Short-term infusion of interleukin-6 does not induce insulin resistance in vivo or impair insulin signalling in rats (original) (raw)
Introduction
Interleukin-6 plays an important role in the acute immune response [1, 2]. In addition, it has recently been implicated in the insulin resistance associated with obesity, impaired glucose tolerance and diabetes [3, 4, 5, 6]. The circulating levels of IL-6 are elevated in these conditions [6, 7] and IL-6 was also shown in prospective studies to be an independent predictor of risk of developing type 2 diabetes irrespective of the amount of body fat [8].
Adipose tissue accounts for 15 to 35% of the total circulating IL-6 levels, making this tissue a major site for IL-6 production and secretion [3, 9]. In addition to secreting IL-6 and other cytokines, the adipocytes are also target cells, as (i) they have been shown to express the different components of IL-6 signalling, such as IL-6 receptors, gp130 and signal transducers and activators of transcription (STATs), and (ii) this signalling system has been shown to be activated upon IL-6 stimulation [10, 11]. Other target tissues for insulin action, such as the liver and skeletal muscle, also express the various components of the IL-6 signalling system [12, 13, 14, 15], supporting the possibility that whole-body insulin sensitivity may be modulated by IL-6. This possibility was corroborated by recent findings that exogenous administration of IL-6 induces insulin resistance in adipose and liver cells [11, 16, 17, 18]. HepG2 cells, as well as primary mouse hepatocytes, had decreased tyrosine phosphorylation of IRS-1 in response to insulin as well as reduced protein kinase B activation in the presence of IL-6 [17]. This effect of IL-6 was attributed to the concomitantly and transiently increased expression of suppressors of cytokine signalling (SOCS-3) [18]. The SOCS-3 gene is also transiently activated in response to IL-6 in muscle cells [19], as well as in adipose cells [16], reaching a peak within 120 min followed by a rapid decline to basal levels (unpublished observations). Surprisingly, insulin also increases SOCS-3 expression with a similar time-course to that of IL-6 and this has been shown to reduce the intracellular signalling of this hormone [20]. Thus, SOCS-3 may play a role in the impaired insulin signalling seen after IL-6 administration and in conjunction with chronic hyperinsulinaemia [20].
In addition to the effects on upstream signalling of insulin, SOCS-3 has also been found to initiate ubiquination and degradation of IRS-1 in HEK293 cells [21]. However, in contrast to the effects of IL-6 in liver cells, short-term (10–120 min) exposure to IL-6 does not significantly reduce the tyrosine phosphorylation of the insulin receptor or the IRS molecules in adipose cells in response to insulin, although SOCS-3 mRNA levels are increased [11, 16] (unpublished observations). However, long-term exposure (≥24 h) does lead to insulin resistance through the down-regulation of IRS-1 and GLUT4, and this is associated with reduced insulin-stimulated glucose transport [11, 16]. In addition, IL-6 has been found to reduce the gene expression and secretion of adiponectin in both 3T3-L1 [22, 23] and human adipocytes [24], and this is another mechanism whereby IL-6 can impair insulin sensitivity.
In addition to the large pool of data supporting a role for IL-6 in insulin resistance, as well as directly demonstrating that this cytokine impairs insulin signalling [11, 16, 17, 18], IL-6 ablation in mice has provided evidence for other effects. Wallenius and co-workers [25] reported that IL-6−/− mice developed maturity-onset obesity, with elevated circulating triglyceride levels in female mice and decreased glucose tolerance. However, since no obese control animals were used in that study, this finding may have been due to effects secondary to the increased obesity. Furthermore, only intracerebro-ventricular IL-6 injections, but not intraperitoneal IL-6 administration, increased energy expenditure and decreased obesity in these mice, suggesting that the primary target for IL-6 is the central nervous system [25]. In addition, a recent study was unable to confirm that lack of IL-6 was associated with obesity in mice [26].
The present study was designed to investigate the potential acute effects of IL-6 on insulin signalling and action in vivo using the euglycaemic–hyperinsulinaemic clamp, which is considered to be the gold standard to measure insulin sensitivity. No such studies have previously been carried out. To examine the potential role of IL-6 on insulin sensitivity and action, it is necessary to have a steady-state concentration of insulin, against which the potential effect of IL-6 can be measured. Previous studies of the effects of IL-6 on glucose metabolism in man have also produced contradictory results [27, 28], possibly a consequence of such non-steady-state experimental conditions.
If SOCS-3 plays a critical role, also under normal in vivo conditions, in mediating a reduced effect of insulin following IL-6, this effect should also be seen after 1 to 2 hours of IL-6 infusion when SOCS-3 levels peak. However, our data clearly show that short-term infusion of IL-6 does not impair the whole-body effect of exogenous insulin on glucose homeostasis or insulin signalling in vivo. These data support other recent findings [11, 29] that IL-6 is predominantly a chronic modulator of insulin action and, in addition, that its effects may be tissue-specific.
Materials and methods
Animals
We studied 18 female Sprague–Dawley rats (B&K Universal, Sollentuna, Sweden) for this study. All animals were in oestrus at the time of study to avoid confounding effects of the hormonal cycle. The animals were housed under controlled conditions (21–22 °C, 55–65% humidity and light from 05.00 to 19.00 hours), with five animals in each cage. Animals were allowed to adjust for at least 5 days before initiation of the studies, and were fed a commercial rat chow with sufficient supply of vitamins and minerals, and free access to tap water. The study was approved by the Animal Ethics Committee of Göteborg University, Sweden.
Hyperinsulinaemic–euglycaemic clamp
Prior to the clamp, the animals were anaesthetised intraperitoneally with thiobutabarbital sodium (125 mg/kg body weight; RBI, Natick, Mass., USA).
The euglycaemic–hyperinsulinaemic glucose clamp technique has been described in detail previously [30]. Briefly, catheters were inserted into the left carotid artery for blood sampling and into the right jugular vein for infusion of glucose and insulin. Body temperature was maintained at 37 °C with a heating blanket. After a bolus injection, insulin (Human Actrapid; Novo Nordisk, Copenhagen, Denmark) was continuously infused at 10 mU·kg−1·min−1. A 20% solution of glucose in isotonic saline was also infused to maintain plasma glucose levels at 6.0 mmol/l. The IL-6 infusion (3 µg in total; human recombinant, Sigma, St. Louis, Mo., USA) was initiated with a bolus dose followed by a constant infusion (5 µl/min), which was started at the time of the insulin infusion. Control animals were infused with saline at the same rate. Glucose concentrations were determined at regular intervals (every 5 min up to 40 min after initiation and then every 10 min). Steady-state glucose levels were reached approximately 40 min after initiation of the infusions and the glucose infusion rate required to maintain euglycaemia was taken as an index of the glucose disposal rate and, thus, insulin action.
Tail blood pressure and pulse were recorded at 0, 90 and 120 min. Blood samples for analyses of insulin, adiponectin, NEFA and IL-6 were taken at 0 and 120 min. A total volume of less than 2 ml was used for these determinations and this was compensated for by the infusion volumes.
The tissues used for gene and protein analyses, i.e. the inguinal adipose tissue, extensor digitorum longus muscle and liver, were taken before the insulin infusion was stopped and were snap-frozen in liquid nitrogen. The tissue was divided into two pieces; one piece was placed in RNA Later for RNA preparation, the other was stored at −80 °C for protein analysis.
Analytical methods
Blood was collected in heparinised microtubes and centrifuged immediately. Plasma concentrations of glucose were enzymatically determined in 15-µl samples on a 2700 SELECT biochemical analyser (YSI, Yellow Springs, Ohio, USA).
Plasma insulin was analysed with a rat insulin RIA kit (Linco Research, St. Charles, Mo., USA) and serum NEFA levels were measured using a kit from Wako Chemicals (Neuss, Germany).
Serum IL-6 and adiponectin levels were measured with ELISA techniques (Diaclone, Besancon, France).
Protein analysis
The adipose tissue used for protein analysis was homogenised in lysis buffer containing 50 mmol/l HEPES, 1% Triton X, 1% NP-40, 10 mmol/l EDTA, 100 mmol/l sodium fluoride, 100 mmol/l sodium pyrophosphate, 10% glycerol, 1 mmol/l benzamidine, 1.5 mmol/l aprotinin, 10.5 mmol/l leupeptin, 1 µmol/l okadaic acid and 5 mmol/l Na3VO4. Samples were incubated for 2 h at 4 °C, centrifuged for 20 min at 20,000 g and then kept at −80 °C until analysed.
The liver tissue was homogenised in the same lysis buffer, centrifuged at 74,000 g for 45 min and the supernatant kept at −80 °C until analysed.
The lysis buffer for the muscle tissue contained 50 mmol/l Tris-HCl, 0.1% Triton X, 1 mmol/l EDTA, 1 mmol/l EGTA, 50 mmol/l sodium fluoride, 5 mmol/l sodium pyrophosphate, 10 mmol/l glycerophosphate, 1 µmol/l okadaic acid, 1 mmol/l benzamidine, 100 µmol/l [4-(2-aminoethyl)-bezenesulphonyl fluoride, HCl], 0.1% β-mercaptoethanol and 1 mmol/l Na3VO4. The homogenate was incubated at +4 °C for 1.5 h and centrifuged at 12,000 g for 15 min. The supernatant was kept at −80 °C until analysed.
The protein concentration was measured with bicinchonic acid (Pierce, Rockford, Ill., USA) for fat and liver and the Bradford assay (Bio-Rad Laboratories, Hercules, Calif., USA) was used for the muscle lysate samples.
Lysate proteins were separated on SDS-PAGE as described [11, 31] and immunoblotted with anti-phosphotyrosine (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), anti-pY705 STAT3 and anti-STAT3 (Cell Signalling Tech, Beverly, Mass., USA) antibodies according to the recommendations of the manufacturer. Protein blots were scanned using a Personal Densitometer (Molecular Dynamics, Sunnyvale, Calif., USA) and analysed using Imagequant software provided by the manufacturer.
Where used, arbitrary units refer to intensity units.
RNA extraction and RT-PCR
The adipose tissue was washed with RNAse-free phosphate-buffered saline and homogenised in a solution containing 4 mol/l guanidinium thiocyanate, 17 mmol/l sarkosyl, 25 mmol/l Na citrate pH 7 and 0.01% antifoam A. An equal volume of cold chloroform was added and the sample was shaken vigorously for 1 min and centrifuged at 12,000 g at −4 °C for 10 min. The water phase was collected and RNA extracted according to Chirgwin et al. [32].
RNA from muscle and liver was extracted using the RNeasy Mini protocol (Qiagen, Hilden, Germany). Muscle tissue was digested with Proteinase K (Sigma) prior to RNA extraction and all samples were treated with DNase I.
We prepared cDNA with TaqMan Reverse Transcription Reagents and the samples were quantified using TaqMan Universal PCR Master Mix and the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA) using 18S as reference as described [33].
Probes and primers for IL-6, IL-6 receptor, adiponectin, GLUT4, SOCS-3 and PGC-1 are available upon request.
Statistical analysis
All statistical analyses were performed in Excel and statistical significances evaluated with paired or unpaired, as appropriate, Student’s t test. A p value of 0.05 was considered significant.
Results
Animal characteristics
As shown in Table 1, the animals used in the control and the IL-6 clamps had the same body weight, and similar insulin levels were reached during the clamps. Circulating IL-6 levels were not measured in the control animals, since an RIA kit recognising only human IL-6 was used. During the IL-6 infusions, high circulating levels were reached (~1 nmol/l) (Table 1). Blood pressure and heart rates were not changed by the IL-6 infusion (data not shown).
Table 1 Animal body weights, plasma insulin and IL-6 levels at 120 min during the euglycaemic–hyperinsulinaemic clamps
Glucose infusion rate
The glucose infusion rates after 90 to 120 min of insulin infusion were similar in the control and IL-6 infusion groups (Fig. 1a).
Fig. 1
Glucose infusion rates, plasma NEFA and adiponectin levels during the hyperinsulinaemic–euglycaemic clamps (10 mU·kg−1·min−1). a. Glucose infusion rate as measured by the amount of glucose required to maintain euglycaemia (_n_=9 in the saline and IL-6 groups respectively). b. Plasma NEFA concentrations at 0 and 120 min (_n_=9 in the saline and IL-6 groups respectively). c. Serum adiponectin levels at 120 min (_n_=9 in the saline and IL-6 groups respectively. * p<0.05; ** p<0.01
NEFA levels
The insulin infusion reduced circulating NEFA levels in the control group, albeit not significantly, whereas NEFA levels tended to increase in the group receiving the IL-6 infusion (_p_=0.07) (Fig. 1b).
Adiponectin levels
Plasma adiponectin levels decreased significantly both in the control and in the group receiving the IL-6 infusion (Fig. 1c). The numerical decrease (levels before minus levels at 120 min) was the same for the two groups of animals, suggesting that it was associated with the increased insulin level during the hyperinsulinaemic–euglycaemic clamp.
Protein expression and tyrosine phosphorylation
STAT3 tyrosine phosphorylation was increased in the fat, muscle and liver of the rats receiving the IL-6 infusion, showing that the IL-6 signalling system was activated in all tissues (Fig. 2a–c).
Fig. 2
Insulin and IL-6 signalling molecules in muscle (a), liver (b) and adipose tissue (c) following the hyperinsulinaemic–euglycaemic clamps. Tyrosine phosphorylation (pY site 705) of STAT3, STAT3 protein and tyrosine phosphorylation (p-Tyr) of IRS and the insulin receptors (IR). Scanned data for (a) muscle (_n_=4), (b) liver (_n_=4), (c) adipose tissue (_n_=7). Results are shown as means ± SEM
There was no consistent difference in the tyrosine phosphorylation (or protein expression, data not shown) of the insulin receptor or the insulin receptor substrates (Fig. 2a–c), showing that IL-6 does not acutely change insulin signalling in vivo under these experimental conditions. This is in accordance with the data for the glucose infusion rates (Fig. 1a).
Gene expression
We also investigated the potential effect of the IL-6 infusion on gene expression of several molecules involved in IL-6 and insulin signalling and action. IL-6 gene expression did not increase significantly in fat and liver, but tended to be increased in muscle (_p_=0.08; Fig. 3a). IL-6 receptor mRNA was not changed in any of the three tissues, nor was GLUT4 or PGC-1 (data not shown). SOCS-3 gene expression was significantly increased in muscle and liver of the IL-6 + insulin infusion group, when compared to insulin alone (Fig. 3b). Adiponectin mRNA levels in the adipose tissue were not significantly altered by infusing IL-6 and insulin as compared to insulin alone (Fig. 3c).
Fig. 3
IL-6 (a) and SOCS-3 (b) mRNA levels in muscle, liver and adipose tissue, and adiponectin (c) mRNA levels in adipose tissue, as determined after the hyperinsulinaemic–euglycaemic clamps using 18S as reference. Results are means ± SEM for 9 rats in each group. * p<0.05; ** p<0.01
Discussion
The present study was designed to investigate if IL-6 rapidly (within 120 min) inhibits the effect of a sustained hyperinsulinaemia on glucose homeostasis in vivo. To this end, the rats were infused with IL-6 or saline for 120 min during a euglycaemic and hyperinsulinaemic clamp. This design was used in order to examine the in vivo effects of the increased SOCS-3 levels in the target tissues for insulin. Previous detailed studies in vitro [17, 18] have shown that the intracellular signalling for insulin is decreased in liver cells in parallel with increased SOCS-3 levels. However, it has not been previously examined whether this perturbation is sufficient to produce insulin resistance in vivo. Somewhat surprisingly, insulin has also been shown to increase SOCS-3 levels in adipose tissue, liver and skeletal muscle to a similar extent as IL-6 and with a similar time-course [20]. Thus, it has been suggested that SOCS-3 may also play a role in modulating insulin sensitivity following chronic hyperinsulinaemia [20].
The present data clearly show that a short-term infusion of IL-6 does not impair the effects of insulin on whole-body glucose disposal in vivo. This is in agreement with previous studies in man, showing a modest or no effect of IL-6 on glucose levels in vivo in the absence of defined hyperinsulinaemia [27, 28, 34].
The fact that the target tissues (adipose cells, skeletal muscle and liver) had activated the intracellular IL-6 signalling cascade is shown by the increased tyrosine phosphorylation of STAT3, a key inducer of the intracellular actions of IL-6 [35]. In agreement with the lack of effect on whole-body insulin action and on glucose disposal, we found no significant or consistent reductions in the tyrosine phosphorylation of the insulin receptors or the IRS molecules. A potential problem for this, however, is that we examined the tyrosine phosphorylation of these molecules as induced by the insulin infusion in vivo. This leads to a greater variability than that seen in in vitro incubations and, thus it may not have been possible to identify minor changes. However, even if this were the case, the variability was small enough not to be associated with whole-body insulin resistance following the IL-6 infusion.
Another potential caveat is that we used a high physiological insulin concentration during the clamps. This may have compensated for a small impairment in insulin action in the liver and adipose tissue considering their normal high sensitivity to insulin. On the other hand, the IL-6 infusions produced ~3 to 400-fold higher plasma concentrations than those normally seen in rats and man [24, 36] and this would favour any potential early inhibitory effects of IL-6. Such concentrations of IL-6 may, in fact, not be unphysiological at the cellular level in vivo, at least in tissues producing IL-6 such as the adipose tissue and the muscle tissue following exercise [37]. We recently measured the interstitial IL-6 concentrations in human adipose tissue in situ using a calibrated microdialysis technique and found concentrations similar to those reached in plasma in the present study [24].
There were only minor or no changes in the genes measured after 120 min: GLUT4, PGC-1, adiponectin, IL-6 or SOCS-3. This may well have been expected considering the short infusion time. However, it must be emphasised that the experimental design only makes it possible to examine effects of IL-6 that are additional to those of insulin itself, since all animals were infused with insulin (and saline or IL-6). This is the most likely reason for the small difference in SOCS-3 gene expression following IL-6, since insulin also increases SOCS-3 mRNA levels to a similar extent as IL-6 and with a similar time-course [20]. The present study, then, suggests that there is no additive or synergistic effect of these hormones under the present experimental conditions.
We [11] and others [16] have previously found that long-term exposure (24 or 48 h) of adipocytes to IL-6 increases IL-6 mRNA levels 4 to 6-fold. In the present short-term study, this was most evident in the skeletal muscle but the physiological consequence of this is currently unclear.
There were only minor changes in NEFA levels in the two groups of animals, although the levels tended to increase following IL-6 infusion. The small antilipolytic effect of insulin may appear surprising but hyperinsulinaemic clamps in non-obese rodents are frequently associated with a minor suppression of NEFA, possibly due to an activation of the sympathetic nervous system. Importantly, however, the circulation in the animals was not compromised, since blood pressure and heart rates remained unchanged during the study.
The available data on the effect of IL-6 on lipolysis is contradictory. IL-6 has been found to reduce lipoprotein lipase activity in the adipose tissue in vivo as well as in 3T3-L1 adipocytes in vitro [38]. It has also been reported that IL-6 stimulated lipolysis in rats [39] and in man [40, 41], whereas no such effect of IL-6 has been found in other studies [42, 43].
Plasma adiponectin levels decreased significantly both in control and in IL-6 treated rats, but the extent of the decrease did not differ between the two groups, suggesting that it was due to the hyperinsulinaemic clamp rather than to IL-6. This was further supported by the findings that there was no difference in adiponectin mRNA levels in the adipose tissue between the two groups where insulin is the common denominator. Hyperinsulinaemic clamps have also previously been shown to lead to decreased serum adiponectin levels in human subjects [44, 45] as well as in monkeys [46] and plasma adiponectin levels correlate negatively with fasting insulin levels [47, 48, 49].
In conclusion, infusion of IL-6 for 120 min in rats during hyperinsulinaemic and euglycaemic conditions did not impair the effect of insulin on whole-body glucose homeostasis in vivo. Furthermore, no short-term effect of IL-6 was found on plasma adiponectin levels or on insulin signalling in adipose tissue, muscle or liver in vivo. These results do not support the idea that SOCS-3 is an important mediator of insulin resistance under in vivo conditions. In contrast, chronically elevated IL-6 levels exert inhibitory effects on insulin signalling and action by suppressing the gene transcription of several important molecules [11, 16]. It is also possible that IL-6 exerts tissue-specific effects [24, 29] and that the major targets for cross-talk between IL-6 and insulin may be the adipose tissue and the liver rather than the skeletal muscle.
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