Hepatol Int (2013) 7:586–591 DOI 10.1007/s12072-012-9412-1
ORIGINAL ARTICLE
Insulin resistance, inflammation, and nonalcoholic fatty liver disease in non-obese adults without metabolic syndrome components Seonah Kim • Jaekyung Choi • Mina Kim
Received: 10 April 2012 / Accepted: 24 October 2012 / Published online: 29 November 2012 Ó Asian Pacific Association for the Study of the Liver 2012
Abstract Purpose The purpose of the present study was to examine the association of insulin resistance and inflammation with nonalcoholic fatty liver disease (NAFLD) in non-obese adults without metabolic syndrome components. Methods This was a cross-sectional study of 759 subjects aged 50 years and older. Diagnosis of NAFLD was based on sonographic evidence of fatty liver without significant alcohol consumption and another cause of chronic liver disease. Subjects without metabolic syndrome components were defined as having none of the following: high blood pressure (C130/85 mmHg), elevated fasting glucose (C100 mg/dl), hypertriglyceridemia (C150 mg/dl), low high-density lipoprotein-cholesterol (men \40 mg/dl; women \50 mg/dl), and abdominal obesity measured by waist circumference C90 cm for men and C80 cm for women. The subjects were divided into quartile groups according to levels of high-sensitivity C-reactive protein (hs-CRP), homeostasis model assessment of insulin resistance (HOMA-IR), and uric acid. Odds ratios (ORs) were computed for each quartile relative to the lowest quartile group. Results After adjustment for age, sex, smoking status, regular exercise, hs-CRP, HOMA-IR, and uric acid, a significant association was found between NAFLD and higher levels of hs-CRP, HOMA-IR, and uric acid. After adjustment for age, sex, smoking status, regular exercise, hs-CRP, HOMA-IR, and uric acid, the Ors (95 % confidence interval) of NAFLD with the highest quartile of
S. Kim J. Choi (&) M. Kim Department of Family Medicine, Konkuk University Medical Center, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, South Korea e-mail: [email protected]
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hs-CRP, HOMA-IR, and uric acid compared with the lowest quartile were 2.58 (1.03–6.50), 2.55 (1.08–6.05), and 5.15 (1.78–14.89), respectively. Conclusions Insulin resistance and inflammation are independently associated with NAFLD in non-obese adults without metabolic syndrome components. Keywords Nonalcoholic fatty liver disease Homeostasis model assessment of insulin resistance High-sensitivity C-reactive protein Uric acid
Introduction Nonalcoholic fatty liver disease (NAFLD) is defined as the accumulation of fat in the liver in the absence of significant alcohol consumption and other causes of chronic liver disease such as viral hepatitis or drugs [1]. This condition encompasses a wide spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis [2]. NAFLD is being identified with rising frequency and is estimated to be an increasingly common problem worldwide in the general population [3, 4]. Nonalcoholic fatty liver disease has been associated with obesity, hyperglycemia, dyslipidemia, and hypertension, and it is currently regarded as the hepatic manifestation of metabolic syndrome [5]. The pathophysiology involved in the development and progression of NAFLD is associated with insulin resistance and inflammation [6]. Although insulin resistance and inflammation have often been considered to be the link with NAFLD, the roles of insulin resistance and inflammation associated with NAFLD in non-obese adults without metabolic syndrome components have been less well established. The purpose of this study was to examine the insulin resistance and
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inflammation associated with NAFLD in non-obese adults without metabolic syndrome components.
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The present study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the institutional review board at Konkuk University Medical Center.
Materials and methods Measurements Study population This was a cross-sectional study designed to identify the association among insulin resistance, inflammation, and NAFLD. The subjects were 3,011 individuals, aged 50 years and older, who visited the health promotion center at Konkuk University Medical Center, Seoul, South Korea, between 1 May 2008 and 30 April 2010, for a general health checkup. Subjects without metabolic syndrome components were defined in terms of the absence of the five following features [7]: high blood pressure (C130/C85 mmHg), elevated fasting glucose (C100 mg/dl), hypertriglyceridemia (C150 mg/dl), low high-density lipoprotein (HDL)cholesterol (men \40 mg/dl; women \50 mg/dl), and abdominal obesity using the Asia Pacific criterion for abdominal obesity [8] (waist circumference C90 cm for men and C80 cm for women). Subjects receiving pharmacologic treatment for hypertension (i.e., ACE inhibitors, angiotensin receptor blockers, a-blockers, b-blockers, calcium channel blockers, or diuretics) were included in the high blood pressure group. Subjects receiving pharmacologic treatment for diabetes (i.e., sulphonylureas, biguanides, a-glucosidase inhibitors, or insulin) were included in the elevated fasting glucose group. We used the definition of ‘‘non-obese’’ (BMI \ 25 kg/m2) proposed by the World Health Organization Western Pacific Region, the International Association for the Study of Obesity, and the International Obesity Task Force [8]. We defined ‘‘nonalcoholic fatty liver disease’’ as the ultrasonographic diagnosis of fatty liver (USFL) without significant alcohol consumption or some other cause of chronic liver disease. A total of 2,252 subjects were excluded for the following reasons: 1,595 subjects were defined as subjects with a metabolic syndrome component; 1,039 subjects were defined as obese; 1,013 subjects reported an alcohol intake of at least 30 g/day (in men) or 20 g/day (in women) [9]; 159 subjects had positive serologic markers for hepatitis B or C virus; 64 subjects displayed elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels ([80 IU/l); 97 subjects had abnormal liver ultrasound findings (indicating liver cirrhosis, intrahepatic or extrahepatic cholelithiasis, or abnormal dilatation of the biliary tree) or were taking medications for hepatitis. Following exclusion of some individuals for multiple reasons, 759 subjects, aged 50–86 years, were identified as eligible participants.
The general health checkup included a medical history, physical examination, lifestyle questionnaire, biochemical measurements, and abdominal ultrasonography. Trained nurses measured sitting blood pressure levels with a standard mercury sphygmomanometer. Height and weight were measured after an overnight fast with the patient wearing a lightweight hospital gown and no shoes. BMI was calculated by dividing the weight in kilograms by the square of the height in meters. Smoking, alcohol intake, and regular exercise were assessed using information from the lifestyle questionnaire. For smoking, individuals were classified as nonsmoker, past smoker, or current smoker. Questions about alcohol intake included the frequency of alcohol consumption on a weekly basis and the usual amount consumed on a daily basis. Regular exercise was defined as engaging in physical activity for a minimum of 30 min at least three times per week. Blood specimens were sampled from the antecubital vein after more than 12 h of fasting. Serum levels of glucose, uric acid, creatinine, triglycerides, HDL, ALT, AST, and c-glutamyltransferase (GGT) were measured using Bayer reagent packs on a 200FR-Neo automated chemistry analyzer (Toshiba, Tokyo, Japan). Measurement techniques included the hexokinase method for glucose, an enzymatic colorimetric assay for serum lipids, and an immunoradiometric assay for insulin (Roche/Hitachi Modular analytics E170; Hitachi, Tokyo, Japan). Insulin resistance was assessed with the homeostasis model assessment of insulin resistance (HOMAIR) according to the following equation: fasting blood insulin (in microunits per milliliter) 9 fasting blood glucose (in mm/l)/22.5. High-sensitivity C-reactive protein (hs-CRP) was analyzed by particle-enhanced immunonephelometry with the BNII System (200FR-Neo; Toshiba). Glomerular filtration rate was estimated from the modification of diet in the renal disease study equation as follows: 186 9 (serum creatinine)-1.154 9 (age)-0.203 9 0.742 (if female) [10]. The clinical laboratory used in the study had been accredited and participated annually in inspections and surveys by the Korean Association of Quality Assurance for Clinical Laboratories. Abdominal ultrasounds were performed with an IU22 3.5-MHz transducer (Philips, Andover, MA, USA) by three experienced radiologists who were unaware of the purpose of the study and blinded to laboratory values. Images were captured in a standard fashion with the patient in the supine position and the right arm raised above the head. Fatty liver
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was diagnosed with ultrasound based on the presence of a diffuse increase of fine echoes in the liver parenchyma compared with the kidney or spleen parenchyma [11]. Ultrasound was performed by the radiologists using live images.
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NAFLD according to each quartile of uric acid were 1.00, 1.39 (0.50–3.87), 2.20 (0.79–6.10), and 5.15 (1.78–14.89). The results are consistent with the suggestion that subjects with higher hs-CRP, HOMA-IR, and uric acid levels are more likely associated with NAFLD than those with lower levels.
Statistical analyses Differences in characteristics between subjects with and without NAFLD were assessed using Student’s t test, Mann-Whitney U test, or v2 test, as appropriate. To explore the associations with NAFLD according to the quartiles of HOMA-IR, hs-CRP, and uric acid, we computed the odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression. The likelihood ratio test for trend was used to determine the relationship between the presence of NAFLD and the level of HOMA-IR, hs-CRP, and uric acid. Multivariate relationships were assessed with adjustments for age, hs-CRP, HOMA-IR, and uric acid as continuous variables and for sex, smoking status, and regular exercise as categorical variables. Commercially available software (PASW, version 17.0; SPSS, Chicago, IL, USA) was used to analyze the data. All reported p-values were two tailed, and statistical significance was set at p \ 0.05.
Results The baseline characteristics of the study subjects are shown in Table 1. When compared with subjects without NAFLD, those with NAFLD comprised significantly more men and had a higher body mass index, larger waist circumference, higher fasting glucose, higher triglycerides, higher AST, higher ALT, higher GGT, and lower HDL. The subjects with NAFLD also had higher hs-CRP (0.21 vs. 1.4 mg/dl, p = 0.037), higher HOMA-IR (1.16 vs. 0.82, p \ 0.001), and higher uric acid (5.6 vs. 4.8 mg/dl, p \ 0.001) than those without NAFLD. Table 2 presents the associations between NAFLD and HOMA-IR quartiles. After adjustment for age, sex, smoking, regular exercise, hs-CRP, and uric acid level, ORs (95 % CI) for NAFLD according to each quartile of HOMA-IR were 1.00, 0.72 (0.25–2.05), 1.41 (0.57–3.49), and 2.55 (1.08–6.05). Table 3 presents the associations between NAFLD and hs-CRP quartiles. After adjustment for age, sex, smoking, regular exercise, HOMA-IR, and uric acid level, ORs (95 % CI) for NAFLD according to each quartile of hs-CRP were 1.00, 1.00 (0.33–2.98), 0.84 (0.30–2.34), and 2.58 (1.03–6.50). Table 4 presents data on the association between NAFLD and uric acid quartiles. After adjustment for age, sex, cigarette smoking, regular exercise, hs-CRP, and HOMA-IR, ORs (95 % CI) for
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Discussion In the present study, NAFLD was independently associated with insulin resistance and inflammation in non-obese adults without metabolic syndrome components. The pathophysiology involved in the development and progression of NAFLD has been less well characterized than the associated risk factors and has been reviewed extensively elsewhere [4]. The pathogenesis of NAFLD seems to be multifactorial, and several mechanisms have been worked out hypothetically [1]. Previous studies have suggested that NAFLD is closely associated with insulin resistance [4, 12]. In the present study, the presence of NAFLD correlated with increased insulin resistance in non-obese adults without metabolic syndrome components. Furthermore, the current data provide evidence that insulin resistance might be an independent risk factor for NAFLD in non-obese adults without metabolic syndrome components who lack confounding factors such as a history of diabetes, hypertension, fasting hyperglycemia, or high blood pressure, high TG, low HDL, and abdominal obesity. Numerous studies have attributed this higher rate to the higher insulin resistance in Asians [13, 14]. C-reactive protein has a short life of around 18 h, and the elevation of serum CRP usually reflects its synthesis in response to a pathological process [15]. Thus, CRP has been considered as a useful nonspecific biochemical marker of chronic inflammation [15]. Several case and control studies have supported an association between elevated serum levels of CRP and the presence of NAFLD [16]. In contrast, some studies failed to show an association of CRP with the histological severity of NAFLD [17]. Presently, the baseline CRP level was independently correlated with NAFLD in non-obese adults without metabolic syndrome components. Serum uric acid concentrations might be closely related to NAFLD, independently of markers of liver injury and inflammation [18]. In the present study, increased serum uric acid concentrations were significantly associated with the presence of NAFLD, independent of insulin resistance and inflammation. Hyperuricemia has been proposed as a compensatory mechanism to counteract oxidative damage in humans [19]. Increased systemic oxidative stress in subjects with NAFLD has been recognized both in animal
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have been identified as various biomarkers for NAFLD, but these have been limited by the lack of reproducibility or inability to diagnose NAFLD [1]. The role of gender has been variable in reported studies, but men are significantly more likely to have NAFLD than women [4]. The preventive effects of estrogen and the sexual dimorphism in liver have been considered the possible mechanisms for this [25, 26]. We confined the age of the study population to over 50 years. The increase in cardiovascular disease risk occurs in the 5th decade, especially in women, corresponding to the onset of menopause. The central distribution and accumulation of adipose tissue as well as the concomitant insulin-resistant dyslipidemic state in
experiments and clinical studies [20]. However, an elevated serum uric acid level may reflect a compensatory mechanism counteracting the increased oxidative stress associated with NAFLD [21]. In contrast, uric acid may be a strong oxidant in certain environments [22] and a part of the inflammatory process [23]. Despite the fact that it is not clear how these opposing roles of uric acid on redox balance are regulated, the urate redox shuttle may explain the paradoxical effects of uric acid on oxidative stress [24], which may further explain why an elevated serum uric acid level is a risk factor for NAFLD. There is no single biochemical marker that can confirm diagnosis of NAFLD. AST, ALT, GGT, and triglyceride
Table 1 Characteristics of the study population
Variables*
No NAFLD (N = 661)
NAFLD (N = 98)
p value
Age (year)
56.7 ± 6.4
57.4 ± 5.5
0.268
Male (%)
325 (49.2)
64 (65.3)
0.003
BMI (kg/m2)
21.9 ± 1.8
23.0 ± 1.4
\0.001
Waist circumference (cm)
78.3 ± 6.2
82.1 ± 5.8
\0.001 0.203
Smoking status (%) SBP systolic blood pressure, DBP diastolic blood pressure, HDL cholesterol high-density lipoprotein cholesterol, AST aspartate aminotransferase, ALT alanine aminotransferase, GGT gamma-glutamyl transferase, eGFR estimated glomerular filtration rate, HOMA-IR homeostasis model assessment of insulin resistance, hs-CRP high-sensitive C-reactive protein, NV normal value *
Data are expressed as mean ± SD, number or proportions (%)
p values obtained from Student’s t test (for continuous variables except GGT), MannWhitney U test (for GGT), and chi-squared test (for categorical variables)
Non-smoker
418 (63.2)
57 (58.2)
Past smoker
143 (21.7)
31 (31.6)
Current smoker
100 (15.1)
10 (10.2)
Regular exercise (%)
211 (34.5)
36 (36.7)
0.597
SBP (mmHg)
116.7 ± 13.0
117.8 ± 11.9
0.410
DBP (mmHg)
76.9 ± 10.0
77.4 ± 8.9
0.614
Fasting glucose (mg/dl) (NV 70–100)
87.6 ± 11.9
91.7 ± 11.6
0.001
Triglycerides (mg/dl) (NV 45–150)
92.4 ± 38.6
114.4 ± 48.9
\0.001
HDL cholesterol (mg/dl) (NV 30–95)
61.6 ± 13.6
56.9 ± 10.8
0.001
AST (IU/l) (NV 7–38) ALT (IU/l) (NV 4–43)
25.9 ± 7.1 21.9 ± 8.8
28.3 ± 7.7 30.8 ± 13.8
0.002 \0.001
GGT (IU/l) (NV 8–48)
27.0 ± 21.8
41.2 ± 43.1
\0.001
Uric acid (mg/dl) (NV 2.5–7.5)
4.8 ± 1.2
5.6 ± 1.2
\0.001
eGFR (ml/min per 1.73 m2)
66.2 ± 7.7
66.3 ± 7.4
0.870
Insulin (lU/dl)
3.7 ± 2.1
5.1 ± 2.9
0.001
HOMA-IR
0.82 ± 0.49
1.16 ± 0.67
\0.001
hs-CRP (mg/dl) (NV 0.01–0.3)
0.12 ± 0.29
0.21 ± 0.36
0.037
Table 2 Odds ratios of nonalcoholic fatty liver disease according to the HOMA-IR quartile Variable*
Quartile 1
Quartile 2
Quartile 3
Quartile 4
p value for trend
HOMA-IR level
0.09–0.50
0.51–0.74
0.75–1.13
1.14–3.41
Crude OR
1
0.70 (0.26–1.90)
1.67 (0.73–3.84)
2.93 (1.34–6.40)
0.001
Model 1
1
0.70 (0.26–1.93)
1.70 (0.74–3.94)
3.11 (1.41–6.87)
0.001
Model 2
1
0.74 (0.26–2.07)
1.66 (0.69–4.00)
2.72 (1.17–6.35)
0.004
Model 3
1
0.72 (0.25–2.05)
1.41 (0.57–3.49)
2.55 (1.08–6.05)
0.009
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for hs-CRP and uric acid *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
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Table 3 Odds ratios of nonalcoholic fatty liver disease according to the hs-CRP quartile p value for trend
Variable*
Quartile 1
hs-CRP level (mg/dl)
0.01–0.02
0.03–0.04
0.05–0.11
0.12–2.34
Crude OR
1
1.07 (0.38–2.97)
1.39 (0.54–3.56)
3.56 (1.49–8.51)
0.001
Model 1
1
1.10 (0.39–3.06)
1.33 (0.52–3.43)
3.29 (1.36–7.94)
0.002
Model 2
1
1.05 (0.37–3.06)
1.13 (0.42–3.05)
3.29 (1.34–8.08)
0.003
Model 3
1
1.00 (0.33–2.98)
0.84 (0.30–2.34)
2.58 (1.03–6.50)
0.019
Quartile 2
Quartile 3
Quartile 4
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for HOMA-IR and uric acid *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
Table 4 Odds ratios of nonalcoholic fatty liver disease according to the uric acid quartile Variable*
Quartile 1
Quartile 2
Quartile 3
Quartile 4
p value for trend
Uric acid level (mg/dl)
1.2–4.0
4.1–4.8
4.9–5.7
5.8–9.8
Crude OR
1
1.10 (0.50–2.40)
2.30 (1.15–4.64)
4.44 (2.30–8.54)
\0.001
Model 1
1
1.09 (0.50–2.38)
2.41 (1.15–5.16)
5.17 (2.35–11.34)
\0.001
Model 2
1
1.14 (0.50–2.59)
2.19 (0.99–4.87)
5.34 (2.37–12.04)
\0.001
Model 3
1
1.39 (0.50–3.87)
2.20 (0.79–6.10)
5.15 (1.78–14.89)
0.002
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for hs-CRP and HOMA-IR *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
postmenopausal women have emerged as important components of a cluster of metabolic abnormalities that are strongly related to coronary heart disease [27]. The present study has several limitations. First, the generalization of the results should be considered with caution. The study subjects were visitors to a health promotion center and might therefore be more health conscious and wealthier than the general populations studied in community-based studies. Second, the diagnosis of NAFLD was based on ultrasonographic examination, which is not sensitive enough to detect mild steatosis. Also, ultrasonography was performed by three radiologists. It is well known that there are intra- and interoperator variabilities in interpreting fatty liver on ultrasonography [28]. However, ultrasonography is a noninvasive and widely available method for the qualitative assessment of fatty infiltration. Thus, ultrasonography is the preferred modality for mass screening for hepatic steatosis and has reasonable sensitivity and specificity [1]. Third, lack of data on levels of serum ceruloplasmin and urinary copper and on history of medications, e.g., steroids and estrogen, might also affect the interpretation of the results. In conclusion, the present study demonstrates that hsCRP, HOMA-IR, and uric acid were independently associated with NAFLD in non-obese adults without metabolic syndrome components. In present study, our results
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demonstrated a significant correlation between NAFLD and the levels of hs-CRP, HOMA-IR, and uric acid in nonobese adults without metabolic syndrome components. We consider that insulin resistance and inflammation may be additional independent risk factors for NAFLD, particularly in non-obese adults without metabolic syndrome components. Additional research should clarify the mechanisms underlying these associations and the interplay of other pathophysiologic factors in the development of NAFLD. Acknowledgements sity in 2012.
This work was supported by Konkuk Univer-
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ORIGINAL ARTICLE
Insulin resistance, inflammation, and nonalcoholic fatty liver disease in non-obese adults without metabolic syndrome components Seonah Kim • Jaekyung Choi • Mina Kim
Received: 10 April 2012 / Accepted: 24 October 2012 / Published online: 29 November 2012 Ó Asian Pacific Association for the Study of the Liver 2012
Abstract Purpose The purpose of the present study was to examine the association of insulin resistance and inflammation with nonalcoholic fatty liver disease (NAFLD) in non-obese adults without metabolic syndrome components. Methods This was a cross-sectional study of 759 subjects aged 50 years and older. Diagnosis of NAFLD was based on sonographic evidence of fatty liver without significant alcohol consumption and another cause of chronic liver disease. Subjects without metabolic syndrome components were defined as having none of the following: high blood pressure (C130/85 mmHg), elevated fasting glucose (C100 mg/dl), hypertriglyceridemia (C150 mg/dl), low high-density lipoprotein-cholesterol (men \40 mg/dl; women \50 mg/dl), and abdominal obesity measured by waist circumference C90 cm for men and C80 cm for women. The subjects were divided into quartile groups according to levels of high-sensitivity C-reactive protein (hs-CRP), homeostasis model assessment of insulin resistance (HOMA-IR), and uric acid. Odds ratios (ORs) were computed for each quartile relative to the lowest quartile group. Results After adjustment for age, sex, smoking status, regular exercise, hs-CRP, HOMA-IR, and uric acid, a significant association was found between NAFLD and higher levels of hs-CRP, HOMA-IR, and uric acid. After adjustment for age, sex, smoking status, regular exercise, hs-CRP, HOMA-IR, and uric acid, the Ors (95 % confidence interval) of NAFLD with the highest quartile of
S. Kim J. Choi (&) M. Kim Department of Family Medicine, Konkuk University Medical Center, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, South Korea e-mail: [email protected]
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hs-CRP, HOMA-IR, and uric acid compared with the lowest quartile were 2.58 (1.03–6.50), 2.55 (1.08–6.05), and 5.15 (1.78–14.89), respectively. Conclusions Insulin resistance and inflammation are independently associated with NAFLD in non-obese adults without metabolic syndrome components. Keywords Nonalcoholic fatty liver disease Homeostasis model assessment of insulin resistance High-sensitivity C-reactive protein Uric acid
Introduction Nonalcoholic fatty liver disease (NAFLD) is defined as the accumulation of fat in the liver in the absence of significant alcohol consumption and other causes of chronic liver disease such as viral hepatitis or drugs [1]. This condition encompasses a wide spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis [2]. NAFLD is being identified with rising frequency and is estimated to be an increasingly common problem worldwide in the general population [3, 4]. Nonalcoholic fatty liver disease has been associated with obesity, hyperglycemia, dyslipidemia, and hypertension, and it is currently regarded as the hepatic manifestation of metabolic syndrome [5]. The pathophysiology involved in the development and progression of NAFLD is associated with insulin resistance and inflammation [6]. Although insulin resistance and inflammation have often been considered to be the link with NAFLD, the roles of insulin resistance and inflammation associated with NAFLD in non-obese adults without metabolic syndrome components have been less well established. The purpose of this study was to examine the insulin resistance and
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inflammation associated with NAFLD in non-obese adults without metabolic syndrome components.
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The present study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the institutional review board at Konkuk University Medical Center.
Materials and methods Measurements Study population This was a cross-sectional study designed to identify the association among insulin resistance, inflammation, and NAFLD. The subjects were 3,011 individuals, aged 50 years and older, who visited the health promotion center at Konkuk University Medical Center, Seoul, South Korea, between 1 May 2008 and 30 April 2010, for a general health checkup. Subjects without metabolic syndrome components were defined in terms of the absence of the five following features [7]: high blood pressure (C130/C85 mmHg), elevated fasting glucose (C100 mg/dl), hypertriglyceridemia (C150 mg/dl), low high-density lipoprotein (HDL)cholesterol (men \40 mg/dl; women \50 mg/dl), and abdominal obesity using the Asia Pacific criterion for abdominal obesity [8] (waist circumference C90 cm for men and C80 cm for women). Subjects receiving pharmacologic treatment for hypertension (i.e., ACE inhibitors, angiotensin receptor blockers, a-blockers, b-blockers, calcium channel blockers, or diuretics) were included in the high blood pressure group. Subjects receiving pharmacologic treatment for diabetes (i.e., sulphonylureas, biguanides, a-glucosidase inhibitors, or insulin) were included in the elevated fasting glucose group. We used the definition of ‘‘non-obese’’ (BMI \ 25 kg/m2) proposed by the World Health Organization Western Pacific Region, the International Association for the Study of Obesity, and the International Obesity Task Force [8]. We defined ‘‘nonalcoholic fatty liver disease’’ as the ultrasonographic diagnosis of fatty liver (USFL) without significant alcohol consumption or some other cause of chronic liver disease. A total of 2,252 subjects were excluded for the following reasons: 1,595 subjects were defined as subjects with a metabolic syndrome component; 1,039 subjects were defined as obese; 1,013 subjects reported an alcohol intake of at least 30 g/day (in men) or 20 g/day (in women) [9]; 159 subjects had positive serologic markers for hepatitis B or C virus; 64 subjects displayed elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels ([80 IU/l); 97 subjects had abnormal liver ultrasound findings (indicating liver cirrhosis, intrahepatic or extrahepatic cholelithiasis, or abnormal dilatation of the biliary tree) or were taking medications for hepatitis. Following exclusion of some individuals for multiple reasons, 759 subjects, aged 50–86 years, were identified as eligible participants.
The general health checkup included a medical history, physical examination, lifestyle questionnaire, biochemical measurements, and abdominal ultrasonography. Trained nurses measured sitting blood pressure levels with a standard mercury sphygmomanometer. Height and weight were measured after an overnight fast with the patient wearing a lightweight hospital gown and no shoes. BMI was calculated by dividing the weight in kilograms by the square of the height in meters. Smoking, alcohol intake, and regular exercise were assessed using information from the lifestyle questionnaire. For smoking, individuals were classified as nonsmoker, past smoker, or current smoker. Questions about alcohol intake included the frequency of alcohol consumption on a weekly basis and the usual amount consumed on a daily basis. Regular exercise was defined as engaging in physical activity for a minimum of 30 min at least three times per week. Blood specimens were sampled from the antecubital vein after more than 12 h of fasting. Serum levels of glucose, uric acid, creatinine, triglycerides, HDL, ALT, AST, and c-glutamyltransferase (GGT) were measured using Bayer reagent packs on a 200FR-Neo automated chemistry analyzer (Toshiba, Tokyo, Japan). Measurement techniques included the hexokinase method for glucose, an enzymatic colorimetric assay for serum lipids, and an immunoradiometric assay for insulin (Roche/Hitachi Modular analytics E170; Hitachi, Tokyo, Japan). Insulin resistance was assessed with the homeostasis model assessment of insulin resistance (HOMAIR) according to the following equation: fasting blood insulin (in microunits per milliliter) 9 fasting blood glucose (in mm/l)/22.5. High-sensitivity C-reactive protein (hs-CRP) was analyzed by particle-enhanced immunonephelometry with the BNII System (200FR-Neo; Toshiba). Glomerular filtration rate was estimated from the modification of diet in the renal disease study equation as follows: 186 9 (serum creatinine)-1.154 9 (age)-0.203 9 0.742 (if female) [10]. The clinical laboratory used in the study had been accredited and participated annually in inspections and surveys by the Korean Association of Quality Assurance for Clinical Laboratories. Abdominal ultrasounds were performed with an IU22 3.5-MHz transducer (Philips, Andover, MA, USA) by three experienced radiologists who were unaware of the purpose of the study and blinded to laboratory values. Images were captured in a standard fashion with the patient in the supine position and the right arm raised above the head. Fatty liver
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was diagnosed with ultrasound based on the presence of a diffuse increase of fine echoes in the liver parenchyma compared with the kidney or spleen parenchyma [11]. Ultrasound was performed by the radiologists using live images.
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NAFLD according to each quartile of uric acid were 1.00, 1.39 (0.50–3.87), 2.20 (0.79–6.10), and 5.15 (1.78–14.89). The results are consistent with the suggestion that subjects with higher hs-CRP, HOMA-IR, and uric acid levels are more likely associated with NAFLD than those with lower levels.
Statistical analyses Differences in characteristics between subjects with and without NAFLD were assessed using Student’s t test, Mann-Whitney U test, or v2 test, as appropriate. To explore the associations with NAFLD according to the quartiles of HOMA-IR, hs-CRP, and uric acid, we computed the odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression. The likelihood ratio test for trend was used to determine the relationship between the presence of NAFLD and the level of HOMA-IR, hs-CRP, and uric acid. Multivariate relationships were assessed with adjustments for age, hs-CRP, HOMA-IR, and uric acid as continuous variables and for sex, smoking status, and regular exercise as categorical variables. Commercially available software (PASW, version 17.0; SPSS, Chicago, IL, USA) was used to analyze the data. All reported p-values were two tailed, and statistical significance was set at p \ 0.05.
Results The baseline characteristics of the study subjects are shown in Table 1. When compared with subjects without NAFLD, those with NAFLD comprised significantly more men and had a higher body mass index, larger waist circumference, higher fasting glucose, higher triglycerides, higher AST, higher ALT, higher GGT, and lower HDL. The subjects with NAFLD also had higher hs-CRP (0.21 vs. 1.4 mg/dl, p = 0.037), higher HOMA-IR (1.16 vs. 0.82, p \ 0.001), and higher uric acid (5.6 vs. 4.8 mg/dl, p \ 0.001) than those without NAFLD. Table 2 presents the associations between NAFLD and HOMA-IR quartiles. After adjustment for age, sex, smoking, regular exercise, hs-CRP, and uric acid level, ORs (95 % CI) for NAFLD according to each quartile of HOMA-IR were 1.00, 0.72 (0.25–2.05), 1.41 (0.57–3.49), and 2.55 (1.08–6.05). Table 3 presents the associations between NAFLD and hs-CRP quartiles. After adjustment for age, sex, smoking, regular exercise, HOMA-IR, and uric acid level, ORs (95 % CI) for NAFLD according to each quartile of hs-CRP were 1.00, 1.00 (0.33–2.98), 0.84 (0.30–2.34), and 2.58 (1.03–6.50). Table 4 presents data on the association between NAFLD and uric acid quartiles. After adjustment for age, sex, cigarette smoking, regular exercise, hs-CRP, and HOMA-IR, ORs (95 % CI) for
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Discussion In the present study, NAFLD was independently associated with insulin resistance and inflammation in non-obese adults without metabolic syndrome components. The pathophysiology involved in the development and progression of NAFLD has been less well characterized than the associated risk factors and has been reviewed extensively elsewhere [4]. The pathogenesis of NAFLD seems to be multifactorial, and several mechanisms have been worked out hypothetically [1]. Previous studies have suggested that NAFLD is closely associated with insulin resistance [4, 12]. In the present study, the presence of NAFLD correlated with increased insulin resistance in non-obese adults without metabolic syndrome components. Furthermore, the current data provide evidence that insulin resistance might be an independent risk factor for NAFLD in non-obese adults without metabolic syndrome components who lack confounding factors such as a history of diabetes, hypertension, fasting hyperglycemia, or high blood pressure, high TG, low HDL, and abdominal obesity. Numerous studies have attributed this higher rate to the higher insulin resistance in Asians [13, 14]. C-reactive protein has a short life of around 18 h, and the elevation of serum CRP usually reflects its synthesis in response to a pathological process [15]. Thus, CRP has been considered as a useful nonspecific biochemical marker of chronic inflammation [15]. Several case and control studies have supported an association between elevated serum levels of CRP and the presence of NAFLD [16]. In contrast, some studies failed to show an association of CRP with the histological severity of NAFLD [17]. Presently, the baseline CRP level was independently correlated with NAFLD in non-obese adults without metabolic syndrome components. Serum uric acid concentrations might be closely related to NAFLD, independently of markers of liver injury and inflammation [18]. In the present study, increased serum uric acid concentrations were significantly associated with the presence of NAFLD, independent of insulin resistance and inflammation. Hyperuricemia has been proposed as a compensatory mechanism to counteract oxidative damage in humans [19]. Increased systemic oxidative stress in subjects with NAFLD has been recognized both in animal
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have been identified as various biomarkers for NAFLD, but these have been limited by the lack of reproducibility or inability to diagnose NAFLD [1]. The role of gender has been variable in reported studies, but men are significantly more likely to have NAFLD than women [4]. The preventive effects of estrogen and the sexual dimorphism in liver have been considered the possible mechanisms for this [25, 26]. We confined the age of the study population to over 50 years. The increase in cardiovascular disease risk occurs in the 5th decade, especially in women, corresponding to the onset of menopause. The central distribution and accumulation of adipose tissue as well as the concomitant insulin-resistant dyslipidemic state in
experiments and clinical studies [20]. However, an elevated serum uric acid level may reflect a compensatory mechanism counteracting the increased oxidative stress associated with NAFLD [21]. In contrast, uric acid may be a strong oxidant in certain environments [22] and a part of the inflammatory process [23]. Despite the fact that it is not clear how these opposing roles of uric acid on redox balance are regulated, the urate redox shuttle may explain the paradoxical effects of uric acid on oxidative stress [24], which may further explain why an elevated serum uric acid level is a risk factor for NAFLD. There is no single biochemical marker that can confirm diagnosis of NAFLD. AST, ALT, GGT, and triglyceride
Table 1 Characteristics of the study population
Variables*
No NAFLD (N = 661)
NAFLD (N = 98)
p value
Age (year)
56.7 ± 6.4
57.4 ± 5.5
0.268
Male (%)
325 (49.2)
64 (65.3)
0.003
BMI (kg/m2)
21.9 ± 1.8
23.0 ± 1.4
\0.001
Waist circumference (cm)
78.3 ± 6.2
82.1 ± 5.8
\0.001 0.203
Smoking status (%) SBP systolic blood pressure, DBP diastolic blood pressure, HDL cholesterol high-density lipoprotein cholesterol, AST aspartate aminotransferase, ALT alanine aminotransferase, GGT gamma-glutamyl transferase, eGFR estimated glomerular filtration rate, HOMA-IR homeostasis model assessment of insulin resistance, hs-CRP high-sensitive C-reactive protein, NV normal value *
Data are expressed as mean ± SD, number or proportions (%)
p values obtained from Student’s t test (for continuous variables except GGT), MannWhitney U test (for GGT), and chi-squared test (for categorical variables)
Non-smoker
418 (63.2)
57 (58.2)
Past smoker
143 (21.7)
31 (31.6)
Current smoker
100 (15.1)
10 (10.2)
Regular exercise (%)
211 (34.5)
36 (36.7)
0.597
SBP (mmHg)
116.7 ± 13.0
117.8 ± 11.9
0.410
DBP (mmHg)
76.9 ± 10.0
77.4 ± 8.9
0.614
Fasting glucose (mg/dl) (NV 70–100)
87.6 ± 11.9
91.7 ± 11.6
0.001
Triglycerides (mg/dl) (NV 45–150)
92.4 ± 38.6
114.4 ± 48.9
\0.001
HDL cholesterol (mg/dl) (NV 30–95)
61.6 ± 13.6
56.9 ± 10.8
0.001
AST (IU/l) (NV 7–38) ALT (IU/l) (NV 4–43)
25.9 ± 7.1 21.9 ± 8.8
28.3 ± 7.7 30.8 ± 13.8
0.002 \0.001
GGT (IU/l) (NV 8–48)
27.0 ± 21.8
41.2 ± 43.1
\0.001
Uric acid (mg/dl) (NV 2.5–7.5)
4.8 ± 1.2
5.6 ± 1.2
\0.001
eGFR (ml/min per 1.73 m2)
66.2 ± 7.7
66.3 ± 7.4
0.870
Insulin (lU/dl)
3.7 ± 2.1
5.1 ± 2.9
0.001
HOMA-IR
0.82 ± 0.49
1.16 ± 0.67
\0.001
hs-CRP (mg/dl) (NV 0.01–0.3)
0.12 ± 0.29
0.21 ± 0.36
0.037
Table 2 Odds ratios of nonalcoholic fatty liver disease according to the HOMA-IR quartile Variable*
Quartile 1
Quartile 2
Quartile 3
Quartile 4
p value for trend
HOMA-IR level
0.09–0.50
0.51–0.74
0.75–1.13
1.14–3.41
Crude OR
1
0.70 (0.26–1.90)
1.67 (0.73–3.84)
2.93 (1.34–6.40)
0.001
Model 1
1
0.70 (0.26–1.93)
1.70 (0.74–3.94)
3.11 (1.41–6.87)
0.001
Model 2
1
0.74 (0.26–2.07)
1.66 (0.69–4.00)
2.72 (1.17–6.35)
0.004
Model 3
1
0.72 (0.25–2.05)
1.41 (0.57–3.49)
2.55 (1.08–6.05)
0.009
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for hs-CRP and uric acid *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
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Table 3 Odds ratios of nonalcoholic fatty liver disease according to the hs-CRP quartile p value for trend
Variable*
Quartile 1
hs-CRP level (mg/dl)
0.01–0.02
0.03–0.04
0.05–0.11
0.12–2.34
Crude OR
1
1.07 (0.38–2.97)
1.39 (0.54–3.56)
3.56 (1.49–8.51)
0.001
Model 1
1
1.10 (0.39–3.06)
1.33 (0.52–3.43)
3.29 (1.36–7.94)
0.002
Model 2
1
1.05 (0.37–3.06)
1.13 (0.42–3.05)
3.29 (1.34–8.08)
0.003
Model 3
1
1.00 (0.33–2.98)
0.84 (0.30–2.34)
2.58 (1.03–6.50)
0.019
Quartile 2
Quartile 3
Quartile 4
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for HOMA-IR and uric acid *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
Table 4 Odds ratios of nonalcoholic fatty liver disease according to the uric acid quartile Variable*
Quartile 1
Quartile 2
Quartile 3
Quartile 4
p value for trend
Uric acid level (mg/dl)
1.2–4.0
4.1–4.8
4.9–5.7
5.8–9.8
Crude OR
1
1.10 (0.50–2.40)
2.30 (1.15–4.64)
4.44 (2.30–8.54)
\0.001
Model 1
1
1.09 (0.50–2.38)
2.41 (1.15–5.16)
5.17 (2.35–11.34)
\0.001
Model 2
1
1.14 (0.50–2.59)
2.19 (0.99–4.87)
5.34 (2.37–12.04)
\0.001
Model 3
1
1.39 (0.50–3.87)
2.20 (0.79–6.10)
5.15 (1.78–14.89)
0.002
Model 1: age and sex adjusted. Model 2: model 1 further adjusted for smoking status and regular exercise. Model 3: model 2 further adjusted for hs-CRP and HOMA-IR *
Odds ratios (ORs) and 95 % confidence intervals were estimated from multivariable logistic regression analysis
p values for trend were obtained from the likelihood ratio test for trend
postmenopausal women have emerged as important components of a cluster of metabolic abnormalities that are strongly related to coronary heart disease [27]. The present study has several limitations. First, the generalization of the results should be considered with caution. The study subjects were visitors to a health promotion center and might therefore be more health conscious and wealthier than the general populations studied in community-based studies. Second, the diagnosis of NAFLD was based on ultrasonographic examination, which is not sensitive enough to detect mild steatosis. Also, ultrasonography was performed by three radiologists. It is well known that there are intra- and interoperator variabilities in interpreting fatty liver on ultrasonography [28]. However, ultrasonography is a noninvasive and widely available method for the qualitative assessment of fatty infiltration. Thus, ultrasonography is the preferred modality for mass screening for hepatic steatosis and has reasonable sensitivity and specificity [1]. Third, lack of data on levels of serum ceruloplasmin and urinary copper and on history of medications, e.g., steroids and estrogen, might also affect the interpretation of the results. In conclusion, the present study demonstrates that hsCRP, HOMA-IR, and uric acid were independently associated with NAFLD in non-obese adults without metabolic syndrome components. In present study, our results
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demonstrated a significant correlation between NAFLD and the levels of hs-CRP, HOMA-IR, and uric acid in nonobese adults without metabolic syndrome components. We consider that insulin resistance and inflammation may be additional independent risk factors for NAFLD, particularly in non-obese adults without metabolic syndrome components. Additional research should clarify the mechanisms underlying these associations and the interplay of other pathophysiologic factors in the development of NAFLD. Acknowledgements sity in 2012.
This work was supported by Konkuk Univer-
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