Cytokines and hormones secreted by adipose tissue can influence energy metabolism and may be directly involved in pathological processes in obesity. Inflammation has long been linked to obesity and insulin resistance. Prior studies have demonstrated that the adipose tissue-derived proinflammatory cytokine TNF-α is a mediator of insulin resistance and provided insight into the pathophysiological link of inflammatory pathways with insulin resistance in obesity (20
). In vitro and in vivo in animal models, TNF-α has been shown to modulate the production of other adipokines, including adiponectin, leptin, and resistin (10
). What are the effects of blocking TNF-α activity on these adipokines in humans? We present evidence here that TNF-α neutralization by etanercept in humans with the metabolic syndrome leads to an increase in total adiponectin but not HMW adiponectin, thereby decreasing the HMW-to-total adiponectin ratio. TNF-α neutralization did not affect circulating leptin levels; however, it tended to decrease circulating resistin. Furthermore, treatment with etanercept was associated with increased skeletal muscle adiposity as measured by CT muscle attenuation. Causes of insulin resistance in the metabolic syndrome are multifactorial, and our study further underscores the notion that TNF-α-mediated inflammation is but only one of the multiple possible mechanisms of insulin resistance in obese humans.
TNF-α exerts its effects by binding to two different cell-surface receptors that have been identified: TNFR1 or TNFR2 (30
). TNF-α-converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase-disintegrin that cleaves and releases the extracellular domain of TNFR1 and TNFR2, thus releasing soluble TNF-α receptors sTNFR1 and sTNFR2 (43
). Etanercept is a soluble TNF-α receptor fusion protein (p75 TNF-α receptor 2 fused to Fc
fragment of human immunoglobulin G1
) that binds TNF-α, blocks its interaction with cell surface receptors, and therefore reduces the biological activity of TNF-α (33
). Etanercept prolongs the half-life of TNF-α (with a subsequent rise in measured serum TNF-α levels), yet etanercept renders TNF-α biologically inactive and unavailable to bind to its receptor. Given the difficulties of interpreting various TNF-α assays (43
), measurements of sTNFR1 and sTNFR2 may be more reliable in the assessment of TNF-α activity. According to the manufacturer, etanercept is not well distributed in adipose tissue.
Hotamisligil and coworkers (17
) have previously found sTNFR2 circulating levels to be significantly elevated in obese female subjects compared with lean control subjects, and their expression levels in adipose tissue of TNFR2 were elevated as well. They showed that TNFR2 expression levels in adipose tissue were strongly correlated with BMI, degree of hyperinsulinemia, and level of TNF-α mRNA expression in fat tissue. In our study, we also found that baseline circulating sTNFR2 correlated with BMI, total body fat, and subcutaneous abdominal fat as did sTNFR1, but we did not find a significant relationship between sTNFR1 or sTNFR2 with insulin levels.
Notably, levels of sTNFR1 and sTNFR2 before etanercept administration correlated with total adiponectin levels but not with HMW adiponectin. In addition, we also found sTNFR1 and sTNFR2 correlated with leptin. Kirchgessner and colleagues (26
) had previously shown that TNF-α regulates leptin secretion posttranslationally in cultured adipocytes and in mice in a secretagogue-like fashion. In contrast, etanercept may not affect leptin in vivo, as it sequesters TNF-α in the circulation and may not be well-distributed in adipose tissue.
Maeda and colleagues (31
) have previously shown in vitro that TNF-α reduced the expression and secretion of adiponectin in 3T3-L1 adipocytes in a dose-dependent manner. Simons and colleagues (48
) have also demonstrated that TNF-α suppressed total adiponectin secretion in cultured human adipocytes in vitro, but they found the amount of secreted HMW complexes were not altered by TNF-α. Consistent with these in vitro results, we have previously shown that total adiponectin increases with TNF-α blockade (4
). Upon further investigation, we now find that etanercept causes an increase in circulating total adiponectin levels but not the HMW form. The baseline significant correlation of total adiponectin, but not HMW adiponectin, to sTNFR1 and sTNFR2 is also consistent with our finding that TNF-α neutralization affected total adiponectin levels and not HMW adiponectin.
Although many studies have demonstrated a significant role for adiponectin in obesity and insulin sensitivity (3
), other studies have shown a lack of a relationship between total circulating adiponectin and obesity or insulin sensitivity (36
). Although thiazolidinedione treatment upregulates mRNA expression and plasma concentrations of adiponectin (6
), another insulin-sensitizing agent, metformin, does not affect adiponectin concentrations (6
). Both metformin and adiponectin can increase hepatic insulin sensitivity via activation of AMP-activated protein kinase (56
). Therefore, thiazolidinediones may increase hepatic insulin sensitivity via raising adiponectin, whereas metformin has direct effects on AMP-activated protein kinase downstream of adiponectin. Levels of HMW adiponectin or the proportion of HMW adiponectin to total adiponectin may possibly be more representative of adiponectin’s biologic activity. Pajvani and colleagues (38
) have previously shown that db/db
mice have a lower proportion of circulating adiponectin in the HMW form but similar total adiponectin levels compared with wild-type mice. In addition, they also demonstrated that diabetic patients have decreased HMW-to-total adiponectin ratios compared with lean controls. Furthermore, they found the ratio of HMW to total adiponectin to correlate better with insulin sensitivity. Thiazolidinediones have been shown to increase HMW adiponectin (38
). In support of these findings, Waki and colleagues (53
) found that mutations in the human adiponectin gene, G84R and G90S, which cause impaired multimerization of adiponectin, are associated with diabetes. T-cadherin has been identified to be a receptor for HMW and hexameric adiponectin (21
). HMW adiponectin levels have been shown to be inversely related to insulin resistance in patients with the metabolic syndrome traits (16
). In our study, the lack of increase in HMW adiponectin and/or the decrease in the HMW-to-LMW ratio may help explain the lack of improvement in insulin sensitivity with etanercept.
In this cohort of patients with the metabolic syndrome, we found that resistin was the adipocytokine that best correlated with insulin resistance. Resistin is a hormone produced by white adipose tissue that is induced during adipocyte differentiation and is reduced by thiazolidinediones (50
). The role of resistin as a mediator of insulin resistance in rodents is well-established (2
). However, whether resistin is involved in glucose regulation in humans remains controversial (1
). On the other hand, resistin has been linked to inflammation in humans (28
). These findings are consistent with our results in which there was a trend of etanercept to lower resistin levels.
In vitro data have shown that TNF-α affects resistin mRNA expression and protein secretion. Fasshauer and colleagues (11
) have found that resistin mRNA expression and protein secretion were inhibited by 70–90% in 3T3-L1 adipocytes after treatment with TNF-α in a time- and dose-dependent fashion. These results were corroborated by Shojima and others (47
) in 3T3-L1 adipocytes. Paradoxically, Kaser and others (22
) have found that TNF-α increased resistin mRNA expression in human peripheral blood mononuclear cells. The latter finding may be more directly applicable to humans, since resistin expression appears to be predominantly in mononuclear cells and low/absent in adipocytes in humans, contrary to rodents (46
). Therefore, our finding of a trend toward a decrease in resistin with etanercept is biologically plausible. Interestingly, the converse also occurs, since human recombinant resistin has been shown to induce the secretion of TNF-α in macrophages. This mutual amplification may play a role in the inflammation-hormonal signaling interaction.
Neutralization of TNF-α resulted in an increase in thigh muscle adiposity as evidenced by decreasing muscle attenuation among patients with the metabolic syndrome treated with etanercept compared with placebo. Because Goodpaster and colleagues (14
) have validated that skeletal muscle attenuation determined by CT is related to muscle lipid content, the decrease in muscle attenuation with etanercept treatment suggests muscle lipid content increased with TNF-α neutralization. The mechanism for the possible increase in muscle lipid content by blocking TNF-α is unknown; however, decreased lipolysis by inhibition of TNF-α within muscle tissue may be a potential mechanism.
In this study, we also showed that muscle attenuation is reduced, indicating more muscle adiposity, in patients with the metabolic syndrome compared with a control population. At baseline, increased indexes of total body and regional fat were associated with muscle attenuation, indicating muscle adiposity is most strongly related to total body and visceral fat in the metabolic syndrome.
In adipose tissue, TNF-α induces lipolysis by decreasing lipoprotein lipase activity and possibly stimulating hormone sensitive lipase. TNF-α decreased transcription of adipocyte lipoprotein lipase gene in vitro (23
). In human adipose tissue, Kern and colleagues (24
) demonstrated that TNF-α expression inversely correlated with lipoprotein lipase activity. Starnes and colleagues (49
) administered recombinant human TNF-α intravenously to patients as part of an antineoplastic trial and found >80% increase in glycerol turnover and >60% increase in free fatty acid turnover, indicating increased whole body lipolysis. TNF-α is expressed in muscle cells, and higher expression of TNF-α occurs in muscle tissue and cultured muscle cells from insulin-resistant and diabetic patients (44
); however, the effect of TNF-α or TNF-α blockade on muscle lipolysis is unknown. Our data showing a possible increase in muscle adiposity by etanercept raises the question whether TNF-α blockade decreases lipolysis within muscle.
Increased triglyceride deposition in skeletal muscle is correlated with insulin resistance (27
). Possible augmentation of muscle lipid content in the etanercept-treated group may be related to the lack of improvement in insulin sensitivity, contrary to animal data. Hotamisligil and colleagues demonstrated an increase in insulin sensitivity using a euglycemic clamp in mice after TNF-α neutralization for 3 days (20
). Perhaps this duration of TNF-α neutralization in rats did not provide enough time for intramyocellular lipids to accumulate. To see if there was a difference in insulin sensitivity after a shorter duration of TNF-α blockade in our study, we analyzed HOMA in the patients that received etanercept at 1 wk after treatment, and there was no improvement when compared with baseline HOMA. Furthermore, single-dose intravenous infusion of a recombinant TNF-α receptor-IgG fusion protein in humans did not improve insulin sensitivity as measured by euglycemic clamp in obese insulin-resistant patients (40
), nor did recombinant human TNF-α neutralizing antibody (CDP571) affect insulin sensitivity in obese type 2 diabetic patients (35
). In contrast to the animal studies, the lack of improvement in insulin sensitivity in humans could also be the result of differences in the regulation of skeletal muscle fatty acid metabolism between rodent models and humans.
Surprisingly, muscle attenuation decreased despite a significant rise in plasma total adiponectin levels with etanercept treatment. Animal data have shown adiponectin can enhance lipid oxidation and reduce muscle triglycerides (12
). Weiss and coworkers (55
) have found obese adolescents to have reduced adiponectin levels, and they found that adiponectin levels were inversely related to intramyocellular lipid accumulation, independent of percentage total body fat and central adiposity. In our current study, etanercept likely directly increased adiponectin through effects of blocking TNF-α but simultaneously increased muscle adiposity by decreasing muscle lipolysis. Because etanercept increased adiponectin and simultaneously increased adiposity, our data suggest that the changes in muscle adiposity resulting from etanercept were not related to an effect of adiponectin. In contrast, the significant correlation between decreased free fatty acids and increased muscle adiposity in response to etanercept supports a potential effect of etanercept to decrease lipolysis and increase muscle adiposity through this mechanism. Furthermore, we saw no overall increase in BMI with etanercept treatment in the primary study; thus, increased muscle adiposity could not be attributed to increased overall body fat. In addition, it is unlikely that muscle attenuation changed because of a change in muscle mass or muscle volume, since total lean body mass measured by DEXA showed no change with etanercept treatment (4
Our study has several potential limitations. The dosing duration was relatively short but adequate to result in significant alterations in adipocytokine concentrations. The effects of longer-term administration of etanercept on glucose homeostasis in humans are not known. Although muscle fat content can be approximated by X-ray attenuation by CT, intramyocellular lipid content was not directly measured. It is uncertain if the muscle attenuation decreased because of extramyocellular fat interspersed within the muscle tissue or lipid within the myocyte.
In conclusion, we investigated the novel effects of etanercept on circulating adipocytokines and muscle adiposity in patients with the metabolic syndrome. We have previously reported that etanercept significantly improved inflammatory markers, including c-reactive protein, total adiponectin, and fibrinogen, but had no effects on insulin resistance in patients with the metabolic syndrome (4
). In this experimental paradigm, we extend these findings to demonstrate a decrease in the HMW-to-total adiponectin ratio and an increase in thigh muscle adiposity among patients with the metabolic syndrome treated with etanercept compared with placebo over a short 4-wk period. These negative effects on HMW adiponectin ratio and on muscle adiposity may tend to counteract potential beneficial effects of etanercept on insulin resistance and explain the absent effect of etanercept on insulin sensitivity in this and other short-term human studies. Etanercept appears to inhibit the TNF-α-mediated inflammatory cascade on obesity, but further long-term studies with more direct endpoints are required to study the effects on adipocytokines and muscle fat and clinical consequences of these changes.