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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-2814
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 7 2688-2695
Copyright © 2007 by The Endocrine Society

Monocyte Chemoattractant Protein-1 in Subcutaneous Abdominal Adipose Tissue: Characterization of Interstitial Concentration and Regulation of Gene Expression by Insulin

Giuseppe Murdolo, Ann Hammarstedt, Madeléne Sandqvist, Martin Schmelz, Christian Herder, Ulf Smith and Per-Anders Jansson

The Lundberg Laboratory for Diabetes Research (G.M., A.H., M.Sa., U.S., P.-A.J.), Department of Molecular and Clinical Medicine, The Sahlgrenska Academy at Göteborg University, S-413 45 Göteborg, Sweden; Department of Internal Medicine (G.M.), Section of Internal Medicine, Endocrine and Metabolic Sciences, Perugia University, I-06122, Perugia, Italy; Department of Anesthesiology and Intensive Care Medicine Mannheim (M.Sc.), University of Heidelberg, 368 D-69120 Heidelberg, Germany; and German Diabetes Clinic (C.H.), German Diabetes Center, Leibniz Center at Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany

Address all correspondence and requests for reprints to: Giuseppe Murdolo, M.D., Ph.D., Department of Internal Medicine, Division of Endocrinology and Metabolism, Via Enrico Dal Pozzo, I-06122 Perugia, Italy. E-mail: giuseppe.murdolo{at}medic.gu.se; gmurdolo{at}tiscalinet.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The chemokine monocyte chemoattractant protein-1 (MCP-1) is implicated in obesity-associated chronic inflammation, insulin resistance, and atherosclerosis.

Objectives: The objectives of this study were to: 1) characterize the interstitial levels and the gene expression of MCP-1 in the sc abdominal adipose tissue (SCAAT), 2) elucidate the response of MCP-1 to acute hyperinsulinemia, and 3) determine the relationship between MCP-1 and arterial stiffness.

Design: Nine lean (L) and nine uncomplicated obese (OB) males were studied in the fasting state and during a euglycemic-hyperinsulinemic clamp combined with the microdialysis technique. Interstitial and serum MCP-1 (iMCP-1 and sMCP-1, respectively) levels, pulse wave analysis, and SCAAT biopsies were characterized at baseline and after hyperinsulinemia.

Results: OB showed elevated sMCP-1 (P < 0.01) but similar iMCP-1 levels as compared with L. Basal iMCP-1 concentrations were considerably higher than sMCP-1 (P < 0.0001), and a gradient between iMCP-1 and sMCP-1 levels was maintained throughout the hyperinsulinemia. At baseline, SCAAT gene expression profile revealed a "co-upregulation" of MCP-1, MCP-2, macrophage inflammatory protein-1{alpha}, and CD68 in OB, and whole-body glucose disposal inversely correlated with the MCP-1 gene expression. After hyperinsulinemia, MCP-1 and MCP-2 mRNA levels significantly increased in L, but not in OB. Finally, sMCP-1 excess in the OB positively correlated with the stiffer vasculature.

Conclusions: These observations demonstrate similar interstitial concentrations and a differential gene response to hyperinsulinemia of MCP-1 in the SCAAT from L and OB individuals. In human obesity, we suggest the SCAAT MCP-1 gene overexpression as a biomarker of an "inflamed" adipose organ and impaired glucose metabolism.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
RECENT STUDIES HAVE shown that adipose tissue (AT) biological functions are dysregulated in the obese (OB) state, leading to a change in the intracellular milieu through increased release of proinflammatory and reduced production of antiinflammatory adipokines ("dysregulated/inflamed fat") (1, 2, 3). The observation that the systemic concentrations of many immune mediators, which are also produced by AT, correlate with estimates of total body fatness (4, 5) has led to the assumption that an increased spillover from the dysregulated fat into the bloodstream triggers the obesity-associated state of chronic low-grade inflammation, as well as contributing to insulin resistance (IR) (6) and atherosclerosis (7). However, the quantitative importance of the AT inflammatory burden to the circulating levels of bioactive immunological proteins remains largely unknown in humans.

The microdialysis (MDA) of sc abdominal adipose tissue (SCAAT) emerges as a validated approach to characterize the AT milieu in vivo, providing important information at the cellular level (8). Novel MDA membranes with high cutoff properties allow sampling of large molecules (like adipokines) within the intercellular water space (9), along with a quantitative estimate of the SCAAT contribution to the circulating levels of a given adipokine.

Monocyte chemoattractant protein-1 (MCP-1), the major ligand of chemokine CC motif receptor 2 (CCR2), is a well-known regulator of monocyte recruitment to sites of inflammation (10). Accordingly, the OB state is associated with increased macrophage infiltration into AT (11, 12, 13, 14, 15), and these resident macrophages may play an important role in the development of the obesity-linked complications (12, 14). AT has also been shown to constitutively synthesize MCP-1 (4, 13), and overexpression of MCP-1 in the AT in transgenic mice is associated with whole-body IR (14, 15). Interestingly, MCP-1 has been also implicated in the modulation of adipocyte metabolism (16, 17) as well as in the impairment of insulin signaling in skeletal muscle (18), and in the pathogenesis of vascular disease (19). So far, increased systemic MCP-1 concentrations in obesity (4) and type 2 diabetes mellitus (20) have been reported, and insulin has also emerged as a putative modulator of MCP-1 (17, 21, 22). In light of these observations, it can be questioned whether in the OB state the circulating MCP-1 excess is a marker or, alternatively, an active mediator of obesity-linked IR and premature atherosclerosis.

The objectives of the present study were to: 1) characterize the systemic levels, as well as the interstitial concentrations and the gene expression of MCP-1 in the SCAAT; 2) elucidate the acute in vivo response to hyperinsulinemia of MCP-1; and 3) investigate the relationship between circulating MCP-1 and arterial stiffness (AS).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We selected 18 male volunteers without known heredity for type 2 diabetes mellitus, recruited through an advertisement in a local newspaper. The participants were enrolled into the study if they met the following eligibility criteria: 1) a healthy state, as determined by medical history, physical examination, and screening laboratory evaluations; 2) a fasting plasma glucose concentration less than 5.6 mM; 3) normal blood pressure (<130/85 mm Hg); 4) absence of changes (≥10%) in body weight during the last year preceding the screening; 5) normal exercise and drinking habits; and 6) no current regular medication.

The selected participants were then divided into two groups: lean (L) [(n = 9) body mass index (BMI) >18.5–24.9 kg/m2)]; and OB [(n = 9) BMI 30–40 kg/m2)].

The study has been approved by the Ethical Committee of Göteborg University and carried out according to the principles of the Declaration of Helsinki. An informed written consent was obtained from all volunteers before their participation in the study.

Study design

All subjects were studied on two different occasions, at 2- to 3-d intervals, in the following order: 1) body composition and basal SCAAT biopsy, and 2) glucose clamp study. For each session, the volunteers were admitted to the research center at 0800 h after an overnight fast, and investigated in a quiet and temperature-controlled room (26 ± 2 C). During the study period, the participants were requested to refrain from any major physical exercise 2 d before each study session.

Body composition and basal SCAAT biopsy. Body composition was assessed by whole-body dual-energy x-ray absorptiometry (DPX-IQ; Lunar Radiation, Madison, WI). SCAAT biopsy was performed in the periumbilical region under local anesthesia (2% lidocaine). Biopsies (1–2 g) were initially washed to remove traces of blood, immediately put in RNA stabilization reagent [RNAlater; Ambion (Europe) Ltd., Huntingdon, UK], and stored at –80 C before RNA extraction using the guanidinium thiocyanate method (23). Another part of the tissue specimen was used for cell size measurement, as reported elsewhere (23).

Glucose clamp study. Figure 1Go depicts the study design. Peripheral indwelling iv catheters were inserted into an antecubital vein (for the infusion of medications) and retrogradely into a wrist vein of the contralateral arm, warmed at 60 C in heating pads (for intermittent blood sampling of arterialized venous blood). After vein cannulation, basal blood samples were drawn, and the study was started with a bolus injection of inulin (50 mg/kg) (Inutest 25%; Fresenius Kabi GmbH, Graz, Austria), followed by a constant iv infusion (100 mg*min–1 for 330 min; see later).


Figure 1
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  		FIG. 1. Glucose clamp study design.

 
MDA. AT MDA for in situ measurement of MCP-1 was performed in the basal state and during insulin infusion. The sc interstitial MCP-1 (iMCP-1) concentrations were estimated as previously described (9). Briefly, custom-made MDA linear probes (Plasmaflo OP-02; Asahi Medical Co. Ltd., Oita, Japan) with a molecular mass cutoff of 3 MDa were inserted without anesthetics into the periumbilical SCAAT with a fine needle. The inlet of MDA membranes was connected to a microinjection pump (CMA Microdialysis AB, Stockholm, Sweden), and perfused at a rate 1.0 µl*min–1 with 1% human albumin (vol/vol) in isotonic saline. After 60 min equilibration, dialysate fractions for MCP-1 and inulin measurements were collected at 60- and 30-min intervals, respectively (Fig. 1Go). The period for the collection of dialysates was paralleled with intermittent blood samplings, and the fractions were stored at –70 C until analyzed.

To assess the actual iMCP-1 levels in vivo, MDA membranes were calibrated in situ to determine the relative recovery rate (RR) (recovery = dialysate concentration/interstitial concentration, with the dimension %) by the external reference technique (24). This calibration technique is based on the fact that the ratio of the RR of two substances is similar regardless of whether the recoveries are assessed under in vivo or in vitro conditions. Therefore, the in vivo RR of a given compound can be estimated by the relationship between the RR in vitro of the molecule of interest, and both the in vitro and in vivo recoveries of a reference substance. Inulin is used as a reference substance because it is a well-characterized polysaccharide that after a 240-min primed-constant infusion shows a full equilibrium between plasma and interstitial fluid (25).

In the present study, the relationship between inulin and MCP-1 in vitro recoveries was 1.15; inulin and MCP-1 RRs in vivo were 20.2 ± 9.8% and 20.3 ± 5.7% (mean ± SD), and 17.6 ± 8.5% and 17.6 ± 3.9% in the L and the OB subjects, respectively. Finally, the actual iMCP-1 concentrations were obtained dividing the dialysate levels by the assessed MCP-1 in vivo recovery.

Euglycemic-hyperinsulinemic clamp (EHC). Insulin sensitivity was evaluated by the EHC technique, essentially as described by De Fronzo et al. (26). Whole-body glucose disposal rate (Mr), normalized per kg of body weight (Mbw) or fat-free mass (Mffm), was determined during a primed-constant infusion of insulin (at the rate of 40 mU/m2*min–1), and calculated from the exogenous glucose infusion rate during the last 40-min period of the steady-state hyperinsulinemia. In addition, insulin action was expressed as the M/I ratio, the ratio of Mr to the steady-state serum insulin (SSSI) concentration.

At time point 270 min, inulin infusion was withdrawn, MDA membranes removed, and the SCAAT biopsy for the gene profile evaluation was taken. Insulin infusion was suspended when the biopsy procedure was completed.

Pulse wave analysis (PWA). The technique of PWA (SphygmoCor version 7.1; AtCor Medical, Sydney, Australia) was used to determine the augmentation index (AIx), a measure of systemic AS (27). Because AIx is influenced by heart rate (28), an index normalized for a heart rate of 75 bpm (AIx@75HR) was used. Larger values of AIx indicate increased wave reflection from the periphery or earlier return of the reflected wave as a result of increased pulse wave velocity (attributable to stiffer vessels).

At baseline, analyses were made twice at 60-min intervals, whereas during the EHC, PWA was recorded every 30 min.

Analytical procedures. sMCP-1 and iMCP-1 levels were quantified by an ELISA assay (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The intraassay and interassay coefficients of variation were less than 10%. Arterialized whole-blood glucose was immediately analyzed enzymatically (Yellow Springs Instrument, Yellow Springs, OH). Serum insulin and C-peptide were evaluated by immunochemiluminometric assay (ADVIA Centaur; Bayer HealthCare Diagnostic Division, Tarrytown, NY). Plasma nonesterified fatty acids (NEFA) were measured by an enzymatic colorimetric method (Wako Chemicals, Neuss, Germany). Inulin concentrations in plasma and dialysates were determined photometrically (9). B-HbA1c, fasting plasma glucose, lipid profile, fibrinogen, and plasminogen activator inhibitor-1 (PAI-1) activity were measured using standard methods at the central laboratory of the Sahlgrenska University Hospital. Homeostasis model assessment of insulin-resistance (HOMA-IR) was calculated as: [fasting insulin (µU/ml) x fasting glucose (mmol/liter)]/22.5.

TaqMan quantitative real-time RT-PCR. TaqMan real-time RT-PCR was used to quantify mRNA expression following the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Single-stranded random-hexamer-primed cDNA was synthesized from the RNA samples. Gene-specific probes and primer pairs for MCP-1/chemokine ligand CCL (CCL)-2, MCP-2/CCL8, macrophage inflammatory protein-1{alpha} (MIP-1{alpha})/CCL3, and CD68 (macrophage marker) were designed using the Primer Express software (Applied Biosystems), and the sequences can be supplied on request. The quantification was performed using the standard protocol of ABI PRISM 7900 HT (Applied Biosystems). For each primer/probe set, a standard curve was generated. Each sample was run in duplicate, and the quantity of a particular gene was normalized to that of 18s rRNA.

Statistical analysis

Data are shown as mean ± SD for parametric and as median (25th percentile; 75th percentile) for non-parametric variables, respectively. Before statistical analysis, non-normally distributed data were log transformed to approximate gaussian distribution, whenever appropriate. Statistical comparisons for groups over time were analyzed by a two-way ANOVA for repeated measures, followed by Tukey’s post hoc. Gene expression differences in SCAAT were assessed using the Wilcoxon and Mann-Whitney U tests. Univariate correlations were analyzed by Pearson and Spearman’s r correlations. Multivariate relationships were assessed by a general linear model. Statistical software from the SAS Institute, Inc. (Cary, NC) was used. P values < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical characteristics, adipocyte cell size, and insulin sensitivity

Table 1Go describes the clinical characteristics of the study subjects. By definition, OB showed increased body fat, central fat mass distribution, and higher HOMA-IR, as compared with L. In addition, elevated fasting triglycerides, total-cholesterol, low-density lipoprotein-cholesterol (LDL-C), as well as higher PAI-1, and fibrinogen levels were also seen in the OB, as compared with the L individuals.


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  		TABLE 1. Principal characteristics of the study subjects

 
The isolated adipocytes from SCAAT basal biopsy showed larger cell diameter and cell weight in OB, as compared with L, implying an increased energy partitioning to the adipose cells.

During the EHC, OB presented significantly lower Mr and M/I values, but higher SSSI and steady-state NEFA levels when compared with the L (Table 2Go).


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  		TABLE 2. Glucose disposal rate and indices of insulin action during the EHC

 
sMCP-1 and iMCP-1 concentrations

OB showed higher sMCP-1 concentrations as compared with L [319.9 (253.4; 371.6) vs. 260.6 pg/ml (237.6; 289); P < 0.01], whereas similar iMCP-1 levels were found [3719.5 (2065; 6657.3) vs. 3787.1 pg/ml (2459.2; 6223.3); P = NS] (Fig. 2Go).


Figure 2
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  		FIG. 2. Box-whisker plots showing sMCP-1 (A) and iMPC-1 (B) concentrations in the L and OB subjects. Boxes represent median (thick line in the middle of the boxes) and interquartile ranges (25th and 75th percentile; thin lines at the bottom and the top of the boxes). Error bars represent 10th and 90th percentiles. Significance is defined as *, P < 0.01.

 
A gradient between iMCP-1 and sMCP-1 was also observed throughout insulin infusion in both groups, but the differences between the subjects were not statistically significant (data not shown). Finally, compared with baseline, hyperinsulinemia was not coupled with significant sMCP-1 changes over time, whereas higher iMCP-1 levels at 120–180 min (P = 0.01) were seen in both groups (data not shown).

Basal sMCP-1 was positively correlated with age, BMI, total fat mass, and LDL-C (data not shown). The multivariate regression analysis revealed age and total fat mass as strong predictors of the sMCP-1, accounting for about 30% and 18% of its variance, respectively (Table 3Go). Moreover, sMCP-1 and iMCP-1 levels were not significantly correlated, nor did iMCP-1 correlate with fat cell size or the measures of adiposity and IR (data not shown).


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  		TABLE 3. Multivariate regression models for the relationship between basal sMCP-1 concentrations (dependent variable) and age alone (model 1), or combined with total fat mass (model 2)

 
SCAAT gene expression profile

The gene expression profile in SCAAT is shown in Table 4Go. At baseline, OB presented a "co-up-regulation" of MCP-1/CCL2, MCP-2/CCL8, MIP-1{alpha}/CCL3, and CD68 gene expression, as compared with L. After the EHC, MCP-1/CCL-2 and MCP-2/CCL8 gene expression was significantly increased in the L, but not in the OB, indicating a differential regulation by insulin and/or IR in these individuals.


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  		TABLE 4. SCAAT gene expression profile characterized in the basal state and after the EHC

 
Notably, SCAAT mRNA levels of CCR2 were below detection both at baseline and after the clamp. In contrast, we found abundant, but similar, CCR4 gene expression in the basal biopsy, and hyperinsulinemia was not coupled with significant changes in the CCR4 mRNA levels.

Interestingly, the basal adipose MCP-1 gene expression did not correlate with both the sMCP-1 and iMCP-1 concentrations (data not shown). Finally, we found a tight correlation between the adipose gene expression pattern and adipocyte cell size, fasting serum insulin as well as the M value (Table 5Go).


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  		TABLE 5. Correlation coefficients (r) between the SCAAT gene expression profile at baseline, adipocyte cell size, fasting serum insulin levels (FSI), and whole-body glucose disposal rate (M value)

 
AS

When compared with the L, OB individuals exhibited significantly higher values of AIx@75HR [0% (–10; 5) vs. –8% (–15; –5); P < 0.0001] as well as a blunted AIx@75HR decrease from the baseline during hyperinsulinemia [–2% (–6; 0) vs. –5% (–10; –2); P < 0.001]. Moreover, in the OB subjects, the larger values of AIx (stiffer vessels) were associated with both increased sMCP-1 (r 0.48; P < 0.01) and higher plasma NEFA (r 0.36; P < 0.05) concentrations.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we characterized, for the first time, MCP-1 concentrations in the bloodstream and at the cellular level by measuring both the gene expression and the interstitial concentrations of MCP-1 in the SCAAT. The results clearly show that the iMCP-1 levels are considerably higher than in serum. We also demonstrate that acute hyperinsulinemia differentially modulates the SCAAT MCP-1 gene expression, as well as the AS in L and OB individuals.

AT is a highly integrated organ, in which different cell types can interact through a complex molecular cross talk. The interest for characterizing the AT cellular milieu in vivo stems from the observations that the obesity-linked state of mild inflammation appears to be triggered by, and to reside in, the inflamed/dysregulated fat (6, 7, 14, 15). However, although AT is a potential systemic source of inflammation, only a limited number of adipose-derived mediators are released into the circulation in substantial amounts, resulting in obesity-associated increased systemic levels. By measuring the arteriovenous difference over the inferior epigastric vein, the trivial contribution of adipose-derived MCP-1 (29), TNF-{alpha} (30), and PAI-1 (31) to the circulating concentrations has been demonstrated.

In this study we found that the temporal variations of the iMCP-1 levels, as well as the mRNA expression, were not paralleled by significant changes in sMCP-1 concentrations, supporting the idea that the sc abdominal fat depot may not be an important contributor of the systemic MCP-1 increase in obesity (29). In addition, we found no significant associations between MCP-1 gene expression, circulating concentrations, and iMCP-1 levels, implying a complex molecular processing of the chemokine at the level of cellular translation, secretion, and/or clearance.

In humans, the increased circulating MCP-1 levels in the OB state have been attributed to the concomitant occurrence of subclinical atherosclerosis rather than obesity per se (32, 33). This possibility is also supported by our data showing a correlation between the AIx with the sMCP-1 levels, further strengthening the hypothesis that sMCP-1 excess in obesity may underlie endothelial dysfunction and, possibly, also a subclinical atherosclerotic burden (34). The finding that age was an independent predictor of sMCP-1 confirms that subclinical atherosclerosis may well represent a confounder in the intricate association between circulating MCP-1, obesity, and aging. Moreover, the lack of association between systemic and iMCP-1 might occur because other potential sources, such as endothelial cells, monocytes (19), and skeletal muscle (35), also produce MCP-1, and the adipose-derived MCP-1 may be "sequestered" in the extracellular and pericellular matrix by glycosaminoglycans and, therefore, does not reach the circulation (17). However, we cannot exclude that the sMCP-1 excess in obesity might also result from the expanded SCAAT through a different retention time in situ or an increased endothelial permeability and delivery to circulation of the iMCP-1.

An interesting, and unexpected, finding in the current study is the differential response to hyperinsulinemia of MCP-1 at the gene level in L and OB subjects. This observation suggests that acute insulinemia up-regulates MCP-1 gene expression in the noninflamed/nondysregulated adipose organ, and, thus, the basal MCP-1 gene overexpression in OB individuals may be a consequence of their ambient hyperinsulinemia. The correlation between MCP-1 expression and fasting insulin is in line with such an assumption. Acute hyperinsulinemia has also inhibited the circulating MCP-1 levels in obesity (22), though it has been suggested that such lowering ability may not be secondary to direct effects of insulin on AT (21). In our study hyperinsulinemia did not affect the sMCP-1 levels, further supporting a differential regulation of MCP-1 at the level of cellular translation. In harmony with this hypothesis, a lack of change in the SCAAT gene expression of MCP-1, despite the reduced plasma concentrations, has recently been reported in severely OB individuals after lifestyle intervention (36).

AT has been proposed to initiate and/or be the initial target organ for insulin where IR develops (37). Here, we showed a "co-up-regulation" of MCP-1, MCP-2, MIP-1{alpha}, and CD68 genes along with larger adipocyte cell size in the SCAAT from OB subjects, implicating the occurrence of an inflamed/dysregulated organ. Remarkably, both the cell size and the SCAAT MCP-1, MCP-2, and MIP-1{alpha} gene levels exhibited a strong and inverse association with the Mr. These findings suggest that the MCP-1 gene up-regulation in SCAAT may be a biomarker of obesity-linked IR and related to an inflamed adipose organ. The inhibitory effect of local cytokines/chemokines on normal preadipocyte development, lipid storage, and, ultimately, adiponectin production can thus impair whole-body insulin sensitivity (3).

MCP-1 effects are mediated through a receptor-ligand interaction in different cell types (10). In mice, CCR2 expression in AT seems to play a pivotal role in obesity-associated IR (11), but very little is currently known about the importance of this receptor pathway in humans (16, 38). In this study we did not detect a robust gene expression of CCR2 in the SCAAT biopsies. In addition, CCR2 was also not detected in the stroma-vascular cells from OB and non-OB Pima Indians (38). Based on these findings, we examined the gene expression of CCR4, another CC motif cognate receptor that also binds MCP-1 (10). The abundance of CCR4 mRNA in the tissue biopsies is in keeping with the concept that, in humans, adipose-derived MCP-1 may exert an autocrine/paracrine effect (17) through a "redundant" receptor-ligand interaction involving CCR4 rather than CCR2. Further investigations are required to unravel the complexity of the MCP-1/CCRs system in AT cell types.

There are few limitations to the present study that need to be considered. First, the characterization of iMCP-1 in the SCAAT may not reflect the activity of the whole-body fat mass because visceral AT has been indicated as the primary source of the circulating MCP-1 excess in obesity (13). However, it should be emphasized that the sc fat in the truncal region appears to contribute more to both the IR and the circulating NEFA pool than do AT depots elsewhere in the body (39). Also, even in the OB state, the intraperitoneal fat depot accounts for only approximately 10% of total fat mass. Therefore, regional differences in production may well have little impact on the total contribution in vivo of visceral fat on systemic MCP-1 excess. In line with this assumption, the lack of MPC-1 arteriovenous concentration differences across visceral fat has recently been reported in morbid OB subjects, by obtaining blood samples from the radial artery and portal vein (40). This finding further underscores the concept that also the visceral depot seems not an important source of secretion for circulating MCP-1 levels in human obesity.

Second, the insertion of the MDA probe causes a minor local injury, which might be reflected in the iMCP-1 levels. However, in unpublished experiments we observed that iMCP-1 concentrations were abundantly detected within 20 min of catheter insertion, and an increase was only seen after 80–100 min. Therefore, the chemokine release evoked by probe placement microtrauma is likely to contribute minimally to the elevated iMCP-1 levels measured initially. This concept is further supported by the control-placebo study we performed in five volunteers (three OB and two L) who also participated in the EHC. During a monitoring period of 6 h after probe implantation, we found that the sc interstitial concentration of IL-1{alpha}, CCL5/Regulated on Activation, Normal T cell Expressed and Secreted, and IL-18, cytokines known to be present at sites of acute inflammation, remained stable or actually decreased. Nonetheless, when compared with the EHC, a similar time course of both iMCP-1 and sMCP-1 was observed (data not shown). These data underline that the probe insertion cannot account for the present findings of elevated iMCP-1 in the AT and that the temporal profile of iMCP-1 reflects a "normal" diurnal pattern of the in situ secretion rather than an insulin or trauma-mediated effect.

In conclusion, our results suggest that the chemokine MCP-1 undergoes a complex cellular processing in SCAAT. In the OB state, MCP-1 gene overexpression is a marker of an inflamed adipose organ and impaired glucose metabolism, whereas circulating MCP-1 excess may underlie the presence of endothelial dysfunction. In contrast, the trivial contribution of the SCAAT to the systemic MCP-1 concentration and the abundant expression of CCR4 implicate adipose-derived MCP-1 as a putative paracrine/autocrine regulator. Finally, the present data do not support a clear cause-and-effect relationship between obesity-linked IR and MCP-1 secretion by the sc abdominal fat depot.


   Acknowledgments
 
The authors thank Mrs. Stina Mikkelsen, Christina Cullberg, and Lena Strindberg for their invaluable technical support and skilled contribution to the study.


   Footnotes
 
This work was supported by the European Community’s FP6 EUGENE2 (Grant LSHM-CT-2004-512013), the Swedish Medical Research Council, the Swedish Diabetes Association, the Novo Nordisk Foundation, the Torsten and Ragnar Söderberg Foundation, and an Albert Renold Fellowship from the European Foundation for the Study of Diabetes (to G.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 24, 2007

Abbreviations: AIx, Augmentation index; AIx@75HR, AIx normalized for a heart rate of 75 bpm; AS, arterial stiffness; AT, adipose tissue; BMI, body mass index; CCL, chemokine ligand CCL; CCR, chemokine CC motif receptor; EHC, euglycemic-hyperinsulinemic clamp; HOMA-IR, homeostasis model assessment of insulin resistance; iMCP-1, interstitial monocyte chemoattractant protein-1; IR, insulin resistance; L, lean; LDL-C, low-density lipoprotein-cholesterol; Mbw, Mr normalized per kilogram of body weight; MCP-1, monocyte chemoattractant protein-1; MDA, microdialysis; Mffm, Mr normalized per kilogram of fat-free mass; MIP-1{alpha}, macrophage inflammatory protein-1 {alpha}; Mr, whole-body glucose disposal rate; NEFA, nonesterified fatty acids; NS, not significant; OB, obese; PAI-1, plasminogen activator inhibitor-1; PWA, pulse wave analysis; RR, relative recovery rate; SCAAT, sc abdominal adipose tissue; sMCP-1, serum monocyte chemoattractant protein-1; SSSI, steady-state serum insulin.

Received December 19, 2006.

Accepted April 12, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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