Objective Severe obesity is a chronic inflammatory disease where various cytokines/adipocytokines play a key role. Pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumour necrosis factor-α (TNFα) are produced by human adipose tissue dependent on the degree of obesity. Mouse studies suggest a key role of adipose tissue-derived IL-6 in hepatic insulin resistance via modification of liver suppressor of cytokine signalling 3 (SOCS-3) expression.
Design and methods We examined the effect of excessive weight loss on systemic levels, subcutaneous and visceral adipose tissue and liver expression of IL-6 and TNFα in 20 severely obese patients undergoing laparoscopic adjustable gastric banding (LAGB). Furthermore, we studied liver expression of SOCS3, an important regulator of insulin resistance, and fat tissue expression of the anti-inflammatory adipocytokine adiponectin and its receptors. Serum and tissue samples were collected before and 6 months after LAGB surgery.
Results IL-6/TNFα mRNA expression before weight loss were similar in subcutaneous and visceral adipose tissue and much higher compared to hepatic expression. Subcutaneous adipose tissue mRNA expression of both pro-inflammatory cytokines, but especially of IL-6 decreased dramatically after extensive weight loss whereas expression of adiponectin and its receptors increased. Weight loss also led to a significant reduction in liver IL-6 expression, whereas liver TNFα mRNA expression did not change. IL-6 and C-reactive protein serum levels decreased after weight loss whereas TNFα serum levels were below the detection limit before and after surgery. These effects were paralleled by reduced hepatic SOCS3 expression and improved insulin resistance 6 months after LAGB surgery.
Conclusion Expression of IL-6 and TNFα mRNA is more pronounced in adipose compared to liver tissue in patients with severe obesity. Our results highlight excessive weight loss as a successful anti-inflammatory strategy.
- weight loss
- non-alcoholic steatohepatitis
Statistics from Altmetric.com
Significance of this study
What is already known about this subject?
Adipose tissue is a major source of pro-inflammatory cytokines in morbid obesity.
Serum levels of various pro-inflammatory cytokines decrease after successful weight loss whereas levels of adiponectin increase.
What are the new findings?
Expression of various pro-inflammatory cytokines is more pronounced in adipose compared to liver tissue in patients with severe obesity.
Adipose tissue may be the major cytokine source in obesity.
Excessive weight loss massively decreases subcutaneous fat expression especially of IL-6 but also of TNFα whereas adiponectin and its receptors (types I and II) expression increases.
How might it impact on clinical practice in the foreseeable future?
Excessive weight loss is an effective anti-inflammatory strategy.
Targeting pro-inflammatory cytokines (IL-6, TNFα) could improve obesity-related inflammation and insulin resistance.
Obesity is associated with a chronic inflammatory response characterised by abnormal cytokine production, increased synthesis of acute-phase reactants and activation of inflammatory signalling pathways. Adipose tissue secretes a variety of bioactive mediators including adipocytokines such as adiponectin, leptin, resistin, pre-B-cell enhancing factor/Nampt/visfatin or classical cytokines such as the pro-inflammatory mediators tumour necrosis factor α (TNFα) and interleukin 6 (IL-6).1 2
A first link between obesity and a pro-inflammatory cytokine (ie, TNFα) came from a study by Hotamisligil et al, who established the concept of a role for TNFα/inflammation in obesity.3 Expression of IL-6 and TNFα, two major pro-inflammatory cytokines, is markedly regulated at the transcriptional level and increased in human fat cells from obese subjects and patients with insulin resistance.4 IL-6 serum levels are elevated in obese, diabetic patients and weight loss results in decreased IL-6 serum levels.5 6 TNFα expression is also enhanced in adipose tissue of obese subjects and reduced TNFα serum levels are observed following weight loss.7
Insulin resistance is a critical factor in obesity-related disorders such as non-alcoholic fatty liver diseases (NAFLDs)1 and is caused by a variety of factors, including soluble mediators derived from immune cells and/or adipose tissue. Serine phosphorylation of the insulin receptor substrate (IRS1) by inflammatory signal transducers such as c-Jun N-terminal kinase (JNK1) or inhibitor of nuclear factor-κB kinase-β (IKKβ) is considered one of the key aspects that disrupts insulin signalling.8 TNFα and IL-6 have both been demonstrated to be involved in the pathophysiology of insulin resistance.4 TNFα, which also upregulates IL-6 production, increases phosphorylation of IRS1 whereas IL-6 inhibits IRS-1 gene transcription.4 Sabio et al reported that JNK1 signalling specifically in adipose tissue consequent to a high-fat diet causes hyperinsulinaemia, hepatic steatosis and hepatic insulin resistance.9 Importantly, this distal effect of adipose tissue on the liver was mediated via increased JNK1-dependent IL-6 secretion from adipocytes, proving that adipose tissue-derived IL-6 regulates distal metabolic effects in the liver.9 Overall, the role of JNK1 in liver and adipose tissue, however, might be opposing as Sabio et al recently demonstrated that mice with specific ablation of JNK1 in hepatocytes present with glucose intolerance, insulin resistance and hepatic steatosis.10 Furthermore, suppressor of cytokine signalling 3 (SOCS3), upregulated by IL-6,11 interferes with insulin signalling through ubiquitin-mediated degradation of IRS1 and 2, resulting in insulin resistance.12 Conversely, inhibition of SOCS3 in obese diabetic mice improves insulin sensitivity, hypertriglyceridaemia and hepatic steatosis.13
Adiponectin is an anti-inflammatory adipocytokine that signals through two receptors, namely adiponectin receptor-1 (adipoR1) and -2 (adipoR2). Obesity is associated with hypoadiponectaemia and adiponectin levels increase after weight loss.2 14 15 16 As the relative contribution of adipose versus liver tissue as sources of IL-6 and TNFα in obesity and obesity-associated diseases remains unclear, we were interested whether extensive weight loss as achieved after bariatric surgery might affect IL-6 and TNFα expression in these tissues. Furthermore, we also studied adipose tissue expression of adiponectin and its receptors.
Material and methods
Subjects and preoperative assessment
Samples were collected between 2003 and 2007. Selection and preoperative assessment of patients considered for the placement of an adjustable gastric banding device was performed at the Department of Medicine, Innsbruck Medical University, Innsbruck, Austria. Some of the patients have been the subject of an earlier report.16 In all patients current and past alcohol intake was less than 20 g per week. All study participants were negative for hepatitis B and C. Additional investigations were performed to exclude the following disorders: autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, haemochromatosis, Wilson's disease, and α1-antitrypsin deficiency. Included patients did not receive any medications including statins. At the time of laparoscopic surgery, we performed an index liver biopsy under laparoscopic guidance and collected visceral and subcutaneous adipose tissue samples. After 6 months, ultrasound-guided follow-up liver and subcutanous adipose tissue biopsies were performed. Blood samples were drawn in the fasting state by venipuncture at the time of index and follow-up biopsies. Routine clinical parameters were measured by automated techniques and specimens for cytokine and mRNA analysis were stored at –80°C until further analysis. The performance of the study was consistent with the principles of the Declaration of Helsinki and was approved by the local ethics committee of Innsbruck Medical University. Written informed consent was obtained from each subject. The baseline characteristics of the participants are shown in Table 1.
Measurement of serum IL-6 and TNFα
Serum IL-6 and TNFα were measured by Quantikine high-sensitivity enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Samples were run in duplicates on one single plate to exclude interassay variability.
Sample preparation and quantitative real-time PCR
Total RNA was extracted and processed as previously described.17 Briefly, tissue was homogenised in Trizol Reagent (Invitrogen, Paisley, UK) using an IKA (Staufen, Germany) homogeniser according to the manufacturer's instructions. RNA was reverse transcribed using moloney murine leukaemia virus (M-MLV) reverse transcriptase (Invitrogen). cDNAs were used for PCR with Eurogentec SYBR Green reagents (Eurogentec, Seraign, Belgium) on a Stratagene MX3000 bioanalyzer (Stratagene, Amsterdam, The Netherlands). Expression levels were calculated using the standard-curve method. A cDNA from stimulated leucocytes served as cDNA standard for all measurements. The abundance of each mRNA was expressed as ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. Sequences of primers are available upon request.
The results are expressed as means±SD. A prospective power analysis has been performed using G*Power free software tool (University of Kiel, Germany). Assuming an effect size dz of 0.6, and α error of 0.05, and a power (1−β) of 0.8 a total sample size of n=19 has been defined. The degree of association between variables was assessed using Spearman's non-parametric correlation. To determine differences between baseline and 6-month follow-up values Student paired t tests were used for normally and Wilcoxon signed rank test for non-normally distributed variables. All statistical tests were performed with PASW statistics 17.0, and independently confirmed by GraphPad Prism version 5.0. All reported p values are two-tailed and a significance level of 5% was used throughout.
All our severely obese study patients underwent laparoscopic adjustable gastric banding (LAGB) surgery. Type 2 diabetes mellitus as defined by a fasting glucose of >126 mg/dl was only present in one patient (5%) before and none of the patients after weight loss. Six months after LAGB surgery we observed a significant weight loss ranging from 19 to 42 kg corresponding to a mean weight loss of 26.1±7.8 kg in the 20 study subjects. Patients' characteristics are listed in Table 1. Surgery-induced weight loss resulted in a significant decrease of alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) (Table 1) serum levels. A significant improvement of biochemical markers for glucose homeostasis such as fasting glucose, insulin levels and HOMA index was also observed after weight reduction (Table 1). Notably, excessive weight loss was paralleled by a significant decrease in serum IL-6 and C-reactive protein levels as well as leucocyte counts (Table 1). Serum TNFα levels were not detectable in our patients before and after weight loss.
Surgery-induced changes in hepatic cytokine expression
RNA was extracted from index and follow-up liver biopsies and changes in hepatic cytokine expression were assayed by quantitative real-time PCR. Hepatic IL-6 mRNA expression decreased significantly 6 months after weight loss (figure 1A; p<0.05) whereas TNFα expression did not change, as shown in figure 1B. SOCS3 mRNA expression as a surrogate of systemic IL-6 bioactivity decreased significantly in the liver within 6 months after LAGB surgery (figure 1C).
Surgery-induced alterations in subcutaneous adipose tissue cytokine expression
Index and follow-up subcutaneous tissue samples were collected simultaneously with liver biopsies and mRNA was extracted as described in Materials and Methods. IL-6 expression was detected in subcutaneous tissue of all study subjects and decreased dramatically (25.9-fold) within 6 months (figure 2A). Moreover, we observed a significant 2.1-fold reduction in subcutaneous adipose tissue TNFα expression (figure 2B). As shown in figure 2C–E, weight loss was paralleled by a significant increase of subcutaneous expression of adiponectin and its two receptors adipoR1 and adipoR2.
Comparative analysis of tissue-specific expression of IL-6 and TNFα
We next examined potential tissue-specific differences in IL-6 and TNFα expression. The same standard and calibrator cDNAs have been used for each qPCR run to quantify comparable levels of tissue-specific mRNAs. As demonstrated in figure 3A IL-6 mRNA levels were 109-fold higher in subcutaneous and 113-fold higher in visceral adipose tissue compared with hepatic IL-6 expression. Subcutaneous and visceral IL-6 levels did not differ significantly (figure 3A). With respect to TNFα, we again observed a 5.7-fold higher TNFα expression in subcutaneous and 4.3-fold higher TNFα levels in visceral adipose tissue compared with hepatic expression (figure 3B). Subcutaneous and visceral TNFα expression levels were not different (figure 3B).
Correlations of subcutaneous IL-6 and TNFα with demographic, biochemical and inflammatory parameters
All correlations are listed in table 2. Subcutaneous IL-6 levels correlated significantly with liver and serum IL-6 levels (rs=0.339, p<0.001; rs=0.342, p=0.036). There were significant positive correlations between subcutaneous IL-6 and subcutaneous TNFα (rs=0.616, p<0.001), BMI (rs=0.459, p=0.003), aspartate aminotransferase (AST) (rs=0.487, p=0.002) and serum CRP levels (rs=0.387, p=0.021). A significantly negative association was found between subcutaneous IL-6 and subcutaneous adiponectin (rs=−0.313, p=0.049). Of note, subcutaneous IL-6 highly significantly correlated with hepatic SOCS3 expression (rs=0.578, p<0.001), underlining the potential biological relevance of subcutaneous adipose IL-6.
Subcutaneous TNFα positively correlated with liver SOCS3 expression (rs=0.350, p=0.026), as well as BMI (rs=0.329, p=0.038). There was also a negative association between subcutaneous TNFα and subcutaneous adiponectin expression (rs=−0.316, p=0.047).
Although there has been a significant correlation of both subcutaneous IL-6 and TNFα with BMI (Table 2), amount of weight loss after surgery did not correlate with tissue cytokine expression and/or liver histology.
IL-6 and TNFα were among the first inflammatory mediators implicated as predictors or pathogenetic markers of insulin resistance.3 8 18–22 We now demonstrate that (1) adipose tissue including subcutaneous adipose tissue is a major source of IL-6 and TNFα compared to hepatic tissue in human obesity; (2) excessive weight loss results in a dramatic decrease especially of IL-6 and TNFα expression with subsequent reduced expression of hepatic SOCS3 expression and improved insulin sensitivity, and hence evidence of hepatic consequences of these alterations in adipose tissue; and (3) weight loss leads in parallel to an increase of adiponectin and its receptors (types I and II) expression in subcutaneous adipose tissue. The observed improvement in systemic inflammatory parameters after extensive weight loss may therefore be explained to a significant extent by this dramatic change in the cytokine/adipocytokine expression profile in the adipose tissue.
Adipose tissue as a source of IL-6 and TNFα in human obesity
Kern et al first described a significant increase in adipose TNFα mRNA levels with increasing adiposity and showed a significant decrease with weight loss.23 Many other studies have demonstrated an increased expression of TNFα in adipose tissue and also a clear correlation with insulin sensitivity.7 24 The first study suggesting that subcutaneous adipose tissue releases IL-6 has been published more than 10 years ago.25 Interestingly, as calculated by the authors from this study, the whole body adipose tissue mass could contribute to 15–35% of the body's total circulating IL-6 making the adipose tissue a key IL-6 producing organ. Fried et al demonstrated that visceral adipose tissue releases around three times more IL-6 into the circulation than subcutaneous adipose tissue.26
Adipose tissue is composed of many different cell types including adipocytes, pre-adipocytes, monocytes/macrophages, stromovascular cells and others. Fain and colleagues found that adipocytes are a minor IL-6 source and cells retained in the tissue matrix after collagenase digestion are the major adipocytokine and IL-6 source.27 The release of IL-6, TNFα and various other mediators such as IL-1 receptor antagonist by adipocytes is approximately 10–12% of that by non-fat cells such as macrophages present in human adipose tissue.28 29 Whereas the visceral adipose tissue is a major IL-6 source, Bastard et al showed that subcutaneous adipose tissue-derived IL-6 is biologically relevant and regulates systemic insulin sensitivity.30 These studies altogether clearly indicate that both subcutaneous and visceral adipose tissues are major sources of IL-6 and TNFα in human obesity.
Liver IL-6 and TNFα expression: the liver is not the major source but a major target organ for IL-6 and TNFα in obesity
Wieckowska et al demonstrated in patients with NAFLD that liver IL-6 expression correlates with the degree of inflammation, fibrosis and systemic insulin resistance.31 The relative contribution of the liver to systemic IL-6 production in obesity (and obesity-related disorders like NAFLD) is still unknown. Our data now suggest that the liver might be a comparably minor source of IL-6 in obesity and obesity-related disorders such as NAFLD, while most of systemic IL-6 might be derived from adipose tissue. All our study patients had moderate-to-severe liver steatosis and 25% evidence of non-alcoholic steatohepatitis (data not shown).32 However, the liver might indeed be the key target organ for these elevated IL-6 and TNFα levels, as continuous IL-6/TNFα exposure affects hepatic insulin resistance via upregulation of SOCS3. In our patients, reduced hepatic SOCS3 expression was linked to an improvement in insulin sensitivity.
Adiponectin and its receptors: increased adipose expression after excessive weight loss
Adiponectin is mainly synthesised by adipocytes.33 It exists as a full-length protein as well as a proteolytic cleavage fragment (globular adiponectin). Serum levels of adiponectin are reduced in individuals with obesity, type 2 diabetes and states of insulin resistance.34 Initial studies suggested that adiponectin exerted anti-inflammatory effects on endothelial cells through the inhibition of TNFα and NFκB activation.35 We and others have demonstrated that adiponectin is also a potent inducer of IL-10.36 In obese animals, treatment with adiponectin decreases hyperglycemia and improves insulin sensitivity. Furthermore, adiponectin receptor type 1- and type 2-deficient mice develop increased liver triglyceride content, liver inflammation and oxidative stress, leading to insulin resistance and marked glucose intolerance.37
Decreased adipose tissue expression of adiponectin has been demonstrated in many clinical studies.38 Several investigators have shown an increase in adipose adiponectin expression after weight loss.14 Weight loss has been found to lead to an increase of adipoR1 expression, whereas adipoR2 expression is in general much lower in adipose tissue.39 Importantly, weight loss and activation of peroxisome proliferator-activated receptor gamma (PPARγ) by its ligands thiazolidinediones lead to increased adiponectin synthesis.40 We now demonstrate in a large patient series that excessive weight loss increases mRNA expression of adiponectin and both type I and type II receptors. Importantly, mRNA expression of adiponectin negatively correlated with body mass index and expression of the pro-inflammatory cytokines IL-6 and TNFα thus again demonstrating that after extensive weight loss there is a clear shift towards an anti-inflammatory cytokine/adipocytokine profile locally in the adipose tissue.
Conclusion and outlook
Our data demonstrate that excessive weight loss decreases IL-6/TNFα in most patients and increases adiponectin and its receptors expression in the adipose tissue. We, however, currently do not know whether less dramatic weight loss might also influence adipose/liver cytokine expression. As the adipose tissue represents an important source of pro-inflammatory cytokines in obesity, the reported ‘massive decrease’ especially of IL-6 in the adipose tissue might play a major role for the improvement of inflammatory parameters after weight loss. Decreased adipose IL-6 and TNFα synthesis might lead to decreased liver SOCS3 expression and thereby positively affect insulin resistance. Our human data are compliant with the mechanistic model developed by Sabio et al implicating adipose tissue IL-6 secretion as an important determinant of hepatic insulin resistance. Anti-TNF antibody administration in a rat model of high-fat diet-induced insulin resistance reversed steatosis and also improved insulin signalling.41 Targeting pro-inflammatory cytokines in future studies might reveal an attractive treatment concept in obesity-related inflammatory disorders.
The authors thank Barbara Enrich for excellent technical assistance. Furthermore, we are indebted to the nursing staff at the Departments of Medicine and Surgery. This study was supported by the Christian-Doppler Research Society.
Competing interests None to declare.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Ethics Committee of Innsbruck Medical University.
Provenance and peer review Not commissioned; externally peer reviewed.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.