Zinc-α2-glycoprotein (ZAG) was found to influence lipolysis in adipose tissue and has recently been proposed as a candidate factor in the regulation of body weight.
To elucidate the association of serum ZAG level with body weight and percentage of body fat in normal, obese subjects and high-fat diet (HFD)-induced obese mice.
The relationship between serum ZAG and obesity-related parameters was studied in 44 human subjects and 36 mice fed standard food and HFD. Furthermore, the effects of ZAG overexpression on adipose tissue of mice was also evaluated by using a liposome transfection method.
Serum ZAG level was significantly lower in obese patients and obese mice in comparison to that in people and mice with normal weight. The further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r=−0.62, P<0.001), body mass index (r=−0.64, P<0.001), waist circumference(r=−0.68, P<0.001), hip circumference (r=−0.60, P<0.001), percentage of body fat (r=−0.52, P=0.03) and fat mass(r=−0.59, P=0.01) in human subjects after adjustment for age and sex. Furthermore, ZAG overexpression in mice reduced body weight and the percentage of epididymal fat. The decreased FAS, ACC1 and DGAT mRNA and the increased HSL mRNA were also observed in epididymal adipose tissue in ZAG overexpression mice.
ZAG is closely linked to obesity. Serum ZAG level is inversely associated with body weight and percentage of body fat. The action of ZAG is associated with downregulated lipogenic enzymes and upregulated lipolytic enzyme expressions in adipose tissue of mice.
Obesity has become a global health problem and it is closely linked to many kinds of diseases such as diabetes, cardiovascular disease and cancer. However, the pathogenesis of obesity still remains unclear. Evidences accumulated over the past decades demonstrate that adipose tissue is not only an energy storage organ, but also plays an important role in monitoring and controlling whole body metabolism by secreting a variety of bioactive molecules, known as adipokines or adipocytokines, in an autocrine, paracrine and/or endocrine manner.1
Zinc-α2–glycoprotein (ZAG) is a 43 kDa glycoprotein, first isolated from human plasma.2 The concentration of ZAG in normal human plasma or serum has been variously reported as between 3.65 and 140 μg ml−1 in different populations using various analytical techniques.3, 4, 5 ZAG was initially reported to be correlated with body weight loss of cancer patients with cachexia, a severe life-threatening wasting syndrome defined by massive depletion of both adipose and skeletal muscle tissues in 1998.6 The concentration of serum ZAG in cancer patients with cachexia was 20-fold higher than that of age and sex-matched normal people.7 In vivo, administration of ZAG to mice caused a highly significant reduction in body weight. Further body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat.8 Loss of adipose tissue may be due to the lipolytic effect of ZAG because incubation of ZAG with adipocytes isolated from murine adipose tissue has been shown to stimulate greatly glycerol release in a dose-dependent manner.6 Interestingly, recent studies performed by Bing et al.9 and Bao et al.10 identified that ZAG is not only expressed in adult mouse white adipose tissue and brown adipose tissue (BAT), but also can be secreted by human SGBS and mouse 3T3-L1 adipocytes. All of these findings suggest that ZAG maybe a new adipokine produced by adipocytes, and it is associated with body fat loss that may be involved in stimulatory action of ZAG in lipolysis of adipose tissue. However, it still remains unclear whether ZAG has similar stimulatory action in lipolysis of adipose tissue in obesity and whether it has any influence in lipogenesis of adipose tissue.
A number of genes are involved in adipocytes lipid accumulation, which include fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and acyl-coenzyme A: diacylglycerol transferase (DGAT). FAS provides nonesterified fatty acid substrate for triglyceride synthesis. ACC catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA, and DGAT catalyzes the addition of the third fatty acyle-CoA moiety to diacylglycerol. It has been reported that increased FAS gene expression in adipose tissue is linked to visceral fat accumulation, impaired insulin sensitivity and increased circulatory fasting insulin.11 FAS activities were significantly higher in obese patients and genetically obese rats than in normal-weight subjects.12, 13 FAS gene promoter activities were 4- to 6-fold higher in adipocytes of obese rats than in those of nonobese rats.14 Overexpressing of DGAT1 in adipose tissue of mice yielded obesity.15 However, hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis. Its physiological role is to hydrolyze the triglyceride stored in adipose tissue into fatty acids and glycerol. It has been shown that overexpression of HSL prevents tryglyceride accumulation in adipocytes.16 Adipose triglyceride lipase and HSL protein expression is decreased in the obese insulin-resistant state,17 and the HSL C-60G promoter polymorphism is associated with increased waist circumference in normal-weight subjects.18
The relationship between ZAG level and weight loss in cachexia and the powerful lipid mobilizing effects of ZAG together with the secretory function of adipocytes have led us to ask whether there is a relationship between ZAG and weight gain in obesity. Does ZAG have any effect on lipogenesis besides its lipolytic function? In this study, we investigate the association of the serum ZAG level with body weight and percentage of body fat in normal, obese subjects and high-fat-induced obese mice. In addition, the expression of FAS, ACC, DGAT and HSL mRNA were also determined by real-time fluorescence quantitative PCR in epididymal adipose tissue of high-fat-induced obese mice with or without transfected ZAG gene to explore the possible targets involved in the action of ZAG.
Materials and methods
A total of 28 overweight or obese subjects (BMI ≧24 kg/m2, mean age: 35.9±11.2 years, male/female: 8/20) and 16 normal-sized control subjects (BMI <24 kg/m2, mean age: 44.0±9.4 years, male/female: 5/11) were recruited for this study. Both overweight or obese subjects and normal volunteers had normal routine blood and urine tests, normal liver, kidney and heart function, and their systolic blood pressure (SBP) and diastolic blood pressure (DBP) were in normal range. Patients with chronic disease history, endocrine diseases and any other disease, which might have influenced our results, were excluded. All subjects were selected from outpatient obesity center in Peking Union Medical College Hospital, and all subjects had anthropometric, clinical (systolic and diastolic pressure, heart rate, electrocardiogram, and so on) and laboratory fasting analyses. Body weight, height, body mass index (BMI), percentage of body fat (fat %), fat mass, free fat mass (FFM) and total body water (TBW) of every subject in light clothing without socks and shoes were obtained by bioelectrical impedance analyzer (TBF-215; Tanita, Akita, Japan). Waist circumference was measured with a soft tape on standing subjects midway between the lowest rib and the iliac crest. Hip circumference was measured over the widest part of the gluteal region, and waist-hip-ratio (WHR) was accordingly calculated. Basal blood samples were taken after an overnight fast. Total cholesterol, triglycerides, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, glucose, creatinine, uric acid, AST, ALT, whole blood routine for WBC, RBC, HB, platelet and urine routine were measured by routine automated laboratory methods. Serum ZAG level was determined by commercially available human zinc-alpha2-glycoprotein ELISA kit (Biovendor Laboratorni Medicina, Modrice, Czech Republic) according to the manufactures instruction. The intraassay and interassay coefficient of variation are 1.7 and 7.3%, respectively.
The general characteristics of two groups are described in Table 1. The study was approved by the ethics committee of Peking Union Medical College Hospital. All participants gave written informed consent before taking part in the study.
Male KM mice (purchased from Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences) were housed individually in the animal center of Peking Union Medical College at an ambient temperature of (22±1°C) under a 12/12 h light/dark cycle with free access to food and drinking water. At the age of 6 weeks, mice weighing 25∼27 g were divided into four distinct groups (n=9 for each group) and were subjected to different diets with the following percent energy: standard food (SF): protein 24%, fat 11% and carbohydrates 65% and high-fat diet (HFD): protein 22%, fat 55% and carbohydrates 23%. The weight of food provided to each mouse was monitored everyday, and the body weight was measured twice a week. We certify that all applicable institutional and governmental regulations concerning the ethical use were followed during this research.
Construction and identification of pcDNA3.1(−)-mZAG expression plasmid
pcDNA3.1(−)-mZAG expression plasmid contained murine ZAG (mZAG) full-length cDNA coding sequences (+1∼+924 bp). The construction process was similar to what has been previously described.19 In brief, total RNA from murine liver of BALB/c mice was extracted using EZNA Total RNA kit (Omega Bio-Tek, Doraville, GA, USA), then reverse transcribed with SuperScript first-strand synthesis system kit (Invitrogen). 2 μl cDNA was amplified with mZAG primers as follows: mZAG forward primer 5′-IndexTermATGGTGCCTGTCCTGCTGTC-3′ (20 bp), mZAG reverse primer: 5′-IndexTermTTACTGAGGCTGAGCTACAAC-3′(21 bp). The cycles consisted of 35 cycles at 95 °C for 40 s, 57 °C for 40 s and 72 °C for 90 s. The product length was 924 bp. The second PCR amplification was performed to get products with EcoRV and HindIII enzyme sites in 5′ and 3′ ends. The second group of PCR products and pcDNA3.1(−) vector both digested by EcoRV and HindIII were ligated by T4 DNA ligase to yield a pcDNA3.1(−)-mZAG expression plasmid, then the plasmid was sequenced.
The above constructed plasmid was digested by the restriction enzymes EcoRV and HindIII. As shown in Figure 1, the predicting digested products appeared. Further in vitro experiments confirmed that ZAG mRNA and protein could be expressed better in 3T3-L1 preadipocytes and cell medium following transfecting pcDNA3.1(−)-mZAG expression plasmid into these cells by real-time quantitative RT-PCR and western blot (data not shown).
Animal in vivo plasmid DNA transfecting
After 5 weeks, the HFD-induced obese mice were divided into three distinct groups: the simple HFD group, ZAG overexpression group (HFD+ZAG) and negative control plasmid group (HFD+NC). Plasmid transfection was performed as previously described.20 Shortly, ZAG expression plasmid (25 μg) or pcDNA3.1(+) negative control plasmid (25 μg) in 150 μl OPTI-MEM medium (a kind of serum-free medium often used in transfection; from Invitrogen, Carlsbad, CA, USA), was mixed thoroughly with lipofactamine 2000 (40 μl) in 150 μl OPTI-MEM medium, and the total 300 μl mixture was incubated at room temperature for 30 min, then injected into mice by tail vein. Mice in the simple HFD group were subjected to the same volume OPTI-MEM medium injection as control. This injection was performed at 0900 hours once every 2 days on seven occasions. After 2 weeks, the mice were killed following overnight fasting. Blood samples were obtained for biochemicals and ZAG assays, and adipose tissue from epididymal were dissected, weighted, then frozen immediately in liquid nitrogen until further FAS and HSL mRNA analysis. The percentage of epididymal fat (epididymal fat %) in mice was calculated by body weight of mice divided by epididymal fat mass.
Western blotting for assays of serum ZAG level in mice
Western blot analysis was performed as previously described.21 In brief, the protein concentration of murine serum was determined using BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Samples containing 10 μg of protein were separated by electrophoresis on 12% SDS–polyacylamide gels. The protein was then transferred to a nitrocellulose membrane (Immobilon-P; Millipore, Billerica, MA, USA) and immunodetection was performed using a mouse monoclonal antibody (ZAG (IE2), SC-21720; Santa Cruz Biotechnology, Santa Cruz, California, USA) at 4 °C overnight at a 1:1000 dilution. Blots were then incubated with a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) at a 1:2000 dilution. Signals were detected by western blotting luminol reagents (Santa Cruz Biotechnology). A similar process was also used to assay serum β-actin in mice as an internal reference. The signal intensity of bands was analyzed using Bandscan software, and the intensities of each ZAG band were normalized to the corresponding β-actin bands.
Real-time fluorescence quantitative PCR analysis for fatty metabolic enzymes expressions in mice adipose tissue
The process of total RNA extraction from mice epididymal adipose tissue and reverse transcription were the same as described above for the construction of pcDNA3.1(−)-mZAG expression plasmid. SYBR Green PCR Master Mix (Applied Biosystems, UK) and ABI 7500 PCR instrument (Applied Biosystems, USA) were used to process real-time fluorescence quantitative PCR. The primer sequences for PCR amplification was shown in Table 1. Total reaction volume of each well is 20 μl in 96-well plate, and each gene was repeated in triplicate. Amplification was proceeded according to the standard thermal cycler protocol, and dissociation curve of every gene demonstrated specific amplification. The mean value of Ct for each sample was used for data analysis. All samples were normalized to the 18S values and the results expressed as fold changes of Ct value relative to control by using the 2−ΔΔCt formula.22
Data are shown as mean±s.d. Before statistical analysis, nonnormally distributed parameters were logarithmically transformed to a normal distribution. Differences between groups were analyzed by one-way ANOVA. Pearson and partial correlation coefficients were used to determine linear association between serum ZAG and other obesity-related parameter variables in human and mice. All statistical computations were run on SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA), and P<0.05 was considered statistically significant.
ZAG level and other characteristics in normal and overweight or obese subjects
The differences of anthropometric and laboratory measurements in normal and overweight or obese subjects are displayed in Table 2. As expected, overweight or obese group had higher weight (P<0.001), BMI (P<0.001), waist circumference (P<0.001), hip circumference (P<0.001), WHR, (P<0.05), percentage of body fat (fat %; P<0.05), fat mass (P<0.05) than the normal group. However, there was no significant difference between these two groups with regard to SBP, DBP, fasting glucose and lipid profiles. Our previous preexperiment primarily found that serum ZAG level in obese patients was much more lower than that in normal-weight people by western blot method (data not shown). Now the same result was achieved by using human zinc-alpha-2-glycoprotein ELISA kit; serum ZAG decreased by 15.1% in overweight or obese group compared to that in normal group (48.9±8.2 vs 57.6±9.1 μg ml−1; P=0.002). The further statistical analysis found an inverse correlation between serum ZAG level and body weight (r=−0.62, P<0.001), BMI (r=−0.64, P<0.001), waist circumference (r=−0.68, P<0.001), hip circumference (r=−0.60, P<0.001), percentage of body fat (r=−0.52, P=0.03) and fat mass (r=−0.59, P=0.01) after adjusting for age and sex in normal and overweight or obese subjects (Figure 2). There is no correlation between ZAG and fasting glucose, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol in human subjects.
The changes of ZAG level and other parameters in SF- and HFD-fed mice
After feeding male mice with HFD for 7 weeks, a marked increase in their body weight (P<0.01), epididymal fat mass (P<0.05), percentage of epididymal fat (epididymal fat %; P<0.05) was first observed when compared with those of the SF-fed mice. Their serum fasting glucose, total cholesterol and LDL-cholesterol also became higher than those of SF-fed mice. Meanwhile, serum ZAG level in HFD-induced obese mice was 32% lower than that in SF-fed mice (0.75±0.07 vs 0.51±0.10, P<0.001; Table 3; Figure 3). These results suggest that these mice became obese after 7 weeks HFD feeding, and the interesting thing was the serum ZAG level decreased greatly in obese mice like that in human subjects. Further analysis showed that a negative correlation between serum ZAG and body weight (r=−0.56, P<0.001), epididymal mass (r=−0.67, P<0.001), percentage of epididymal fat (r=−0.65, P<0.001) and increased weight (r=−0.57, P<0.001) in SF- and HFD-fed mice (Figure 4). There was no relationship between ZAG and fasting glucose, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol in mice.
Effects of ZAG on body weight, percentage of epididymal fat and biochemicals parameters in HFD-induced mice
To further demonstrate the relationship between ZAG and body weight, obese mice were transfected with mouse ZAG expression plasmid seven times over a 2-week period. The results showed that serum ZAG level in mice receiving ZAG gene transfection increased by 98 and 87%, respectively, when compared with two controls (OPTI-MEM medium treated mice (HFD group) and pcDNA3.1(+) negative control plasmid treated mice (HFD+NC group)) as detected by western blot (1.01±0.16 vs 0.51±0.10, 1.01±0.16 vs 0.54±0.01, P<0.001), suggesting ZAG indeed overexpressed in HFD+ZAG mice and plasmid DNA transfection by lipid transfection reagent was successful (Figure 3). Subsequently, overexpression of ZAG in HFD mice first produced a decrease in final body weight (40.0±2.6 vs 45.7±4.1, P<0.01) with a reduction of 12.7% and increased weight (comparison to initial weight; 12.8±2.4 vs 18.7±3.9, P<0.001) with a reduction of 31.7% without a change in food intake. Overexpression of ZAG in HFD mice also caused a great reduction in epididymal fat mass (a decrease of 54.9%, P<0.01) and percentage of epididymal fat (a decrease of 47.5%, P<0.001; Table 3). Among serum glucose and lipid profiles parameters, only fasting glucose decreased significantly after transfecting ZAG gene to mice (P<0.01). These results demonstrate that ZAG overexpression results in a reduction of body weight, epididymal fat mass, percentage of epididymal fat in HFD-fed obese mice.
Effects of ZAG on fatty metabolic enzymes mRNA expressions in adipose tissue of HFD-induced mice
To explore possible targets involved in the action of ZAG, the lipogenic enzymes FAS, ACC1 and DGAT1 mRNA and the lipolytic enzyme HSL mRNA were determined by real-time fluorescence quantitative PCR in epididymal adipose tissue of HFD-fed obese mice with or without transfected ZAG gene. As shown in Table 4, FAS, ACC1 and DGAT1 mRNA in epididymal adipose tissue of HFD-fed mice increased 1.21-, 1.82-, 1.23-fold, respectively, compared to that in SF-fed mice (P<0.05, P<0.01). After transfecting ZAG gene into HFD-fed mice, FAS, ACC1 and DGAT1 mRNA decreased 73.8, 73.4 and 70.0%, respectively, compared to that in HFD mice (P<0.01, P<0.001). In contrast to the above lipogenic enzymes mRNA changes, HSL mRNA in epididymal adipose tissue of HFD-fed mice reduced by 30.0% in comparison to that in SF-fed mice (P<0.01). Transfection of ZAG gene to HFD-fed mice resulted in a significant increase in HSL mRNA (P<0.001), 4.11-fold higher than that of HFD-fed mice. These results showed that lipogenic enzymes FAS, ACC1 and DGAT1 mRNA level downregulated and the lipolytic enzyme HSL mRNA level upregulated with an increase of ZAG expression in epididymal adipose tissue of HFD-fed mice (Table 4).
It has been reported that cachectic patients with weight loss have an elevated level of ZAG, which appears to parallel the weight loss.23, 24 A linear relationship was observed between the level of serum and urinary ZAG and weight loss in cancer patients.25 Further studies demonstrated that loss of body weight resulted entirely from the loss of body fat, and loss of adipose tissue may have been due to the strong lipolytic effect of ZAG in adipose tissue. However, little is known about ZAG function in weight-gain obesity. In this study, the concentration of serum ZAG in normal and overweight or obese subjects was first measured by a commercially available human serum ZAG ELISA assay kit, and an interesting and significant decrease in serum ZAG level (P=0.002) in overweight or obese subjects was observed. Statistical analysis further showed that serum ZAG level was associated negatively with body weight (r=−0.62), BMI (r=−0.64), waist circumference (r=−0.68), hip circumference (r=−0.60), percentage of body fat (r=−0.52) and fat mass (r=−0.59) in human subjects. Consistent with our findings in human studies, our experiments performed in mice also found a marked decrease in the serum ZAG level in HFD-induced obese mice and an inverse correlation between serum ZAG and body weight, epididymal fat mass, epididymal fat% and increased body weight (all P<0.001). All these findings imply that serum ZAG was linked not only to weight loss in cachectic cancer patient, but also to weight gain in obesity patients and obese mice. Studies performed by Marrades et al.26 and Dahlman et al.27 at the tissue level revealed ZAG expression was downregulated by 70 and 69%, respectively, in subcutaneous abdominal adipose tissue of obese subjects compared to lean subjects by real-time PCR analysis and affymetrix microassay technique. Moreover, a statistically significant positive correlation between ZAG gene expression and serum adiponectin and a negative correlation with the plasma levels of leptin and waist circumference were found in obese subjects,26 suggesting that ZAG may be a candidate factor in the regulation of body weight at the tissue level, and adiponectin and leptin are also involved in this process.
The relationship between ZAG and body weight was further supported by our experiments in transfected ZAG gene obese mice, where ZAG overexpression was found to result in a reduction of body weight, epididymal fat mass and epididymal fat % in HFD-fed obese mice without a change in food intake. The similar conclusion was also to be drawn by Hirai et al.6 and Russell et al.8 who demonstrated that administration ZAG to exbreeder mice and ob/ob mice both induced a rapid and dose-dependent reduction in body weight without a reduction in food and water intake. Loss of body weight could be attributed entirely to the loss of body fat. In contrast to administration ZAG to obese mice, Rolli et al.28 inactivated both ZAG alleles by gene targeting in mice, then subjected these ZAG deficient mice to standard or lipid rich food regimens for 20 weeks. The results showed that ZAG knock out (ZAG−/−) mice gained significantly more weight than ZAG+/+ control mice fed by both standard and lipid rich food. All of these findings together with the evidence of the reduced serum level of ZAG in obesity patients and obese mice indicate that there is a close relationship between ZAG and body weight.
As ZAG is associated with body weight in human and mice, administration of ZAG resulted in a reduction of body weight and body fat in normal and obese mice, what are the possible targets involved in the action of ZAG? Thus, four key enzymes in lipid metabolism, FAS, ACC1, DGAT and HSL mRNA were determined in epididymal adipose tissue of HFD-induced obese mice with or without transfected ZAG gene by real-time PCR method. Our results showed that the lipogenic enzymes FAS, ACC1, DGAT mRNA decreased and HSL mRNA increased significantly in epididymal adipose tissue after transfecting ZAG gene into mice. As these four enzymes are key enzymes during lipogenesis and lipolysis and they are related with obesity, this result suggested lipid metabolism including lipogenesis and lipolysis are involved in ZAG-induced reduction in body fat of obese mice. It has been reported that in vivo, administration of ZAG to mice induced a reduction of body fat together with an increased serum free fatty acid, glycerol level and an elevated oxygen uptake in interscapular BAT, providing evidence of increased lipolysis, lipid mobilization and utilization.6, 29 To our knowledge, in this study, we are the first to report that the action of ZAG to reduce body weight and body fat in obese mice is involved in inhibiting lipogenesis besides stimulating lipolysis. Experiments performed in vitro demonstrated that incubation of ZAG with adipocytes isolated from murine adipose tissue has been shown to stimulate lipolysis in a dose-dependent manner,6 suggesting the lipolytic action of ZAG may be direct. In our animal experiment, whether the action of ZAG in FAS, ACC1, DGAT and HSL mRNA expression is direct or indirect still needs further study.
ZAG overexpression caused a reduction of fasting glucose in HFD-fed obese mice. Similar results were also obtained by Hirai et al.6 who revealed decreased fasting glucose in the administration of ZAG to exbreeder mice. However, no relationship was found between serum ZAG and fasting glucose in normal and obese subjects. The effect of ZAG on glucose metabolism still needs further investigation.
Finally, in this study, we first amplified mZAG full-length cDNA coding sequences from liver of BALB/c mice and constructed mZAG expression plasmid pcDNA3.1(−)-mZAG. DAN sequence analysis revealed 100% homology with mZAG cDNA. Experiments performed in vitro and in vivo both confirmed that ZAG mRNA and protein could be better expressed in cell lysis, cell medium and serum of mice using real-time RT-PCR and western blot method. Thus, mZAG expression plasmid provided us a good tool for studying ZAG biological function in the further studies.
In conclusion, ZAG is closely linked to obesity. ZAG level is inversely associated with body weight and percentage of body fat. The action of ZAG is correlated with reduced FAS, ACC1, DGAT and increased HSL expression in adipose tissue of mice.
Conflict of interest
The authors declare no conflict of interest.
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This research was supported by the national natural science foundation of China (No.: 30540036 and 30771026)
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Biological Trace Element Research (2019)
Frontiers in Endocrinology (2019)
Neuroscience Bulletin (2019)
Molecular Metabolism (2019)
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