1Department of Endocrinology and Metabolism C, Aarhus Amtssygehus, Aarhus University Hospital and Faculty of Health Sciences, Aarhus University, DK-8000 Aarhus C; and Departments of 2Internal Medicine and Endocrinology, 3Infectious Diseases, and 4Radiology, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark
Submitted 8 May 2003 ; accepted in final form 18 July 2003
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ABSTRACT |
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human immunodeficiency virus-associated lipodystrophy syndrome; tumor necrosis factor-; interleukin-6; interleukin-8; cytokines; fat redistribution; insulin resistance
Within recent years, the perception of the AT being solely an energy storage organ has extended to include an endocrine organ producing numerous proteins and substances with various biological functions (5, 54). Abnormal production of AT-derived proteins is suggested to play a role in the pathogenesis of insulin resistance and the metabolic syndrome seen in relation to obesity (27, 40, 61). Among the substances secreted by AT are cytokines such as TNF-, IL-6, IL-8 (2, 11), and the more recently discovered protein adiponectin (37, 47). TNF-
and IL-6 have been shown to attenuate insulin sensitivity in vivo and in vitro (2729, 48, 49), and the plasma concentration of IL-6 independently predicts future risk of developing Type 2 diabetes mellitus (46). Serum IL-8 levels are increased in diabetic patients (62), and IL-8 has been suggested to be involved in the pathogenesis of atherosclerosis (6).
Adiponectin is an adipocyte-specific protein found to be inversely correlated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease (4, 30, 35, 56). Apart from possessing anti-inflammatory and anti-atherogenic properties (43, 44), adiponectin is suggested to be a modulator of insulin sensitivity (30, 36). Administration of adiponectin to diabetic mice resulted in improved insulin sensitivity (9), and several studies have shown positive correlations between plasma adiponectin and insulin sensitivity (30, 56). Decreased plasma adiponectin concentrations preceded the development of hyperglycemia and insulin resistance in an animal model of diabetes (31), and low plasma adiponectin concentration is associated with an increased risk of developing type 2 diabetes (36). Thus increasing evidence is supporting the notion that adiponectin is an enhancer of insulin sensitivity.
In patients with congenital and acquired lipodystrophy, Haque et al. (26) found a strong negative correlation between serum adiponectin and fasting insulin. Furthermore, Yamauchi et al. (59) showed partial correction of insulin resistance in lipoatrophic mice treated with adiponectin, suggesting that adiponectin is one possible mediator of insulin resistance associated with lipodystrophy. Recently, low levels of serum adiponectin in HIV-infected patients with HALS has been demonstrated (1, 41, 53). It remains unclear, however, how the level of circulating adiponectin is downregulated in obesity and in lipodystrophic patients. Recent in vitro studies have shown that cytokines, including TNF- and IL-6, may inhibit the gene expression and secretion of adiponectin in 3T3-L1 adipocytes (21) as well as in human AT (10). Thus production of adiponectin might be regulated by locally released cytokines in AT.
In the present study, we investigated the implications of adipocytokines for the level of adiponectin in HALS patients. The plasma level and AT gene expression of adiponectin were investigated in HIV-infected male patients with and without HALS. To investigate whether changes in adiponectin may be mediated by cytokines, the levels of TNF-, IL-6, and IL-8 were determined both in AT and in the circulation. Furthermore, the in vitro effect of TNF-
and IL-8 on adiponectin gene expression was investigated.
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SUBJECTS AND METHODS |
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For the in vitro study, we used subcutaneous AT from 10 healthy women [mean body mass index (BMI) 23.3 ± 0.9 kg/m2] undergoing liposuction at a plastic surgery clinic.
Both studies were approved by the local ethics committee, and all subjects provided written informed consent.
Experimental design of the in vivo study. Instructions were given to abstain from strenuous exercise for 3 days before the metabolic assessments. All subjects underwent a 120-min hyperinsulinemic euglycemic clamp. Subcutaneous fat biopsies were taken from the abdominal region at baseline.
Measurements of body composition. BMI was calculated as body weight (kg) divided by squared height (m2). In the supine position, waist circumference was measured at the level of umbilicus and hip circumference in the horizontal plane at the level of the maximal extensions of the buttocks. Body composition was evaluated by dual-energy X-ray absorptiometry (DEXA; Norland, Fort Atkinson, WI). To estimate the amount of fat in the trunk (chest, abdomen, and pelvis) and in the extremities a whole body scan was performed. The proximal limitation of the leg region was placed through the hip joints at an angle of 45°. Percentage of limb fat (LF) was calculated as the extremity fat mass (total fat in arms and legs) expressed as percentage of total fat mass. The DEXA scans were done in random order, and the operator was unaware of the assignment of the patients to study groups. To assess the distribution of visceral AT (VAT) and subcutaneous abdominal AT (SAT) a single-slice computed tomography scan at the level of L4 was performed in 16 HALS subjects and in 15 non-HALS subjects. The area of AT was measured in square centimeters.
Hyperinsulinemic euglycemic clamp. In the morning after an overnight fast, two intravenous cannulas were inserted, one in a dorsal hand vein and the other in an antecubital vein. The hand vein was used for blood sampling, and the antecubital vein was used for infusion. Blood samples were drawn at baseline. Insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) infusion was started with a stepwise decline in infusion rate every 3rd min from 100 to 80 to 60 to 40 mU · m2 · min1, after which (+9 to 120 min) the insulin infusion rate was fixed at 40 mU · m2 · min1. Plasma glucose concentration was kept at 5 mM by adjusting the infusion rate of 20% glucose. During the clamp, glucose levels were determined every 5 min, and blood samples for assessing plasma insulin levels were taken every 15 min. The last 30 min of the clamp (time 90120 min) were defined as the hyperinsulinemic steady-state period. The amount of glucose infusion (mg · kg lean body wt1 · min1) required to maintain euglycemia during this predefined steady-state period was taken as an index of insulin action (M value).
AT biopsies. At baseline, AT biopsies were taken from the subcutaneous abdominal region (periumbilically), as previously described (33a). By use of local anesthesia (5 mg/ml lidocaine), the biopsies were taken by needle aspiration (liposuction). The AT was washed thoroughly with isotonic saline and then frozen in liquid nitrogen and kept at 80°C for later RNA extraction.
AT fragments in cultures. The AT was minced into fragments, and organ culture was performed as previously described (11). The fragments were preincubated for 24 h. Then, the medium was replaced, and TNF- (10 ng/ml) or IL-8 (1 µg/ml) was added. The incubation was continued for 48 h. Incubations were performed as duplicates. After culturing, the AT fragments were frozen in liquid nitrogen and kept at 80°C for later RNA extraction.
Analytic techniques. Immediately after sampling, whole blood glucose levels were determined pairwise on two calibrated HemoCue B-Glucose Analyzers (HemoCue, Ängelholm, Sweden) with an intra-assay coefficient of variation (CV) of 3.5% and an interassay CV of 3.3%. Plasma glucose was calculated using the equations of Fogh-Andersen and D'Orazio (23). Plasma insulin concentrations were determined by a 1235 AutoDELPHIA automatic immunoassay system (Wallac Oy, Turku, Finland). The intra-assay CV was 4.5% and the interassay CV 7%. Plasma adiponectin was measured by radioimmunoassay (RIA; Linco Research, St. Charles, MO), and the intra-assay CV was 2.6% (n = 12). IL-6 and TNF- plasma concentrations were measured using human enzyme-linked immunosorbent assays (ELISA; Quantikine HS ELISA, R&D Systems Europe, Abingdon, UK). Plasma IL-8 was determined with a human Biotrak ELISA kit (Amersham International, Buckinghamshire, UK).
Isolation of RNA. Total RNA was isolated using the TRIzol reagent (GIBCO-BRL, Life Technologies, Roskilde, Denmark). RNA was quantified by measuring absorbency at 260 and 280 nm, and the integrity of the RNA was checked by visual inspection of the two ribosomal RNAs 18S and 28S on an agarose gel.
Real-time RT-PCR measurement of adiponectin mRNA. RNA was reverse transcribed with reverse transcriptase and random hexamer primers at 23°C for 10 min at 42°C for 60 min and at 95°C for 10 min, according to the manufacturer's instructions (GeneAmp RNA PCR Kit; Perkin Elmer Cetus, Norwalk, CT). Then, PCR-mastermix containing the specific primers Hot Star Taq DNA polymerase, and SYBR-green PCR buffer was added. All samples were determined in duplicate.
The following primers were used: human TNF- primers 5'-CGAGTGACAAGCCTGTAGC and 5'-GGTGTGGGTGAGGAGCACAT, human IL-6 primers 5'-AAATGCCAGCCTGCTGACGAAC and 5'-AACAACAATCTGAGGTGCCCATGCTAC, human IL-8 primers 5'-AACTTCTCCACAACCCTCTG and 5'-TTGGCAGCCTTCCTGATTTC, and human adiponectin primers 5'-CATGACCAGGAAACCACGACT and 5'-TGAATGCTGAGCGGTAT. As a housekeeping gene,
-actin was amplified using the primers 5'-ACGGGGTCACCCACACTGTGC and 5'-CTAGAAGCATTTG-CGGTGGACGATG. Real-time quantitization of adiponectin to
-actin mRNA was performed using a SYBR-green PCR assay and an iCycler PCR machine (Bio-Rad Laboratories, Hercules, CA) as previously described (12). In brief, adiponectin mRNA and
-actin mRNA were amplified in separate tubes at 95°C for 10 min and thereafter repeating cycles comprising 95°C for 30 s and 57°C for 30 s and extension at 72°C for 60 s. During the extension step, increase in fluorescence was measured in real time. Data were obtained as CT values (threshold cycle). CT was defined as the cycle number at which the fluorescence reached 10 times the standard deviation of the baseline fluorescence. Relative gene expression was calculated using the formula 1/2(CT adiponectin CT
-actin), essentially as described in the User Bulletin No. 2, 1997 from Perkin Elmer (Perkin Elmer Cetus).
Statistical analysis. Because adiponectin mRNA and serum adiponectin as well as the cytokines were not normally distributed according to the Kolmogorov-Smirnov test, we used a Mann-Whitney test to test the statistical significance of differences between control and HALS values. For comparison of mean values before and after the hyperinsulinemic clamp, a Wilcoxon signed-rank test was used. Correlation between variables was tested by Pearson's correlation coefficient after logarithmic transformation when necessary. A backward multivariate linear regression analysis was performed to evaluate the most important determinants of insulin sensitivity. Values are presented as means ± SE, and a P value <0.05 was considered statistically significant. For analyses the SPSS statistical package was used (SPSS, Chicago, IL).
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RESULTS |
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HALS patients were slightly older and had a slightly higher BMI (24.6 ± 0.6 vs. 22.5 ± 0.8 kg/m2, P < 0.05; Table 1). As expected, the HALS patients had significantly higher waist-to-hip ratio (1.01 ± 0.01 vs. 0.92 ± 0.01, P < 0.001), more visceral AT (203 ± 26 vs. 68 ± 15 cm2, P < 0.001), and a lower percentage of LF (36.2 ± 1.3 vs. 46.1 ± 1.8%, P < 0.001) than non-HALS patients. However, there was no difference in total fat mass between the two groups (Table 1). These results confirmed the presence of lipodystrophy in HALS patients.
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HALS patients were characterized by being insulin resistant, having twice as high fasting plasma insulin levels (89 ± 13 vs. 41 ± 6 pM, P < 0.001) and a lower M-value (5.54 ± 0.50 vs. 8.24 ± 0.51 mg · kg lean body wt1 · min1, P < 0.01). However, both groups were normoglycemic (Table 1).
Total plasma cholesterol was significantly higher in HALS patients (6.3 ± 0.4 vs. 5.0 ± 0.2 mM, P < 0.01), whereas there were no differences in plasma HDL or plasma triglyceride between the two groups.
Adiponectin mRNA expression and plasma adiponectin. The level of adiponectin mRNA in SAT was 52% lower in HALS patients compared with control subjects, which was very close to reaching statistical significance (13.5 ± 2.1 vs. 28.1 ± 6.1, P = 0.06; Fig. 1A).
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We found significantly reduced baseline plasma adiponectin levels, corresponding to adiponectin mRNA expression, in HALS patients (7.34 ± 1.28 vs. 12.46 ± 2.02 µg/ml, P < 0.05; Fig. 1B). After a 120-min hyperinsulinemic clamp, adiponectin levels were 6.85 ± 1.18 and 11.92 ± 1.71 µg/ml, P < 0.05, respectively (data not shown).
The two groups compared were not matched on BMI; however, when BMI was controlled for (by dividing the values with BMI), the differences in adiponectin mRNA (0.55 ± 0.09 vs. 1.34 ± 0.30 m2/kg, P < 0.05) and plasma adiponectin concentrations (0.30 ± 0.05 vs. 0.59 ± 0.10 µg · ml1 · kg1 · m2, P < 0.05) between HALS and non-HALS persisted (data not shown).
Cytokines in AT and in plasma. To investigate whether adipocytokines might play a role in the reduced mRNA and protein level of adiponectin in AT of HALS patients, we investigated the gene expression of various cytokines in AT. The expression of TNF- and IL-8 mRNA was significantly increased in HALS patients (Fig. 2). Similarly, IL-6 mRNA expression was three times higher in HALS patients compared with controls; however, this difference did not reach statistical significance. The circulating levels of these cytokines did not differ between the two groups (Table 2).
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Correlations between adiponectin and insulin sensitivity and fat redistribution. A positive correlation was found between insulin sensitivity determined by the M value and plasma adiponectin (r = 0.55, P < 0.01; Fig. 3A) as well as between the M value and adiponectin mRNA in AT (r = 0.40, P < 0.05). Interestingly, plasma adiponectin and adiponectin mRNA were found to be closely correlated (r = 0.52, P < 0.01; Fig. 3B).
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Plasma adiponectin was highly correlated with different measures of fat redistribution such as VAT and LF (r = 0.42, P < 0.05 and r = 0.61, P < 0.01; Table 3). The well-known correlations between plasma adiponectin and HDL-cholesterol and triglycerides were also found in this study (Table 3).
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To determine which fat depot was the most important predictor of plasma adiponectin, we performed multiple linear regression analysis with plasma adiponectin as the dependent variable. LF turned out to be the most important predictor (partial r = 0.64, P < 0.001), whereas VAT and BMI were excluded from the analysis (backward linear regression analysis). Adding M value to the analysis did not change the fact that LF was explaining the major part of variation in plasma adiponectin (r2 = 0.41).
The positive correlation found between plasma adiponectin and M value was maintained after adjustment for BMI and SAT; however, the association was weakened after adjustment for fat redistribution in terms of VAT and LF (data not shown).
Correlations between adiponectin and cytokines. Plasma TNF- was negatively correlated with plasma adiponectin (r = 0.39, P < 0.05; Fig. 4A) and with AT adiponectin mRNA (r = 0.36, P < 0.05; Table 4). Furthermore, AT TNF-
mRNA levels were negatively correlated with plasma adiponectin (r = 0.33, P < 0.05) but not with adiponectin mRNA [r = 0.21, not significant (NS)]. Among the other cytokines, a negative correlation was found between AT IL-8 mRNA and plasma adiponectin (r = 0.38, P < 0.05) and a negative but statistically nonsignificant correlation between IL-8 mRNA and adiponectin mRNA in AT (r = 0.26, NS). No correlations were found between IL-6 and adiponectin either in gene expression or in plasma levels (data not shown).
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AT IL-8 mRNA was positively correlated with plasma TNF- and AT TNF-
mRNA (r = 0.47 and r = 0.51, respectively, P < 0.01). Moreover, AT IL-6 mRNA was positively correlated with both TNF-
mRNA (r = 0.41, P < 0.05) and IL-8 mRNA (r = 0.53, P < 0.01) in AT (data not shown). Plasma TNF-
was positively correlated with AT TNF-
mRNA (r = 0.48, P < 0.01; data not shown).
Among the mRNA levels of cytokines in AT, IL-8 mRNA turned out to be a significant predictor of plasma adiponectin (partial r = 0.38, P < 0.05) when backward multiple linear regression analysis was used, whereas TNF- mRNA and IL-6 mRNA in AT were excluded when IL-8 mRNA was adjusted for (data not shown).
In vitro effects of TNF- on adiponectin mRNA expression. To determine whether cytokines may directly inhibit adiponectin in human AT, we incubated AT fragments with 10 ng/ml TNF-
or 1 µg/ml IL-8. Adiponectin mRNA levels were significantly reduced by incubation with TNF-
(0.32 ± 0.09 vs. 0.06 ± 0.01, n = 10, P < 0.05; Fig. 4B), whereas IL-8 did not affect adiponectin mRNA expression in AT fragments (0.55 ± 0.16 vs. 0.42 ± 0.06, n = 5, P < 0.05; data not shown).
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DISCUSSION |
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In accord with recent in vitro studies (10, 20, 32), this study shows that adiponectin gene expression and secretion are inhibited by TNF-. Similarly, IL-6 has been shown to attenuate adiponectin levels in vitro (10, 21), and in vivo studies have shown plasma levels of adiponectin and IL-6 to be inversely correlated (10, 19). These findings suggest that cytokines are possible regulators of adiponectin production. Furthermore, cytokines, including TNF-
, IL-6, and IL-8, are thought to be implicated in the health complications of obesity (6, 15, 60). Positive correlations have been found between abdominal obesity particularly and circulating levels of TNF-
(33), IL-6 (8), and IL-8 (11). In the obese state, the mRNA expression of these cytokines in AT have been found to be increased (Bruun JM, personal communication). Apart from being dysregulated in obesity, cytokines have also been implicated in HAART-associated lipodystrophy. A HAART-induced proinflammatory response with dysregulated TNF-
synthesis is suggested to play a pathogenic role in the fat redistribution (34). This hypothesis was further strengthened when Mynarcik et al. (42) showed elevated levels of soluble type 2 TNF-
receptor in HIV-infected patients with lipodystrophy and when increased TNF-
gene expression was demonstrated in subcutaneous AT of HALS patients (7). In accord with these findings, we found significantly increased levels of TNF-
mRNA expression in AT from HALS subjects. However, we extend the finding to include increased gene expression of the cytokines IL-6 and IL-8 in AT of HALS patients as well, which further highlights possible cytokine dysregulation in these patients. The plasma levels of these cytokines were slightly but not significantly increased in HALS patients. From the negative correlations found between TNF-
and IL-8 in AT and plasma adiponectin, it might be hypothesized that these cytokines are directly involved in the reduced gene expression of adiponectin in AT and thereby the reduced plasma adiponectin found in HALS patients. Causality cannot, however, be determined directly from the present cross-sectional study.
Recently, Suzawa et al. (50) showed that the cytokines IL-1 and TNF- inhibit the expression and activity of the nuclear receptor peroxisome proliferator-activated receptor-
(PPAR
). PPAR
is highly expressed in AT, and it is an important factor for adipocyte differentiation and for insulin sensitivity. Furthermore, several studies have shown that PPAR
activation increases adiponectin synthesis (3, 16, 38). Thus inhibition of the PPAR
system in AT could hypothetically be a mechanism by which enhanced levels of cytokines in AT might play a role in the development of lipodystrophy, insulin resistance, and reduced levels of adiponectin. This hypothesis is in accord with a study by Bastard et al. (7) showing reduced expression of transcription factors known to activate PPAR
transcription and increased TNF-
expression in AT of HALS patients compared with controls. Bastard et al. further demonstrated characteristic changes in the morphology of subcutaneous AT of HALS subjects. The major finding was clusters of small adipocytes suggested to be the result of an increased number of young, regenerating adipocytes. This change in differentiation state of the adipocytes was associated with altered expression of transcription factors, and it might play a role in the dysregulation of adipocytokine gene expression found in HALS subjects.
In agreement with several recent studies (1, 4, 35, 41, 53, 56), we found a close correlation between plasma adiponectin and insulin sensitivity determined by the M value and a negative correlation with VAT, possibly indicating that low levels of adiponectin play a role in the reduced insulin sensitivity observed in HALS subjects. Recent animal and in vitro studies strongly support a direct role of adiponectin in enhancing insulin sensitivity, glucose uptake, and fatty acid oxidation (52, 57, 58).
Among the fat redistribution variables, LF was the strongest predictor of both insulin sensitivity and plasma adiponectin. This finding confirms recent studies showing that the relative amounts of LF and insulin sensitivity are positively correlated (41, 51, 55). Furthermore, our data on LF and plasma adiponectin are consistent with the data from Tong et al. (53) demonstrating extremity fat to be an independent contributor to plasma adiponectin. Whether the production of adiponectin is higher in femoral AT than in other fat depots is still unknown; however, gluteal AT and SAT have been shown to express more adiponectin than VAT (22). The close correlation between LF and plasma adiponectin could, alternatively, be caused by LF being the best discriminator for the HALS/non-HALS state, and LF would then be just a marker for a still-unidentified lipodystrophy-related factor affecting plasma adiponectin. The pathogenesis of HALS is still unclear; however, the highly active antiretroviral agents, including protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors, are suggested to play a role (14, 39). Recent studies indicate that the PIs inhibit PPAR expression and thereby AT development (13, 18). Because the expression of adiponectin is highest in differentiated adipocytes (47), reduced adiponectin expression could be a direct consequence of PI-induced reduced PPAR
activity. Low plasma adiponectin levels, however, seem to be a consequence of the lipodystrophic state independent of the reason for the lipodystrophy as shown by Haque et al. (26).
Yamauchi et al. (59) showed that replacement with either leptin or adiponectin could partially restore the metabolic abnormalities seen in lipoatrophic mice and that replacement with both leptin and adiponectin was necessary to fully normalize the metabolic syndrome. It is therefore tempting to speculate that adiponectin could be used as a therapeutic agent in the treatment of the metabolic abnormalities seen in HALS patients. Because adiponectin has anti-inflammatory properties (17), it might even directly reduce the high levels of cytokines in adipose tissue of HALS patients.
In conclusion, HIV-infected patients with HAART-induced lipodystrophy have substantially reduced levels of plasma adiponectin and reduced AT adiponectin mRNA. Furthermore, enhanced mRNA levels of the adipocytokines TNF-, IL-6, and IL-8 were found in SAT of HALS patients. Increased adipocytokine expression might play a role for the reduced levels of adiponectin found in HALS patients. Because low levels of adiponectin are closely associated with metabolic abnormalities such as insulin resistance, we hypothesize that the HAART-induced lipodystrophy and metabolic aberrations may be partly due to activation of cytokines in adipose tissue.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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