ORIGINAL ARTICLE
METABOLIC SYNDROME AND RELATED DISORDERS Volume X, Number X, 2015 Mary Ann Liebert, Inc. Pp. 1–7 DOI: 10.1089/met.2015.0018
Insulin Resistance but Not Visceral Adiposity Index Is Associated with Liver Fibrosis in Nondiabetic Subjects with Nonalcoholic Fatty Liver Disease Cemal Nuri Ercin, MD,1 Teoman Dogru, MD,1 Halil Genc, MD,2 Gurkan Celebi, MD,1 Fatih Aslan, MD,3 Hasan Gurel, MD,1 Muammer Kara, MD,4 Erdim Sertoglu, MD,5 Serkan Tapan, MD,6 Sait Bagci, MD,1 Manfredi Rizzo, MD,7 and Alper Sonmez, MD 8
Abstract
Background: Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, type 2 diabetes mellitus, and dyslipidemia. It is well known that the presence of visceral fat increases the risk for metabolic complications of obesity, especially NAFLD. The visceral adiposity index (VAI), a novel marker of visceral fat dysfunction, shows a strong association with insulin resistance and also cardiovascular and cerebrovascular events. However, there is conflicting data regarding the association between VAI and NAFLD. Our aim was to assess the relationship between VAI, insulin resistance, adipocytokines, and liver histology, in nondiabetic subjects with NAFLD. Methods: A total of 215 male patients with biopsy-proven NAFLD were included. Among this group, serum levels of adiponectin, tumor necrosis factor-a (TNF-a, interleukin-6 (IL-6), and high-sensitivity C-reactive protein (hsCRP) were measured in 101 patients whose blood samples were available. Results: High gamma-glutamyl transferase (GGT), high total cholesterol (TC), high triglycerides (TGs), low highdensity lipoprotein cholesterol (HDL-C), and presence of metabolic syndrome were significantly associated with higher VAI, although only higher GGT and TC were independent factors on multiple linear regression analysis. On the other hand, no significant association was found between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels. The multivariate analysis of variables in patients with (n = 124) and without (n = 91) fibrosis showed that only higher homeostasis model assessment of insulin resistance value was independently associated with liver fibrosis. Conclusions: Our findings suggest that VAI is not related to the severity of hepatic inflammation or fibrosis in nondiabetic patients with NAFLD. The lack of association between the adipocytokines and VAI also implies that the VAI may not be a significant indictor of the adipocyte functions. factors for the development of NAFLD.3 Adipose tissue, as an endocrine organ, produces various proteins and soluble factors called adipokines, and these adipokines are considered a main link between obesity, IR, and related metabolic disorders, like NAFLD.4 It is well known that fat tissue is not uniformly distributed in the body. Hence, the distribution of fat appears more important than total fat mass in obesity.5 The presence of predominantly upper body fat increases the risk for metabolic complications of obesity, including hepatic steatosis.6 Visceral fat, a precursor
Introduction
N
onalcoholic fatty liver disease (NAFLD) represents the most common hepatic disorder in western countries. It has a broad spectrum of manifestations ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) and cirrhosis.1 It is now well accepted that NAFLD is the hepatic manifestation of the metabolic syndrome.2 Insulin resistance (IR), obesity, type 2 diabetes mellitus (T2DM), and dyslipidemia are the most important risk 1
Gulhane School of Medicine, Department of Gastroenterology, Ankara, Turkey. _ Izmir Military Hospital2, Izmir, Turkey. _ Department of Gastroenterology, Katip Celebi University, Izmir, Turkey. 4 _ Department of Gastroenterology, Haydarpasa Training Hospital, Istanbul, Turkey. 5 Medical Biochemistry, Anittepe Military Dispensary, Ankara, Turkey. 6 Medical Biochemistry, Gulhane School of Medicine, Ankara, Turkey. 7 Department of Clinical Medicine and Emerging Diseases, Palermo University, Palermo, Italy. 8 Department of Endocrinology, Gulhane School of Medicine, Ankara, Turkey. 2_ 3
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of increased lipolysis and elevated free fatty acid flow and metabolism, is believed to be a more important factor in causing IR than overall obesity or subcutaneous fat.7–9 In fact, many studies have indicated that free fatty acid, leptin, interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) from adipocytes in the visceral fat participate in the development of metabolic syndrome, including IR.10,11 Waist circumference (WC) is a major clinical parameter used for the indirect evaluation of increased visceral fat. The strong and consistent relationships between visceral adiposity and metabolic risk are the basis for including larger WC as part of the Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP) criteria for metabolic syndrome.12 Nevertheless, WC alone does not help to distinguish subcutaneous and visceral fat mass.13 This is a considerable drawback, given that visceral fat, but not subcutaneous fat, plays a decisive role in the genesis of cardiovascular sequelae.14–16 The visceral adiposity index (VAI), a novel marker of visceral fat dysfunction, showed a strong association with IR and visceral adipose tissue. An independent association of VAI with both cardiovascular and cerebrovascular events has also been reported.17 On the other hand, there is very scarce and conflicting data regarding the relationship of VAI with NAFLD.18,19 Thus, in this study, we assessed the relationship between VAI, IR, adipocytokines, and liver histology in subjects with NAFLD. To prevent any interference of confounding factors for IR and inflammation, we studied a specifically selected group having no additional disorders, such as hypertension and T2DM.
Fasting blood specimens were collected from all participants after an 8-hr overnight fast. The samples were centrifuged for 15 min at 4000 rpm, aliquoted, and immediately frozen at -80C for analyses until examination. All samples were run in the same assay. FPG, total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) levels were measured by the enzymatic colorimetric method with an Olympus AU2700 autoanalyzer using reagents from Olympus Diagnostics (Olympus Life Science Europa GmbH, Hamburg, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald formula.20 The serum basal insulin level was measured in duplicate by the chemiluminescence method using reagents from Roche Diagnostics (Mannheim, Germany). IR was calculated by modified homeostasis model assessment of IR (HOMA-IR), with the following formula: HOMA-IR = fasting plasma insulin (mU/mL) · FPG (mg/dL)/405. HOMAIR was originally reported by Matthews et al.,21 and this index has been shown to be well correlated with the results of the euglycemic–hyperinsulinemic clamp method to determine IR. The VAI score was calculated using the following sexspecific equations, where TG and HDL-C levels are expressed in mmol/L17:
Material and Methods
The high sensitivity C-reactive protein (hsCRP) level was determined in serum by the immune turbidimetric fixed-rate method using an Olympus AU- 2700 autoanalyzer (Hamburg, Germany). Intra-assay coefficient of variation (CV) and interassay CV were 5.8% and 3.1%, respectively. The minimum detectable concentration for hsCRP was 0.07 mg/L. Plasma adiponectin levels were determined by the enzymelinked immunosorbent assay (ELISA) method (Human Adiponectin ELISA Kit, TECO medical AG, Sissach, Switzerland). The minimum detectable concentration for adiponectin was 0.6 ng/mL. Intra-assay CV ranged from 2.35% to 4.66%, whereas interassay CV ranged from 5.7% to 6.72% for adiponectin. Plasma TNF-a and IL-6 levels were determined by ELISA (Human TNF-a High Sensitivity ELISA and Human IL-6 High Sensitivity ELISA, eBioscience, Vienna, Austria). The minimum detectable concentrations for TNF-a and IL-6 were 0.13 pg/mL and 0.03 pg/mL, respectively. The calculated overall intra-assay CV values for TNF-a and IL-6 were 8.5% and 4.9%, whereas the calculated overall interassay CV for TNF-a and IL-6 were 9.8% and 6.0%, respectively. Measurements were carried out using an ELISA plate reader (Bio-Tek Synergy HT, BioTek Instruments Inc., Winooski, VT). A 2-hr OGTT was performed in each patient with the standard 75 grams of glucose. Glucose tolerance status was determined according to the classification of the American Diabetes Association (ADA) in which FPG levels up to 99 mg/dL are considered normal and T2DM is defined by a FPG level of 126 mg/dL or greater or a 2-hr plasma glucose levels of 200 mg/dL or greater.22 Metabolic syndrome was defined by NCEP ATP III criteria; WC ‡ 102 cm in males or ‡88 cm in females, systolic/ diastolic blood pressure >130/85 mmHg or receiving drug
Patient population We enrolled 215 male patients with biopsy-proven NAFLD. Inclusion criteria were persistently (at least 6 months) elevated aminotransferases, ultrasonographic presence of bright liver without any other liver or biliary tract disease, and liver histology compatible with a diagnosis of NASH or SS. Patients were excluded if they had a history of alcohol consumption >140 grams/week, as assessed by a detailed interview extended to family members; positive testing for hepatitis B virus or hepatitis C virus; body mass index (BMI) ‡40 kg/m2; positive serum markers of autoimmune or celiac disease; abnormal copper metabolism or thyroid function tests; a diagnosis of overt T2DM [fasting plasma glucose (FPG) ‡126 mg/dL or ‡200 mg/dL at 2 hr on a standard oral glucose tolerance test (OGTT)] and systemic hypertension; and exposure to occupational hepatotoxins or drugs known to be steatogenic or to affect glucose and lipid metabolism. The study was approved by the local ethics committee of Gulhane School of Medicine. All participants gave their written informed consent to the study, which was conducted according to the Helsinki Declaration.
Clinical and laboratory data All participants provided a medical history and underwent a clinical examination. The weight and height of the participants were measured with a calibrated scale after the patients had removed their shoes and any heavy clothing. BMI was computed as body weight/(height2). WC was measured as the midpoint between the lower costal margin and the level of the anterior superior iliac crests.
Males: VAI ¼ ðWC=ð39:68 þ (1:88 · BMI))) · (TG=1:03) · (1:31=HDL) Females: VAI ¼ ðWC=ð36:58 þ (1:89 · BMI))) · (TG=0:81) · (1:52=HDL)
VISCERAL ADIPOSITY INDEX IN NAFLD
treatment, TGs ‡150 mg/dL, HDL-C 33–66%) 3 (>66%) Hepatocellular ballooning 0 1 2 Lobular inflammation 0 1 2 3 Fibrosis 0 1 2 3 NAFLD activity score (NAS %) 0–2 3–4 5–8
Values 32.11 (20–53)* 28.31 (21.2–38.3)* 94.34 (86–126)* 50.04 (15–148)* 104.48 (19–330)* 69.10 (19–455)* 204.45 – 44.67 ** 197.52 (22–774)* 124.22 – 37.28** 40.84 (27–60)* 92.97 (58–121)* 15.53 (2.12–53.78)* 3.58 (0.33–13.57)* 3.04 (0.24–12.01)* 11.4464.33** 0.4960.32** 4.4562.16** 2.8862.87** 9.3 28.4 95 (44.5) 72 (33.5) 48 (22) 53 (24.7) 129 (60) 33 (15.3) 23 (10.7) 130 (60.5) 62 (28.8) 0 91 112 9 3
(42.3) (52.1) (4.2) (1.4)
51 (23.7) 81 (37.7) 83 (38.6)
*Mann–Whitney U-test. **Independent sample t-test. BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gammaglutamyl transferase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; VAI, visceral adiposity index; TNF-a, tumor necrosis factor-a; IL-6, interleukin6; hsCRP, high-sensitivity C-reactive protein; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score.
VAI and steatosis, hepatocellular ballooning, lobular inflammation, fibrosis, and NAS (Table 2). In subgroup analysis, there were no significant associations between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels.
Factors associated with histological features The univariate and multivariate comparisons of variables between patients with (n = 124) and without (n = 91) fibrosis
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Table 2.
Univariate and Multivariate Analysis of Factors Associated with VAI Univariate analysis
Variable Age (years) BMI (kg/m2) WC (cm) AST (IU/L) ALT (IU/L) GGT (IU/L) TC (mg/dL) TGs (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) FPG (mg/dL) Insulin (mU/mL) HOMA-IR Presence of metabolic syndrome Smoking Histology Steatosis grade Hepatocellular ballooning Lobular inflammation Stage of fibrosis NAFLD activity score (NAS)
Multivariate analysis
b
SE
P value
b
SE
- 0.003 - 0.042 0.002 0.001 - 0.003 0.006 0.017 0.017 0.002 - 0.150 - 0.021 0.028 0.093 0.995 0.366
0.024 0.050 0.021 0.007 0.003 0.003 0.003 0.000 0.004 0.019 0.013 0.016 0.066 0.501 0.325
0.903 0.408 0.920 0.921 0.280 0.049 < 0.001 < 0.001 0.682 < 0.001 0.105 0.080 0.157 0.048 0.261
- 0.021
0.017
0.235
0.005 0.014
0.002 0.002
0.018 < 0.001
- 0.004
0.012
0.739
- 0.087 - 0.005 - 0.151 - 0.036 - 0.070
0.187 0.235 0.244 0.233 0.189
0.641 0.984 0.538 0.878 0.711
P value
VAI, visceral adiposity index; b, b coefficient; SE, standard error of b; IU, international units; BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score.
are reported in Table 3. Univariate logistic regression analysis showed that the following features were linked to fibrosis: WC (P = 0.043), aspartate aminotransferase (AST) (P = 0.011), alanine aminotransferase (ALT (P = 0.027), insulin (P = 0.005), HOMA-IR index (P = 0.003), steatosis (P = 0.001), hepatocellular ballooning (P < 0.001), lobular inflammation (P < 0.001), and NAS (P < 0.001). On the other hand, only the HOMA-IR value (P = 0.010) was independently associated with fibrosis in multivariate analysis.
Relationship of adipocytokines with liver histology Circulating IL-6 levels were significantly higher in subjects with fibrosis (n = 66) compared to subjects without fibrosis (n = 35) (P = 0.007). In correlation analysis, IL-6 levels were significantly correlated to lobular inflammation and fibrosis (r = 0.342, P < 0.001 and r = 0.367, P < 0.001, respectively). On the other hand, no significant association was found between levels of hsCRP, adiponectin, and TNFa and histopathological findings.
Discussion In the present study, we investigated the relationship of VAI with IR, liver histology, and circulating adipocytokines in nondiabetic male subjects with NAFLD. To the best of our knowledge, this is the largest study that has investigated this relationship in this clinically relevant condition. The findings of our study demonstrate that VAI is not associated with IR, adipocytokines, and the severity of hepatic inflammation or fibrosis in NAFLD. As far as we know, there are only two studies that investigate the VAI in subjects with NAFLD. Petta et al.18
evaluated 142 consecutive NAFLD patients by liver biopsy, regarding clinical and metabolic measurements, including IR with HOMA-IR index, serum levels of TNF-a, IL-6, adiponectin, leptin, and VAI. By multiple linear regression analysis, VAI was found to be independently associated with higher a HOMA index and degree of fibrosis. In addition, an independent association was found between higher VAI and lower adiponectin levels. However, no significant correlation was found among NAS, histologic activity of NAFLD, and VAI. They concluded that VAI is an expression of both qualitative and quantitative adipose tissue dysfunction and, together with IR, is independently associated with significant fibrosis in NAFLD patients.18 Another study performed by Vongsuvanh et al.19 examined 190 adults with biopsy-proven NAFLD and 129 healthy controls. On multivariate analysis, NAFLD diagnosis and FPG were independently associated with VAI. VAI increased across control, steatosis, and NASH groups; however, this association was no stronger than the increase in WC across groups. VAI was not associated with steatosis, lobular inflammation, or fibrosis, but WC was associated with fibrosis. VAI and WC correlated with an increasing number of metabolic syndrome components and with metabolic syndrome diagnosis. However, no significant association was found between VAI and circulating adiponectin levels. They concluded that the VAI score was not associated with the severity of hepatic inflammation or fibrosis and thus cannot be recommended as a surrogate marker of liver injury in patients with NAFLD.19 On the other hand, in the present study, VAI was positively correlated with GGT, TC, TGs levels, and the presence of metabolic syndrome and negatively correlated with
VISCERAL ADIPOSITY INDEX IN NAFLD
Table 3.
Variable Age (years) BMI (kg/m2) WC (cm) AST (IU/L) ALT (IU/L) GGT (IU/L) TC (mg/dL) TGs (mg/dL) LDL-C (mg/dL) HDL–C (mg/dL) FPG (mg/dL) Insulin (mU/mL) HOMA-IR VAI Metabolic syndrome present (%) Smoking present (%)
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The Univariate and Multivariate Analysis of Variables Between Patients With and Without Fibrosis Without fibrosis (n = 91) 31.76 – 5.52 28 – 2.62 98.18 – 5.85 47.22 – 21.09 97.43 – 43.09 69.94 – 58.615 204.41 – 39.29 192.77 – 119.81 125.64 – 33.03 40.19 – 6.69 91.96 – 12.41 13.43 – 7.33 3.03 – 1.70 3.0 – 2.1 7
(32) (28.2) (98)* (42) (90) (69) (203) (161) (123) (40) (92) (11.3) (2.47) (2.57)
With fibrosis (n = 124) 32.36 – 6.61 28.53 – 3.11 101.19 – 7.51 52.11 – 20.64 109.65 – 48.52 67.97 – 39.331 204.48 – 48.40 201.01 – 123.50 123.19 – 40.22 41.31 – 6.95 93.71 – 10.07 17.07 – 9.89 3.98 – 2.49 3.07 – 2.19 13
27
34
(32) (28.1) (100) (49) (103) (68) (210.5) (161.5) (128) (41) (94) (14.59) (3.44) (2.41)
Univariate analysis (P)
Multivariate analysis [OR (95% CI)]
P
0.657* 0.302* 0.043* 0.011* 0.027* 0.695* 0.991** 0.501* 0.625** 0.210* 0.383* 0.005* 0.003* 0.726* 0.486
1.015 (0.965–1.068)
0.561
1.001 (0.954–1.049)
0.977
1.246 (1.054–1.472)
0.010
0.718
*Mann–Whitney U-test. ** Independent sample t-test. OR, odds ratio; CI, confidence interval; BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; VAI, visceral adiposity index.
HDL-C levels in univariate analysis. However, no association with BMI, WC, and IR as assessed by the HOMA equation could be detected. To ensure that our findings were independent of key confounders, we performed multivariate analysis using those factors significant on univariate analysis. Inclusion of all relevant risk factors resulted in an independent association of GGT and TC levels with VAI. On the other hand, no significant association was found between VAI and steatosis, hepatocellular ballooning, lobular inflammation, NAS score, and fibrosis. Only higher HOMAIR value was independently associated with liver fibrosis in multivariate analysis. Moreover, no significant association was found between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels. We think that the lack of relationship between VAI and NAFLD within our cohort may be due to several reasons. First, some participants were hypertensive and also diabetic in the study by Petta et al.18 It is well known that TGs and HDL-C levels that are the components of VAI are affected by these metabolic disorders. In addition, circulating concentrations of adipokines and measures of insulin sensitivity are easily affected by these metabolic confounders.24,25 Second, NAFLD is strongly associated with obesity, hypertension, and glucose tolerance abnormalities.2,3,26 It has been reported that all of these metabolic problems are also risk factors for T2DM. Although it was stated in the study by Petta et al. that some of the patients were diabetic at baseline, there is no information regarding the glucose tolerance status of the other subjects. In light of these clear data, we think that some of the study participants may still have overt glucose dysregulation or T2DM without implementation of the glucose tolerance test. This issue is important because NAFLD is strongly associated with T2DM, and blood adipokine and insulin levels are altered in subjects with disordered glucose me-
tabolism.25 Finally, no information about the drug use (antihypertensive or hypoglycemic) of the subjects could be seen in the text. We know that circulating adipokines and measures of insulin sensitivity are easily affected from the medications started for the metabolic problems mentioned above.27–29 For this reason, we carefully excluded those patients taking hypoglycemic drugs such as thiazolidinediones and metformin, which have been shown to significantly affect circulating adipokine levels. Consequently, we think that some of the previous findings regarding the VAI in NAFLD might be affected by these confounders. Hence, in a recent study performed in 139 morbidly obese 139 patients who underwent bariatric surgery, IR and metabolic syndrome were the main factors associated with VAI.30 Adipokines play an important role in the pathogenesis of IR and NAFLD through complex and interactive paracrine and endocrine mechanisms.31 The relationship of adipokines with NAFLD has been extensively investigated in several previous studies,32,33 but the published reports have yielded conflicting results regarding the role of adipokines in the pathogenesis of NAFLD.34,35 In the present study, we found a significant association of IL-6 with lobular inflammation and fibrosis in NAFLD. So, our results are in line with current scientific knowledge regarding this issue.36,37 On the other hand, there were no associations between hsCRP, adiponectin, TNF-a, and liver histology in our cohort. Consequently, we suggest that the discrepant findings of these studies were likely due to differences in the populations of patients enrolled, specifically with respect to the age, number of participants, and co-morbid conditions such as diabetes and hypertension. Several limitations must be acknowledged in this study. First, despite the sample size and the strict inclusion criteria, the findings obtained are not representative for all subjects with NAFLD. However, as mentioned above, we think that
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the design of our study was a requirement for the goals to be achieved. Second, even if the HOMA-IR index has a reasonable linear correlation with the glucose clamp technique and was used in several population studies, another limitation of this work is due to the fact that the HOMA index does not help to clarify whether IR is central or peripheral. Third, our study population represents a group with a mild NAFLD phenotype, with most subjects having grade 0–1 fibrosis. Whether our results can be generalized to a more severe NAFLD cohort remains to be determined. Last, all participants were men, and that it remains to be determined if these results are similar also in women. In conclusion, our findings further support the data that VAI is not an expression of adipose tissue dysfunction and is not associated with the severity of hepatic inflammation or fibrosis in NAFLD.
14. 15.
16. 17.
18.
Author Disclosure Statement No competing financial interests exist.
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Address correspondence to: Cemal Nuri Ercin, Associate Professor Gulhane School of Medicine Department of Gastroenterology General Tevfik Saglam Cad Etlik-Kecioren Ankara 06180 Turkey E-mail: [email protected]
METABOLIC SYNDROME AND RELATED DISORDERS Volume X, Number X, 2015 Mary Ann Liebert, Inc. Pp. 1–7 DOI: 10.1089/met.2015.0018
Insulin Resistance but Not Visceral Adiposity Index Is Associated with Liver Fibrosis in Nondiabetic Subjects with Nonalcoholic Fatty Liver Disease Cemal Nuri Ercin, MD,1 Teoman Dogru, MD,1 Halil Genc, MD,2 Gurkan Celebi, MD,1 Fatih Aslan, MD,3 Hasan Gurel, MD,1 Muammer Kara, MD,4 Erdim Sertoglu, MD,5 Serkan Tapan, MD,6 Sait Bagci, MD,1 Manfredi Rizzo, MD,7 and Alper Sonmez, MD 8
Abstract
Background: Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, type 2 diabetes mellitus, and dyslipidemia. It is well known that the presence of visceral fat increases the risk for metabolic complications of obesity, especially NAFLD. The visceral adiposity index (VAI), a novel marker of visceral fat dysfunction, shows a strong association with insulin resistance and also cardiovascular and cerebrovascular events. However, there is conflicting data regarding the association between VAI and NAFLD. Our aim was to assess the relationship between VAI, insulin resistance, adipocytokines, and liver histology, in nondiabetic subjects with NAFLD. Methods: A total of 215 male patients with biopsy-proven NAFLD were included. Among this group, serum levels of adiponectin, tumor necrosis factor-a (TNF-a, interleukin-6 (IL-6), and high-sensitivity C-reactive protein (hsCRP) were measured in 101 patients whose blood samples were available. Results: High gamma-glutamyl transferase (GGT), high total cholesterol (TC), high triglycerides (TGs), low highdensity lipoprotein cholesterol (HDL-C), and presence of metabolic syndrome were significantly associated with higher VAI, although only higher GGT and TC were independent factors on multiple linear regression analysis. On the other hand, no significant association was found between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels. The multivariate analysis of variables in patients with (n = 124) and without (n = 91) fibrosis showed that only higher homeostasis model assessment of insulin resistance value was independently associated with liver fibrosis. Conclusions: Our findings suggest that VAI is not related to the severity of hepatic inflammation or fibrosis in nondiabetic patients with NAFLD. The lack of association between the adipocytokines and VAI also implies that the VAI may not be a significant indictor of the adipocyte functions. factors for the development of NAFLD.3 Adipose tissue, as an endocrine organ, produces various proteins and soluble factors called adipokines, and these adipokines are considered a main link between obesity, IR, and related metabolic disorders, like NAFLD.4 It is well known that fat tissue is not uniformly distributed in the body. Hence, the distribution of fat appears more important than total fat mass in obesity.5 The presence of predominantly upper body fat increases the risk for metabolic complications of obesity, including hepatic steatosis.6 Visceral fat, a precursor
Introduction
N
onalcoholic fatty liver disease (NAFLD) represents the most common hepatic disorder in western countries. It has a broad spectrum of manifestations ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) and cirrhosis.1 It is now well accepted that NAFLD is the hepatic manifestation of the metabolic syndrome.2 Insulin resistance (IR), obesity, type 2 diabetes mellitus (T2DM), and dyslipidemia are the most important risk 1
Gulhane School of Medicine, Department of Gastroenterology, Ankara, Turkey. _ Izmir Military Hospital2, Izmir, Turkey. _ Department of Gastroenterology, Katip Celebi University, Izmir, Turkey. 4 _ Department of Gastroenterology, Haydarpasa Training Hospital, Istanbul, Turkey. 5 Medical Biochemistry, Anittepe Military Dispensary, Ankara, Turkey. 6 Medical Biochemistry, Gulhane School of Medicine, Ankara, Turkey. 7 Department of Clinical Medicine and Emerging Diseases, Palermo University, Palermo, Italy. 8 Department of Endocrinology, Gulhane School of Medicine, Ankara, Turkey. 2_ 3
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of increased lipolysis and elevated free fatty acid flow and metabolism, is believed to be a more important factor in causing IR than overall obesity or subcutaneous fat.7–9 In fact, many studies have indicated that free fatty acid, leptin, interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) from adipocytes in the visceral fat participate in the development of metabolic syndrome, including IR.10,11 Waist circumference (WC) is a major clinical parameter used for the indirect evaluation of increased visceral fat. The strong and consistent relationships between visceral adiposity and metabolic risk are the basis for including larger WC as part of the Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP) criteria for metabolic syndrome.12 Nevertheless, WC alone does not help to distinguish subcutaneous and visceral fat mass.13 This is a considerable drawback, given that visceral fat, but not subcutaneous fat, plays a decisive role in the genesis of cardiovascular sequelae.14–16 The visceral adiposity index (VAI), a novel marker of visceral fat dysfunction, showed a strong association with IR and visceral adipose tissue. An independent association of VAI with both cardiovascular and cerebrovascular events has also been reported.17 On the other hand, there is very scarce and conflicting data regarding the relationship of VAI with NAFLD.18,19 Thus, in this study, we assessed the relationship between VAI, IR, adipocytokines, and liver histology in subjects with NAFLD. To prevent any interference of confounding factors for IR and inflammation, we studied a specifically selected group having no additional disorders, such as hypertension and T2DM.
Fasting blood specimens were collected from all participants after an 8-hr overnight fast. The samples were centrifuged for 15 min at 4000 rpm, aliquoted, and immediately frozen at -80C for analyses until examination. All samples were run in the same assay. FPG, total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) levels were measured by the enzymatic colorimetric method with an Olympus AU2700 autoanalyzer using reagents from Olympus Diagnostics (Olympus Life Science Europa GmbH, Hamburg, Germany). Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald formula.20 The serum basal insulin level was measured in duplicate by the chemiluminescence method using reagents from Roche Diagnostics (Mannheim, Germany). IR was calculated by modified homeostasis model assessment of IR (HOMA-IR), with the following formula: HOMA-IR = fasting plasma insulin (mU/mL) · FPG (mg/dL)/405. HOMAIR was originally reported by Matthews et al.,21 and this index has been shown to be well correlated with the results of the euglycemic–hyperinsulinemic clamp method to determine IR. The VAI score was calculated using the following sexspecific equations, where TG and HDL-C levels are expressed in mmol/L17:
Material and Methods
The high sensitivity C-reactive protein (hsCRP) level was determined in serum by the immune turbidimetric fixed-rate method using an Olympus AU- 2700 autoanalyzer (Hamburg, Germany). Intra-assay coefficient of variation (CV) and interassay CV were 5.8% and 3.1%, respectively. The minimum detectable concentration for hsCRP was 0.07 mg/L. Plasma adiponectin levels were determined by the enzymelinked immunosorbent assay (ELISA) method (Human Adiponectin ELISA Kit, TECO medical AG, Sissach, Switzerland). The minimum detectable concentration for adiponectin was 0.6 ng/mL. Intra-assay CV ranged from 2.35% to 4.66%, whereas interassay CV ranged from 5.7% to 6.72% for adiponectin. Plasma TNF-a and IL-6 levels were determined by ELISA (Human TNF-a High Sensitivity ELISA and Human IL-6 High Sensitivity ELISA, eBioscience, Vienna, Austria). The minimum detectable concentrations for TNF-a and IL-6 were 0.13 pg/mL and 0.03 pg/mL, respectively. The calculated overall intra-assay CV values for TNF-a and IL-6 were 8.5% and 4.9%, whereas the calculated overall interassay CV for TNF-a and IL-6 were 9.8% and 6.0%, respectively. Measurements were carried out using an ELISA plate reader (Bio-Tek Synergy HT, BioTek Instruments Inc., Winooski, VT). A 2-hr OGTT was performed in each patient with the standard 75 grams of glucose. Glucose tolerance status was determined according to the classification of the American Diabetes Association (ADA) in which FPG levels up to 99 mg/dL are considered normal and T2DM is defined by a FPG level of 126 mg/dL or greater or a 2-hr plasma glucose levels of 200 mg/dL or greater.22 Metabolic syndrome was defined by NCEP ATP III criteria; WC ‡ 102 cm in males or ‡88 cm in females, systolic/ diastolic blood pressure >130/85 mmHg or receiving drug
Patient population We enrolled 215 male patients with biopsy-proven NAFLD. Inclusion criteria were persistently (at least 6 months) elevated aminotransferases, ultrasonographic presence of bright liver without any other liver or biliary tract disease, and liver histology compatible with a diagnosis of NASH or SS. Patients were excluded if they had a history of alcohol consumption >140 grams/week, as assessed by a detailed interview extended to family members; positive testing for hepatitis B virus or hepatitis C virus; body mass index (BMI) ‡40 kg/m2; positive serum markers of autoimmune or celiac disease; abnormal copper metabolism or thyroid function tests; a diagnosis of overt T2DM [fasting plasma glucose (FPG) ‡126 mg/dL or ‡200 mg/dL at 2 hr on a standard oral glucose tolerance test (OGTT)] and systemic hypertension; and exposure to occupational hepatotoxins or drugs known to be steatogenic or to affect glucose and lipid metabolism. The study was approved by the local ethics committee of Gulhane School of Medicine. All participants gave their written informed consent to the study, which was conducted according to the Helsinki Declaration.
Clinical and laboratory data All participants provided a medical history and underwent a clinical examination. The weight and height of the participants were measured with a calibrated scale after the patients had removed their shoes and any heavy clothing. BMI was computed as body weight/(height2). WC was measured as the midpoint between the lower costal margin and the level of the anterior superior iliac crests.
Males: VAI ¼ ðWC=ð39:68 þ (1:88 · BMI))) · (TG=1:03) · (1:31=HDL) Females: VAI ¼ ðWC=ð36:58 þ (1:89 · BMI))) · (TG=0:81) · (1:52=HDL)
VISCERAL ADIPOSITY INDEX IN NAFLD
treatment, TGs ‡150 mg/dL, HDL-C 33–66%) 3 (>66%) Hepatocellular ballooning 0 1 2 Lobular inflammation 0 1 2 3 Fibrosis 0 1 2 3 NAFLD activity score (NAS %) 0–2 3–4 5–8
Values 32.11 (20–53)* 28.31 (21.2–38.3)* 94.34 (86–126)* 50.04 (15–148)* 104.48 (19–330)* 69.10 (19–455)* 204.45 – 44.67 ** 197.52 (22–774)* 124.22 – 37.28** 40.84 (27–60)* 92.97 (58–121)* 15.53 (2.12–53.78)* 3.58 (0.33–13.57)* 3.04 (0.24–12.01)* 11.4464.33** 0.4960.32** 4.4562.16** 2.8862.87** 9.3 28.4 95 (44.5) 72 (33.5) 48 (22) 53 (24.7) 129 (60) 33 (15.3) 23 (10.7) 130 (60.5) 62 (28.8) 0 91 112 9 3
(42.3) (52.1) (4.2) (1.4)
51 (23.7) 81 (37.7) 83 (38.6)
*Mann–Whitney U-test. **Independent sample t-test. BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gammaglutamyl transferase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; VAI, visceral adiposity index; TNF-a, tumor necrosis factor-a; IL-6, interleukin6; hsCRP, high-sensitivity C-reactive protein; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score.
VAI and steatosis, hepatocellular ballooning, lobular inflammation, fibrosis, and NAS (Table 2). In subgroup analysis, there were no significant associations between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels.
Factors associated with histological features The univariate and multivariate comparisons of variables between patients with (n = 124) and without (n = 91) fibrosis
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Table 2.
Univariate and Multivariate Analysis of Factors Associated with VAI Univariate analysis
Variable Age (years) BMI (kg/m2) WC (cm) AST (IU/L) ALT (IU/L) GGT (IU/L) TC (mg/dL) TGs (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) FPG (mg/dL) Insulin (mU/mL) HOMA-IR Presence of metabolic syndrome Smoking Histology Steatosis grade Hepatocellular ballooning Lobular inflammation Stage of fibrosis NAFLD activity score (NAS)
Multivariate analysis
b
SE
P value
b
SE
- 0.003 - 0.042 0.002 0.001 - 0.003 0.006 0.017 0.017 0.002 - 0.150 - 0.021 0.028 0.093 0.995 0.366
0.024 0.050 0.021 0.007 0.003 0.003 0.003 0.000 0.004 0.019 0.013 0.016 0.066 0.501 0.325
0.903 0.408 0.920 0.921 0.280 0.049 < 0.001 < 0.001 0.682 < 0.001 0.105 0.080 0.157 0.048 0.261
- 0.021
0.017
0.235
0.005 0.014
0.002 0.002
0.018 < 0.001
- 0.004
0.012
0.739
- 0.087 - 0.005 - 0.151 - 0.036 - 0.070
0.187 0.235 0.244 0.233 0.189
0.641 0.984 0.538 0.878 0.711
P value
VAI, visceral adiposity index; b, b coefficient; SE, standard error of b; IU, international units; BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score.
are reported in Table 3. Univariate logistic regression analysis showed that the following features were linked to fibrosis: WC (P = 0.043), aspartate aminotransferase (AST) (P = 0.011), alanine aminotransferase (ALT (P = 0.027), insulin (P = 0.005), HOMA-IR index (P = 0.003), steatosis (P = 0.001), hepatocellular ballooning (P < 0.001), lobular inflammation (P < 0.001), and NAS (P < 0.001). On the other hand, only the HOMA-IR value (P = 0.010) was independently associated with fibrosis in multivariate analysis.
Relationship of adipocytokines with liver histology Circulating IL-6 levels were significantly higher in subjects with fibrosis (n = 66) compared to subjects without fibrosis (n = 35) (P = 0.007). In correlation analysis, IL-6 levels were significantly correlated to lobular inflammation and fibrosis (r = 0.342, P < 0.001 and r = 0.367, P < 0.001, respectively). On the other hand, no significant association was found between levels of hsCRP, adiponectin, and TNFa and histopathological findings.
Discussion In the present study, we investigated the relationship of VAI with IR, liver histology, and circulating adipocytokines in nondiabetic male subjects with NAFLD. To the best of our knowledge, this is the largest study that has investigated this relationship in this clinically relevant condition. The findings of our study demonstrate that VAI is not associated with IR, adipocytokines, and the severity of hepatic inflammation or fibrosis in NAFLD. As far as we know, there are only two studies that investigate the VAI in subjects with NAFLD. Petta et al.18
evaluated 142 consecutive NAFLD patients by liver biopsy, regarding clinical and metabolic measurements, including IR with HOMA-IR index, serum levels of TNF-a, IL-6, adiponectin, leptin, and VAI. By multiple linear regression analysis, VAI was found to be independently associated with higher a HOMA index and degree of fibrosis. In addition, an independent association was found between higher VAI and lower adiponectin levels. However, no significant correlation was found among NAS, histologic activity of NAFLD, and VAI. They concluded that VAI is an expression of both qualitative and quantitative adipose tissue dysfunction and, together with IR, is independently associated with significant fibrosis in NAFLD patients.18 Another study performed by Vongsuvanh et al.19 examined 190 adults with biopsy-proven NAFLD and 129 healthy controls. On multivariate analysis, NAFLD diagnosis and FPG were independently associated with VAI. VAI increased across control, steatosis, and NASH groups; however, this association was no stronger than the increase in WC across groups. VAI was not associated with steatosis, lobular inflammation, or fibrosis, but WC was associated with fibrosis. VAI and WC correlated with an increasing number of metabolic syndrome components and with metabolic syndrome diagnosis. However, no significant association was found between VAI and circulating adiponectin levels. They concluded that the VAI score was not associated with the severity of hepatic inflammation or fibrosis and thus cannot be recommended as a surrogate marker of liver injury in patients with NAFLD.19 On the other hand, in the present study, VAI was positively correlated with GGT, TC, TGs levels, and the presence of metabolic syndrome and negatively correlated with
VISCERAL ADIPOSITY INDEX IN NAFLD
Table 3.
Variable Age (years) BMI (kg/m2) WC (cm) AST (IU/L) ALT (IU/L) GGT (IU/L) TC (mg/dL) TGs (mg/dL) LDL-C (mg/dL) HDL–C (mg/dL) FPG (mg/dL) Insulin (mU/mL) HOMA-IR VAI Metabolic syndrome present (%) Smoking present (%)
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The Univariate and Multivariate Analysis of Variables Between Patients With and Without Fibrosis Without fibrosis (n = 91) 31.76 – 5.52 28 – 2.62 98.18 – 5.85 47.22 – 21.09 97.43 – 43.09 69.94 – 58.615 204.41 – 39.29 192.77 – 119.81 125.64 – 33.03 40.19 – 6.69 91.96 – 12.41 13.43 – 7.33 3.03 – 1.70 3.0 – 2.1 7
(32) (28.2) (98)* (42) (90) (69) (203) (161) (123) (40) (92) (11.3) (2.47) (2.57)
With fibrosis (n = 124) 32.36 – 6.61 28.53 – 3.11 101.19 – 7.51 52.11 – 20.64 109.65 – 48.52 67.97 – 39.331 204.48 – 48.40 201.01 – 123.50 123.19 – 40.22 41.31 – 6.95 93.71 – 10.07 17.07 – 9.89 3.98 – 2.49 3.07 – 2.19 13
27
34
(32) (28.1) (100) (49) (103) (68) (210.5) (161.5) (128) (41) (94) (14.59) (3.44) (2.41)
Univariate analysis (P)
Multivariate analysis [OR (95% CI)]
P
0.657* 0.302* 0.043* 0.011* 0.027* 0.695* 0.991** 0.501* 0.625** 0.210* 0.383* 0.005* 0.003* 0.726* 0.486
1.015 (0.965–1.068)
0.561
1.001 (0.954–1.049)
0.977
1.246 (1.054–1.472)
0.010
0.718
*Mann–Whitney U-test. ** Independent sample t-test. OR, odds ratio; CI, confidence interval; BMI, body mass index; WC, waist circumference; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; TC, total cholesterol; TGs, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; FPG, fasting plasma glucose; HOMA-IR, homeostasis model assessment for insulin resistance; VAI, visceral adiposity index.
HDL-C levels in univariate analysis. However, no association with BMI, WC, and IR as assessed by the HOMA equation could be detected. To ensure that our findings were independent of key confounders, we performed multivariate analysis using those factors significant on univariate analysis. Inclusion of all relevant risk factors resulted in an independent association of GGT and TC levels with VAI. On the other hand, no significant association was found between VAI and steatosis, hepatocellular ballooning, lobular inflammation, NAS score, and fibrosis. Only higher HOMAIR value was independently associated with liver fibrosis in multivariate analysis. Moreover, no significant association was found between VAI and adiponectin, TNF-a, IL-6, and hsCRP levels. We think that the lack of relationship between VAI and NAFLD within our cohort may be due to several reasons. First, some participants were hypertensive and also diabetic in the study by Petta et al.18 It is well known that TGs and HDL-C levels that are the components of VAI are affected by these metabolic disorders. In addition, circulating concentrations of adipokines and measures of insulin sensitivity are easily affected by these metabolic confounders.24,25 Second, NAFLD is strongly associated with obesity, hypertension, and glucose tolerance abnormalities.2,3,26 It has been reported that all of these metabolic problems are also risk factors for T2DM. Although it was stated in the study by Petta et al. that some of the patients were diabetic at baseline, there is no information regarding the glucose tolerance status of the other subjects. In light of these clear data, we think that some of the study participants may still have overt glucose dysregulation or T2DM without implementation of the glucose tolerance test. This issue is important because NAFLD is strongly associated with T2DM, and blood adipokine and insulin levels are altered in subjects with disordered glucose me-
tabolism.25 Finally, no information about the drug use (antihypertensive or hypoglycemic) of the subjects could be seen in the text. We know that circulating adipokines and measures of insulin sensitivity are easily affected from the medications started for the metabolic problems mentioned above.27–29 For this reason, we carefully excluded those patients taking hypoglycemic drugs such as thiazolidinediones and metformin, which have been shown to significantly affect circulating adipokine levels. Consequently, we think that some of the previous findings regarding the VAI in NAFLD might be affected by these confounders. Hence, in a recent study performed in 139 morbidly obese 139 patients who underwent bariatric surgery, IR and metabolic syndrome were the main factors associated with VAI.30 Adipokines play an important role in the pathogenesis of IR and NAFLD through complex and interactive paracrine and endocrine mechanisms.31 The relationship of adipokines with NAFLD has been extensively investigated in several previous studies,32,33 but the published reports have yielded conflicting results regarding the role of adipokines in the pathogenesis of NAFLD.34,35 In the present study, we found a significant association of IL-6 with lobular inflammation and fibrosis in NAFLD. So, our results are in line with current scientific knowledge regarding this issue.36,37 On the other hand, there were no associations between hsCRP, adiponectin, TNF-a, and liver histology in our cohort. Consequently, we suggest that the discrepant findings of these studies were likely due to differences in the populations of patients enrolled, specifically with respect to the age, number of participants, and co-morbid conditions such as diabetes and hypertension. Several limitations must be acknowledged in this study. First, despite the sample size and the strict inclusion criteria, the findings obtained are not representative for all subjects with NAFLD. However, as mentioned above, we think that
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the design of our study was a requirement for the goals to be achieved. Second, even if the HOMA-IR index has a reasonable linear correlation with the glucose clamp technique and was used in several population studies, another limitation of this work is due to the fact that the HOMA index does not help to clarify whether IR is central or peripheral. Third, our study population represents a group with a mild NAFLD phenotype, with most subjects having grade 0–1 fibrosis. Whether our results can be generalized to a more severe NAFLD cohort remains to be determined. Last, all participants were men, and that it remains to be determined if these results are similar also in women. In conclusion, our findings further support the data that VAI is not an expression of adipose tissue dysfunction and is not associated with the severity of hepatic inflammation or fibrosis in NAFLD.
14. 15.
16. 17.
18.
Author Disclosure Statement No competing financial interests exist.
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Address correspondence to: Cemal Nuri Ercin, Associate Professor Gulhane School of Medicine Department of Gastroenterology General Tevfik Saglam Cad Etlik-Kecioren Ankara 06180 Turkey E-mail: [email protected]
