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Endocrinology, doi:10.1210/en.2003-1336
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Endocrinology Vol. 145, No. 5 2273-2282
Copyright © 2004 by The Endocrine Society

Comparison of the Release of Adipokines by Adipose Tissue, Adipose Tissue Matrix, and Adipocytes from Visceral and Subcutaneous Abdominal Adipose Tissues of Obese Humans

John N. Fain, Atul K. Madan, M. Lloyd Hiler, Paramjeet Cheema and Suleiman W. Bahouth

Departments of Molecular Sciences (J.N.F., P.C.), Surgery (A.K.M., M.L.H.), and Pharmacology (S.W.B.), College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: John N. Fain, Department of Molecular Sciences, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163. E-mail: jfain{at}utmem.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of this study was to examine the source of adipokines released by the visceral and sc adipose tissues of obese humans. Human adipose tissue incubated in primary culture for 48 h released more prostaglandin E2, IL-8, and IL-6 than adiponectin, whereas the release of plasminogen activator inhibitor 1 and hepatocyte growth factor was less than that of adiponectin but greater than that of leptin. IL-10 and TNF{alpha} were released in amounts less than those of leptin, whereas vascular endothelial growth factor and IL1-ß were released in much lower amounts. The accumulation of adipokines was also examined in the three fractions (adipose tissue matrix, isolated stromovascular cells, and adipocytes) obtained by collagenase digestion of adipose tissue. Over 90% of the adipokine release by adipose tissue, except for adiponectin and leptin, could be attributed to nonfat cells. Visceral adipose tissue released greater amounts of vascular endothelial growth factor, IL-6, and plasminogen activator inhibitor 1 compared with abdominal sc tissue. The greatly enhanced total release of TNF{alpha}, IL-8, and IL-10 by adipose tissue from individuals with a body mass index of 45 compared with 32 was due to nonfat cells. Furthermore, most of the adipokine release by the nonfat cells of adipose tissue was due to cells retained in the tissue matrix after collagenase digestion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENTLY, THE CONCEPT of adipose tissue as an endocrine organ has become accepted (1, 2, 3, 4, 5). Leptin and adiponectin are proteins secreted by the adipocytes of adipose tissue, and there is an inverse relationship between circulating adiponectin and leptin (6). These hormones are often referred to as adipokines because they are secreted by the adipocytes of adipose tissue. A variety of other factors are also released by adipose tissue in vitro including cytokines such as IL-6 (7) and IL-8 (8), which when released by adipose tissue have been called adipocytokines or adipokines. We use the term adipokine for any substance released by adipose tissue. Although leptin and adiponectin are released to the blood as hormones, other adipokines appear to be paracrine factors whose release by adipose tissue may not contribute to circulating levels.

The concentration in blood of many adipokines, hormones, and acute-phase proteins is altered in human obesity. Leptin is elevated, whereas plasma adiponectin is reduced in obese humans (1, 2, 3, 4, 5, 6). C-reactive protein (CRP) (9, 10, 11, 12, 13, 14, 15) is an example of an acute-phase protein whose circulating level is higher in obese than in nonobese individuals. Blood levels of IL-10 (15), IL-6 (11, 12, 13, 14, 15, 16, 17, 18, 19), IL-8 (18, 20), plasminogen activator inhibitor 1 (PAI-1) (17, 21, 22, 23, 24), TNF{alpha} (11, 25), and hepatocyte growth factor (HGF) (26) have all been reported to be elevated in obesity. There is some controversy about circulating levels of TNF{alpha} because several groups found either no detectable TNF{alpha} (27) or no increase of circulating TNF{alpha} in obesity (10, 18, 19, 28). However, there was greater release of TNF{alpha} by adipose tissue (27, 28) as well as elevated levels of TNF{alpha} mRNA in adipose tissue from obese humans (27). Furthermore, the studies of Mohamed-Ali et al. (29) indicated that there was release of IL-6 but not of TNF{alpha} by sc adipose tissue in vivo.

Previously, we demonstrated that substantial amounts of connective tissue and blood vessels remain after collagenase digestion of human adipose tissue (30). We describe this fraction as tissue matrix, and it accounts for almost 70% of the total protein retrieved in the three fractions obtained by collagenase digestion of human adipose tissue. The remainder of the protein was equally divided between the isolated stromovascular (SV) cells that do not contain enough lipid to float and adipocytes that do float. Although there was no appreciable release of leptin by the tissue matrix or the SV cells, these two fractions accounted for over 95% of the prostacyclin or prostaglandin E2 (PGE2) released over a 48-h incubation per gram of adipose tissue (30). Similar results were seen with respect to resistin release (31). The hypothesis we wanted to examine was whether the source of other adipokines, which are defined as factors released by adipose tissue, is the adipocytes or the nonfat cells present in adipose tissue. The first aim was to compare adipokine release by tissue with that by adipocytes, the isolated SV cells, and the undigested adipose tissue matrix of human adipose tissue. The second aim was to examine the time course for the release of adipokines by human sc and visceral adipose tissue explants and adipocytes in primary culture from morbidly obese [body mass index (BMI) >40] individuals. The third aim was to compare release by tissue and adipocytes from individuals over 48 h with an average BMI of 45 vs. that by tissue and adipocytes from individuals with an average BMI of 32. The fourth aim was to compare the relative release of adipokines by visceral compared with sc adipose tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abdominal sc and visceral adipose tissue were obtained from women who were undergoing open abdominal surgery (abdominoplasty) or who were undergoing laparoscopic gastric bypass with Roux-en-Y gastroenterostomy surgery for the treatment of morbid obesity. Body fat content was determined using bioelectrical impedance (Tanita TBF-310GS Body Composition Analyzer/Scale, Tanita Corp., Arlington Heights, IL). Each experimental replication involved tissue from a separate individual. The study had the approval of the local institutional review board, and all patients involved gave their informed consent. The patients were on a clear liquid diet the day before surgery, but they had not been on any type of dietary restriction before surgery.

Samples of visceral and abdominal sc adipose tissue were immediately transported to the laboratory. The handling of tissue and cells was performed under aseptic conditions. The tissue was cut with scissors into small pieces (10–20 mg). All the studies used explants of adipose tissue that had been incubated in buffer plus albumin (3 ml/g of tissue) for approximately 30 min to reduce contamination of the tissue with blood cells and soluble factors. At the conclusion of the 30-min incubation, the tissue explants were centrifuged for 30 sec at 400 x g to remove blood cells and pieces of tissues containing insufficient adipocytes to float. The explants were separated from the medium plus the sedimented cells and resuspended in fresh buffer. The explants (500 mg/5 ml) were then incubated in duplicate for 48 h in suspension culture under aseptic conditions.

One gram of cut tissue, again in duplicate, was incubated in 2 ml of incubation medium containing 1.3 mg of bacterial collagenase in a rotary water bath shaker (100 rpm) for 2 h. The collagenase digest was then separated from undigested tissue matrix by filtration through 200-µm mesh fabric. Five milliliters of medium were then added back to the digestion tubes and used to wash the undigested matrix on the filter mesh. This wash solution was combined with the collagenase digest, and SV cells were separated from adipocytes and medium by centrifugation in 15-ml tubes for 1 min at 400 x g. The SV cells are defined as those cells isolated by collagenase digestion that do not float. The SV cells and adipocytes were each suspended in 5 ml of fresh buffer and centrifuged for 10 sec at 400 x g. The medium was removed. The undigested tissue matrix on the nylon mesh, the SV cells, and the adipocytes were then incubated in a volume of 5 ml for the indicated periods.

The serum-free buffer for incubation of adipose tissue and adipocytes was as previously described (30, 31). The pH of the buffer was adjusted to 7.4 and then filtered through a 0.2-µm filter. Aliquots of the medium were taken and stored at –20 C for measurement of release to the medium. Leptin and adiponectin were determined on all samples using RIA kits from Linco Research, Inc. (St. Charles, MO) and by ELISA using reagents from R&D Systems, Inc. (Minneapolis, MN). Lipolysis was measured as glycerol (32). Lactate was measured using lactate dehydrogenase and PGE2 as described by Parfenova et al. (33). IL-6, IL-8, IL-10, TNF{alpha}, HGF, and IL-1ß were measured using ELISA kits from the Central Laboratory of The Netherlands Red Cross that are distributed by Research Diagnostics (Flanders, NJ) or DuoSet ELISA development kits from R&D Systems, Inc. PAI-1 and vascular endothelial growth factor (VEGF) were measured using ELISA kits from American Diagnostica (Greenwich, CT) and Pierce Biotechnology (Rockford, IL), respectively.

All values are shown as the mean or the mean ± SEM. Pearson correlation coefficients were determined using the GraphPad Prism Program (GraphPad Software, Inc., San Diego, CA) assuming a Gaussian population and a two-tailed P value.

BSA powder (Bovuminar, containing <0.05 mol of fatty acid/mol of albumin) was obtained from Intergen (Purchase, NY). Bacterial collagenase Clostridium histolyticum (type 1) was obtained from Worthington Biochemical Corp. (Lakewood, NJ; lot CLS1-4197-MOB3773-B, 219 U/mg). Other chemicals were from Sigma Chemical (St. Louis, MO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Release at 4 h vs. that at 48 h by tissue explants and adipocytes
In the studies shown in Table 1Go, we examined the time course for release of adipokines by tissue explants from 12 obese individuals with an average BMI of 42. Comparison of release on a molar basis indicated that 25-fold more PGE2, 5-fold more IL-8, and 2-fold more IL-6 than adiponectin was released by adipose tissue explants over the first 4 h of incubation in serum-free medium containing 1% albumin. Leptin release was 10% of that seen for adiponectin over 4 h. PAI-1 and HGF were released in amounts greater than those of leptin, whereas IL-10 and TNF{alpha} were released in smaller amounts than of leptin. Even smaller amounts of VEGF and IL-1ß were released over 4 h.


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  		TABLE 1. Comparison of the release of adipokines and other factors at 4 h with that at 24 and 48 h by human adipose tissue

 
We also compared the release of adipokines to the medium by explants of human adipose tissue over 24 and 48 h (Table 1Go). The most striking difference was the marked decrease in TNF{alpha} release between 4 and 48 h. In fact, there was a net disappearance of TNF{alpha} because total release over 24 h was 180 fmol/g, whereas release over the first 4 h was 201 fmol/g. Adiponectin release per 4 h over 24 and 48 h was only 23% and 12%, respectively, of that seen over the first 4 h. The release of leptin per 4 h over 48 h was 46% of that during the first 4 h, whereas that of HGF was 26% of that seen during the first 4 h. There were statistically insignificant changes in the rates of PGE2, IL-8, IL-6, IL-10, VEGF, and IL-1ß release per 4 h over 48 h. In contrast, there was a substantial (>8-fold) up-regulation of PAI-1 release between 4 and 48 h of incubation.

The time course for release by human adipocytes of adipokines at 4, 24, and 48 h is shown in Table 2Go. Data for IL-10 release are not shown because its release was so low during the first 4 h that it could not be accurately determined. Human adipocytes released at least 3-fold more PGE2 over the first 4 h of incubation than of adiponectin (Table 2Go). The accumulation of IL-8 in the medium was only slightly less than that of adiponectin at 4 h but substantially higher over 48 h because of the up-regulation of its release by adipocytes between 4 and 48 h (4.7-fold). Leptin release was only 6% of that for adiponectin over the first 4 h. PAI-1 was released in amounts only slightly less than those of leptin over 4 h but there was a 5.5-fold increase in PAI-1 accumulation per 4 h over 4–48 h. HGF, VEGF, TNF{alpha}, and IL-1ß were released in amounts far less than those of leptin and their release was down-regulated over 4–48 h. Leptin release by adipocytes was up-regulated over 4–48 h, whereas adiponectin release was down-regulated. Up- or down-regulation are used here to refer to changes in the net accumulation of adipokines in the medium that could involve changes in the rate of release as well as that of degradation or both.


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  		TABLE 2. Comparison of the release of adipokines and other factors at 4 h with that at 24 and 48 h by human adipocytes

 
The major difference in adipokine release between 4 and 48 h in adipocytes vs. explants was the up-regulation of leptin release in adipocytes compared with down-regulation in explants (Tables 1Go and 2Go). The release of PAI-1 was up-regulated between 4 and 48 h in both adipocytes and adipose tissue explants, whereas that of adiponectin, TNF{alpha}, and HGF was down-regulated to about the same extent in tissue explants and isolated adipocytes. Minor differences were the greater up-regulation of PGE2 and IL-8 release over 4–48 h in adipocytes as contrasted with explants as well as down-regulation of VEGF release in adipocytes but not in adipose tissue explants.

Effect of BMI on adipokine release by adipocytes and tissue explants from visceral and sc adipose tissue
We compared the release of 11 adipokines as well as that of glycerol and lactate over 48 h of incubation by visceral adipose tissue explants and adipocytes from eight gastric bypass subjects with an average BMI of 45 and eight abdominoplasty subjects with an average BMI of 32. There was no difference between the age or fasting blood glucose of the two groups of patients (Table 3Go). However, the abdominoplasty patients had a significantly lower BMI (–29%), waist measurement (–19%), weight (–26%), and total body fat mass (–43%).


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  		TABLE 3. Comparison of abdominoplasty and gastric bypass patients

 
Leptin, PAI-1, adiponectin, and VEGF release over 48 h were significantly greater by visceral adipose tissue explants expressed per gram of adipose tissue from individuals with a BMI of 32 compared with that of explants from subjects with a BMI of 45 (Table 4Go). The Pearson correlation coefficients were calculated for release of PAI-1, adiponectin, and VEGF by visceral adipose tissue explants vs. BMI for all 16 subjects shown in Table 4Go. For PAI-1 the correlation coefficient was –0.55 (P = 0.03), for adiponectin it was –0.81 (P = 0.001), and for VEGF it was –0.77 (P = 0.001). There were no significant differences in release of other substances per gram of tissue except for lactate formation by tissue explants, which was greater in visceral adipose tissue from individuals with a BMI of 32.


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  		TABLE 4. Comparison between the release of adipokines by explants of human visceral adipose tissue and isolated adipocytes as affected by BMI

 
The release of adipokines by visceral adipocytes was much lower than that by explants of visceral adipose tissue (Table 4Go). However, the reduced release of adiponectin, PAI-1, and VEGF at a BMI of 45 compared with 32 was also seen in adipocytes. For visceral adipocytes, the correlation coefficient was –0.56 (P = 0.023) for adiponectin, –0.48 (P = 0.06) for VEGF, and –0.55 (P = 0.03) for PAI-1 accumulation vs. BMI of the fat donors.

Data for adipokine release by sc adipose tissue and adipocytes from the same individuals are shown in Table 5Go. The data differed from those by visceral adipose tissue explants (Table 4Go) where TNF{alpha}, IL-8, and IL-10 release were unaffected by BMI. However, TNF{alpha}, IL-10, and IL-8 release were markedly higher by sc adipose tissue explants from subjects with at a BMI of 45 compared with explants from individuals with a BMI of 32. PAI-1 release was unaffected by BMI in sc adipose tissue explants (Table 5Go), whereas it was elevated in visceral adipose tissue explants from individuals with a BMI of 32 compared with 45. Adipocytes from sc adipose tissue differed from visceral adipocytes with respect to effects of BMI because release of PGE2 was significantly higher and TNF{alpha} lower in sc adipocytes from individuals with a BMI of 32 (Table 5Go).


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  		TABLE 5. Comparison between the release of adipokines by explants of human sc adipose tissue and isolated adipocytes as affected by BMI

 
Comparison of adipokine release by visceral compared with sc adipose tissue explants
The statistically significant differences between release of adipokines by explants of sc compared with that by explants of visceral adipose tissue from subjects with an average BMI of 32 are shown in Fig. 1Go based on paired comparisons. Visceral adipose tissue explants released more VEGF, IL-6, PAI-1, and PGE2 than did explants of sc adipose tissue. However, the differences in release between sc vs. visceral adipocytes were small except for PAI-1, and none were statistically significant (Fig. 1Go).



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  		FIG. 1. Comparison of adipokine release by visceral vs. sc adipose tissue explants and adipocytes. The data are shown as the percentage increase ±SE of eight paired replications for adipokine release by explants of visceral adipose tissue or adipocytes derived from visceral adipose tissue over that by sc adipose tissue explants or adipocytes from the same individuals (average BMI: 32). Statistically significant differences are indicated as follows: **, P < 0.025; ***, P < 0.01.

 
Comparison of adipokine release by tissue matrix, SV cells, and adipocytes
The much smaller formation of most adipokines by adipocytes as contrasted to tissue explants from the same individuals (Tables 4Go and 5Go) suggested that either collagenase digestion has deleterious effects on adipokine formation or there is much less formation by adipocytes than by the other cells present in adipose tissue. To correct for effects of collagenase digestion, we compared the release of adipokines as well as those of lactate and glycerol by the cells of the tissue matrix, the cells liberated by collagenase digestion passing through a 200-µm mesh filter that do not float (SV cells) and adipocytes. The data are uncorrected for breakage of adipocytes during collagenase digestion or for losses during the isolation and washing of the cell tissue digest. Recovery in the three fractions obtained after collagenase digestion of adipokine release ranged from 73–85% for leptin, IL-8, PGE2, VEGF, and lactate formation. IL-1ß accumulation over 48 h by the three fractions was 107%, and TNF{alpha} was 118% of that by tissue, suggesting some up-regulation, whereas that of glycerol was 64%, and IL-6 was 61%, suggesting some reduction in their formation or increased degradation due to collagenase treatment. The recovery of adiponectin, HGF, IL-10, and PAI-1 ranged between 45 and 51% indicating relative loss of their net accumulation as a result of collagenase digestion.

The data in Table 6Go also indicate that leptin is made only by mature adipocytes that float. In contrast, lipolysis, which is based on glycerol release and immunoreactive adiponectin release, occurred at levels 94 and 64% of that by the nonfat cells, suggesting that cells in the adipose tissue matrix carry out some lipolysis and release adiponectin. PAI-1 was released by adipocytes in amounts that were 25% of that by the nonfat cells, whereas lactate formation by adipocytes was 16% of that by matrix plus SV cells. IL-8 formation by adipocytes was 12% of that by the matrix plus SV cells, whereas formation of TNF{alpha}, VEGF, IL-6, PGE2, IL-1ß, HGF, or IL-10 by adipocytes was 8% or less than that by matrix plus SV cells.


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  		TABLE 6. Comparison of adipokine release with that of glycerol and lactate by the three fractions obtained after collagenase digestion of human adipose tissue

 
The question of whether comparing the release of adipokines by matrix, SV cells, and adipocytes was biased by measurements made at 48 h was examined in another series of experiments. We compared the formation of adipokines at 24 and 48 h by the adipose tissue matrix, SV cells, and adipocytes with that by tissue explants (Fig. 2Go). There was little difference between formation by the tissue matrix, SV cells, or adipoctyes as a percentage of that by tissue explants at 24 compared with 48 h. The one exception was an increase in leptin formation by adipocytes between 24 and 48 h and a lesser decrease over the same time period in PAI-1 formation by matrix. The total recovery of formation by the three fractions obtained by collagenase digestion was comparable to that in the experiments shown Table 6Go. However, there was a greater recovery of adiponectin (85% of tissue at either 24 or 48 h), a greater formation of IL-1ß by SV cells, and a decrease in PAI-1 formation by adipocytes. The data in Fig. 2Go indicate that measurements of release by the different cell fractions at 48 h are as reliable a way to estimate relative contribution of the different cell fractions to total release as measurements at 24 h.



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  		FIG. 2. Comparison of adipokine release at 24 and 48 h by the three fractions obtained after collagenase digestion of human adipose tissue. The values for adipocytes, the SV, and the undigested tissue matrix are expressed as the percentage of the activity per gram of adipose tissue that was taken for digestion from the same experiments. The values are the mean ± SEM of the data for 12 experiments (six with sc and six with visceral adipose tissue) from individuals with an average BMI of 43.

 
In both the studies shown in Fig. 2Go and Table 6Go, leptin was made primarily by adipocytes, whereas adiponectin was also released by the adipose tissue matrix. Lactate formation like that of PAI-1, IL-8, VEGF, PGE2, IL-10, HGF, and IL-6 was primarily an activity seen with the adipose tissue matrix cells. TNF{alpha} was the only adipokine whose accumulation over 48 h was greater by SV cells than by the cells of the tissue matrix (Table 6Go).

Comparison of release by tissue vs. circulating levels of adipokines and other factors
One way to examine whether adipose tissue contributes to circulating levels of the putative adipokines is to compare their release in picomoles/kilogram over 4 h of adipose tissue with their circulating levels in picomoles/liter reported for very obese individuals (BMI >=38). The data in Table 7Go indicate that the release by adipose tissue of adipokines can be divided into several categories. Some adipokines are present in blood at very low concentrations but over 4 h are released by adipose tissue in amounts far greater than their circulating levels. These adipokines (IL-6, IL-8, PGE2, IL-10, IL-1ß, TNF{alpha}, VEGF, and HGF) are probably paracrine factors because their plasma concentrations are very low. However, the rate of release of IL-6, IL-8, and PGE2 is so high that they could potentially contribute to circulating levels. PAI-1 is in a separate category because its circulating concentration in picomoles/liter is 24- to 12,500-fold higher than those of IL-6, IL-8, PGE2, IL-10, IL-1ß, TNF{alpha}, VEGF, or HGF. Therefore, it is more likely that PAI-1 release by adipose tissue could contribute to circulating levels of PAI-1.


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  		TABLE 7. Comparison of adipokine release by pooled visceral and sc adipose tissue over 4 h with circulating levels of the same adipokines

 
Leptin and adiponectin are circulating hormones released primarily by adipose tissue, and their ratios of release divided by circulating levels are 0.13 and 0.04. If we assume that humans with a BMI of 45 have 56 kg of fat and a plasma volume of 6 liters, this gives a ratio of 0.11 for plasma volume in liters divided by fat in kilograms. Thus, over 6 h the release of leptin, if it occurred in vivo at rates comparable to that seen in vitro over 4 h, would be comparable to the blood concentration of leptin.

Total release of adipokines at a BMI of 45 compared with 32 based on release over 48 h
Another way to compare release of adipokines is to express the data as total release of adipokines by adipose tissue and adipocytes over 48 h rather than per gram of adipose tissue. The data in Fig. 3Go are shown as release at a BMI of 45 divided by that at a BMI of 32. The data are the average of the pooled values for visceral and sc adipose tissue or adipocytes. Two major conclusions can be drawn from the data shown in Fig. 3Go. The first is that the release of TNF{alpha}, IL-8, and IL-10 are markedly greater in adipose tissue from women with a BMI of 45 compared with those by adipose tissue explants from women with a BMI of 32. The second is that these marked differences are not seen in adipocytes except for TNF{alpha}. There were 46 and 75% elevations in release of IL-8 and IL-1ß, respectively, by adipocytes from women with a BMI of 45, but for most adipokines total release by adipocytes was reduced at a BMI of 45 compared with 32 (Fig. 3Go). These comparisons are approximations because we have no estimate of the relative contribution of visceral compared with sc adipose tissue to the total body fat content.



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  		FIG. 3. Total release of adipokines at BMI of 45 compared with that at BMI of 32. The total release of adipokines was obtained by averaging the data for visceral and sc adipose tissue explants or adipocytes per gram from Tables 4Go and 5Go. The data for release at a BMI of 45 were multiplied by 1.75 to correct for the 75% greater amount of fat (56 kg) in these individuals compared with that (32 kg) in those with a BMI of 32. Acrp30, Adiponectin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies suggest that human adipose tissue in massively obese individuals is a major site for the synthesis and release of adipokines and other factors. It was surprising that the release of PGE2, IL-8, or IL-6 was much greater than that of adiponectin at all time periods. Although the release of PAI-1 by adipose tissue explants was smaller than that of adiponectin at short time periods, it was greater over 48 h of incubation. The very high rates of release of PGE2, IL-8, IL-6, and PAI-1 suggest that their release by adipose tissue could theoretically contribute to the blood levels of these factors. Total IL-8 release (release per gram of tissue times the amount of fat) was 4-fold greater by adipose tissue from individuals with a BMI of 45 compared with tissue from those with a BMI of 32. However, although Bruun et al. (18) found that plasma levels of IL-8 were elevated in obese subjects after a 24-wk period of weight loss that reduced BMI by 15% there was actually an elevation rather than a decrease in plasma IL-8.

It is established that adiponectin and leptin are hormones released by adipose tissue to the circulation. Possibly PAI-1 is also a hormone released by adipose tissue because its circulating level is comparable to that of leptin in obese individuals and PAI-1 release by adipose tissue explants in vitro is greater than that of leptin. However, Yudkin et al. (39) found no evidence for release of PAI-1 by human sc adipose tissue based on in vivo measurements. Furthermore, Bastard et al. (40) found an increase in PAI-1 protein as well as mRNA in human sc adipose tissue after obese patients lost 5.8 kg on a very low-calorie diet. Our data are in agreement because we found a higher total PAI-1 release by adipocytes from individuals with a BMI of 32 compared with those with a BMI of 45 and no difference in the release of PAI-1 from adipose tissue in vitro. Alessi et al. (41) found that accumulation of hepatic fat was more closely correlated with plasma PAI-1 than was the amount of adipose tissue. The most likely explanation for the elevation of circulating PAI-1 in obesity is still an elevation in TNF{alpha} secretion that has been seen in obese mice (42).

The present results indicate that most of the so-called adipokines released by adipose tissue in vitro come from cells other than mature adipocytes. It should be noted that we incubated the adipose tissue explants with shaking for 30 min before the experiments to remove factors released during cutting the tissue as well as circulating blood cells. The cells that make most of the adipokines in washed adipose tissue explants are not macrophages or the so-called preadipocytes that are readily released from the tissue during collagenase digestion. Rather, they are the cells in the human adipose tissue matrix that are resistant to dissociation during collagenase digestion and release far more adiponectin, PAI-1, IL-8, VEGF, IL-6, IL-1ß, PGE2, IL-10, and HGF than the SV cells. TNF{alpha} is the exception because its release by SV cells was twice that by the matrix cells.

The undigested tissue matrix of human adipose tissue has been a neglected entity because digestion of rodent tissue with collagenase results in relatively little undigested material that does not pass through a 200-µm mesh filter. This is not the case with regard to human adipose tissue where there are large amounts of undigested matrix containing blood vessels and connective tissue as well as other cells imbedded within the matrix that are not released by collagenase digestion. Samad et al. (43) examined the localization of PAI-1 mRNA in murine adipose tissue and found a significant signal only in the smooth muscle cells within the vascular wall. However, after treatment of mice with endotoxin, PAI-1 mRNA was detected in SV cells and adipocytes (at a concentration 66% of that in SV cells). In human SV cells, the release of PAI-1 was about one third that by adipocytes over 48 h, and the release by adipocytes was 25% of that by the combined SV cells and the tissue matrix. This represented a greater (2- to 10-fold) contribution by adipocytes to total release of PAI-1 over 48 h than was seen with respect to release of IL-8, VEGF, IL-6, IL-1ß, PGE2, IL-10, or HGF.

It is established that abdominal obesity is associated with an increased risk of coronary heart disease as well as metabolic complications such as diabetes (44, 45). However, it is still unclear what is responsible for the greater risk of fat accumulation in the abdominal region as contrasted to overall adiposity. Studies on the regional differences in adipokine formation and release by human adipose tissue have reported that leptin release by human sc adipose tissue was greater than that of visceral adipose tissue (46, 47), whereas the reverse was seen with respect to IL-6 (7). Our data were comparable for IL-6 release by tissue explants but not by adipocytes, where we found no differences. Leptin release by sc adipose tissue explants was also greater than that by visceral adipose tissue explants from subjects with a BMI of 45 but not by explants in those with a BMI of 32. There is controversy with regard to PAI-1 and TNF{alpha} with differences being seen in some studies but not in others as reviewed by Arner (48). TNF{alpha} release was greater by sc adipose tissue or adipocytes than by that from visceral tissue or adipocytes when tissue was obtained from humans with a BMI of 42 but not from those with a BMI of 32 in our studies. These data suggest that BMI can affect regional differences in release of adipokines.

Alessi et al. (49) found an enhanced release of PAI-1 by explants of visceral adipose tissue compared with that by sc adipose tissue from individuals with a mean BMI of 27. Their results were comparable to ours using tissue from individuals with an average BMI of 32 in that PAI-1 secretion was greater by visceral adipose tissue and secretion by adipocytes was about 30% of that by tissue. The greatest difference in our studies between visceral and sc adipose tissue release of adipokines was that of VEGF, which was 400% greater by visceral adipose tissue. This and the increased PGE2 secretion by visceral adipose tissue have not been previously reported.

The increased release of IL-6, VEGF, and PGE2 by explants but not by adipocytes of visceral adipose tissue compared with sc suggests that visceral adipose tissue has more nonfat cells that produce these factors than is the case for sc adipose tissue. The increased release of PAI-1 by visceral explants was also seen in adipocytes, but the difference was cut in half. Bastelica et al. (50) suggested that PAI-1 was released by the nonfat cells of human adipose tissue rather than adipocytes and that stromal cells are more numerous in the visceral than in sc adipose tissue. The elevated blood levels of, TNF{alpha} (11, 25), IL-8 (18, 20), and IL-6 (11, 12, 13, 14, 15, 16, 17, 18, 19, 29) in obese individuals may reflect increased release of these adipokines by human adipose tissue because of the high levels of their release in vitro. This is supported by our data that their release by adipose tissue explants was greatly enhanced at a BMI of 45 compared with 32. Circulating IL-10 is elevated in obesity (15), and we found a correlation between BMI and release of IL-10 in sc but not in visceral adipose tissue. However, the release of IL-10 was far smaller than that of IL-8 or IL-6, and it appears less likely that IL-10 release by adipose tissue contributes to circulating levels of IL-10.

There are no reports of elevations of circulating levels of PGE2, VEGF, or IL-1ß in obese humans. Furthermore, our data did not indicate any increases in the release of PGE2, VEGF, or IL-1ß in adipose tissue from morbidly obese individuals with a BMI of 45 compared with their release by fat from individuals with a BMI of 32. IL-18 is another cytokine whose concentration in blood is some 100-fold higher than those of IL-6, IL-8, IL-10, or HGF and elevated in obesity (51). However, we measured the release of IL-18 by human adipose tissue explants and found that it was approximately one tenth that of IL-1ß (our unpublished results). This suggests that the elevated levels of this cytokine seen in obesity do not reflect release by adipose tissue.

The release of TNF{alpha} by both adipose tissue and adipocytes over 48 h is probably a reflection of their release during the first hours of incubation because there was no net increase in their release between 4 and 48 h. It is unclear why net accumulation of TNF{alpha} decreases after 4 h of incubation as this could reflect either an inhibition of TNF{alpha} formation or enhanced rate of degradation. Our data are quite different from those of Gesta et al. (52), who reported a substantial up-regulation of TNF{alpha} in the adipocytes that were isolated from adipose tissue explants after 24 or 48 h of incubation in medium containing serum. However, our data are in agreement with prior reports of greater release of TNF{alpha} (27, 28) as well as greater amounts of TNF{alpha} mRNA (27) in sc adipose tissue from obese humans. Most of this TNF{alpha} release was due to nonfat cells, but there was also enhanced release by adipocytes isolated from sc adipose tissue of massively obese individuals (BMI of 45).

One problem in the interpretation of data obtained by measuring release of factors by adipose tissue and fractions derived from adipose tissue is the possibility that manipulation of the tissue may alter the rate of adipokine release. Ruan et al. (53) found that there was a marked up-regulation over 24 h in the level of IL-6 gene expression as well as release by adipocytes after the removal of adipose tissue from mice. In our studies the release of IL-6 during the first 4 h of incubation was comparable to the release of IL-6 per 4 h in human adipose tissue or adipocytes incubated for 48 h. In contrast, we saw a marked up-regulation of PAI-1 over 48 h in both human adipose tissue and adipocytes. The major differences in release of factors between 4 and 48 h between human adipocytes isolated by collagenase digestion and adipose tissue was a significant up-regulation over time in PGE2 and IL-8 release and down-regulation of VEGF release in adipocytes but not in adipose tissue. The release of the other adipokines did not significantly change between 4 and 48 h of incubation or was affected to the same extent in both adipocytes and adipose tissue making it unlikely that collagenase digestion affected their rate of release over time. The possibility must be kept open, however, that rapid changes in gene expression might occur during the less than 1 h that it takes for the removal and mincing of the adipose tissue. We have done studies examining the release of IL-6 after a 2-h incubation of adipose tissue explants and found that it was slightly but not significantly greater per h than the rate per h over 48 h (our unpublished experiments). Our data suggest that if there is any effect of removal and mincing of human adipose tissue on subsequent release of IL-6 it is rapid in onset and sustained over 48 h.

In conclusion, we have shown that human adipose tissue in primary culture releases more PGE2, IL-8, and IL-6 than of adiponectin or leptin to the medium. The release of PAI-1 and HGF was less than that of adiponectin over 4 h but greater than that of leptin. IL-10 and TNF{alpha} were released in amounts less than those of leptin, whereas VEGF and IL1-ß were released in much lower amounts. Over 90% of adipokine release by adipose tissue, except for adiponectin and leptin, was due to nonfat cells. Although PAI-1 was released to the medium by adipocytes in amounts 30% of that by the tissue matrix the release of all other adipokines by adipocytes was less than 15% of that by the tissue matrix. Furthermore, the greater release of VEGF, IL-6, and PAI-1 by visceral adipose tissue as opposed to abdominal sc adipose tissue was due to the nonfat cells of the tissue. The greatly enhanced total release of TNF{alpha}, IL-8, and IL-10 over 48 h by adipose tissue from individuals with a BMI of 45 compared with a BMI of 32 is primarily due to the nonfat cells present in the adipose tissue.


   Footnotes
 
This work was supported by the Van Vleet Chair of Excellence and the Vascular Biology Center of the University of Tennessee College of Medicine.

Abbreviations: BMI, Body mass index; CRP, C-reactive protein; HGF, hepatocyte growth factor; PAI-1, plasminogen activator inhibitor 1; PGE2, prostaglandin E2; SV, stromovascular; VEGF, vascular endothelial growth factor.

Received October 6, 2003.

Accepted for publication January 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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