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Department of General Surgery, Nutrition Research Institute Maastricht and University Hospital Maastricht, 6202 AZ Maastricht, The Netherlands
Address all correspondence and requests for reprints to: F. M. H. van Dielen, Department of General Surgery, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. E-mail: f.vandielen{at}ah.unimaas.nl.
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Abstract |
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Weight loss was achieved by gastric restrictive surgery in 27 morbidly obese patients. Preoperative as well as 3-, 6-, 12-, and 24-month postoperative plasma concentrations of inflammatory mediators macrophage inhibitory factor, plasminogen activator inhibitor-1, lipopolysaccharide binding protein, 
-1 acid glycoprotein, C-reactive protein, soluble TNF receptors 55 and 75, and leptin were measured.
Macrophage inhibitory factor levels remained low normal for 6 months, during weight loss, after which they significantly increased to normal levels at 24 months postoperatively. The other inflammatory mediators remained elevated up to minimally 3 months postoperatively; thereafter they decreased significantly. Both TNF receptors remained elevated up to at least 12 months postoperatively to decrease significantly at 2 yr postoperatively.
This study demonstrates that during weight loss, after gastric restrictive surgery, inflammatory mediators remain elevated for at least 3 months postoperatively, suggesting initially an ongoing inflammatory state. However, 2 yr after surgery, the inflammatory mediators reach near normal values.
These findings may be an explanation for the reduced comorbidity seen in morbidly obese patients after gastric restrictive surgery.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) in the development of the obesity-related morbidity such as insulin resistance and cardiovascular disease (4). Moreover, in vivo and in vitro studies have indicated an involvement of macrophage inhibitory factor (MIF) in the pathophysiology of insulin resistance (5). In a previous study, we demonstrated a correlation between levels of inflammatory mediators, acute-phase proteins, and body weight (6). Although a causative relation has not been found yet, it is suggested that elevated levels of C-reactive protein (CRP) are prognostic for the development of cardiovascular disease (7, 8). Also, other proteins related to inflammation are suggested to be associated with the development of obesity-related diseases such as plasminogen activator inhibitor-1 (PAI-1) and MIF (9, 10).
Increased plasma levels of inflammatory markers and acute phase proteins were present without physical evidence of acute or chronic inflammation in morbidly obese (6). It is tempting to speculate that the metabolic stress caused by morbid obesity is responsible for the acute phase response seen in these patients.
Several studies demonstrated that weight loss leads to reduced obesity-related comorbidity (11, 12, 13). Based on the above-mentioned findings, we hypothesized that weight loss, after gastric restrictive surgery, resulting in a reduction of the metabolic stress, leads to a decrease in inflammatory mediators and acute-phase proteins in morbidly obese subjects.
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Subjects and Methods |
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. Weight loss was expressed as percentage excess weight loss (EWL), which could be calculated with the following formula: (preoperative weight – weight after reduction)/(preoperative weight – ideal weight) * 100%.
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VBG
In our hospital the procedure was performed as initially described by Mason (14). Briefly, a small pouch of the stomach (approximately 15–20 ml) was created with a four-row linear stapler (TA-90B, United States Surgical Corp., Norwalk, CT) precisely to the angle of His, and a Dacron band of 5.0 cm in circumference placed through the window formed by a circular stapler (Premium Plus CEEA 31 mm, United States Surgical Corp.), leaving a very small opening for food to pass from the small pouch to the remaining stomach. Because of the small capacity of the gastric pouch, the amount of ingested food is considerably limited, leading to extensive weight loss (15).
Laparoscopic gastric banding (LapBand)
The LapBand (BioEnterics, Inamed, Carpinteria, CA) is a new surgical technique to reduce body weight. The LapBand is made of soft silicone and equipped with an elastic balloon that can be inflated to the desired volume by means of injection postoperatively. After inflation the outlet diameter will be reduced, leading to diminished food intake and consequently to weight loss. This procedure was initially performed as described by Belachew et al. (16). In short, the LapBand was placed laparoscopically around the stomach. For definitive positioning of the band, a 15-ml calibration balloon advanced by the anesthetist and pulled up to the gastroesophageal junction was placed right below the cardia. Under this balloon the band was closed, and with three or four sutures, the ventral aspect of the greater curvature of the stomach is fixed to the pouch to ensure a stable anterior position of the band. Six weeks postoperatively the LapBand was insufflated when weight loss was insufficient (less than 6 kg). During the study period, the LapBand was insufflated as often as needed, up to a maximum of 4.5 ml, to induce sufficient weight loss (approximately 1 kg weight loss per week).
Reagents and materials
Monoclonal antibodies (mAbs) specifically directed against soluble TNF receptor 55 (TNFR55) and soluble TNF receptor 75 (TNFR75) were obtained as described elsewhere (17). Polyclonal rabbit antisera anti-TNFR55 and anti-TNFR75 were obtained by immunizing rabbits with TNFR55 and TNFR75, respectively. Both mAbs 4G1 and 4F8 to leptin, were kindly provided by Dr. R. Devos (Hoffmann La-Roche, Welwyn Garden City, UK).
Human recombinant lipopolysaccharide binding protein (LBP), used as standard, was produced by transfected Chinese hamster ovary cells, kindly provided by Dr. P. Tobias (Research Institute of Scripps Clinic, La Jolla, CA). Polyclonal antibodies to human LBP were obtained by immunizing rabbits with human LBP. Human CRP was obtained from Dade Behring (Deerfield, IL); rabbit antihuman CRP and rabbit antihuman CRP-horseradish peroxidase were purchased from Dako (Glostrup, Denmark).
Human -1 acid glycoprotein (AGP) was obtained from Sigma (St. Louis, MO) and rabbit antihuman AGP from Dako. BSA was purchased from Sigma. Recombinant human leptin and MIF, a mAb antihuman MIF and a polyclonal antibody against human MIF were purchased from R&D Systems (Minneapolis, MN). Peroxidase-conjugated streptavidin was purchased from Dakopatts (Glostrup, Denmark) and 3, 3', 5, 5'-tetramethylbenzidine (TMB) substrate from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Immunomaxisorp plates (Nunc, Roskilde, Denmark) were used for ELISAs.
Immunoassays
Plasma concentrations of both soluble TNF receptors, leptin, LBP, CRP, AGP, MIF, and PAI-1 concentrations were measured using sandwich ELISAs. TNFR55, TNFR75, LBP, CRP, and AGP were quantified as described elsewhere (6, 17, 18). The ELISAs for TNFR55, TNFR75, and LBP had a detection limit of approximately 100 pg/ml. The detection limit for CRP and AGP was approximately 10 pg/ml. The detection of plasma leptin levels was described elsewhere (19). In short, 96-wells plates were coated overnight at 4 C with an antihuman leptin antibody and diluted plasma samples as well as a dilution series of recombinant human leptin were added to the plate. Bound leptin was detected with a second antihuman leptin antibody, followed by peroxidase-conjugated goat antimurine IgA and TMB. The detection limit of this leptin assay is 0.04 ng/ml.
PAI-1 concentrations were measured using an ELISA, kindly provided by Dr. T. Kooistra (Leiden University Medical Center, Leiden, The Netherlands). In short, microtiter strip plates were coated with a high-affinity mAb PAI-1 3–3B against PAI-1. Detection for PAI-1 was carried out using a horseradish peroxidase-labeled antibody rabbit anti-PAI-1 followed by TMB. For MIF quantification, plates were coated with a mAb against human MIF. After adding the samples, detection was carried out with a biotinylated mAb against human MIF, followed by peroxidase-conjugated streptavidin and substrate.
All plasma samples were analyzed in the same run. When plasma concentrations exceeded the upper detection limit of the assay, samples were additionally diluted and analyzed in a separate run with an overlap to correct for interassay variation. The intra- and interassay coefficients of variance of the various assays were less than 10%.
Statistical analysis
Data are given as mean and SD except for MIF and PAI-1 because of no normal distribution. Statistical analysis was performed nonparametrically and two sided. The Wilcoxon signed ranks test was used to analyze differences between preoperative and postoperative values, within the morbidly obese subjects.
Pearson correlation coefficients were computed between body mass index (BMI) and leptin on the different time points. Statistical analyses were performed using the SPSS 10.0.7 statistical package (SPSS Inc. (Chicago, IL). P < 0.05 was denoted as statistically significant.
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Results |
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Table 1
summarizes the characteristics of the total of 27 morbidly obese patients studied. No differences in preoperative BMI was observed between both types of operation technique. As shown in Fig. 1A, BMI significantly decreased after surgery from 46.7 ± 5.8 kg/m2 (mean ± SD) preoperatively to 33.0 ± 4.8 at 24 months postoperatively (P < 0.001). BMI decreased most strikingly in the first 3 months postoperatively. As shown in Table 2, the relative EWL also was more in the period 0–3 months, compared with 4–6 months postoperatively. In the following 7- to 12-month period, the increase of EWL was significantly lower in comparison with the 0- to 6-month period. From 12 to 24 months, postoperative EWL hardly changed.
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, the reduction in leptin levels was most pronounced in the first 3 months, in which the strongest decrease in EWL was observed.
In our previous study, we also demonstrated a correlation between body weight and acute-phase proteins. The acute-phase proteins MIF and PAI-1 are, besides leptin, also produced by adipose tissue (20, 21, 22). Next to this, MIF is considered to be related to insulin resistance (5) as well as to the development of atherosclerosis (9). PAI-1 is demonstrated to contribute to the increased susceptibility to atherogenesis, described in insulin-resistant patients with obesity (23, 24). In this context MIF and PAI-1 levels were measured in weight-losing morbidly obese subjects. Interestingly, in contrast to leptin levels, plasma MIF levels remained stable for the first 6 months postoperatively at a relatively low level. Thereafter MIF levels significantly increased to reach levels of lean individuals of 0.71 ± 0.58 ng/ml at 24 months (Fig. 2A). PAI-1 levels significantly decreased rapidly after surgery from 23.0 ± 14.0 ng/ml preoperatively to 7.5 ± 4.5 ng/ml at 12 months postoperatively (Fig. 2B). PAI-1 levels displayed an initial strong reduction, being restricted to the first 3 months postoperatively. After 12 months PAI-1 levels remained rather stable, although with a wide SD, at 9.2 ± 15.6 ng/ml up to 24 months postoperatively.
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In addition, the plasma levels of the acute-phase proteins LBP, CRP, and AGP were measured (Fig. 3). LBP levels did not change significantly during the first 6 months postoperatively. However, at 12 and 24 months postoperatively, LBP levels decreased significantly (P < 0.05; 107.6 ± 77.4 and 78.9 ± 39.4 µg/ml, respectively), compared with preoperative values (134.7 ± 98.2). Similarly, CRP levels did not change up to 6 months postoperatively. Only after an EWL of more than 40% could a significant reduction in CRP levels be observed (P < 0.05).
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The effect of weight loss on both soluble TNF receptors
Both soluble TNF receptor levels did not change during the first year postoperatively. However, after 24 months TNFR55 was significantly decreased (P < 0.05), compared with preoperative values, whereas TNFR75 remained unchanged. The levels of both soluble TNF receptors at 24 months postoperatively were still significantly higher, compared with levels in lean subjects (6).
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Discussion |
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Previously it has been demonstrated that inflammatory mediators are elevated in obese subjects (6, 27, 28). In recent studies a regulatory role for leptin on the immune response has been proposed (29, 30, 31, 32). In this context, the elevated plasma leptin concentrations in morbidly obese patients may modulate the immunological homeostasis, leading to increased concentrations of acute-phase proteins and other inflammatory mediators, characteristic for a chronic inflammatory state. However, Hukshorn et al. (33) demonstrated that injections with high concentrations of recombinant leptin, in obese patients with already elevated plasma leptin levels, did not affect plasma levels of different acute-phase proteins, indicating that in obese individuals leptin does not directly regulate acute-phase protein levels.
Another important protein produced by adipose tissue is MIF, an acute-phase protein that is shown in animal models to be released by anterior pituitary cells in response to endotoxin (34). Recent animal and in vitro studies suggested that MIF is linked to the development of atherosclerosis (9, 10). These findings might suggest that high MIF concentrations are to be expected in morbidly obese subjects. In contrast, here it is demonstrated that MIF levels in morbidly obese subjects are low, although still in the normal range, and increase postoperatively with decreasing body weight.
An explanation for these decreased MIF levels in morbidly obese subjects appears to be the reduced insulin sensitivity, often seen in morbidly obese subjects. The intracellular glucose level was found to be critical for the MIF protein content in adipose tissue.
In this context MIF mRNA expression of both epididymal fat pads of Tokushima fatty rats and Wistar fatty rats was found to be down-regulated, whereas plasma MIF levels of Wistar fatty rats increased upon treatment with pioglitazone, an insulin sensitizer (5). These data suggest that the interplay between glucose and insulin is central to the regulation of MIF concentrations, leading to an increase of plasma MIF levels with enhanced insulin sensitivity. Because it is known that, with an increase in body weight, insulin sensitivity decreases (35), these data might explain the unexpected low concentrations of MIF measured in morbidly obese subjects.
Furthermore, MIF is demonstrated to sustain macrophage survival and function by suppressing activation-induced, p53-dependent apoptosis (36). This finding combined with the preoperative low concentrations of MIF found in this report suggest an impaired macrophage function in morbidly obese subjects. If so, this might be an explanation for the observation that morbidly obese patients demonstrate more postoperative complications, compared with lean subjects (37, 38). However, further studies are necessary to unravel the pathophysiological role of MIF in morbidly obese subjects.
Although mainly produced in the stromal cell, besides leptin and MIF, PAI-1 is also adipose tissue derived (36). Mitchell et al. (36) demonstrated that PAI-1 expression was 5-fold higher in the visceral fat that in the sc fat. Next to this, Janssen and Ross (39) demonstrated that during weight loss the reduction in visceral fat was faster than in sc fat. In line with these data, the present results show that PAI-1 levels decreased rapidly with a decrease in body weight.
On the other hand, despite significant weight loss and concurrent decrease of plasma leptin levels, the levels of the acute-phase proteins LBP and CRP and both TNF receptors remained elevated up to 6 months or longer. The acute-phase protein AGP, like PAI-1, showed a rapid decrease after surgery. Two years after gastric restrictive surgery, all acute-phase proteins were significantly reduced.
Different explanations can be proposed for the initially sustained elevation of inflammatory mediators. First, the effect of the operation and the subsequent healing process might be a possible explanation for the enhanced inflammatory state during the first 6 months postoperatively. However, various studies demonstrate that the highest CRP levels occur 12–48 h after surgery (40, 41, 42) and will remain elevated only for a period of maximum 12 d postoperatively (42). Second, a nonalcoholic steatohepatitis might be an explanation for the sustained elevation of inflammatory mediators. Rapid weight loss can result in a mild increase in inflammatory lesions (hepatitis) (43). Increased concentration of intracellular fatty acids, as has been observed during rapid weight loss, could explain these inflammatory lesions in the liver after weight loss. Such elevated levels of free fatty acids may be directly toxic for the liver or lead to oxidative stress. However, severe nonalcoholic steatohepatitis and hepatic failure are seldom described after gastroplasty or gastric bypass (44).
A third possible explanation for the prolonged elevation of inflammatory mediators might be an enhanced metabolic stress response due to relative starvation. It is demonstrated in very malnourished anorexia nervosa patients that TNF and IL-1ß were elevated, compared with healthy controls. After refeeding these inflammatory mediators returned to normal levels (19, 45, 46, 47). If we assume that extensive weight loss after gastric restrictive surgery is comparable with starvation, this could be an explanation for the prolonged elevation of inflammatory mediators. We consider therefore that from the moment onward that the body weight is stabilized at approximately 12 months postoperatively, the metabolic instability will improve, leading to a reduction in inflammatory mediators. This was reflected by the reduced CRP levels at 24 months after gastric restrictive surgery. Taken together, CRP levels were postoperatively not directly correlated with BMI but rather also influenced by the disturbed metabolism.
Next to the acute-phase proteins, soluble TNFR55 and TNFR75 were measured. In earlier reports, levels of soluble TNF receptors were demonstrated to be of value in characterizing an inflammatory response (48, 49). In an earlier report we demonstrated an increase in both soluble TNF receptors with increasing body weight (6). Whether this elevation of soluble TNF receptor levels is caused by elevated levels of TNF, produced by adipocytes, is as yet unknown. If so, soluble TNF receptor levels would decrease with decreasing body weight due to a reduction in body fat. However, in this report we demonstrate a sustained elevation, compared with healthy controls, up to 1 yr postoperatively for both soluble TNF receptors, despite the weight loss. The fact that the studied subjects were still obese (mean BMI was 33.0 kg/m2), even at 24 months after gastric restrictive surgery, might be an explanation for the sustained elevation of both soluble TNF receptors.
An increasing number of studies demonstrate a central role for inflammatory processes in the pathogenesis of cardiovascular disease and insulin resistance (8, 50). Plasma levels of several markers of inflammation have been found to be associated with an increased cardiovascular risk in a variety of clinical settings. Especially CRP and PAI-1 are important prognostic factors for the development of cardiovascular disease (24, 51). As demonstrated in this report, during weight loss due to gastric restrictive surgery, almost all inflammatory mediators measured eventually decrease. Next to this, it is demonstrated by others that after gastric restrictive surgery, obesity-related comorbidities like cardiovascular disease and diabetes mellitus are strongly reduced (12, 13).
So hypothetically the decreased inflammatory mediators after weight loss in morbidly obese patients, as shown in this study, may be related to an improvement of comorbidities in these patients.
In summary, this study demonstrates that after gastric restrictive surgery, BMI and leptin levels significantly decrease, whereas MIF levels increase. Despite the extensive weight loss, both soluble TNF receptors as well as the acute phase proteins LBP and CRP remained elevated up to 6 months postoperatively, suggesting an ongoing inflammatory state in these obese subjects. However, 2 yr after gastric restrictive surgery, when the body weight has stabilized, the levels of all inflammatory mediators were strongly decreased.
Hypothetically this improved metabolic state might be an explanation for the reduced obesity-related comorbidity after successful surgical treatment for morbid obesity.
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Acknowledgments |
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Footnotes |
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Abbreviations: AGP, 1 Acid glycoprotein; BMI, body mass index; CRP, C-reactive protein; EWL, excess weight loss; LBP, lipopolysaccharide binding protein; mAb, monoclonal antibody; MIF, macrophage inhibitory factor; PAI-1, plasminogen activator inhibitor-1; TMB, 3, 3', 5, 5'-tetramethylbenzidine; TNFR55, TNF receptor 55; TNFR75, TNF receptor 75; VBG, vertical banded gastroplasty.
Received December 11, 2003.
Accepted May 6, 2004.
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References |
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receptors in obese subjects. Int J Obes Relat Metab Disord 22:1239–1243[CrossRef][Medline]
. Biochem Biophys Res Commun 235:94–98[CrossRef][Medline]
(TNF-) and soluble TNF receptors in patients with anorexia nervosa. J Clin Endocrinol Metab 84:1226–1228 and total parenteral nutrition-induced anorexia. Surgery 118:756–760;discussion 760–762[CrossRef][Medline]
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