Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Subcutaneous Adipose Tissue Zinc-a2-Glycoprotein is Associated with Adipose Tissue and Whole-Body Insulin Sensitivity Miroslav Balaz1, Marek Vician2, Zuzana Janakova1, Timea Kurdiova1, Martina Surova1, Richard Imrich1, Zuzana Majercikova1, Adela Penesova1, Miroslav Vlcek1, Alexander Kiss1, Vitazoslav Belan3, Iwar Klimes1, Juraj Olejnik2, Daniela Gasperikova1, Christian Wolfrum4, Barbara Ukropcova1 and Jozef Ukropec1
Objective: To examine the regulatory aspects of zinc-a2-glycoprotein (ZAG) association with obesityrelated insulin resistance. Methods: ZAG mRNA and protein were analyzed in subcutaneous adipose tissue (AT) and circulation of lean, obese, prediabetic, and type 2 diabetic men; both subcutaneous and visceral AT were explored in lean and extremely obese. Clinical and ex vivo findings were corroborated by results of in vitro ZAG silencing experiment. Results: Subcutaneous AT ZAG was reduced in obesity, with a trend to further decrease with prediabetes and type 2 diabetes. ZAG was 3.3-fold higher in subcutaneous than in visceral AT of lean individuals. All differences were lost in extreme obesity. Obesity-associated changes in AT were not paralleled by alterations of circulating ZAG. Subcutaneous AT ZAG correlated with adiposity, adipocyte hypertrophy, whole-body and AT insulin sensitivity, mitochondrial content, expression of GLUT4, PGC1a, and adiponectin. Subcutaneous AT ZAG and adipocyte size were the only predictors of insulin sensitivity, independent on age and BMI. Silencing ZAG resulted in reduced adiponectin, IRS1, GLUT4, and PGC1a gene expression in primary human adipocytes. Conclusions: ZAG in subcutaneous, but not in visceral AT, was markedly reduced in obesity. Clinical, cellular, and molecular evidence indicate that ZAG plays an important role in modulating whole-body and AT insulin sensitivity. Obesity (2014) 00, 00-00. doi:10.1002/oby.20764
Introduction White adipose tissue (AT), our largest energy depot, is a highly dynamic secretory organ, producing a whole spectrum of biologically active substances. Adipokines, proteins synthesized and secreted by adipocytes, modulate many physiological processes in different tissues and organs (1-3). Changes in AT secretory profile such as found in obesity may severely impact the whole-body metabolism, leading to the development of metabolic disease (4-6).
Previous studies have shown that AT-derived zinc-a2-glycoprotein (ZAG) may play a pivotal role in the regulation of adiposity (7-9). Initially, ZAG was identified as a product of secretory epithelial cells and several forms of malignant tumors and has been proposed to serve as a potential cancer marker (10-13). Later, ZAG was shown to be identical to a lipid-mobilizing factor responsible for a loss of fat mass in patients with cancer cachexia (14), and AT ZAG was shown to be elevated proportionally to the degree of weight loss (8). ZAG has been found to be produced by differentiated human adipocytes
1
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia. Correspondence: Jozef Ukropec ([email protected]) Department of Surgery, Slovak Medical University, Bratislava, Slovakia 3 Radiology Clinic, University Hospital, Bratislava, Slovakia 4 Institute of Food, Nutrition & Health, Swiss Federal Institute of Technology, ETH Zurich, Schwerzenbach, Switzerland
2
Funding agencies: This work was supported by EFSD New Horizons, 7FP-EC-LipidomicNET #202272, VEGA 2/0198/11 and 2/0132/10, National Scholarship Programme of the Slovak Republic. Disclosure: The authors declare no conflict of interest. Author Contributions: MB: data generation, analysis, and interpretation, drafting the manuscript, figures generation; MV, JO: muscle sample collection, clinical data collection & interpretation; ZJ, TK, MS: sample collection, clinical data generation, critical reading of the manuscript; RI, AP, MV: sample collection, critical reading of the manuscript; ZM, AK: data generation, analysis, interpretation; VB: clinical data generation, analysis; DG, IK: critical reading of the manuscript; CW: in vitro study design, critical reading of the manuscript; BU, JU: study conception and design, data interpretation, critical reading of the manuscript. All authors have read and approved the final version of the manuscript. Additional Supporting Information may be found in the online version of this article. Received: 17 February 2014; Accepted: 4 April 2014; Published online 00 Month 2014. doi:10.1002/oby.20764
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Obesity | VOLUME 00 | NUMBER 00 | MONTH 2014
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Obesity
ZAG in Obesity and Metabolic Disease Balaz et al.
TABLE 1 Clinical characteristics of study cohort 1
Sedentary healthy lean to obese diabetic men (Cohort 1) Lean/overweight (n 5 27)
Obese (n 5 26)
Prediabetic (n 5 25)
T2D (n 5 15)
27/0 36.8 6 1.8 23.3 6 0.4 18.4 6 0.9 88.0 6 1.7 94.5 6 3.0 1.07 6 0.99 4.18 6 0.16 1.36 6 0.05 2.34 6 0.12 0.57 6 0.04 0.11 6 0.01 0.15 6 0.01 4.7 6 0.08 5.4 6 0.25
26/0 37.6 6 1.5 30.2 6 0.5a 28.3 6 0.9a 105.0 6 1.4a 116.7 6 2.2a 1.39 6 0.14 4.58 6 0.15 1.24 6 0.05 2.68 6 0.12 0.62 6 0.04 0.15 6 0.01 0.08 6 0.01a 4.9 6 0.15 5.4 6 0.28
25/0 44.2 6 1.9a,b 31.6 6 0.6a 30.9 6 0.7a 111.3 6 1.9a 120.7 6 2.4a 1.95 6 0.19 5.03 6 0.24a 1.26 6 0.05 2.90 6 0.19a 0.71 6 0.04 0.16 6 0.02a 0.07 6 0.01a 5.6 6 0.13 8.0 6 0.33a,b
15/0 50.5 6 2.1a,b 31.5 6 1.0a 30.9 6 1.1a 109.8 6 2.8a 119.8 6 4.2a 2.27 6 0.33a,b 5.21 6 0.21a 1.25 6 0.05 2.94 6 0.16 0.64 6 0.08 0.18 6 0.03a 0.05 6 0.01a 9.0 6 1.7a,b,c -
Gender (M/F) Age (years) BMI (kg/m2) Body fat (%) Waist circumference (cm) Adipocyte diameter (lm) Triglycerides (mM) Total cholesterol (mM) HDL cholesterol (mM) LDL cholesterol (mM) FFA fasted state (mM) FFA during EHC (mM) M-value/insulin (mg/kgBW/min/lU/ml) Fasting glycemia (mM) 2 h glycemia in oGTT (mM)
FFA, free fatty acids; M-value/insulin, insulin sensitivity index determined using EHC (euglycemic-hyperinsulinemic clamp) and normalized to steady-state insulinemia; T2D, type 2 diabetic. a Significantly different from alean/overweight; bobese; cprediabetic group; p < 0.05.
and secreted into the culture medium at levels comparable to those of adiponectin (15,16). Similar expression pattern for these two adipokines with AT expansion was observed (16). In vitro treatment with either macrophage-conditioned media or specifically with tumor necrosis factor-alpha down-regulated ZAG expression and secretion in primary human adipocytes (17), indicating a role of inflammation in the reduction of ZAG in the AT of obese individuals. Importantly, administration of ZAG to either lean (NMRI) or obese (ob/ob) mice induced a selective reduction in body fat with no effect on lean body mass and food intake (18). In addition, the direct anti-obesity effect of orally or intravenously administered ZAG was demonstrated in rodents (7,19-21). Furthermore, ZAG-deficient mice are susceptible to weight gain when fed a high fat diet, which is associated with decreased lipolysis, unresponsive to b3-adrenoreceptor agonists (22). Collectively, available evidence indicates that reduction of ZAG in AT of obese individuals compromises the lipid-mobilizing capacity and might participate in the development of metabolic disease (16). Thus, the primary aim of our study was to investigate the possible links between ZAG, whole-body, and AT insulin sensitivity, with emphasis to better understand processes participating in the regulation of adipocyte hypertrophy and capacity to store, oxidize, and release lipids. Clinical observations are complemented by ex vivo AT analyses and in vitro studies on primary human adipocytes.
Methods Study Cohorts This study was approved by the Local Ethics Committee and conforms to the ethical guidelines of the 2000 Helsinki declaration. All
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study participants provided witnessed written informed consent before entering the study.
Sedentary Lean and Obese Nondiabetic, Prediabetic, and Type 2 Diabetic Men (Cohort 1) Ninety-three middle-aged sedentary men were assigned to lean, obese, prediabetic, and type 2 diabetic (T2D) subgroups, according to their BMI, fasting, and/or 2 h glycemia. Newly diagnosed untreated patients with T2D were recruited. Patients with chronic disease or regular use of pharmacotherapy were not eligible to participate. All patients underwent complex metabolic phenotyping. The characteristics of study cohort 1 are summarized in Table 1.
Lean and Morbidly Obese Patients (Cohort 2) Subcutaneous and visceral AT samples were collected from thirtyfour lean and morbidly obese patients with normal or impaired glucose tolerance and T2D during the laparoscopic surgery due to inguinal hernia (lean) or gastric banding (extremely obese). The characteristics of study cohort 2 are summarized in Table 2.
Clinical Phenotyping Euglycemic-Hyperinsulinemic Clamp and Indirect Calorimetry. Insulin sensitivity was determined using the euglycemichyperinsulinemic clamp (EHC) as previously described (cohort 1) (23). In brief, human insulin (Novo Nordisk, Denmark) was infused in a primed-continuous fashion at the rate of 1 mU.kg21min21. Blood glucose was measured in 5-minute intervals and maintained
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Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
TABLE 2 Clinical characteristics of study cohort 2
Lean and extremely obese patients (Cohort 2)
Gender (M/F) Age (years) BMI (kg/m2) Triglycerides (mM) Total cholesterol (mM) HDL cholesterol (mM) FFA fasted state (mM) Fasting glycemia (mM) 2h glycemia in oGTT (mM)
Lean (n 5 8)
Obese (n 5 9)
Prediabetic (n 5 10)
T2D (n 5 7)
3/5 37.9 6 1.6 21.7 6 1.3 1.14 6 0.16 4.32 6 0.25 1.13 6 0.07 0.93 6 0.17 4.79 6 0.17 5.70 6 0.64
1/8 43.0 6 4.0 48.5 6 4.2a 1.63 6 0.21 5.07 6 0.47 1.18 6 0.07 1.03 6 0.09 4.76 6 0.17 6.46 6 0.51
4/6 44.1 6 2.6 49.8 6 3.5a 1.96 6 0.24a,a 5.08 6 0.35 1.04 6 0.08 1.20 6 0.10 5.51 6 0.26 7.92 6 0.85
3/4 47.4 6 4.4 44.6 6 4.7a 2.07 60.20a,a 5.29 6 0.57 1.07 6 0.05 0.80 6 0.09 10.32 6 1.41a,a,b 12.05 6 1.05a,a,b
FFA, free fatty acids; T2D, type 2 diabetic. oGTT was used to diagnose type 2 diabetes in four patients. Significantly different from alean/overweight; bobese; cprediabetic group; p < 0.05.
at euglycemia using variable infusion rate of 20% glucose. The whole-body insulin sensitivity index (M-value/insulin) was calculated from the steady-state glucose infusion rate required to maintain euglycemia and expressed per kilogram body weight per minute and normalized to the steady-state insulinemia (lU/ml). The EHC protocol included a 20-minute indirect calorimetry measurement in the fasted state (Ergostik, Geratherm-Respiratory, Germany).
Oral Glucose Tolerance Test. After an overnight fast, blood samples were drawn before and 30, 60, 90, and 120 min after ingestion of 75 g glucose and used to determine levels of plasma glucose, insulin and ZAG. Magnetic Resonance Imaging and 1H Spectroscopy.
Abdominal fat content and distribution and hepatic lipid content were assessed as previously described (cohort 1) (23).
Tissue Collection In cohort 1, subcutaneous AT samples were taken by needle biopsy from abdominal region in the fasted state as described previously (6). In cohort 2, subcutaneous (abdominal) and visceral (omental) AT samples were obtained during laparoscopic surgery. Tissue samples were immediately cleaned from blood and connective tissue and (i) frozen in liquid nitrogen; (ii) fixed in paraformaldehyde; and (iii) used to isolate adipocytes. Adipocyte diameter was determined by image analysis of collagenase-isolated adipocytes (ImageTool, UTHSCSA, USA). Average diameter of 100 cells from each adipocyte suspension was calculated (cohort 1).
Immunohistochemistry AT samples were fixed in 4% buffered paraformaldehyde and embedded into paraffin. Immunohistochemical staining was performed on 6 lm tissue sections. Endogenous peroxidase was inactivated by incubating with 0.3% H2O2 in methanol. Nonspecific binding was blocked with normal goat serum (Gibco, USA). Sections
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were incubated with anti-AZGP1 antibody (13399-1-AP, 1:100; Proteintech Group, USA) containing 4% NGS, 0.5% Triton-X 100, 0.1% sodium azide for 24h/4 C. Washing was followed by incubaR 555 conjugated secondary antibody diluted tion with Alexa FluorV 1:500 in PBS with 0.1% Triton-X 100 (Abcam, UK). ZAG protein immunoreactivity was inspected with an epifluorescence microscope (Carl Zeiss, Germany).
Cell Culture Experiment Primary human preadipocytes obtained from abdominal subcutaneous AT were cultured as previously described (24). Differentiation of confluent preadipocytes was induced by supplementing the adipocyte medium (DMEM-HamF12, 33 lM biotine, 17 lM D-panthotenate, 66 nM insulin, 1 nM T3, 10 lg/ml transferrin, 50 lg/ml gentamycin, and 100 U/ml penicillin/streptomycin) with rosiglitazone (1 lM), dexamethasone (1 lM), and isobutyl-methylxanthine (0.25 mM). Adipocyte medium supplemented with differentiation cocktail was changed every other day until day 7, when primary human adipocytes were transfected with human ZAG siRNA (30 nM) or nontargeting control (On-Target plus; Thermo Scientific, USA) using Lipofectamine (Invitrogen, USA). After 24 h, the antibiotic-free transfection mix was replaced by adipocyte medium and cells were kept in culture until day 12. Medium was replaced every other day.
Western Blot Tissue lysates were prepared from powdered subcutaneous AT homogenized in ice-cold RIPA buffer containing protease inhibitor cocktail (Complete, Roche, Switzerland), aprotinin, pepstatin A, leupeptin, and phosphatase inhibitor cocktails 2 and 3 (Sigma, USA). Proteins (40 lg) were separated on 10% SDS-PAGE and transferred to a PVDF membrane (Immobilon-FL, Millipore, USA). Membranes were incubated in ZAG primary antibody (1:1000; Santa Cruz, USA). Signal of the secondary antibody (IRDye-800CW; Li-Cor, USA) was detected with the Odyssey Infrared Imaging System (Li-
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Cor). ZAG protein levels were normalized to signal of GAPDH (1:1000; Millipore), which was stably expressed.
RNA Isolation and Real-Time PCR Total RNA from AT and primary human adipocytes was extracted R mini kit (Qiausing Trizol (Invitrogen) and purified by the RNeasyV gen, USA). DNase treatment was included (Qiagen, Germany). cDNA was produced using the high-capacity RNA-to-cDNA kit (Applied Biosystems, USA). Gene expression was measured by real-time PCR R Gene Expression Assays (Applied using either pre-designed TaqManV Biosystems) or Sybr-green master mix and specific pairs of primers designed (cross exon-exon junction) with PrimerExpress 3.0 (Table S1; Applied Biosystems). Gene expression was determined in duplicate on 7900HT-fast (Applied Biosystems). The 18S rRNA in the AT samples; GAPDH, and RPL13a in primary human adipocytes were stably expressed and used as internal reference genes (Table S1). PCR efficiency was optimized for every set of primers.
Mitochondrial Content DNA was isolated according to manufacturer’s instructions (Trizol, Invitrogen). Mitochondrial DNA content was assessed by real-time PCR using TaqMan gene expression assays targeting mitochondrial gene MT-ND1 (mitochondrial NADH-dehydrogenase subunit 1) and genomic 18s rRNA (Table S1).
Biochemical Assays Fasting serum was used for biochemical analyses. ZAG and adiponectin concentrations were determined with ELISA (Biovendor, Czech Republic). Glucose was measured using the glucose oxidase method (Beckman Coulter, USA), insulin with IRMA (Immunotech, France); GH, triglycerides, total and HDL-cholesterol with diagnostic assays from Roche (Switzerland), and free fatty acids with a kit from Randox (UK).
Statistical Analyses Differences between the two groups were analyzed using Student’s t-test and differences between more than two experimental groups with repeated measure ANOVA and Tukey post hoc test. Nonparametric tests were used where appropriate. Associations between ZAG mRNA, adiposity, insulin sensitivity, glycemia, and AT morphologic, metabolic, and endocrine properties were assessed using the Spearman’s correlation test. Multiple regression analysis was used to determine the best predictors of the whole-body and AT insulin sensitivity independent of age and BMI (JMP, SAS Institute, USA). The data are reported as mean 6 SEM, with P < 0.05 indicating statistical significance.
Results First, we studied the possible role of ZAG in modulating the progression of obesity-related metabolic disease by quantifying serum levels and subcutaneous AT ZAG mRNA and protein. We clearly showed that subcutaneous AT of lean healthy men expressed about 65-82% more ZAG mRNA and 47-62% more protein than their age-matched counterparts from any of the obese groups (Figure 1A). The presence of neither prediabetes nor newly diagnosed T2D was associated with any additional significant changes in subcutaneous AT ZAG mRNA or protein (Figure 1A) in this middle-aged sedentary population with
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ZAG in Obesity and Metabolic Disease Balaz et al.
the first degree of obesity (Table 1). However, a tendency toward the gradual decrease paralleling the decline in insulin sensitivity was evident (Figure 1A, Table 1). Immunohistochemical staining of ZAG in AT sections confirmed the aforementioned results (Figure 1B). ZAG immunoreactivity was in significant quantities found only in adipocytes of lean healthy individuals. In obese individuals, its presence was limited to few scattered areas and ZAG never surrounded a larger area of lipid droplet (Figure 1B). Obesity-associated down-regulation of AT ZAG was not paralleled by changes in serum levels (Figure 1A), indicating that subcutaneous AT is unlikely to significantly contribute to circulating levels of this adipokine. Next, we determined the differences in obesity- and diabetes-related changes of ZAG mRNA and protein in subcutaneous and visceral AT from lean and extremely obese individuals (Table 2). In lean individuals, expression of ZAG gene was 3.3-fold higher in subcutaneous than in visceral AT (Figure 1C). This was paralleled by higher protein content found in western blot as well as on the subcutaneous AT sections (Figure 1D, 1E). It is important to note that morbidly obese individuals with BMI > 45 kg/m2 had strongly reduced ZAG mRNA in subcutaneous AT (Figure 1C). ZAG protein content also seemed to be reduced (Figure 1D). However, no obesity-related alterations of ZAG mRNA or protein were observed in visceral AT (Figure 1C, 1D). Prediabetes or T2D was not associated with changes in ZAG mRNA in neither subcutaneous nor visceral AT of morbidly obese patients. Similar to cohort 1, obesityassociated regulation of AT ZAG was not paralleled by alteration in circulating levels (Figure 1F). As expected, subcutaneous AT ZAG mRNA was strongly negatively associated with parameters of obesity (BMI), overall and abdominal adiposity (percentage of body fat, waist circumference), abdominal subcutaneous and in lesser extent also with abdominal visceral AT cross-sectional area, circulating lipids, and hepatic lipid accumulation (Table 3). Negative associations of ZAG mRNA with all these noxious prognostic markers of metabolic disease was counterbalanced by positive associations with beneficial metabolic parameters related to the whole-body (M-value/insulin; Figure 2A) and AT insulin sensitivity (degree of insulin-induced suppression of circulating free fatty acids during EHC; Figure 2B), resting energy expenditure, glucose tolerance, as well as with levels of circulating adiponectin and growth hormone (Table 3) and expression of GLUT4, PGC1a, and mitochondrial content in subcutaneous AT (Figures 2D2F). More importantly, associations with LDL cholesterol, wholebody and AT insulin sensitivity, 2 h glycemia, AT mitochondrial content, and expression of GLUT4 were at least in part independent on BMI (Table 3). It is important to point out that ZAG protein content in subcutaneous AT was also positively associated with AT insulin sensitivity (Figure 2C) and expression of GLUT4 mRNA (R 5 0.49, P < 0.05). Moreover, multiple regression analysis revealed that ZAG mRNA and adipocyte size were the strongest age- and BMI-independent predictors of whole-body insulin sensitivity explaining almost 20% of overall variability (P 5 0.0005). AT ZAG expression was also the strongest predictor of the AT-specific, age-, and BMI-independent in vivo insulin sensitivity, explaining 8% of the variability (P 5 0.017). Our clinical and ex vivo AT analyses indicated that ZAG could be an obesity-independent predictor of whole-body and AT insulin sensitivity acting by modulating lipid metabolism and/or insulin sensitivity in AT. To support this notion, we silenced ZAG in primary
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Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Figure 1 Regulation of serum, subcutaneous and visceral adipose tissue zinc-a2-glycoprotein (ZAG) in obesity, prediabetes, and type 2 diabetes. (A) Serum and subcutaneous adipose tissue levels of ZAG mRNA and protein in sedentary lean (n 5 21), obese (n 5 21), prediabetic (n 5 18), and type 2 diabetic (n 5 15) men (cohort 1). Representative western blot shows adipose tissue ZAG protein content in age-matched lean (L), obese (O), prediabetic (P), and type 2 diabetic (D) patients (n 5 6 per group; cohort 1). (B) Representative images of the fluorescent immunostaining of ZAG in subcutaneous adipose tissue from a lean, obese, prediabetic, and type 2 diabetic individual. (C) Expression of ZAG gene in subcutaneous (SAT) and visceral (VAT) adipose tissue from lean (n 5 7) and extremely obese (n 5 9), prediabetic (n 5 10), and type 2 diabetic individuals (n 5 6) (cohort 2). (D) The representative western blot shows adipose tissue ZAG protein content in subcutaneous (S) and visceral (V) adipose tissue of three lean and three obese individuals (cohort 2). (E) Fluorescent immunostaining of ZAG in subcutaneous (SAT) and visceral R 555-conjugated secondary antibody confirming specificity of (VAT) adipose tissue from lean individual. Negative control staining with Alexa FluorV ZAG immunostaining was included. Marker line equals 100 lm. (F) Serum ZAG protein in lean and extremely obese nondiabetic (obese), prediabetic, and type 2 diabetic individuals (n 5 6 per group; cohort 2). ***P < 0.001, *P < 0.05 compared to lean; #P < 0.05 compared to SAT.
human adipocytes. First, we observed that silencing reduced both ZAG mRNA and protein (P < 0.001) by more than 80%, without affecting the adipocyte differentiation capacity. More importantly, ZAG silencing was accompanied by 35% reduction of adiponectin mRNA, 26% reduction in expression of PGC1a (P < 0.05) and a marked 41% and 54% decrease in IRS1 (P 5 0.05) and GLUT 4 mRNA (P < 0.05), respectively (Figure 3).
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Discussion The major finding of our study is that the whole-body as well as AT-specific insulin sensitivity and AT mitochondrial content were positively associated with ZAG expression in subcutaneous AT, and that silencing ZAG gene partly reversed this positive connections in primary human adipocytes. In addition, we found that obesity-
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ZAG in Obesity and Metabolic Disease Balaz et al.
TABLE 3 Associations of ZAG gene expression with parameters of adiposity, markers of metabolic disease, whole-body insulin sensitivity, and adipose tissue morphology, molecular and metabolic phenotype
Sedentary men (Cohort 1) ZAG mRNA SAT adjusted to BMIb
ZAG mRNA SAT
BMI Waist circumference Body fat by bioelectric impedance Fat cell size Subcutaneous adiposity by MRI Visceral adiposity by MRI Hepatic lipid content by 1H-MRS Fasting triglycerides LDL-cholesterol HDL-cholesterol Fasting free fatty acids Free fatty acids insulin-suppressed during EHC Fasting glycemia 2 h glycemia in oGTT C-peptide M-value corrected to steady-state insulinemia Resting energy expenditure corrected LBM Growth hormone Fasting adiponectinemia GLUT4 mRNAa PGC1a mRNAa Mitochondrial content MT-ND1/18s rRNA
R
P
R
P
20.81 20.74 20.79 20.66 20.71 20.41 20.42 20.40 20.37 0.39 20.27 20.40 20.36 20.36 20.48 0.73 0.47 0.27 0.61 0.67 0.44 0.46
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Subcutaneous Adipose Tissue Zinc-a2-Glycoprotein is Associated with Adipose Tissue and Whole-Body Insulin Sensitivity Miroslav Balaz1, Marek Vician2, Zuzana Janakova1, Timea Kurdiova1, Martina Surova1, Richard Imrich1, Zuzana Majercikova1, Adela Penesova1, Miroslav Vlcek1, Alexander Kiss1, Vitazoslav Belan3, Iwar Klimes1, Juraj Olejnik2, Daniela Gasperikova1, Christian Wolfrum4, Barbara Ukropcova1 and Jozef Ukropec1
Objective: To examine the regulatory aspects of zinc-a2-glycoprotein (ZAG) association with obesityrelated insulin resistance. Methods: ZAG mRNA and protein were analyzed in subcutaneous adipose tissue (AT) and circulation of lean, obese, prediabetic, and type 2 diabetic men; both subcutaneous and visceral AT were explored in lean and extremely obese. Clinical and ex vivo findings were corroborated by results of in vitro ZAG silencing experiment. Results: Subcutaneous AT ZAG was reduced in obesity, with a trend to further decrease with prediabetes and type 2 diabetes. ZAG was 3.3-fold higher in subcutaneous than in visceral AT of lean individuals. All differences were lost in extreme obesity. Obesity-associated changes in AT were not paralleled by alterations of circulating ZAG. Subcutaneous AT ZAG correlated with adiposity, adipocyte hypertrophy, whole-body and AT insulin sensitivity, mitochondrial content, expression of GLUT4, PGC1a, and adiponectin. Subcutaneous AT ZAG and adipocyte size were the only predictors of insulin sensitivity, independent on age and BMI. Silencing ZAG resulted in reduced adiponectin, IRS1, GLUT4, and PGC1a gene expression in primary human adipocytes. Conclusions: ZAG in subcutaneous, but not in visceral AT, was markedly reduced in obesity. Clinical, cellular, and molecular evidence indicate that ZAG plays an important role in modulating whole-body and AT insulin sensitivity. Obesity (2014) 00, 00-00. doi:10.1002/oby.20764
Introduction White adipose tissue (AT), our largest energy depot, is a highly dynamic secretory organ, producing a whole spectrum of biologically active substances. Adipokines, proteins synthesized and secreted by adipocytes, modulate many physiological processes in different tissues and organs (1-3). Changes in AT secretory profile such as found in obesity may severely impact the whole-body metabolism, leading to the development of metabolic disease (4-6).
Previous studies have shown that AT-derived zinc-a2-glycoprotein (ZAG) may play a pivotal role in the regulation of adiposity (7-9). Initially, ZAG was identified as a product of secretory epithelial cells and several forms of malignant tumors and has been proposed to serve as a potential cancer marker (10-13). Later, ZAG was shown to be identical to a lipid-mobilizing factor responsible for a loss of fat mass in patients with cancer cachexia (14), and AT ZAG was shown to be elevated proportionally to the degree of weight loss (8). ZAG has been found to be produced by differentiated human adipocytes
1
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia. Correspondence: Jozef Ukropec ([email protected]) Department of Surgery, Slovak Medical University, Bratislava, Slovakia 3 Radiology Clinic, University Hospital, Bratislava, Slovakia 4 Institute of Food, Nutrition & Health, Swiss Federal Institute of Technology, ETH Zurich, Schwerzenbach, Switzerland
2
Funding agencies: This work was supported by EFSD New Horizons, 7FP-EC-LipidomicNET #202272, VEGA 2/0198/11 and 2/0132/10, National Scholarship Programme of the Slovak Republic. Disclosure: The authors declare no conflict of interest. Author Contributions: MB: data generation, analysis, and interpretation, drafting the manuscript, figures generation; MV, JO: muscle sample collection, clinical data collection & interpretation; ZJ, TK, MS: sample collection, clinical data generation, critical reading of the manuscript; RI, AP, MV: sample collection, critical reading of the manuscript; ZM, AK: data generation, analysis, interpretation; VB: clinical data generation, analysis; DG, IK: critical reading of the manuscript; CW: in vitro study design, critical reading of the manuscript; BU, JU: study conception and design, data interpretation, critical reading of the manuscript. All authors have read and approved the final version of the manuscript. Additional Supporting Information may be found in the online version of this article. Received: 17 February 2014; Accepted: 4 April 2014; Published online 00 Month 2014. doi:10.1002/oby.20764
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Obesity | VOLUME 00 | NUMBER 00 | MONTH 2014
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Obesity
ZAG in Obesity and Metabolic Disease Balaz et al.
TABLE 1 Clinical characteristics of study cohort 1
Sedentary healthy lean to obese diabetic men (Cohort 1) Lean/overweight (n 5 27)
Obese (n 5 26)
Prediabetic (n 5 25)
T2D (n 5 15)
27/0 36.8 6 1.8 23.3 6 0.4 18.4 6 0.9 88.0 6 1.7 94.5 6 3.0 1.07 6 0.99 4.18 6 0.16 1.36 6 0.05 2.34 6 0.12 0.57 6 0.04 0.11 6 0.01 0.15 6 0.01 4.7 6 0.08 5.4 6 0.25
26/0 37.6 6 1.5 30.2 6 0.5a 28.3 6 0.9a 105.0 6 1.4a 116.7 6 2.2a 1.39 6 0.14 4.58 6 0.15 1.24 6 0.05 2.68 6 0.12 0.62 6 0.04 0.15 6 0.01 0.08 6 0.01a 4.9 6 0.15 5.4 6 0.28
25/0 44.2 6 1.9a,b 31.6 6 0.6a 30.9 6 0.7a 111.3 6 1.9a 120.7 6 2.4a 1.95 6 0.19 5.03 6 0.24a 1.26 6 0.05 2.90 6 0.19a 0.71 6 0.04 0.16 6 0.02a 0.07 6 0.01a 5.6 6 0.13 8.0 6 0.33a,b
15/0 50.5 6 2.1a,b 31.5 6 1.0a 30.9 6 1.1a 109.8 6 2.8a 119.8 6 4.2a 2.27 6 0.33a,b 5.21 6 0.21a 1.25 6 0.05 2.94 6 0.16 0.64 6 0.08 0.18 6 0.03a 0.05 6 0.01a 9.0 6 1.7a,b,c -
Gender (M/F) Age (years) BMI (kg/m2) Body fat (%) Waist circumference (cm) Adipocyte diameter (lm) Triglycerides (mM) Total cholesterol (mM) HDL cholesterol (mM) LDL cholesterol (mM) FFA fasted state (mM) FFA during EHC (mM) M-value/insulin (mg/kgBW/min/lU/ml) Fasting glycemia (mM) 2 h glycemia in oGTT (mM)
FFA, free fatty acids; M-value/insulin, insulin sensitivity index determined using EHC (euglycemic-hyperinsulinemic clamp) and normalized to steady-state insulinemia; T2D, type 2 diabetic. a Significantly different from alean/overweight; bobese; cprediabetic group; p < 0.05.
and secreted into the culture medium at levels comparable to those of adiponectin (15,16). Similar expression pattern for these two adipokines with AT expansion was observed (16). In vitro treatment with either macrophage-conditioned media or specifically with tumor necrosis factor-alpha down-regulated ZAG expression and secretion in primary human adipocytes (17), indicating a role of inflammation in the reduction of ZAG in the AT of obese individuals. Importantly, administration of ZAG to either lean (NMRI) or obese (ob/ob) mice induced a selective reduction in body fat with no effect on lean body mass and food intake (18). In addition, the direct anti-obesity effect of orally or intravenously administered ZAG was demonstrated in rodents (7,19-21). Furthermore, ZAG-deficient mice are susceptible to weight gain when fed a high fat diet, which is associated with decreased lipolysis, unresponsive to b3-adrenoreceptor agonists (22). Collectively, available evidence indicates that reduction of ZAG in AT of obese individuals compromises the lipid-mobilizing capacity and might participate in the development of metabolic disease (16). Thus, the primary aim of our study was to investigate the possible links between ZAG, whole-body, and AT insulin sensitivity, with emphasis to better understand processes participating in the regulation of adipocyte hypertrophy and capacity to store, oxidize, and release lipids. Clinical observations are complemented by ex vivo AT analyses and in vitro studies on primary human adipocytes.
Methods Study Cohorts This study was approved by the Local Ethics Committee and conforms to the ethical guidelines of the 2000 Helsinki declaration. All
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study participants provided witnessed written informed consent before entering the study.
Sedentary Lean and Obese Nondiabetic, Prediabetic, and Type 2 Diabetic Men (Cohort 1) Ninety-three middle-aged sedentary men were assigned to lean, obese, prediabetic, and type 2 diabetic (T2D) subgroups, according to their BMI, fasting, and/or 2 h glycemia. Newly diagnosed untreated patients with T2D were recruited. Patients with chronic disease or regular use of pharmacotherapy were not eligible to participate. All patients underwent complex metabolic phenotyping. The characteristics of study cohort 1 are summarized in Table 1.
Lean and Morbidly Obese Patients (Cohort 2) Subcutaneous and visceral AT samples were collected from thirtyfour lean and morbidly obese patients with normal or impaired glucose tolerance and T2D during the laparoscopic surgery due to inguinal hernia (lean) or gastric banding (extremely obese). The characteristics of study cohort 2 are summarized in Table 2.
Clinical Phenotyping Euglycemic-Hyperinsulinemic Clamp and Indirect Calorimetry. Insulin sensitivity was determined using the euglycemichyperinsulinemic clamp (EHC) as previously described (cohort 1) (23). In brief, human insulin (Novo Nordisk, Denmark) was infused in a primed-continuous fashion at the rate of 1 mU.kg21min21. Blood glucose was measured in 5-minute intervals and maintained
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TABLE 2 Clinical characteristics of study cohort 2
Lean and extremely obese patients (Cohort 2)
Gender (M/F) Age (years) BMI (kg/m2) Triglycerides (mM) Total cholesterol (mM) HDL cholesterol (mM) FFA fasted state (mM) Fasting glycemia (mM) 2h glycemia in oGTT (mM)
Lean (n 5 8)
Obese (n 5 9)
Prediabetic (n 5 10)
T2D (n 5 7)
3/5 37.9 6 1.6 21.7 6 1.3 1.14 6 0.16 4.32 6 0.25 1.13 6 0.07 0.93 6 0.17 4.79 6 0.17 5.70 6 0.64
1/8 43.0 6 4.0 48.5 6 4.2a 1.63 6 0.21 5.07 6 0.47 1.18 6 0.07 1.03 6 0.09 4.76 6 0.17 6.46 6 0.51
4/6 44.1 6 2.6 49.8 6 3.5a 1.96 6 0.24a,a 5.08 6 0.35 1.04 6 0.08 1.20 6 0.10 5.51 6 0.26 7.92 6 0.85
3/4 47.4 6 4.4 44.6 6 4.7a 2.07 60.20a,a 5.29 6 0.57 1.07 6 0.05 0.80 6 0.09 10.32 6 1.41a,a,b 12.05 6 1.05a,a,b
FFA, free fatty acids; T2D, type 2 diabetic. oGTT was used to diagnose type 2 diabetes in four patients. Significantly different from alean/overweight; bobese; cprediabetic group; p < 0.05.
at euglycemia using variable infusion rate of 20% glucose. The whole-body insulin sensitivity index (M-value/insulin) was calculated from the steady-state glucose infusion rate required to maintain euglycemia and expressed per kilogram body weight per minute and normalized to the steady-state insulinemia (lU/ml). The EHC protocol included a 20-minute indirect calorimetry measurement in the fasted state (Ergostik, Geratherm-Respiratory, Germany).
Oral Glucose Tolerance Test. After an overnight fast, blood samples were drawn before and 30, 60, 90, and 120 min after ingestion of 75 g glucose and used to determine levels of plasma glucose, insulin and ZAG. Magnetic Resonance Imaging and 1H Spectroscopy.
Abdominal fat content and distribution and hepatic lipid content were assessed as previously described (cohort 1) (23).
Tissue Collection In cohort 1, subcutaneous AT samples were taken by needle biopsy from abdominal region in the fasted state as described previously (6). In cohort 2, subcutaneous (abdominal) and visceral (omental) AT samples were obtained during laparoscopic surgery. Tissue samples were immediately cleaned from blood and connective tissue and (i) frozen in liquid nitrogen; (ii) fixed in paraformaldehyde; and (iii) used to isolate adipocytes. Adipocyte diameter was determined by image analysis of collagenase-isolated adipocytes (ImageTool, UTHSCSA, USA). Average diameter of 100 cells from each adipocyte suspension was calculated (cohort 1).
Immunohistochemistry AT samples were fixed in 4% buffered paraformaldehyde and embedded into paraffin. Immunohistochemical staining was performed on 6 lm tissue sections. Endogenous peroxidase was inactivated by incubating with 0.3% H2O2 in methanol. Nonspecific binding was blocked with normal goat serum (Gibco, USA). Sections
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were incubated with anti-AZGP1 antibody (13399-1-AP, 1:100; Proteintech Group, USA) containing 4% NGS, 0.5% Triton-X 100, 0.1% sodium azide for 24h/4 C. Washing was followed by incubaR 555 conjugated secondary antibody diluted tion with Alexa FluorV 1:500 in PBS with 0.1% Triton-X 100 (Abcam, UK). ZAG protein immunoreactivity was inspected with an epifluorescence microscope (Carl Zeiss, Germany).
Cell Culture Experiment Primary human preadipocytes obtained from abdominal subcutaneous AT were cultured as previously described (24). Differentiation of confluent preadipocytes was induced by supplementing the adipocyte medium (DMEM-HamF12, 33 lM biotine, 17 lM D-panthotenate, 66 nM insulin, 1 nM T3, 10 lg/ml transferrin, 50 lg/ml gentamycin, and 100 U/ml penicillin/streptomycin) with rosiglitazone (1 lM), dexamethasone (1 lM), and isobutyl-methylxanthine (0.25 mM). Adipocyte medium supplemented with differentiation cocktail was changed every other day until day 7, when primary human adipocytes were transfected with human ZAG siRNA (30 nM) or nontargeting control (On-Target plus; Thermo Scientific, USA) using Lipofectamine (Invitrogen, USA). After 24 h, the antibiotic-free transfection mix was replaced by adipocyte medium and cells were kept in culture until day 12. Medium was replaced every other day.
Western Blot Tissue lysates were prepared from powdered subcutaneous AT homogenized in ice-cold RIPA buffer containing protease inhibitor cocktail (Complete, Roche, Switzerland), aprotinin, pepstatin A, leupeptin, and phosphatase inhibitor cocktails 2 and 3 (Sigma, USA). Proteins (40 lg) were separated on 10% SDS-PAGE and transferred to a PVDF membrane (Immobilon-FL, Millipore, USA). Membranes were incubated in ZAG primary antibody (1:1000; Santa Cruz, USA). Signal of the secondary antibody (IRDye-800CW; Li-Cor, USA) was detected with the Odyssey Infrared Imaging System (Li-
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Cor). ZAG protein levels were normalized to signal of GAPDH (1:1000; Millipore), which was stably expressed.
RNA Isolation and Real-Time PCR Total RNA from AT and primary human adipocytes was extracted R mini kit (Qiausing Trizol (Invitrogen) and purified by the RNeasyV gen, USA). DNase treatment was included (Qiagen, Germany). cDNA was produced using the high-capacity RNA-to-cDNA kit (Applied Biosystems, USA). Gene expression was measured by real-time PCR R Gene Expression Assays (Applied using either pre-designed TaqManV Biosystems) or Sybr-green master mix and specific pairs of primers designed (cross exon-exon junction) with PrimerExpress 3.0 (Table S1; Applied Biosystems). Gene expression was determined in duplicate on 7900HT-fast (Applied Biosystems). The 18S rRNA in the AT samples; GAPDH, and RPL13a in primary human adipocytes were stably expressed and used as internal reference genes (Table S1). PCR efficiency was optimized for every set of primers.
Mitochondrial Content DNA was isolated according to manufacturer’s instructions (Trizol, Invitrogen). Mitochondrial DNA content was assessed by real-time PCR using TaqMan gene expression assays targeting mitochondrial gene MT-ND1 (mitochondrial NADH-dehydrogenase subunit 1) and genomic 18s rRNA (Table S1).
Biochemical Assays Fasting serum was used for biochemical analyses. ZAG and adiponectin concentrations were determined with ELISA (Biovendor, Czech Republic). Glucose was measured using the glucose oxidase method (Beckman Coulter, USA), insulin with IRMA (Immunotech, France); GH, triglycerides, total and HDL-cholesterol with diagnostic assays from Roche (Switzerland), and free fatty acids with a kit from Randox (UK).
Statistical Analyses Differences between the two groups were analyzed using Student’s t-test and differences between more than two experimental groups with repeated measure ANOVA and Tukey post hoc test. Nonparametric tests were used where appropriate. Associations between ZAG mRNA, adiposity, insulin sensitivity, glycemia, and AT morphologic, metabolic, and endocrine properties were assessed using the Spearman’s correlation test. Multiple regression analysis was used to determine the best predictors of the whole-body and AT insulin sensitivity independent of age and BMI (JMP, SAS Institute, USA). The data are reported as mean 6 SEM, with P < 0.05 indicating statistical significance.
Results First, we studied the possible role of ZAG in modulating the progression of obesity-related metabolic disease by quantifying serum levels and subcutaneous AT ZAG mRNA and protein. We clearly showed that subcutaneous AT of lean healthy men expressed about 65-82% more ZAG mRNA and 47-62% more protein than their age-matched counterparts from any of the obese groups (Figure 1A). The presence of neither prediabetes nor newly diagnosed T2D was associated with any additional significant changes in subcutaneous AT ZAG mRNA or protein (Figure 1A) in this middle-aged sedentary population with
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the first degree of obesity (Table 1). However, a tendency toward the gradual decrease paralleling the decline in insulin sensitivity was evident (Figure 1A, Table 1). Immunohistochemical staining of ZAG in AT sections confirmed the aforementioned results (Figure 1B). ZAG immunoreactivity was in significant quantities found only in adipocytes of lean healthy individuals. In obese individuals, its presence was limited to few scattered areas and ZAG never surrounded a larger area of lipid droplet (Figure 1B). Obesity-associated down-regulation of AT ZAG was not paralleled by changes in serum levels (Figure 1A), indicating that subcutaneous AT is unlikely to significantly contribute to circulating levels of this adipokine. Next, we determined the differences in obesity- and diabetes-related changes of ZAG mRNA and protein in subcutaneous and visceral AT from lean and extremely obese individuals (Table 2). In lean individuals, expression of ZAG gene was 3.3-fold higher in subcutaneous than in visceral AT (Figure 1C). This was paralleled by higher protein content found in western blot as well as on the subcutaneous AT sections (Figure 1D, 1E). It is important to note that morbidly obese individuals with BMI > 45 kg/m2 had strongly reduced ZAG mRNA in subcutaneous AT (Figure 1C). ZAG protein content also seemed to be reduced (Figure 1D). However, no obesity-related alterations of ZAG mRNA or protein were observed in visceral AT (Figure 1C, 1D). Prediabetes or T2D was not associated with changes in ZAG mRNA in neither subcutaneous nor visceral AT of morbidly obese patients. Similar to cohort 1, obesityassociated regulation of AT ZAG was not paralleled by alteration in circulating levels (Figure 1F). As expected, subcutaneous AT ZAG mRNA was strongly negatively associated with parameters of obesity (BMI), overall and abdominal adiposity (percentage of body fat, waist circumference), abdominal subcutaneous and in lesser extent also with abdominal visceral AT cross-sectional area, circulating lipids, and hepatic lipid accumulation (Table 3). Negative associations of ZAG mRNA with all these noxious prognostic markers of metabolic disease was counterbalanced by positive associations with beneficial metabolic parameters related to the whole-body (M-value/insulin; Figure 2A) and AT insulin sensitivity (degree of insulin-induced suppression of circulating free fatty acids during EHC; Figure 2B), resting energy expenditure, glucose tolerance, as well as with levels of circulating adiponectin and growth hormone (Table 3) and expression of GLUT4, PGC1a, and mitochondrial content in subcutaneous AT (Figures 2D2F). More importantly, associations with LDL cholesterol, wholebody and AT insulin sensitivity, 2 h glycemia, AT mitochondrial content, and expression of GLUT4 were at least in part independent on BMI (Table 3). It is important to point out that ZAG protein content in subcutaneous AT was also positively associated with AT insulin sensitivity (Figure 2C) and expression of GLUT4 mRNA (R 5 0.49, P < 0.05). Moreover, multiple regression analysis revealed that ZAG mRNA and adipocyte size were the strongest age- and BMI-independent predictors of whole-body insulin sensitivity explaining almost 20% of overall variability (P 5 0.0005). AT ZAG expression was also the strongest predictor of the AT-specific, age-, and BMI-independent in vivo insulin sensitivity, explaining 8% of the variability (P 5 0.017). Our clinical and ex vivo AT analyses indicated that ZAG could be an obesity-independent predictor of whole-body and AT insulin sensitivity acting by modulating lipid metabolism and/or insulin sensitivity in AT. To support this notion, we silenced ZAG in primary
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Figure 1 Regulation of serum, subcutaneous and visceral adipose tissue zinc-a2-glycoprotein (ZAG) in obesity, prediabetes, and type 2 diabetes. (A) Serum and subcutaneous adipose tissue levels of ZAG mRNA and protein in sedentary lean (n 5 21), obese (n 5 21), prediabetic (n 5 18), and type 2 diabetic (n 5 15) men (cohort 1). Representative western blot shows adipose tissue ZAG protein content in age-matched lean (L), obese (O), prediabetic (P), and type 2 diabetic (D) patients (n 5 6 per group; cohort 1). (B) Representative images of the fluorescent immunostaining of ZAG in subcutaneous adipose tissue from a lean, obese, prediabetic, and type 2 diabetic individual. (C) Expression of ZAG gene in subcutaneous (SAT) and visceral (VAT) adipose tissue from lean (n 5 7) and extremely obese (n 5 9), prediabetic (n 5 10), and type 2 diabetic individuals (n 5 6) (cohort 2). (D) The representative western blot shows adipose tissue ZAG protein content in subcutaneous (S) and visceral (V) adipose tissue of three lean and three obese individuals (cohort 2). (E) Fluorescent immunostaining of ZAG in subcutaneous (SAT) and visceral R 555-conjugated secondary antibody confirming specificity of (VAT) adipose tissue from lean individual. Negative control staining with Alexa FluorV ZAG immunostaining was included. Marker line equals 100 lm. (F) Serum ZAG protein in lean and extremely obese nondiabetic (obese), prediabetic, and type 2 diabetic individuals (n 5 6 per group; cohort 2). ***P < 0.001, *P < 0.05 compared to lean; #P < 0.05 compared to SAT.
human adipocytes. First, we observed that silencing reduced both ZAG mRNA and protein (P < 0.001) by more than 80%, without affecting the adipocyte differentiation capacity. More importantly, ZAG silencing was accompanied by 35% reduction of adiponectin mRNA, 26% reduction in expression of PGC1a (P < 0.05) and a marked 41% and 54% decrease in IRS1 (P 5 0.05) and GLUT 4 mRNA (P < 0.05), respectively (Figure 3).
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Discussion The major finding of our study is that the whole-body as well as AT-specific insulin sensitivity and AT mitochondrial content were positively associated with ZAG expression in subcutaneous AT, and that silencing ZAG gene partly reversed this positive connections in primary human adipocytes. In addition, we found that obesity-
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TABLE 3 Associations of ZAG gene expression with parameters of adiposity, markers of metabolic disease, whole-body insulin sensitivity, and adipose tissue morphology, molecular and metabolic phenotype
Sedentary men (Cohort 1) ZAG mRNA SAT adjusted to BMIb
ZAG mRNA SAT
BMI Waist circumference Body fat by bioelectric impedance Fat cell size Subcutaneous adiposity by MRI Visceral adiposity by MRI Hepatic lipid content by 1H-MRS Fasting triglycerides LDL-cholesterol HDL-cholesterol Fasting free fatty acids Free fatty acids insulin-suppressed during EHC Fasting glycemia 2 h glycemia in oGTT C-peptide M-value corrected to steady-state insulinemia Resting energy expenditure corrected LBM Growth hormone Fasting adiponectinemia GLUT4 mRNAa PGC1a mRNAa Mitochondrial content MT-ND1/18s rRNA
R
P
R
P
20.81 20.74 20.79 20.66 20.71 20.41 20.42 20.40 20.37 0.39 20.27 20.40 20.36 20.36 20.48 0.73 0.47 0.27 0.61 0.67 0.44 0.46
