fatty acid, and cholesterol did not differ between subjects with or without NAFLD (Table 1). With regard to medications that subjects were taking, the subjects with NAFLD had higher medication rates compared with the subject without NAFLD for treatment of hypertension, diabetes, and hyperlipidemia (p < 0.001, p = 0.034, and p < 0.001, respectively). Furthermore, one subject with NAFLD and four subjects without NAFLD (0.50% and 0.59%, respectively) received steroid therapy for inflammatory disease such as arthritis. In addition, there were no subjects with conditions associated with steatosis such as genetic disease, parenteral nutrition, and malnutrition.
3.1. Characteristics of the participants with and without NAFLD The characteristics of the study subjects with and without NAFLD are shown in Table 1. Of the 871 subjects included, 200 subjects (22.9%) had NAFLD. Compared with the subjects without NAFLD, the subjects with NAFLD were predominantly male; had a greater BMI and body fat percentage (BFP); showed a higher prevalence of hypertension, diabetes, and hyperlipidemia; and presented higher levels of glucose, glycated hemoglobin A1c, total cholesterol, LDL-cholesterol, triglyceride, AST, ALT, and GGT, and lower level of HDL-cholesterol. A significantly higher level of leptin, lower level of adiponectin, and higher L/A ratio were observed in the subjects with NAFLD as compared with the subjects without NAFLD. IL-6 level was lower in the subjects with NAFLD than in the subjects without NAFLD (p < 0.001). In addition, the subjects with NAFLD had higher baPWV compared with the subjects without NAFLD. There were no differences in age and alcohol 3
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Fig. 1. Correlation between L/A ratio and severity of hepatic steatosis in NAFLD. (A, B) Serum leptin level correlated positively and serum adiponectin level correlated negatively with the severity of hepatic steatosis. (C) Consequently, L/A ratio correlated positively with the severity of hepatic steatosis. (D–F) The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. The correlation coefficient was calculated using Spearman’s correlation test.
3.2. Relationship between L/A ratio and NAFLD
3.3. Relationship between baPWV and severity of steatosis in NAFLD
Serum leptin level increased with increasing severity of steatosis (r = 0.302, p < 0.001), while serum adiponectin level decreased with increasing severity of steatosis (r = −0.412, p < 0.001) (Fig. 1A and B). Consequently, L/A ratio increased with increasing severity of steatosis (r = 0.482, p < 0.001) (Fig. 1C). The L/A ratio showed positive correlations with BMI (r = 0.588, p < 0.001) and BFP (r = 0.603, p < 0.001), and a negative correlation with age (r = −0.131, p < 0.001) (Fig. 1D–F). We examined the gender differences in the L/A ratio in an exploratory analysis. The L/A ratio was higher in women than men regardless of the presence (1.4 ± 0.8 vs. 2.3 ± 2.4, p < 0.001) or absence (0.6 ± 0.3 vs. 0.9 ± 0.6, p < 0.001) of NAFLD (Fig. 2A). Although BMI in subjects with NAFLD was higher in both men and women compared with subjects without NAFLD (p < 0.001, respectively), men had higher BMI than women without NAFLD (22.8 ± 2.6 kg/m2 vs. 21.6 ± 3.0 kg/m2, p < 0.001), and there was no difference in BMI between men and women with NAFLD (Fig. 2B). On the other hand, BFP was higher in women than men regardless of the presence (24.6 ± 5.0% vs. 36.1 ± 6.3%, p < 0.001) or absence (19.2 ± 5.7% vs. 28.7 ± 6.6%, p < 0.001) of NAFLD in the same manner as L/A ratio (Fig. 2C).
There was a weak positive correlation between baPWV and severity of steatosis (r = 0.100, p = 0.003) (Fig. 3A). Only in women, baPWV was higher in subjects with NAFLD compared with subjects without NAFLD (1435.5 ± 378.9 cm/sec vs. 1509.9 ± 330.7 cm/sec, p < 0.05), and there was no difference in baPWV between men and women regardless of the presence or absence of NAFLD (1521.5 ± 338.2 cm/sec vs. 1509.9 ± 330.7 cm/sec, p = 0.914 and 1496.9 ± 411.5 cm/sec vs. 1435.5 ± 378.9 cm/sec, p = 0.097, respectively) (Fig. 3B). BaPWV was strongly correlated with age (r = 0.803, p < 0.001), while no relation was found between baPWV and L/A ratio (r = −0.058, p = 0.086) (Fig. 3C and D).
3.4. Correlations between IL and 6 and L/A ratio or other measured factors Although IL-6 level was lower in the subjects with NAFLD than in the subjects without NAFLD (Table 1), Il-6 level was very weakly correlated with leptin level and L/A ratio (r = 0.133, p < 0.001 and r = 0.130, p < 0.001, respectively) (Fig. 4A, C), whereas there was no correlation between IL and 6 level and adiponectin level (Fig. 4B). On the other hand, IL-6 level was also correlated with BMI, BFP, and age, and baPWV in particular (r = 0.351, p < 0.001) (Fig. 4D–G).
Fig. 2. Gender difference in the L/A ratio. (A) Women had higher a L/A ratio compared with men regardless of the presence or absence of NAFLD. (B) Both men and women had a higher BMI in subjects with NAFLD than subjects without NAFLD. However, men had a higher BMI than women in subjects without NAFLD. BMIs in subjects with NAFLD in both men and women were not significantly different. (C) On the contrary, women had a higher BFP compared with men regardless of the presence or absence of NAFLD in the same manner as L/A ratio. ***P < 0.001. 4
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Fig. 3. Relationship between baPWV and NAFLD. (A) BaPWV correlated weakly with the severity of hepatic steatosis. (B) BaPWVs between men and women were not significantly different in subjects both with or without NAFLD. Only in women, baPWV was higher in subjects with NAFLD than subjects without NAFLD. (C) BaPWV was strongly correlated with age. (D) On the contrary, baPWV was not correlated with L/A ratio. The correlation coefficient was calculated using Spearman’s correlation test. *P < 0.05.
Fig. 4. Correlations between IL and 6 and L/A ratio or other measured factors. The correlation coefficient between IL and 6 and measured factors were calculated using Spearman’s correlation test (A-G). Il-6 level was very weakly correlated with leptin level (A) and L/A ratio (C), whereas there was no correlation between IL and 6 level and adiponectin level (B). On the other hand, IL-6 was also correlated with BMI (E), BFP (F), and age (G), and baPWV (D) in particular. 5
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Fig. 5. Correlations between L/A ratio and serum lipid profiles or other biochemistry. The correlation coefficients between L/A ratio and biochemical components were calculated using Spearman’s correlation test (A-I). L/A ratio was correlated best with triglyceride level (D). Furthermore, L/A ratio was negatively correlated with HDL-cholesterol level (B). On the other hand, L/A ratio was very weakly correlated with LDL-cholesterol level (C), and there was no significant correlation between L/A ratio and total cholesterol level (A). Overall, L/A ratio was also correlated with the ratio of total cholesterol to HDL-cholesterol (E). In relation to other parameters, L/A ratio was weakly correlated with glycated hemoglobin A1c, ALT, and GGT (F, H, I), and there was no correlation between L/A ratio and AST (G).
steatosis. However, arterial stiffness was strongly correlated with age, and there was no relationship between arterial stiffness and L/A ratio. Leptin and adiponectin might be associated with NAFLD development and progression [14,27]. In fact, patients with NAFLD or NASH have shown a higher L/A ratio or lower adiponectin-to-leptin (A/L) ratio, correlated with lobular necrosis and fibrosis of the liver [44–46]. Moreover, L/A ratio correlates with obesity [47], metabolic syndrome [22,48–51], insulin resistance [23,24,52], and atherosclerosis [25,53], which are often accompanied by NAFLD. Indeed, in our study, subjects with NAFLD showed a higher L/A ratio and arterial stiffness compared with subjects without NAFLD in the general population. Previous studies have shown that the L/A ratio in women is higher than that in men in the general population, and in patients with metabolic syndrome or obesity [47,51]. These gender-related differences of the L/A ratio are considered to be due to the direct effect of sex hormones on adipokine synthesis [54]. Our data also showed a higher L/A ratio in women than in men with or without NAFLD. Furthermore, although the L/A ratio was associated with both BMI and BFP, the L/A ratio was more correlated with BFP than BMI. Therefore, the gender difference of L/A ratio is presumed to be partly due to the body fat composition. Epidemiological studies have suggested that NAFLD is associated with higher risk of atherogenic CVD development [4,5]. However, the causal relationship between NAFLD and CVD risk has not been clarified, because there are many relative factors such as heterogeneity of NAFLD, genetic variants, metabolic factors, and lifestyle difference [6]. In regard to CVD risk, arterial stiffness is associated with age, and is an independent predictor of cardiovascular morbidity and mortality [55–57]. In our study, arterial stiffness evaluated by baPWV was correlated with the severity of hepatic steatosis; however, baPWV was actually more strongly correlated with age. Moreover, baPWV was not
3.5. Correlations between L/A ratio and serum lipid profiles or other biochemistry Regarding the correlation between L/A ratio and lipids, the L/A ratio was correlated best with triglyceride level (r = 0.342, p < 0.001) (Fig. 5D). Moreover, the L/A ratio was negatively correlated with HDLcholesterol level (r = −0.314, p < 0.001) (Fig. 5B). On the other hand, the L/A ratio very weakly correlated with LDL-cholesterol level (r = 0.117, p < 0.001) (Fig. 5C), and there was no significant correlation between the L/A ratio and total cholesterol level (r = 0.066, p = 0.053) (Fig. 5A). Overall, the L/A ratio was also correlated with the ratio of total cholesterol to HDL-cholesterol (Fig. 5E). With respect to the relation with other parameters, the L/A ratio was weakly correlated with glycated hemoglobin A1c, ALT, and GGT (Fig. 5F, H, I), and there was no correlation between L/A ratio and AST (Fig. 5G). 4. Discussion The present study investigated the relationship between severity of steatosis in NAFLD and L/A ratio and arterial stiffness in an unselected community-dwelling population using a cross-sectional study design. In this study, the prevalence of NAFLD diagnosed by ultrasonography was 22.9%. The prevalence in our study was within the range of previous results [41–43]. The participants with NAFLD had a higher L/A ratio than those without NAFLD, and the L/A ratio increased with increased severity of steatosis. In addition, the L/A ratio was higher in women than men regardless of the presence or absence of NAFLD. Furthermore, L/A ratio was positively correlated with BMI and BFP, i.e., obesity, and L/A ratio was negatively correlated with age. On the other hand, arterial stiffness evaluated by baPWV was also associated with severity of 6
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suitable as a marker of atherosclerosis in NAFLD than L/A ratio. On the other hand, dyslipidemia is one of the risk factors of atherosclerosis and cardiovascular disease. Regarding the relationship between L/A ratio and lipid profiles in our study, the L/A ratio was positively correlated with triglyceride level and the ratio of total cholesterol to HDL-cholesterol, and negatively correlated with HDL-cholesterol. Thus, from these results of correlations between L/A ratio and lipid profiles, we consider that L/A ratio might be related to CVD risk in NAFLD. Furthermore, L/A ratio is associated with the risk of obesity-related cancers such as breast and endometrium cancer [76–80]. On the other hand, the association between L/A ratio and hepatocarcinogenesis has not been reported. Therefore, prospective studies are needed to investigate the relationship between L/A ratio and hepatocellular carcinoma in NAFLD patients. There are several limitations to our study. First, this study was limited by the cross-sectional design. Thus, longitudinal studies must be considered in the future to investigate these causal associations among the measured items. Second, the diagnosis of fatty liver was made based on ultrasonography examination without liver biopsy. While liver biopsy remains the standard for confirming the diagnosis, staging fibrosis, grading activity, and judging response to treatment, liver biopsy is an invasive procedure. In our study based on mass surveillance, it was impractical to perform liver biopsy in the general population. Instead, a common ultrasonographic definition of fatty liver was established and used as a noninvasive modality [40,81]. Third, our survey was limited to Japanese subjects, and hence possible ethnic differences were not considered.
associated with L/A ratio in our study. With regard to this point, a previous report showed that baPWV was not correlated with visceral fat area, while arterial elasticity had a strong correlation with visceral fat area [58]. Thus, it is considered that baPWV, which is not correlated with visceral fat area, was not correlated with L/A ratio in our study. In addition, another report indicated that, like our findings, there was no association between aortic stiffness and liver steatosis in obese patients [59]. On the other hand, a few reports showed the relationship between L/A ratio and carotid intima-media thickness [25,26] or CVD risk in men [53]. These differences in results between our study and other reports might be due to the differences in age and ethnicity, and in evaluation endpoints such as baPWV, arterial elasticity, and carotid intima-media thickness. Therefore, further studies are needed to clarify the L/A ratio and CVD risk, especially in NAFLD. Dietary intake and medications have been reported as factors affecting the expression of adipokines [60,61]. With regard to dietary intake, Mediterranean diets, low-carbohydrate diets, and low-fat diets induce body weight loss and improve cardiometabolic risk parameters such as blood pressure, serum lipids, leptin, and adipokine levels [62,63]. Mediterranean diets are rich in vegetables, nuts, and olive oil, and low in red meat, providing increased monounsaturated fatty acids and decreased saturated fatty acids [64]. Therefore, when evaluating leptin and adiponectin levels, it is recommended that meals for the participants be standardized. However, due to the nature of the community health survey with over 1000 participants in our study, it was impossible to standardize their daily diet. Alternatively, according to the evaluation of daily nutrition intake using BDHQ, the subjects with NAFLD had a significantly higher intake of daily energy and carbohydrates compared with the subjects without NAFLD, but daily intake of protein and total fat did not differ between the subjects with or without NAFLD (Table 1). Therefore, we consider that daily fat intake had the least influence on the evaluation of L/A ratio in our study. On the other hand, certain kinds of medications for hyperlipidemia (e.g., ezetimibe) [65,66], diabetes (e.g., sodium-glucose cotransporter type-2 inhibitors), [67,68], and hypertension (e.g., renin-angiotensin system inhibitors) [69] influence the expression levels of leptin and adiponectin. In our study, although we asked participants if they were taking any medications for hyperlipidemia, diabetes, and hypertension, we could not analyze each type of medication in detail. In addition, it has been reported that antipsychotics [70] and oral contraceptives [71] influence the expression levels of leptin and adiponectin. Unfortunately, we could not analyze these medications in this study due to the lack of interview information. Thus, in our study, the influence of medications was not completely eliminated from the evaluation of L/A ratio. On the other hand, although steroid therapy influences hepatic steatosis, very few participants had taken steroid medication (0.57% of all participants), and there was no difference in the number of participants who had taken steroid therapy between subjects with or without NAFLD (0.50% and 0.59%, respectively). Therefore, inclusion of the participants with steroid therapy might have had little impact on the evaluation of L/A ratio in our study. Leptin has a proinflammatory and atherogenic function, whereas adiponectin has an anti-inflammatory and protective function to regulate vascular homeostasis in the pathophysiological condition leading to atherosclerosis [72]. Indeed, L/A ratio has been suggested as a biomarker of obesity- and metabolic syndrome-associated CVD risk [73]. Leptin is also involved in immune response and exerts proinflammatory activity linked with proinflammatory cytokines such as IL-6, tumor necrosis factor-alpha, and interleukin-17 (IL-17) [60,74]. Moreover, IL6 is associated with the prevalence and severity of atherosclerosis in NAFLD [75]. In our study, although IL-6 level was lower in the participants with NAFLD compared with the participants without NAFLD (Table 1), IL-6 level was very weakly correlated with L/A ratio (Fig. 4C). Moreover, IL-6 level was correlated with baPWV, which is closely related to atherosclerosis (Fig. 4D). Thus, according to our findings and those in a previous report, IL-6 level might be more
5. Conclusion We found that L/A ratio and arterial stiffness were associated with the severity of steatosis in NAFLD in a Japanese community population. L/A ratio was correlated with body fat composition with gender difference, and arterial stiffness was correlated with age, respectively, whereas there was no correlation between L/A ratio and arterial stiffness. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the underlying mechanisms for increased atherosclerosis in NAFLD. Further studies with longitudinal observations are needed to investigate the involvement of L/A ratio in the progression of NAFLD. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We are extremely grateful to all the participants in the Iwaki Health Promotion Project and the entire staff of the project, who conducted the interview and collected the data. We appreciate Mr. Jeffery G. Stocker’s contribution to proofreading. Author contributions KM, TE, NS, SN, and SF contributed to study conception and design. KM, TE, NS, GI, MK, TH, CI, KS, and SN contributed to data acquisition. KM, TE, HT, MM, and NT contributed to data interpretation. KM analyzed the data and drafted the manuscript. SN is the guarantor of this work and takes responsibility for the integrity and accuracy of the data. Funding This work was supported by JST COI Grant Number JPMJCE1302. 7
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Ethical approval [32]
This study was approved by the Ethics Committee of the Hirosaki University School of Medicine, and written informed consent was obtained from all participants.
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