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Endocrinology Vol. 147, No. 6 2690-2695
Copyright © 2006 by The Endocrine Society

Increased Levels of Acylation-Stimulating Protein in Interleukin-6-Deficient (IL-6–/–) Mice

I. Wernstedt, B. Olsson, M. Jernås, S. Paglialunga, L. M. S. Carlsson, U. Smith, K. Cianflone, K. Wallenius and V. Wallenius

Research Center for Endocrinology and Metabolism (I.W., B.O., M.J., L.M.S.C., K.W., V.W.), Wallenberg Laboratory (I.W., V.W.), Lundberg Laboratory for Diabetes Research (U.S.), Departments of Internal Medicine and Surgery (V.W.), Sahlgrenska Academy, Sahlgrenska University Hospital, SE-41345 Goteborg, Sweden; and Centre de Recherche de l’Hôpital Laval, Université Laval (S.P., K.C.), Québec, Canada G1V 4G5

Address all correspondence and requests for reprints to: Dr. Ville Wallenius, Department of Surgery, Sahlgrenska University Hospital, Bruna Stråket 11, SE-41345 Goteborg, Sweden. E-mail: ville.wallenius{at}medic.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-6-deficient (IL-6–/–) mice develop obesity at 6–7 months of age. To elucidate the mechanisms of this mature-onset obesity, global gene expression profiles of 3-month-old preobese IL-6–/– were compared with those of IL-6+/+ mice using DNA arrays. Genes that were up-regulated in IL-6–/– mice included the factors transthyretin and properdin in white adipose tissue and adipsin in muscle. These factors have been shown to influence the formation of acylation-stimulating protein (ASP), a cleavage product of complement C3. ASP stimulates the synthesis of triacylglycerol in adipocytes, and ASP-deficient mice are resistant to diet-induced obesity. In line with the increases in transthyretin, properdin, and adipsin, ASP levels in serum were increased by 31–54% in IL-6–/– compared with IL-6+/+ mice. Furthermore, IL-6 replacement treatment in IL-6–/– mice decreased ASP levels significantly by 25–60%. In conclusion, ASP levels are increased in preobese IL-6–/– mice. This increase may result in increased triacylglycerol formation and uptake in IL-6–/– adipocytes and thereby contribute to the development of obesity in IL-6–/– mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IL-6 IS A MULTIFUNCTIONAL, immune-modulating cytokine that has lately gathered interest as one of the adipocytokines, i.e. adipocyte-derived circulating factors, with relevance to morbidity in obesity. IL-6 has been suggested to have important functions in glucose and lipid metabolism, on the one hand, as a physiological signal from exercising muscle for metabolic adaptation to increased energy demand (1, 2) and, on the other, as a pathophysiological mediator of inflammation and metabolic disease (3).

We reported that IL-6-deficient (IL-6–/–) mice develop mature-onset obesity at 6–7 months of age (4). There may be several mechanisms contributing to this. We and others have shown that intracerebroventricular, but not peripheral sc, administration of IL-6 increases the metabolic rate (4, 5), and chronic administration of IL-6 intracerebroventricularly decreases fat mass in rats (6, 7). Thus, the lack of IL-6 centrally in IL-6–/– mice may lead to decreased metabolic rate and increased fat mass. This assumption was corroborated by our finding of a negative correlation between cerebrospinal fluid IL-6 levels and fat mass in obese humans, indicating a relative lack of IL-6 centrally with increasing obesity (8). However, in IL-6–/– mice, peripheral IL-6 treatment was able to partly reverse the obesity. Although IL-6 was given sc, this effect may well be related to central nervous system effects of IL-6, because of, for instance, increased sensitivity and/or increased uptake over the blood-brain barrier of IL-6 in IL-6–/– mice (4). It has been shown by several investigators that IL-6 has lipolytic effects in both animal and human models (9, 10, 11, 12).

To our knowledge, the only putative mechanism that has been described for the lipolytic effect of IL-6 in mice and adipocytes is suppression of lipoprotein lipase (LPL) activity (13). To search for additional antiobesity mechanisms of IL-6, global gene expression was analyzed in white adipose tissue (WAT) and muscle from preobese male IL-6–/– and IL-6+/+ mice. One group of genes that was regulated encodes proteins that are important for the formation of acylation-stimulating protein (ASP), a cleavage product of complement C3. ASP stimulates the uptake of glucose and lipids into adipose tissue and contributes to the development of obesity in some animal models (14, 15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The IL-6–/– mice were generated by Kopf et al. (16). To reduce genetic heterogeneity, the IL-6–/– genotype was moved onto the C57BL/6 background by eight backcrosses, resulting in mice genetically consisting of more than 99.5% C57BL/6. Littermate IL-6+/+ mice were used as controls in all experiments. Animals were maintained under standardized nonbarrier conditions. Principles of laboratory animal care were followed as well as specific national laws, and all animal procedures were approved by local ethic committees on animal care (Gothenburg, Sweden).

DNA microarray
All mice were tested against acute inflammation before collection of tissues for DNA array experiments by measurement of the acute phase protein serum amyloid A using a kit (Tridelta, Wicklow, Ireland). This was performed to exclude ongoing inflammation in either group of animals considering that IL-6–/– mice are in part immune compromised (16). Based on serum amyloid A measurements, tissue from seven mice per group were selected for DNA array experiments. The mice used for DNA array experiments did not differ significantly in body weight or leptin levels [body weight, 28.3 ± 0.98 vs. 29.6 ± 0.74 g (P = 0.29); serum leptin, 3.69 ± 1.04 vs. 5.39 ± 0.850 pg/ml (P = 0.23); respectively, IL-6–/– vs. IL-6+/+]. WAT from the gonadal depot and muscle tissue from the quadriceps femoris muscle were collected from IL-6–/– and IL-6+/+ mice. For WAT, individual DNA microarrays were run for each mouse. For muscle, duplicate microarrays of four and three pooled samples were used for both groups of mice (in total, seven mice per group). RNA preparation and DNA microarray analysis, using mouse-specific DNA arrays (Affymetrix, Inc., Santa Clara, CA), were performed as previously described (17). Of the original seven WAT samples per group, two in the IL-6–/– group had to be excluded due to poor technical quality of the arrays, leaving seven arrays in the IL-6–/– group and five in the IL-6–/– group to be analyzed further.

Singleplex real-time-PCR
Biopsies of gonadal adipose tissue from 3- to 4-month-old IL-6+/+ and IL-6–/– mice (n = 9 and 8, respectively) were immediately frozen in liquid nitrogen, then stored at –80 C until analysis. Total RNA was isolated using an RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA) and additionally purified using a ribonuclease-free deoxyribonuclease set (Qiagen). The quality of the RNA was verified by agarose gel electrophoresis before it was reverse transcribed to cDNA by TaqMan RT reagents (Applied Biosystems, Foster City, CA). Properdin, adipsin, factor B, and transthyretin were analyzed using predesigned TaqMan Assays-on-Demand (singleplex assays, Applied Biosystems) with the reference sequence m36B4 as the endogenous control. TaqMan reverse transcriptase reagents, TaqMan Universal PCR Master Mix (Applied Biosystems), and reaction conditions were used according to the manufacturer’s instructions. The relative expression levels were estimated using the comparative threshold cycle method.

ASP assay
Serum concentrations of ASP were measured by dot blot, as described below. Blood samples were collected by puncture of descending aorta or from the tail vein and were treated with a protease inhibitor mixture (Complete, Roche, Bromma, Sweden). Samples were centrifuged to obtain serum, which was then immediately frozen and stored at –20 C until analysis. To precipitate globular proteins, including C3, the serum was acidified with concentrated HCl to a final concentration of 1 M, mixed, and incubated at room temperature for 1 h. Precipitate was then removed by centrifugation (18). Samples were neutralized with 9 N NaOH, diluted in 1.5 vol 1.333% dithiothreitol in 0.222 M Tris-HCl (pH 6.8) buffer, and immediately applied to a nitrocellulose membrane using a dot-blot apparatus (96 well; Bio Dot, Bio-Rad Laboratories, Hercules, CA) together with a human ASP standard ranging from 0.3125–1.5 nM. Each well was washed three times with Tris-buffered saline (TBS)-Tween [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.05% Tween 20]. Membranes were then blocked for 30 min with 3% BSA in TBS-Tween buffer, washed twice with TBS-Tween, then incubated in a 1:500 dilution of a rabbit antimouse ASP antibody, prepared by immunization of rabbits with 10 sc injections of a mouse ASP peptide containing the 10 carboxyl-terminal amino acids (HRRDHVLGLA). A secondary goat antirabbit IgG alkaline phosphatase conjugate was applied at a 1:1500 dilution for 1.5 h. After repeated washing in TBS, membranes were developed using an alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories), then computer scanned and quantified using Image software (Scion Corp., Frederick, MD).

IL-6 treatment
Recombinant IL-6 (R&D Systems, Inc., Minneapolis, MN) in sterile PBS containing 0.1% BSA or vehicle only was injected sc into 4-month-old female IL-6+/+ and IL-6–/– mice. In total, the treated mice received 300 ng IL-6, divided into six injections of 50 ng IL-6 over 4 d. The first treatment was received at 1700 h on d 1, and the last at 2000 h on d 4. Blood samples were collected at 1400 h on d 4 as described above. ASP levels after vehicle and IL-6 treatments were compared with pretreatment ASP levels (n = 4 + 4 for IL-6+/+ and n = 5 + 5 for IL-6–/– mice).

Measurement of free fatty acids and glucose
EDTA-plasma collected from overnight fasted 5-month-old female IL-6+/+ and IL-6–/– mice were immediately frozen at –80 C until analysis. Glucose levels were analyzed using Infinity Glucose reagent (Sigma-Aldrich Corp., St. Louis, MO), and free fatty acids levels were determined using the nonesterified fatty acid C, acyl coenzyme A synthetase-acyl coenzyme oxidase method (Wako Chemicals, Nordic Biolabs, Täby, Sweden) on a Konelab 20 autoanalyzer (Thermo Clinical Labsystems, Vantaa, Finland).

Measurement of adipocyte size
Adipose tissue from the inguinal fat depot was excised and placed in medium 199 medium (Invitrogen Life Technologies, Inc., Gaithersburg, MD). Adipose cells were isolated by collagenase digestion, and adipose cell size was measured by microscopic sizing, as previously reported (19).

Statistical analysis
Values are presented as the mean ± SEM. For single-animal WAT DNA arrays, Student’s t test for unpaired samples was used to compare the expression of genes between IL-6–/– and IL-6+/+ mice, except for those groups [transthyretin and {alpha}2-HS (Heremans-Schmid) glycoprotein] containing samples that were classified as undetectable on the DNA arrays, where an independent samples nonparametric t test using SPSS software (version 11.5; SPSS, Inc., Chicago, IL) was used (Table 1Go). Only genes that showed significant up-regulation by at least 50% or down-regulation by at least 30% in IL-6–/– mice were initially analyzed. For the pooled sample muscle DNA arrays, average gene expressions in the two pools of each genotype were compared. Comparisons where the direction of regulation for one genotype was uniform in both pools were used. For comparison of ASP levels in young vs. old and IL-6–/– vs. IL-6+/+ mice, two-way ANOVA was used (Fig. 1AGo). Linear regression was used for the correlations in Fig 1CGo. The effect of IL-6 treatment within IL-6+/+ or IL-6–/– mice were evaluated using the nonparametric Wilcoxon log-rank test. All other comparisons between groups were made using Student’s t test. P < 0.05 was considered significant.


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  		TABLE 1. Genes of importance for ASP formation and lipid metabolism that were regulated in 3-month-old IL-6–/– mice, compared with IL-6+/+ mice (n = 7 for IL-6+/+, n = 5 for IL-6–/–)

 

Figure 1
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  		FIG. 1. A, ASP levels in young (3 months old) preobese and old (13 months old) obese male IL-6–/– and IL-6+/+ mice. B, ASP levels in young (5–6 months old) preobese female IL-6–/– and IL-6+/+ mice. C, Linear correlations for ASP vs. DXA fat in IL-6+/+ and IL-6–/– mice (r = 0.73, P = 0.016, n = 10 and r = 0.127, P = 0.764, n = 8, respectively). D, Effect of 4 d of IL-6 treatment (six doses of 50 ng, sc) to young (4 months old) preobese female IL-6+/+ and IL-6–/– mice on circulating ASP levels. *, P < 0.05, by t test; #, P < 0.05, by Wilcoxon’s log-rank test; ##, P < 0.01, by two-way ANOVA (IL-6–/– vs. IL-6+/+ mice).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In total, on the DNA arrays 50 genes were found to be up-regulated and 35 down-regulated in WAT of young preobese IL-6–/– mice compared with IL-6+/+ mice. In muscle, 24 genes were up-regulated and 22 down-regulated in the young IL-6–/– mice. Among these, the expression of several factors of potential importance for formation of ASP were regulated, i.e. the negative acute phase protein transthyretin (TTR), and properdin and complement factor H showed significant up-regulation in WAT of IL-6–/– mice (Table 1Go). Another factor of importance for ASP formation, adipsin (or factor D), was increased in muscle, but not in WAT (Table 1Go). The expression of TTR in control IL-6+/+ mice was low on the DNA microarrays and classified as undetectable by the Affymetrix algorithm in all but one (six of seven) animals. In IL-6–/– mice, in contrast, it was classified as present in all but one (four of five) animals. Another example of a negative acute phase protein expressed only in IL-6–/– WAT was {alpha}2-HS glycoprotein (Table 1Go). Several other ASP-related genes, e.g. complement factor C3, factor B, adipsin, and Crry, were expressed in WAT, but did not differ between IL-6–/– and IL-6+/+ mice (Table 1Go). LPL mRNA expression was not changed in IL-6–/– compared with IL-6+/+ mice (Table 1Go).

The finding, of increased TTR and properdin expression on DNA arrays was confirmed by singleplex real-time PCR, normalized to m36B4, which was used as a reference gene in a separate reaction [TTR, 11.0 ± 6.7 vs. 2.23 ± 1.25 (IL-6–/– vs. IL-6+/+, P = 0.046); properdin, 0.67 ± 0.05 vs. 0.50 ± 0.05 (IL-6–/– vs. IL-6+/+, P = 0.02)].

To elucidate the net effect of these changes in gene expression on circulating ASP levels, we measured ASP in serum. ASP levels were increased by 54% and 38% in 3-month-old preobese and 13-month-old obese male IL-6–/– mice, respectively (Fig. 1AGo: P = 0.056 for 3-month-old and P = 0.02 for 13-month-old male IL-6–/– vs. IL-6+/+ mice, by Student’s t test; P < 0.01 for IL-6–/– vs. IL-6+/+, by two-way ANOVA) and by 31% in 5- to 6-month-old preobese female IL-6–/– mice compared with IL-6+/+ mice (Fig. 1BGo; P = 0.038, by Student’s t test). In addition, ASP levels in IL-6+/+ mice correlated with body weight (r = 0.688; P = 0.028; n = 10; not shown) and fat mass determined by dual-energy x-ray absorptiometry (DXA; Fig. 1CGo; r = 0.73, P = 0.016; n = 10), whereas in IL-6–/– mice there were no such correlations for either body weight or fat mass (body weight: r = –0.028; P = 0.947, n = 8, not shown; DXA fat: r = 0.127, P = 0.764, n = 8; Fig. 1Go), because ASP was increased even in nonobese mice. Because ASP has been shown to potently stimulate both uptake of dietary TG and TG storage in adipocytes, the increased ASP levels would suggest an increased TG uptake and storage in adipocytes of IL-6–/– mice.

IL-6 treatment with daily sc injections to IL-6–/– mice for 4 d decreased ASP levels in serum by approximately 25% and 60% compared with baseline and PBS/BSA-treated mice, respectively (P < 0.05 for both). In IL-6+/+ mice, the suppression of ASP was not significant. These data are in line with an inhibitory effect of IL-6 treatment on ASP formation (Fig. 1DGo).

Analysis of adipocyte size by microscopic sizing of freshly isolated cells from the inguinal fat depot in 7- to 8-month-old IL-6+/+ and IL-6–/– males showed a significant increase in cell diameter as well as cell weight in IL-6–/– mice compared with IL-6+/+ mice (Fig. 2Go). Moreover, free fatty acid (FFA) levels in plasma of 5- to 6-month-old nonobese female IL-6–/– mice were decreased after overnight fasting compared with those in IL-6+/+ mice (Table 2Go). Glucose levels were significantly higher in fasted IL-6–/– mice than in IL-6+/+ mice (Table 2Go).


Figure 2
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  		FIG. 2. Adipocyte cell weight (A) and adipocyte cell diameter (B) in 7- to 8-month-old male IL-6+/+ and IL-6–/– mice (n = 10, respectively). **, P < 0.05, by Student’s t test.

 

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  		TABLE 2. Fasting serum glucose and FFA levels in 5-month-old IL-6–/– and IL-6+/+ mice (n = 4 for IL-6+/+, n = 5 for IL-6–/–)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-6–/– mice display mature-onset obesity. We and others have previously shown that single central IL-6 injections to rats increase energy expenditure acutely (4, 5), and chronic central treatment with IL-6 specifically decreases fat mass (6, 7). It has also been shown that IL-6 treatment induces lipolysis (9, 10, 11, 12). The aim of the present study was to search for antiobesity mechanisms of IL-6. For this purpose, global gene expression analysis was performed and revealed changed expression of a number of genes in WAT and muscle of IL-6–/– compared with IL-6+/+ mice. Of these, we focused on a group of genes that have in common that they may influence the formation of ASP, which has been shown to have important effects on adipocyte TG formation.

Among factors influencing ASP formation, we found increased expression of TTR, properdin, and complement factor H in WAT of IL-6–/– mice. Of these, properdin and factor H have been reported to be expressed by adipocytes as components of the alternative pathway of complement activation. TTR, a negative, acute phase reactant normally released from the liver, has to our knowledge not been reported to be expressed in WAT. In this study it was not detectable in the majority of IL-6+/+ WAT samples, whereas it was expressed in the majority of IL-6–/– WAT samples. The same was observed for another liver-derived, negative, acute phase protein, the insulin receptor-tyrosine kinase inhibitor, or {alpha}-2 HS glycoprotein, which was also detected only in WAT from IL-6–/–, but not IL-6+/+, mice. This suggests that a complete lack of IL-6, by which these negative, acute phase reactants are normally suppressed in the liver, allows for ectopic expression of the genes in adipose tissue where they are not normally expressed. Expression of other acute phase proteins in WAT, e.g. C-reactive protein, has also been found in humans, where IL-6 may be of importance for its regulation (20).

The present experiments do not provide information about which cell types in WAT express the above-described IL-6-regulated, acute phase genes. All of these genes, including TTR, have been reported to be of importance for regulation of ASP formation in vitro, whereas there are to our knowledge no reports of increased ASP formation mediated by these factors in vivo. Figure 3Go shows a schematic presentation of ASP formation and the involvement of TTR and properdin (21). Properdin and TTR stimulate, whereas complement factor H inhibits, ASP formation. C3 converts spontaneously to C3a and C3b. C3b combines with factor B to form C3bB, and adipsin then cleaves factor B to generate C3bBb convertase, which cleaves C3 to C3a and C3b. The generated C3b can combine with factor B to start another cycle, whereas C3a is cleaved by carboxypeptidase to produce C3adesArg, which is identical with ASP. Properdin binds to and stabilizes, whereas factor H destabilizes, C3bBb convertase. We found adipsin to be up-regulated in muscle, but not in WAT. Muscle is a known site of adipsin production. It is possible that muscle-produced adipsin may be released into the circulation and can contribute to conversion of B to Bb and Ba in the circulation or adipose tissue, or it could have this effect locally within muscle. TTR stimulates the conversion of C3 to ASP, and it has been reported in vitro that ASP production is increased severalfold by the presence of TTR and chylomicrons (22). It has been demonstrated that TTR acts as a transporter of a signal, retinol (23). Retinol, which is then converted to the active form, retinoic acid, acts as a transcription factor to enhance C3 expression, C3 secretion, and, consequently, ASP production (23, 24). Several other ASP-related genes, e.g. complement factor C3, factor B, adipsin (factor D), and Crry, were expressed, but showed no regulation in WAT.


Figure 3
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  		FIG. 3. Schematic presentation of the effect of IL-6 deficiency on factors involved in the regulation of ASP formation. Complement factor C3 converts spontaneously and/or via catalysis to C3a and C3b. C3b complexes with factor B. Factor B is then cleaved by adipsin to form C3bBb convertase and Ba. The C3bBb convertase is stabilized by properdin, whereas it is destabilized by the inhibitory complement factor H. Stable C3bBb convertase cleaves C3 to C3a and C3b. Finally, the C-terminal arginine of C3a is cleaved by carboxypeptidase to form ASP (C3adesArg). Transthyretin stimulates ASP formation via a mechanism involving 1) retinol transfer from chylomicrons to adipocytes, 2) conversion of retinol to retinoic acid (RA), a transcription factor, 3) increased C3 expression and secretion, and 4) activation of ASP production. Factors that are regulated in IL-6–/– mice are marked with an arrow and in bold type. It is also possible that IL-6 may have effects via the central nervous system on ASP formation. RBP, Retinol-binding protein; R, retinol; CNS, central nervous system.

 
To elucidate the net effect of these changes in gene expression on circulating ASP levels, we measured ASP in serum of young and old IL-6–/– and IL-6+/+ mice. We found that ASP levels were increased 31–54% in both genders of IL-6–/– mice at different ages. Statistical analysis by two-way ANOVA indicated that the difference in ASP between male IL-6–/– and IL-6+/+ mice was attributable to genotype rather than age, although this difference did not quite reach significance by t test in young male mice alone (P = 0.056). In young nonobese female IL-6–/– mice, however, ASP levels were significantly increased compared with those in IL-6+/+ mice. These data together with the original findings of ASP-related gene regulations in young male mice support our hypothesis, i.e. that ASP is already increased in preobese IL-6–/– mice. Furthermore, ASP levels in IL-6+/+ mice correlated well with body weight and DXA fat mass, whereas in IL-6–/– mice there were no such correlations, supporting the idea that the lack of IL-6 causes dysregulation of ASP production. ASP levels were altered even though no change in expression of the ASP precursor complement C3 occurred in IL-6–/– mice.

IL-6 treatment with a total of 300 ng IL-6 over 4 d significantly decreased ASP levels in serum of IL-6–/– mice, in line with an inhibitory effect of IL-6 on ASP formation. This short-term IL-6 treatment did not affect body weight in either IL-6+/+ or IL-6–/– mice. We have shown previously that continuous IL-6 treatment of IL-6–/– mice for a prolonged period decreases fat mass and body weight (4). This was most likely an effect of IL-6 in the central nervous system, possibly mediated via the sympathetic nervous system (25). In line with this, it is possible that the presently reported effect of IL-6 on ASP formation may be of central nervous origin (Fig. 3Go). The present data also suggest that the effect of IL-6 deficiency (and IL-6 treatment) on ASP is specific and not secondary to changes in, for example, fat mass in IL-6–/– mice.

ASP has been shown to potently stimulate both uptake of dietary TG and TG storage in adipocytes. Thus, the increased ASP levels in the absence of IL-6 suggest that IL-6–/– mice may have increased TG uptake and storage in adipocytes. To test for differences in TG uptake between IL-6–/– and IL-6+/+ mice, we made several attempts to measure TG clearance after an intragastric TG load. Triglyceride levels in blood did not, however, increase significantly in either IL-6+/+ or IL-6–/– mice (not shown). This is in line with previous reports, which have shown that mice on the C57BL/6 background clear TG very efficiently from the circulation. This has also been proposed as a reason for the reported discrepancies in postprandial TG clearance in different strains of ASP knockout models (21). Therefore, we looked at indirect measures of TG uptake in adipocytes, adipocyte size, and FFAs in plasma. Analysis of adipocyte size by microscopic sizing of freshly isolated cells from the inguinal fat depot of 7- to 8-month-old IL-6–/– and IL-6+/+ males showed a significant increase in cell diameter and cell weight in IL-6–/– mice compared with IL-6+/+ mice (Fig. 2Go). Moreover, FFA levels in plasma of IL-6–/– mice were decreased after overnight fasting in nonobese female IL-6–/– mice vs. IL-6+/+ mice, indicating a decreased rate of lipolysis, which is well in line with previous reports of the effects of IL-6 on lipolysis (9, 10, 11, 12). This is also consistent with the reported inhibitory effect of increased ASP levels on hormone-sensitive lipase activity in adipose tissue and with the reported inhibitory effect of ASP on FFA turnover in adipose tissue (26). The importance of ASP for adipocyte lipid accumulation is indicated by several examples. Up-regulation of ASP has been shown in obese mice as well as in obese humans (21, 27, 28). C3–/– mice, which lack ASP, are lean and resistant to diet-induced obesity (14, 29). ASP deficiency can even attenuate the obese phenotype of leptin-deficient ob/ob mice (15). Depending on the genetic background, ASP-deficient mice either have or do not have delayed postprandial TG clearance (21).

Glucose levels were significantly higher in young preobese (5 months old) IL-6–/– mice compared with IL-6+/+ mice, which confirms our previous findings in older obese IL-6–/– mice (4). In this case, the mice were of equal body weight and fat mass, and therefore, the increase in glucose levels was not attributable to differences in adiposity. In light of the present finding of increased ASP levels in IL-6–/– mice, this may be consistent with the decreased plasma glucose levels observed in ASP-deficient mice (21). In vitro ASP has been shown to potently increase glucose uptake by adipocytes (30). Thus, this would suggest increased glucose uptake in adipocytes of IL-6–/– mice. The mechanism for the increased fasting plasma glucose level in IL-6–/– mice is not known, and this finding may be surprising in light of the above-mentioned ASP effect on adipocyte glucose uptake and other studies that suggested IL-6 as an adipocyte-derived inducer of insulin resistance (3). Recent data indicate, however, that IL-6 may have a tissue-specific action on insulin function, increasing insulin sensitivity in muscle and decreasing it in liver and adipose tissue (31, 32, 33).

The present data indicate that there may be several routes by which ASP formation can be regulated in vivo. To our knowledge this is the first report in vivo indicating an increase in ASP by up-regulation of properdin and TTR in adipose tissue. The increase in complement factor H, which inhibits ASP formation, was of lesser magnitude and may be a compensatory change balancing an additional increase in ASP levels.

To summarize, this is to our knowledge the first report to 1) show down-regulation of ASP by IL-6 and increased ASP levels in IL-6–/– mice; 2) imply that properdin and TTR in WAT, and adipsin in muscle, may influence ASP formation in vivo; and 3) suggest that up-regulation of ASP precedes and/or is a primary mechanism contributing to the development of obesity in IL-6–/– mice. This may also provide a new mechanism for how dysregulation of acute phase cytokines can influence lipid storage in adipose tissue.


   Acknowledgments
 
We thank Prof. John-Olov Jansson for valuable comments on the manuscript.


   Footnotes
 
This work was supported by the Swedish Research Council (Grant 521-2003-9116), the Swedish Society for Medical Research (Grant A200200346), the Adlerbertska Science Foundation, and Canadian Institutes for Health Research Grant OOP69600 (to K.C.). K.C. holds the Canada Research Chair in Adipose Tissue.

I.W., B.O., M.J., S.P., LMS.C., U.S., K.C., K.W., and V.W. have nothing to declare.

First Published Online March 2, 2006

Abbreviations: ASP, Acylation-stimulating protein; DXA, dual-energy x-ray absorptiometry; FFA, free fatty acid; HS, Heremans-Schmid; LPL, lipoprotein lipase; TBS, Tris-buffered saline; TG, triacylglycerol; TTR, transthyretin; WAT, white adipose tissue.

Received September 7, 2005.

Accepted for publication February 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pedersen BK, Steensberg A, Keller P, Keller C, Fischer C, Hiscock N, van Hall G, Plomgaard P, Febbraio MA 2003 Muscle-derived interleukin-6: lipolytic, anti-inflammatory and immune regulatory effects. Pflugers Arch 446:9–16[Medline]
  2. Wallenius K, Jansson JO, Wallenius V 2003 The therapeutic potential of interleukin-6 in treating obesity. Expert Opin Biol Ther 3:1061–1070[CrossRef][Medline]
  3. Fernandez-Real JM, Ricart W 2003 Insulin resistance and chronic cardiovascular inflammatory syndrome. Endocr Rev 24:278–301[Abstract/Free Full Text]
  4. Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, Ohlsson C, Jansson JO 2002 Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 8:75–79[CrossRef][Medline]
  5. Rothwell NJ, Busbridge NJ, Lefeuvre RA, Hardwick AJ, Gauldie J, Hopkins SJ 1991 Interleukin-6 is a centrally acting endogenous pyrogen in the rat. Can J Physiol Pharmacol 69:1465–1469[Medline]
  6. Li G, Klein RL, Matheny M, King MA, Meyer EM, Scarpace PJ 2002 Induction of uncoupling protein 1 by central interleukin-6 gene delivery is dependent on sympathetic innervation of brown adipose tissue and underlies one mechanism of body weight reduction in rats. Neuroscience 115:879–889[CrossRef][Medline]
  7. Wallenius K, Wallenius V, Sunter D, Dickson SL, Jansson JO 2002 Intracerebroventricular interleukin-6 treatment decreases body fat in rats. Biochem Biophys Res Commun 293:560–565[CrossRef][Medline]
  8. Stenlof K, Wernstedt I, Fjallman T, Wallenius V, Wallenius K, Jansson JO 2003 Interleukin-6 levels in the central nervous system are negatively correlated with fat mass in overweight/obese subjects. J Clin Endocrinol Metab 88:4379–4383[Abstract/Free Full Text]
  9. Lyngso D, Simonsen L, Bulow J 2002 Metabolic effects of interleukin-6 in human splanchnic and adipose tissue. J Physiol 543:379–386[Abstract/Free Full Text]
  10. Metzger S, Hassin T, Barash V, Pappo O, Chajek-Shaul T 2001 Reduced body fat and increased hepatic lipid synthesis in mice bearing interleukin-6-secreting tumor. Am J Physiol 281:E957–E965
  11. Nonogaki K, Fuller GM, Fuentes NL, Moser AH, Staprans I, Grunfeld C, Feingold KR 1995 Interleukin-6 stimulates hepatic triglyceride secretion in rats. Endocrinology 136:2143–2149[Abstract]
  12. van Hall G, Steensberg A, Sacchetti M, Fischer C, Keller C, Schjerling P, Hiscock N, Moller K, Saltin B, Febbraio MA, Pedersen BK 2003 Interleukin-6 stimulates lipolysis and fat oxidation in humans. J Clin Endocrinol Metab 88:3005–3010[Abstract/Free Full Text]
  13. Greenberg AS, Nordan RP, McIntosh J, Calvo JC, Scow RO, Jablons D 1992 Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3–L1 adipocytes: a possible role for interleukin 6 in cancer cachexia. Cancer Res 52:4113–4116[Abstract/Free Full Text]
  14. Murray I, Havel PJ, Sniderman AD, Cianflone K 2000 Reduced body weight, adipose tissue, and leptin levels despite increased energy intake in female mice lacking acylation-stimulating protein. Endocrinology 141:1041–1049[Abstract/Free Full Text]
  15. Xia Z, Sniderman AD, Cianflone K 2002 Acylation-stimulating protein (ASP) deficiency induces obesity resistance and increased energy expenditure in ob/ob mice. J Biol Chem 277:45874–45879[Abstract/Free Full Text]
  16. Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, Kohler G 1994 Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368:339–342[CrossRef][Medline]
  17. Olsson B, Bohlooly YM, Brusehed O, Isaksson OG, Ahren B, Olofsson SO, Oscarsson J, Tornell J 2003 Bovine growth hormone-transgenic mice have major alterations in hepatic expression of metabolic genes. Am J Physiol 285:E504–E511
  18. Saleh J, Blevins JE, Havel PJ, Barrett JA, Gietzen DW, Cianflone K 2001 Acylation stimulating protein (ASP) acute effects on postprandial lipemia and food intake in rodents. Int J Obes Relat Metab Disord 25:705–713[CrossRef][Medline]
  19. Sopasakis VR, Sandqvist M, Gustafson B, Hammarstedt A, Schmelz M, Yang X, Jansson PA, Smith U 2004 High local concentrations and effects on differentiation implicate interleukin-6 as a paracrine regulator. Obes Res 12:454–460[Medline]
  20. Ouchi N, Kihara S, Funahashi T, Nakamura T, Nishida M, Kumada M, Okamoto Y, Ohashi K, Nagaretani H, Kishida K, Nishizawa H, Maeda N, Kobayashi H, Hiraoka H, Matsuzawa Y 2003 Reciprocal association of C-reactive protein with adiponectin in blood stream and adipose tissue. Circulation 107:671–674[Abstract/Free Full Text]
  21. Cianflone K, Xia Z, Chen LY 2003 Critical review of acylation-stimulating protein physiology in humans and rodents. Biochim Biophys Acta 1609:127–143[Medline]
  22. Scantlebury T, Maslowska M, Cianflone K 1998 Chylomicron-specific enhancement of acylation stimulating protein and precursor protein C3 production in differentiated human adipocytes. J Biol Chem 273:20903–20909[Abstract/Free Full Text]
  23. Scantlebury T, Sniderman AD, Cianflone K 2001 Regulation by retinoic acid of acylation-stimulating protein and complement C3 in human adipocytes. Biochem J 356:445–452[CrossRef][Medline]
  24. Kalant D, Maslowska M, Scantlebury T, Wang HW, Cianflone K 2003 Control of lipogenesis in adipose tissue and the role of acylation stimulating protein. Can J Diab 27:154–171
  25. Wernstedt I, Edgley A, Berndtsson A, Faldt J, Bergstrom G, Wallenius V, Jansson JO 2 February 2006 Reduced stress- and cold-induced increase in energy expenditure in IL-6 deficient mice. Am J Physiol [Epub ahead of print: http://ajpregu.physiology.org/cgi/reprint/00514.2005v1]
  26. Van Harmelen V, Reynisdottir S, Cianflone K, Degerman E, Hoffstedt J, Nilsell K, Sniderman A, Arner P 1999 Mechanisms involved in the regulation of free fatty acid release from isolated human fat cells by acylation-stimulating protein and insulin. J Biol Chem 274:18243–18251[Abstract/Free Full Text]
  27. Koistinen HA, Vidal H, Karonen SL, Dusserre E, Vallier P, Koivisto VA, Ebeling P 2001 Plasma acylation stimulating protein concentration and subcutaneous adipose tissue C3 mRNA expression in nondiabetic and type 2 diabetic men. Arterioscler Thromb Vasc Biol 21:1034–1039[Abstract/Free Full Text]
  28. Maslowska M, Vu H, Phelis S, Sniderman AD, Rhode BM, Blank D, Cianflone K 1999 Plasma acylation stimulating protein, adipsin and lipids in non-obese and obese populations. Eur J Clin Invest 29:679–686[CrossRef][Medline]
  29. Murray I, Sniderman AD, Cianflone K 1999 Mice lacking acylation stimulating protein (ASP) have delayed postprandial triglyceride clearance. J Lipid Res 40:1671–1676[Abstract/Free Full Text]
  30. Maslowska M, Sniderman AD, Germinario R, Cianflone K 1997 ASP stimulates glucose transport in cultured human adipocytes. Int J Obes Relat Metab Disord 21:261–266[CrossRef][Medline]
  31. Klover PJ, Clementi AH, Mooney RA 2005 Interleukin-6 depletion selectively improves hepatic insulin action in obesity. Endocrinology 146:3417–3427[Abstract/Free Full Text]
  32. Rotter V, Nagaev I, Smith U 2003 Interleukin-6 (IL-6) induces insulin resistance in 3T3–L1 adipocytes and is, like IL-8 and tumor necrosis factor-{alpha}, overexpressed in human fat cells from insulin-resistant subjects. J Biol Chem 278:45777–45784[Abstract/Free Full Text]
  33. Weigert C, Hennige AM, Brodbeck K, Haring HU, Schleicher ED 2005 Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt. Am J Physiol 289:E251–E257



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