J Bone Miner Metab DOI 10.1007/s00774-015-0687-x
ORIGINAL ARTICLE
Circulating sclerostin and Dickkopf‑1 levels in patients with nonalcoholic fatty liver disease Stergios A. Polyzos1,2 · Athanasios D. Anastasilakis3 · Jannis Kountouras1 · Polyzois Makras4 · Athanasios Papatheodorou5 · Panagiotis Kokkoris5 · Grigorios T. Sakellariou6 · Evangelos Terpos7
Received: 6 February 2015 / Accepted: 9 May 2015 © The Japanese Society for Bone and Mineral Research and Springer Japan 2015
Abstract There is increasing evidence for bone-liver interplay. The main aim of this study was to determine serum sclerostin and Dickkopf (DKK)-1 levels in patients with nonalcoholic fatty liver disease (NAFLD) and their association with the disease severity. Patients with biopsyproven NAFLD, 13 with nonalcoholic simple steatosis (SS) and 14 with steatohepatitis (NASH), and 20 gender-, age-, body mass index- and waist circumference-matched controls were enrolled. Serum sclerostin, DKK-1, bone turnover markers, vitamin D, insulin and standard biochemical and hematologic parameters were measured; lumbar spinal dualenergy X-ray absorptiometry was performed. We observed that there was a progressive decline in serum sclerostin levels from the controls (76.1 ± 6.8) to SS (53.5 ± 6.4) and NASH (46.0 ± 8.1 pmol/l) patients (p = 0.009); in adjusted
* Stergios A. Polyzos [email protected] 1
Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, 13 Simou Lianidi, 551 34 Thessaloniki, Macedonia, Greece
2
Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
3
Department of Endocrinology, 424 General Military Hospital, Thessaloniki, Macedonia, Greece
4
Department of Endocrinology and Diabetes, 251 Hellenic Air Force and VA General Hospital, Athens, Greece
5
Department of Medical Research, 251 Hellenic Air Force and VA General Hospital, Athens, Greece
6
Department of Rheumatology, 424 General Military Hospital, Thessaloniki, Macedonia, Greece
7
Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece
pairwise comparisons, sclerostin was significantly higher in the controls than in NASH patients (p = 0.012). Although serum DKK-1 did not differ between groups (p = 0.135), there was a trend toward U-shaped distribution (controls 35.8 ± 2.8; SS 27.3 ± 2.9; NASH 36.8 ± 4.4 pmol/l). Higher DKK-1 levels were independently associated with NASH. Regarding specific histological lesions, DKK-1 levels were marginally lower in NAFLD patients with lower (≤33 %) than higher (>33 %) steatosis grade (27.7 ± 3.1 and 38.8 ± 4.7 pmol/l, respectively; p = 0.049). No other significant difference was observed within histological lesions. In conclusion, serum sclerostin levels were lower in NASH patients than in controls. DKK-1 levels were independently associated with NASH in NAFLD patients. The potential importance of these findings indicates a possible bone-liver interaction and warrants further investigation. Keywords Bone turnover markers · Dickkopf-1 · Nonalcoholic fatty liver disease · Nonalcoholic steatohepatitis · Sclerostin
Introduction Nonalcoholic fatty liver disease (NAFLD), a global public health problem of increasing significance [1], ranges from nonalcoholic simple steatosis (SS) to nonalcoholic steatohepatitis (NASH), characterized by inflammation and/or fibrosis that may lead to liver cirrhosis and hepatocellular carcinoma [2]. NAFLD is regarded as the hepatic component of metabolic syndrome, given that insulin resistance (IR) essentially contributes to its pathogenesis [2]. NAFLD prevalence is increasing in parallel with the epidemics of obesity and type 2 diabetes mellitus (T2DM) and is linked with cardiovascular disease (CVD) [1].
13
NAFLD has also been linked to low bone mineral density (BMD) and osteoporosis in both pediatric and adult populations, although data are still limited and the mechanisms linking them are poorly understood [3, 4]. Interplay between hepatic and bone metabolism, possibly bidirectional, appears to exist: hepatic metabolism may affect and be affected by bone metabolism. Experimental data showed a complex system of interaction among the liver, adipose tissue and bone, which reciprocally modulate the function of each other. Dysregulation of this liver-adipose tissuebone interaction predisposes to the development of obesity, T2DM, NAFLD and osteoporosis [5]. IR has also been proposed to play a critical intermediary role in the crosstalk between NAFLD and low bone mass [6]. Sclerostin and Dickkopf (DKK)-1 are negative regulators of the canonical Wingless (Wnt)/β-catenin signaling, thus inhibiting osteoblastic bone formation with significant effect on bone metabolism [7, 8]. Specifically, both sclerostin and DKK-1 inhibit the Wnt signaling pathway, which in turn results in the inhibition of osteoblast differentiation and bone formation [7]. Inactivating monoclonal antibodies for either sclerostin or DKK-1 are currently under investigation for the treatment of metabolic bone diseases [9]. Apart from bone metabolism, the Wnt/β-catenin pathway also exists in other systems, thereby being possibly associated with components of insulin resistance (IR) syndrome, including hyperlipidemia, T2DM and atherosclerosis [10– 13]. However, to date no study has reported on circulating sclerostin and DKK-1 levels in NAFLD, which is regarded as the hepatic manifestation of IR syndrome [14]. The primary aim of this study was the evaluation of serum sclerostin and DKK-1 levels in patients with biopsyproven NAFLD, untreated for metabolic bone disease, and their association with the disease severity. The secondary aim was the association of serum sclerostin, DKK-1, bone turnover markers and 25-hydroxy vitamin D [25(OH)D] with NAFLD-related factors.
Materials and methods Patients This was a single-center, cross-sectional study. NAFLD patients and controls were consecutively recruited on an outpatient basis at the Second Medical Clinic (Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece). Determination of eligibility was based on medical history, physical examination and liver function tests [serum aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), total and direct bilirubin] and liver ultrasound imaging performed during the screening
13
J Bone Miner Metab
visit. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local ethics committee. Informed consent was obtained from all individual participants included in the study. Inclusion criteria for NAFLD patients were: (1) age >18 years; (2) bright liver on ultrasound imaging and abnormal liver function tests for at least 6 months before liver biopsy; (3) patient’s consent for liver biopsy. Individuals of similar gender, age, body mass index (BMI) and waist circumference (WC) without NAFLD, living in the same region, who underwent regular check-ups for professional needs were recruited as controls. Inclusion criteria for the controls were: (1) age >18 years; (2) no history of abnormal liver ultrasound imaging or abnormal liver function tests; (3) currently normal liver ultrasound imaging; (4) currently normal liver function tests. Controls did not undergo a liver biopsy because of obvious ethical considerations. Exclusion criteria for both NAFLD patients and controls, as described in detail elsewhere [15], were: (1) ethanol consumption more than 20 g/day; (2) liver cirrhosis; (3) other liver disease (viral hepatitis, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis and overlap syndromes, drug-induced liver disease, hemochromatosis, Wilson’s disease, α1-antitrypsin deficiency); (4) type 1 diabetes mellitus; (5) pancreatitis; (6) uncontrolled hypothyroidism or hyperthyroidism; (7) adrenal insufficiency; (8) renal failure; (9) thrombotic disorders; (10) metabolic bone diseases other than osteoporosis; (11) cancer; (12) pregnancy; (13) addiction to any drug; (14) ever use of antiosteoporotic agents, including calcium and/or vitamin D supplements, calcitonin, selective estrogen receptor modulators, bisphosphonates, strontium ralenate, denosumab, parathyroid hormone or teriparatide; (15) use of the following medications within a 12-month period before screening: estrogens, progestins, glucocorticoids, thiazolidinediones, insulin, sibutramine, orlistat, rimonabant, vitamin E, ursodeoxycholic acid, ferrum, interferon, tamoxifene, amiodarone, biologic agents, any antibiotic, and any medication against tuberculosis, epilepsy or viruses; (16) intravenous glucose administration or parenteral nutrition within a 1-month period before screening. Methods Morning (8−9 a.m.) fasting blood samples were collected 1–2 h prior to liver biopsy. AST, ALT, GGT, total alkaline phosphatase (TALP), triglycerides, high-density lipoprotein cholesterol (HDL-C), creatinine, glucose, calcium and phosphate, white blood cells and subpopulations, and erythrocyte sedimentation rate (ESR) were measured immediately after blood drawing, with standard methods: Olympus
J Bone Miner Metab
AU2700 (Olympus, Hamburg, Germany) for biochemical tests; Beckman Coulter LH 750 (Nyon, Switzerland) for hematologic tests; and Ves Matic 20 (Menarini Diagnostics, Rungis, France) for ESR. Sera were also immediately frozen at −30 °C for the measurement of sclerostin, DKK1, bone-specific alkaline phosphatase (BALP), carboxyterminal telopeptides of type I collagen (CTX), 25(OH)D and insulin, measured in one batch at the end of the study. Serum sclerostin, DKK-1, CTX and BALP were measured with enzyme-linked immunosorbent assay (ELISA). The assay characteristics were: sclerostin (Biomedica, Vienna, Austria) intra-assay coefficient of variation (CV) 5–7 %, inter-assay CV 3−10 %; DKK-1 (Biomedica, Vienna, Austria) intra-assay CV 7–8 %, inter-assay CV 9–12 %; CTX (Immunodiagnostic Systems, Boldon, UK) intra-assay CV 1.7–3.0 %, inter-assay CV 2.5–10.9 %; BALP (Immunodiagnostic Systems, Boldon, UK) intra-assay CV 1.7–4.1 %, inter-assay CV 3.8–4.8 %. The 25(OH)D and insulin were measured with immuno-chemiluminescence (ICMA) on an Immulite 2500 automated immunoassay system (Siemens Healthcare Diagnostics, Deerfield, IL, USA); intra-assay and total CV were 2.9–5.5 % and 6.3–12.9 % for 25(OH)D and 3.3–5.5 % and 4.1–7.3 % for insulin, respectively. Liver biopsy was performed under computed tomography guidance by an experienced radiologist and interpreted by two experienced pathologists. NAFLD patients were subdivided into those with SS or NASH according to the criteria of the NAFLD Activity Score (NAS) [16]. Steatosis grade, fibrosis stage, lobular and portal inflammation, and ballooning were categorized based on the classification of the NASH Clinical Research Network [16]. Regarding fibrosis stage, cirrhosis (grade 4) was not included (exclusion criterion). BMD was measured at baseline by dualenergy X-ray absorptiometry (DXA) using a DPX-IQ densitometer (Lunar Corp., Madison, WI, USA). BMI was calculated by the formula: body weight (kg)/ height2 (m2). IR was quantified by the homeostasis model of assessment-insulin resistance (HOMA-IR) using the formula HOMA-IR = glucose (mmol/l) × insulin (μU/ml)/22.5 [17]. Corrected calcium was calculated by the formula: corrected calcium (mg/dl) = Ca (mg/ dL) + 0.8 × [4.0 − albumin (g/dl)]. The AST-to-ALT ratio was also calculated. Statistical analysis Continuous data are presented as mean ± standard error of the mean (SEM). Categorical data are presented as frequencies. The Kolmogorov-Smirnov test was used to check the normality of distributions of continuous variables. The chi-square test was used for between-group comparisons in case of categorical variables. Spearman’s coefficient (rs) was used for binary correlations.
In case of continuous variables, between-group comparisons were performed with independent samples T test or Mann-Whitney test when two groups were compared or one-way analysis of variance (ANOVA) or Kruskal-Wallis test when more than two groups were compared. In case of a statistically significant difference in the ANOVA or Kruskal-Wallis test, Bonferroni post hoc adjustment was used for multiple pairwise comparisons. Analysis of covariance (ANCOVA) was used to adjust betweengroup comparisons for covariates. Multiple binary logistic regression analysis (method “enter”) was used within NAFLD patients to identify whether sclerostin or DKK-1 was independently associated with NASH (dependent variable SS = 0 or NASH = 1) or specific histological lesions (e.g., for steatosis grade: ≤33 % = 0 or >33 % = 1, etc.). In ANCOVA and logistic regression analyses, we included variables possibly related in univariate analyses, but also tried to avoid colinearity; for example, we separately added BMI, WC, HOMA-IR, triglycerides or HDL-C in the models to avoid colinearity since all these parameters are closely related in IR syndrome. For the needs of these analyses, variables that did not follow normal distribution were logarithmically transformed. Statistical analysis was performed with SPSS 21.0 for Macintosh (SPSS Inc., Chicago, IL). Significance was set at p
ORIGINAL ARTICLE
Circulating sclerostin and Dickkopf‑1 levels in patients with nonalcoholic fatty liver disease Stergios A. Polyzos1,2 · Athanasios D. Anastasilakis3 · Jannis Kountouras1 · Polyzois Makras4 · Athanasios Papatheodorou5 · Panagiotis Kokkoris5 · Grigorios T. Sakellariou6 · Evangelos Terpos7
Received: 6 February 2015 / Accepted: 9 May 2015 © The Japanese Society for Bone and Mineral Research and Springer Japan 2015
Abstract There is increasing evidence for bone-liver interplay. The main aim of this study was to determine serum sclerostin and Dickkopf (DKK)-1 levels in patients with nonalcoholic fatty liver disease (NAFLD) and their association with the disease severity. Patients with biopsyproven NAFLD, 13 with nonalcoholic simple steatosis (SS) and 14 with steatohepatitis (NASH), and 20 gender-, age-, body mass index- and waist circumference-matched controls were enrolled. Serum sclerostin, DKK-1, bone turnover markers, vitamin D, insulin and standard biochemical and hematologic parameters were measured; lumbar spinal dualenergy X-ray absorptiometry was performed. We observed that there was a progressive decline in serum sclerostin levels from the controls (76.1 ± 6.8) to SS (53.5 ± 6.4) and NASH (46.0 ± 8.1 pmol/l) patients (p = 0.009); in adjusted
* Stergios A. Polyzos [email protected] 1
Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, 13 Simou Lianidi, 551 34 Thessaloniki, Macedonia, Greece
2
Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
3
Department of Endocrinology, 424 General Military Hospital, Thessaloniki, Macedonia, Greece
4
Department of Endocrinology and Diabetes, 251 Hellenic Air Force and VA General Hospital, Athens, Greece
5
Department of Medical Research, 251 Hellenic Air Force and VA General Hospital, Athens, Greece
6
Department of Rheumatology, 424 General Military Hospital, Thessaloniki, Macedonia, Greece
7
Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece
pairwise comparisons, sclerostin was significantly higher in the controls than in NASH patients (p = 0.012). Although serum DKK-1 did not differ between groups (p = 0.135), there was a trend toward U-shaped distribution (controls 35.8 ± 2.8; SS 27.3 ± 2.9; NASH 36.8 ± 4.4 pmol/l). Higher DKK-1 levels were independently associated with NASH. Regarding specific histological lesions, DKK-1 levels were marginally lower in NAFLD patients with lower (≤33 %) than higher (>33 %) steatosis grade (27.7 ± 3.1 and 38.8 ± 4.7 pmol/l, respectively; p = 0.049). No other significant difference was observed within histological lesions. In conclusion, serum sclerostin levels were lower in NASH patients than in controls. DKK-1 levels were independently associated with NASH in NAFLD patients. The potential importance of these findings indicates a possible bone-liver interaction and warrants further investigation. Keywords Bone turnover markers · Dickkopf-1 · Nonalcoholic fatty liver disease · Nonalcoholic steatohepatitis · Sclerostin
Introduction Nonalcoholic fatty liver disease (NAFLD), a global public health problem of increasing significance [1], ranges from nonalcoholic simple steatosis (SS) to nonalcoholic steatohepatitis (NASH), characterized by inflammation and/or fibrosis that may lead to liver cirrhosis and hepatocellular carcinoma [2]. NAFLD is regarded as the hepatic component of metabolic syndrome, given that insulin resistance (IR) essentially contributes to its pathogenesis [2]. NAFLD prevalence is increasing in parallel with the epidemics of obesity and type 2 diabetes mellitus (T2DM) and is linked with cardiovascular disease (CVD) [1].
13
NAFLD has also been linked to low bone mineral density (BMD) and osteoporosis in both pediatric and adult populations, although data are still limited and the mechanisms linking them are poorly understood [3, 4]. Interplay between hepatic and bone metabolism, possibly bidirectional, appears to exist: hepatic metabolism may affect and be affected by bone metabolism. Experimental data showed a complex system of interaction among the liver, adipose tissue and bone, which reciprocally modulate the function of each other. Dysregulation of this liver-adipose tissuebone interaction predisposes to the development of obesity, T2DM, NAFLD and osteoporosis [5]. IR has also been proposed to play a critical intermediary role in the crosstalk between NAFLD and low bone mass [6]. Sclerostin and Dickkopf (DKK)-1 are negative regulators of the canonical Wingless (Wnt)/β-catenin signaling, thus inhibiting osteoblastic bone formation with significant effect on bone metabolism [7, 8]. Specifically, both sclerostin and DKK-1 inhibit the Wnt signaling pathway, which in turn results in the inhibition of osteoblast differentiation and bone formation [7]. Inactivating monoclonal antibodies for either sclerostin or DKK-1 are currently under investigation for the treatment of metabolic bone diseases [9]. Apart from bone metabolism, the Wnt/β-catenin pathway also exists in other systems, thereby being possibly associated with components of insulin resistance (IR) syndrome, including hyperlipidemia, T2DM and atherosclerosis [10– 13]. However, to date no study has reported on circulating sclerostin and DKK-1 levels in NAFLD, which is regarded as the hepatic manifestation of IR syndrome [14]. The primary aim of this study was the evaluation of serum sclerostin and DKK-1 levels in patients with biopsyproven NAFLD, untreated for metabolic bone disease, and their association with the disease severity. The secondary aim was the association of serum sclerostin, DKK-1, bone turnover markers and 25-hydroxy vitamin D [25(OH)D] with NAFLD-related factors.
Materials and methods Patients This was a single-center, cross-sectional study. NAFLD patients and controls were consecutively recruited on an outpatient basis at the Second Medical Clinic (Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece). Determination of eligibility was based on medical history, physical examination and liver function tests [serum aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), total and direct bilirubin] and liver ultrasound imaging performed during the screening
13
J Bone Miner Metab
visit. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local ethics committee. Informed consent was obtained from all individual participants included in the study. Inclusion criteria for NAFLD patients were: (1) age >18 years; (2) bright liver on ultrasound imaging and abnormal liver function tests for at least 6 months before liver biopsy; (3) patient’s consent for liver biopsy. Individuals of similar gender, age, body mass index (BMI) and waist circumference (WC) without NAFLD, living in the same region, who underwent regular check-ups for professional needs were recruited as controls. Inclusion criteria for the controls were: (1) age >18 years; (2) no history of abnormal liver ultrasound imaging or abnormal liver function tests; (3) currently normal liver ultrasound imaging; (4) currently normal liver function tests. Controls did not undergo a liver biopsy because of obvious ethical considerations. Exclusion criteria for both NAFLD patients and controls, as described in detail elsewhere [15], were: (1) ethanol consumption more than 20 g/day; (2) liver cirrhosis; (3) other liver disease (viral hepatitis, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis and overlap syndromes, drug-induced liver disease, hemochromatosis, Wilson’s disease, α1-antitrypsin deficiency); (4) type 1 diabetes mellitus; (5) pancreatitis; (6) uncontrolled hypothyroidism or hyperthyroidism; (7) adrenal insufficiency; (8) renal failure; (9) thrombotic disorders; (10) metabolic bone diseases other than osteoporosis; (11) cancer; (12) pregnancy; (13) addiction to any drug; (14) ever use of antiosteoporotic agents, including calcium and/or vitamin D supplements, calcitonin, selective estrogen receptor modulators, bisphosphonates, strontium ralenate, denosumab, parathyroid hormone or teriparatide; (15) use of the following medications within a 12-month period before screening: estrogens, progestins, glucocorticoids, thiazolidinediones, insulin, sibutramine, orlistat, rimonabant, vitamin E, ursodeoxycholic acid, ferrum, interferon, tamoxifene, amiodarone, biologic agents, any antibiotic, and any medication against tuberculosis, epilepsy or viruses; (16) intravenous glucose administration or parenteral nutrition within a 1-month period before screening. Methods Morning (8−9 a.m.) fasting blood samples were collected 1–2 h prior to liver biopsy. AST, ALT, GGT, total alkaline phosphatase (TALP), triglycerides, high-density lipoprotein cholesterol (HDL-C), creatinine, glucose, calcium and phosphate, white blood cells and subpopulations, and erythrocyte sedimentation rate (ESR) were measured immediately after blood drawing, with standard methods: Olympus
J Bone Miner Metab
AU2700 (Olympus, Hamburg, Germany) for biochemical tests; Beckman Coulter LH 750 (Nyon, Switzerland) for hematologic tests; and Ves Matic 20 (Menarini Diagnostics, Rungis, France) for ESR. Sera were also immediately frozen at −30 °C for the measurement of sclerostin, DKK1, bone-specific alkaline phosphatase (BALP), carboxyterminal telopeptides of type I collagen (CTX), 25(OH)D and insulin, measured in one batch at the end of the study. Serum sclerostin, DKK-1, CTX and BALP were measured with enzyme-linked immunosorbent assay (ELISA). The assay characteristics were: sclerostin (Biomedica, Vienna, Austria) intra-assay coefficient of variation (CV) 5–7 %, inter-assay CV 3−10 %; DKK-1 (Biomedica, Vienna, Austria) intra-assay CV 7–8 %, inter-assay CV 9–12 %; CTX (Immunodiagnostic Systems, Boldon, UK) intra-assay CV 1.7–3.0 %, inter-assay CV 2.5–10.9 %; BALP (Immunodiagnostic Systems, Boldon, UK) intra-assay CV 1.7–4.1 %, inter-assay CV 3.8–4.8 %. The 25(OH)D and insulin were measured with immuno-chemiluminescence (ICMA) on an Immulite 2500 automated immunoassay system (Siemens Healthcare Diagnostics, Deerfield, IL, USA); intra-assay and total CV were 2.9–5.5 % and 6.3–12.9 % for 25(OH)D and 3.3–5.5 % and 4.1–7.3 % for insulin, respectively. Liver biopsy was performed under computed tomography guidance by an experienced radiologist and interpreted by two experienced pathologists. NAFLD patients were subdivided into those with SS or NASH according to the criteria of the NAFLD Activity Score (NAS) [16]. Steatosis grade, fibrosis stage, lobular and portal inflammation, and ballooning were categorized based on the classification of the NASH Clinical Research Network [16]. Regarding fibrosis stage, cirrhosis (grade 4) was not included (exclusion criterion). BMD was measured at baseline by dualenergy X-ray absorptiometry (DXA) using a DPX-IQ densitometer (Lunar Corp., Madison, WI, USA). BMI was calculated by the formula: body weight (kg)/ height2 (m2). IR was quantified by the homeostasis model of assessment-insulin resistance (HOMA-IR) using the formula HOMA-IR = glucose (mmol/l) × insulin (μU/ml)/22.5 [17]. Corrected calcium was calculated by the formula: corrected calcium (mg/dl) = Ca (mg/ dL) + 0.8 × [4.0 − albumin (g/dl)]. The AST-to-ALT ratio was also calculated. Statistical analysis Continuous data are presented as mean ± standard error of the mean (SEM). Categorical data are presented as frequencies. The Kolmogorov-Smirnov test was used to check the normality of distributions of continuous variables. The chi-square test was used for between-group comparisons in case of categorical variables. Spearman’s coefficient (rs) was used for binary correlations.
In case of continuous variables, between-group comparisons were performed with independent samples T test or Mann-Whitney test when two groups were compared or one-way analysis of variance (ANOVA) or Kruskal-Wallis test when more than two groups were compared. In case of a statistically significant difference in the ANOVA or Kruskal-Wallis test, Bonferroni post hoc adjustment was used for multiple pairwise comparisons. Analysis of covariance (ANCOVA) was used to adjust betweengroup comparisons for covariates. Multiple binary logistic regression analysis (method “enter”) was used within NAFLD patients to identify whether sclerostin or DKK-1 was independently associated with NASH (dependent variable SS = 0 or NASH = 1) or specific histological lesions (e.g., for steatosis grade: ≤33 % = 0 or >33 % = 1, etc.). In ANCOVA and logistic regression analyses, we included variables possibly related in univariate analyses, but also tried to avoid colinearity; for example, we separately added BMI, WC, HOMA-IR, triglycerides or HDL-C in the models to avoid colinearity since all these parameters are closely related in IR syndrome. For the needs of these analyses, variables that did not follow normal distribution were logarithmically transformed. Statistical analysis was performed with SPSS 21.0 for Macintosh (SPSS Inc., Chicago, IL). Significance was set at p
