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gene expression in human subcutaneous adipose tissue
Department of Infectious Diseases, The Copenhagen Muscle Research Centre, Rigshospitalet, DK-2100 Copenhagen, Denmark
Submitted 18 June 2003 ; accepted in final form 30 September 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) are found in patients with hyperinsulinemia. Insulin stimulates release of IL-6 from adipocyte cultures, and it stimulates IL-6 gene expression in insulin-resistant, but not control, rat skeletal muscle. In addition, TNF- may be involved in the pathogenesis of insulin resistance. Therefore, we studied the effect of insulin on IL-6 and TNF- gene expression in human skeletal muscle and adipose tissue. Nine healthy young volunteers participated in the study. They underwent a 6-h hyperinsulinemic euglycemic clamp at a fixed insulin infusion rate, with blood glucose clamped at fasting level. Blood samples drawn at 0, 1, 2, 3, 4, 5, and 6 h were analyzed for IL-6 and TNF-. Muscle and fat biopsies, obtained at 0, 2, 4, and 6 h, were analyzed for IL-6 and TNF- mRNA with real-time PCR. IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, in adipose tissue (ANOVA P = 0.027), whereas there was no significant effect of insulin on skeletal muscles. Plasma IL-6 increased during insulin stimulation. TNF- mRNA increased 2.4-, 1.4-, and 2.2-fold in adipose tissue (ANOVA P = 0.001) and decreased 0.74-, 0.64-, and 0.68-fold in muscle tissue (ANOVA P = 0.04). Plasma levels of TNF- were constant. In conclusion, the finding that insulin stimulates IL-6 and TNF- gene expression in adipose tissue only and inhibits the TNF- production in skeletal muscles suggests a differential regulation of muscle- and adipose tissue-derived IL-6 and TNF-. cytokines; euglycemic clamp
(TNF-) have been found in patients with obesity and/or insulin resistance (12, 13, 15, 17, 30). IL-6 knockout mice develop late-onset obesity and insulin resistance, which can be partially reversed by IL-6 replacement (31), indicating that IL-6 is not causing insulin resistance but, rather, reversing this condition. Insulin resistance is associated with high levels of IL-6, and therefore the possibility exists that insulin stimulates the production of IL-6. TNF- induces insulin resistance in rodents (11), whereas its role in insulin resistance and glucose turnover in humans remains to be determined. During the past few years, it has been demonstrated that working skeletal muscles produce and release IL-6 but not TNF- (18, 28). In addition, Carey et al. (3) recently demonstrated an increased IL-6 gene expression in insulin-resistant rat muscle, but no change in control animals, after insulin stimulation (3), suggesting that muscle-derived IL-6 could impair the TNF- gene expression in muscle tissue. Furthermore, adipose tissue contributes to the production of IL-6 (7, 8, 13, 16) and TNF- (4, 10), and a correlational relationship exists between circulating levels of IL-6 and TNF- on one hand and truncal fat mass on the other (19). In an in vitro study (29), Vicennati et al. demonstrated that insulin induced stimulation of IL-6 secretion from human adipocytes, and a recent study (6) demonstrated that insulin enhanced IL-6 transcription in fully differentiated murine 3T3-L1 adipocytes. Although high levels of TNF- mRNA in muscle tissue have been demonstrated in patients with insulin resistance (22), it is less clear whether a causal relationship exists between insulin and TNF-. Thus we hypothesized that in vivo insulin stimulation would induce IL-6 gene expression in adipose tissue, but not in skeletal muscles, in healthy humans and that insulin might be involved in the regulation of TNF-. |
MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nine healthy human males [mean age 25.8 yr (range 22–36 yr); mean body mass index 23.6 kg/m2 (range 20.2–26.6 kg/m2)] with an unremarkable medical past were included after giving oral and written informed consent. Before the study, all nine subjects had a thorough clinical examination. Blood samples for renal, hepatic, and thyroid function, hemoglobin, white blood cell counts, electrolytes, and plasma glucose were analyzed as well. All tests were normal. The study was approved by the Scientific Ethics Committee of Copenhagen and Frederiksberg Municipalities (KF 01–204/02).
Study Design
All subjects received a 6-h hyperinsulinemic euglycemic clamp. On the study day, the subjects reported to the laboratory after an overnight fast. A peripheral catheter was placed in an antecubital vein for blood sampling and in the contralateral antecubital vein for infusion of insulin and glucose. A peripheral catheter was placed in a dorsal hand vein; this hand was then wrapped in a heating blanket to obtain arterialized venous blood for measurement of glucose and potassium. The electrocardiogram was continually monitored; heart rate and tympanic temperature were recorded every hour.
Insulin [Actrapid, Novo Nordisk Insulin; 100 U/ml] was infused continuously at an infusion rate at 0.08 U·min–1·m–2. Glucose (200 g/1,000 ml) was infused by a computer-controlled infusion pump at rates adjusted to maintain blood glucose at baseline levels (fasting level). To maintain potassium at the baseline value, isotonic saline with potassium (51 meq/l) was infused continuously during the study. Arterialized blood was analyzed at intervals of 10 min for glucose and potassium concentrations. Venous blood sampling for cytokines and insulin concentrations was drawn at baseline and every hour (0, 1, 2, 3, 4, 5, and 6 h). Muscle biopsies obtained from musculus quadriceps, vastus lateralis, and abdominal subcutaneous adipose tissue were taken at baseline and every second hour (0, 2, 4, and 6 h).
Measurements
Cytokines in plasma. Samples were drawn into tubes containing EDTA and centrifuged. Plasma was stored at –80°C until analyzed. Plasma concentrations of IL-6 and TNF- were measured by the enzyme-linked immunosorbent assay (ELISA). Cytokine determinations were measured in duplicate, and mean concentrations were calculated. All ELISA kits were high-sensitivity kits from R&D Systems (Minneapolis, MN).
Tissue biopsies. Abdominal subcutaneous adipose tissue and muscle tissue (vastus lateralis) were obtained by use of the percutaneous biopsy technique with suction and were immediately frozen in liquid nitrogen and stored at –80°C until analyzed for IL-6 and TNF- mRNA, as described in RNA extraction.
RNA extraction. Adipose and muscle tissue RNA extraction was performed with TRIzol (Life Technologies) according to the manufacturer's directions. Briefly, 50–100 mg of the tissue were dissolved in 1 ml of TRIzol and homogenized on a Brinkman Polytron (version PT 2100) on setting 26. Samples were allowed to sit for a few minutes for a triglyceride phase to form. The lower aqueous phase was transferred to a fresh tube, and 100 µl of chloroform-isoamyl alcohol (24:1) were added and vigorously shaken. Samples were allowed to sit for 5 min and then were spun at 12,000 g for 15 min at 4°C, after which the upper aqueous phase was transferred to a new tube. The aqueous phase was mixed with 0.5 ml of isopropanol, and samples were placed at –20°C for 1 h. Samples were centrifuged at 12,000 g for 15 min at 4°C, and the resulting pellet was washed with 0.5 ml of 75% ethanol in diethyl pyrocarbonate (DEPC)-treated water. After centrifugation at 6,000 g for 10 min, pellets were redissolved in 15 µl of DEPC-treated water and allowed to dissolve on ice, after which samples were ready for reverse transcription.
Reverse transcription. One microgram of total RNA was reverse transcribed using the Applied Biosystems Taqman RT-Kit.
Real-time PCR. IL-6 and TNF- gene expressions were analyzed using semiquantitative real-time PCR with an ABI PRISM 7900 sequence detector (PE Biosystems). 18S served as the internal reference gene. We used the predeveloped, primer-limited assay reagents for 18S rRNA and TNF- determination. The IL-6 primers and probe sequences used were obtained from Starkie et al. (25). All PCR reagents were obtained from Applied Biosystems.
An 81-bp fragment was amplified using IL-6 forward primer: 5'-GGTACATCCTCGACGGCATCT-3'; IL-6 reverse primer: 5'-GTGCCTCTTTGCTGCTTTCAC-3'; and IL-6 probe: 5'-FAM-TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-TAMRA-3'. A reagent mixture of 75 µl was made up for each sample with 1x MasterMix, 900 nM IL-6 forward primer, 300 nM reverse primer, 100 nM IL-6 probe, 1 x 18S mix (primers and probe), and 50–100 ng of sample, to a final volume of 75 µl with water. Each sample was run in triplicate in a reaction volume of 20 µl for 50 cycles by use of standard real-time PCR cycling conditions. All samples were run in triplicate and normalized to a relative standard curve, and the transcript quantity of genes was then normalized to 18s rRNA.
Arterialized blood gas analysis. Concentrations of blood potassium and blood glucose were measured immediately on an ABL 700 (Radiometer).
Insulin. Samples were drawn into tubes containing EDTA and immediately centrifuged. Plasma was stored at –80°C until analyzed using an ELISA technique (DAKO).
Statistical Analysis
Data were analyzed using parametrical methods, and P < 0.05 was considered statistically significant. Plasma cytokine (TNF- and IL-6) concentrations and gene expression of IL-6 and TNF- from muscle and fat were log-transformed before analysis, and data are expressed as the geometric mean ± geometric SE. Glucose and insulin concentrations are given as means ± SE. Two-way ANOVA for repeated measures was used to evaluate the effect of clamp time. If a significant interaction was found, a paired t-test was used to detect changes from preclamp. Analysis was performed with SYSTAT.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA levels are expressed as ratios of mRNA to 18S rRNA (Figs. 1 and 3). IL-6 gene expression in abdominal subcutaneous adipose tissue increased during the insulin clamp (ANOVA P < 0.03). IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, the values not being significantly different from each other. There was no change in IL-6 mRNA in skeletal muscle (Fig. 1).
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The concentrations of circulating levels of IL-6 increased continuously during the insulin clamp (ANOVA P < 0.001; Fig. 2).
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TNF- gene expression in adipose tissue increased significantly during the clamp (ANOVA P = 0.001) and decreased in muscle tissue (ANOVA P = 0.04). TNF- mRNA in adipose tissue increased 2.4-, 1.4-, and 2.2-fold at 2, 4, and 6 h, respectively, with no significant difference between values. In muscle tissue, TNF- mRNA slightly decreased 0.74-, 0.64-, and 0.68-fold at 2, 4, and 6 h, respectively, with no significant difference between values (Fig. 3). There was no significant change in circulating levels of TNF- (Fig. 4). A control experiment was performed in which saline was infused for 3 h to a group of healthy volunteers. Abdominal fat biopsies were obtained at the four corresponding time points. Relative to prevalues, IL-6 mRNA and TNF- mRNA remained unchanged over time (unpublished data).
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Plasma concentrations of insulin were enhanced 
800-fold at 1 h and kept constant throughout the experiment (Fig. 5). Plasma glucose concentration was kept constant at the individual fasting level (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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differently in adipose tissue and muscle. Thus insulin stimulates TNF- gene expression in fat but induces a small inhibition of muscle TNF- mRNA. The finding that circulating levels of IL-6 increased as well indicates a release of IL-6 from subcutaneous adipose tissue, although it cannot be ruled out that IL-6 could be released from other sites as well. In contrast, plasma TNF- does not change, probably because its effect is mainly local or because a small release from fat tissue is counteracted by a reduced release of muscle TNF-. Several studies have demonstrated enhanced levels of IL-6 in patients with obesity and type 2 diabetes (5, 13, 14, 20, 24, 32). In a population-based study, plasma concentrations of IL-6 have been shown to predict total and cardiovascular mortality (21). Aging is associated with increased levels of IL-6 (2), and it has been proposed that IL-6 is the mediator that links the acute-phase response to visceral obesity, insulin resistance, and atherosclerosis (32). These studies demonstrate, however, only a putative association between IL-6 and disease. It is clear from the present study that insulin stimulates IL-6 gene expression in fat tissue, a finding that is substantiated by in vitro studies (6, 29). Thus, increased levels of IL-6 in relation to obesity and type 2 diabetes may rather be a cause of increased insulin levels than the inducer of hyperinsulinemia. Wallenius et al. (31) demonstrated that an IL-6-deficient mouse, vs. a wild-type control mouse, developed mature-onset obesity and insulin resistance. When mice were treated with IL-6, there was a pronounced decrease in body weight in the transgenic but not in the wild-type mice. In addition, we recently demonstrated that recombinant human IL-6 enhances lipolysis in healthy humans (9) and induces strong anti-inflammatory effects (26, 27).
Also, circulating levels of TNF- are elevated in obesity and diabetes (12, 15), and there is an increased TNF- gene expression in muscle (10) and adipose tissue (13) in patients with insulin resistance. In contrast to IL-6, TNF- has been demonstrated to induce insulin resistance in rodents. Given that TNF- induces IL-6 (18), it is possible that insulin stimulates adipose tissue gene expression of IL-6 via a stimulation of TNF-. Because insulin also inhibits muscle TNF- gene expression, and because TNF- in the circulation does not increase, the net effect of circulating cytokines is an increase in anti-inflammatory cytokines. In a recent clinical trial (1), it was demonstrated that intensive insulin treatment to maintain blood glucose at or below 6.1 mM (110 mg/dl) reduces mortality and morbidity of critically ill surgical patients. The effect on mortality was achieved mainly by a reduction in late deaths (after 5 days of admission) due to a septic focus (1). In addition, a euglycemic hyperinsulinemic clamp (compared with saline infusion) in volunteers who had received an endotoxin dose at 2 ng/kg induced an increase in the IL-6 response to endotoxin (23). Thus the latter study, in accord with data presented here, demonstrates an effect of insulin on circulating levels of IL-6, although the use of supraphysiological concentrations of insulin on adipose tissue IL-6 production in the present study cannot easily be extrapolated into a clinical state of hyperinsulinemia, such as in insulin resistance.
In conclusion, we have demonstrated that supraphysiological concentrations of insulin, given to healthy young subjects, stimulate IL-6 and TNF- gene expression in adipose tissue, and we have seen a corresponding rise in circulating IL-6 but no effect on circulating TNF-. In addition, we observed a decrease in the gene expression of TNF- in muscle tissue. The differential effects of insulin on IL-6 and TNF- gene expression in adipose and muscle tissue indicate that fat- and muscle-derived IL-6 and TNF- may be regulated by different mechanisms. In addition, the net effect of circulating cytokines is an increase in anti-inflammatory cytokines (IL-6), which indicates a positive metabolic role of insulin.
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported by grants from the Danish National Research Foundation (no. 504–14), the Novo Nordisk Foundation, Lundbeckfonden Rigshospitalet, and Copenhagen Hospital Corporation, Civil engineer Frode V. Nyegaard og Hustrus Fond, Danfoss.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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expression in, and release from, contracting human skeletal muscle. Am J Physiol Endocrinol Metab 283: E1272–E1278, 2002.This article has been cited by other articles:
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