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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 4833-4839
Copyright © 2004 by The Endocrine Society

Somatostatin Is Expressed and Secreted by Human Adipose Tissue upon Infection and Inflammation

Dalma Seboek, Philippe Linscheid, Henryk Zulewski, Igor Langer, Mirjam Christ-Crain, Ulrich Keller and Beat Müller

Department of Research (D.S., P.L.), Division of Endocrinology, Diabetology and Clinical Nutrition (H.Z., M.C.-C., U.K., B.M.), and Department of Visceral Surgery (I.L.), University Hospitals, CH-4031 Basel, Switzerland

Address all correspondence and requests for reprints to: Dalma Seboek, Department of Research, University Hospitals, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail: Dalma.Seboek{at}unibas.ch.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Somatostatin (SRIF) is a well-known neuroendocrine secretion product. SRIF expression and secretion are induced after inflammation in murine macrophages and in endotoxin-injected sheep and pigs. Because adipocytes have been demonstrated to produce numerous cytokines and peptide hormones, we investigated the expression of SRIF and its receptors (SSTR1–5) in human adipose tissue after inflammatory stimulation in vitro and in tissues from patients with septic disease.

Preadipocyte-derived adipocytes, mesenchymal stem cell-derived adipocytes, and mature explanted adipocytes expressed SRIF-mRNA after endotoxin [lipopolysaccharide (LPS)] or IL-1ß treatments. LPS- and IL-1ß-mediated SRIF-mRNA induction was blocked by pretreatment with dexamethasone. Using cocultures and quantitative real-time PCR, we demonstrate adipocyte SRIF induction by secretion factors from activated peripheral blood mononuclear cell-derived macrophages. In contrast to basal adipocytes, SRIF protein was detected in culture supernatants of LPS-treated and of combined TNF{alpha}/IL-1ß/LPS-treated adipocytes. SRIF protein was visualized by immunohistochemistry in explanted minced adipose tissue after overnight incubation in culture medium supplemented with combined IL-1ß and LPS. In septic patients, expression of SRIF-mRNA and SRIF protein was found in visceral, but not in sc, adipose tissue. Adipocyte mRNA abundance of SSTR 1–5 was differentially regulated by inflammatory treatments.

Thus, human visceral adipose tissue secretes SRIF during inflammation and sepsis and expresses several SSTRs. It is tempting to speculate that visceral adipose tissue-derived SRIF plays a modulatory role in the immunological and metabolic response to inflammation.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SRIF) WAS described initially as a secretory product of the hypothalamus acting as a potent inhibitor of GH secretion (1). Subsequently, high densities of SRIF-producing neuroendocrine cells have been localized throughout the central and peripheral nervous systems, in the endocrine pancreas and in the gut, and to a lower extent in the thyroid, adrenals, submandibular glands, kidneys, prostate, and placenta (2, 3, 4). SRIF expression and secretion was also described in murine inflammatory and immune cells after activation (2). In the gastrointestinal tract, SRIF inhibits the secretion of numerous peptide hormones (insulin, glucagon, gastrin, and cholecystokinin), gastric emptying, gallbladder contraction, and exocrine gut secretion (3, 5). The induction of SRIF by inflammatory cytokines IL-1ß, TNF{alpha}, and IL-6 was demonstrated in vitro in models of rat diencephalic cells (6, 7). SRIF-mRNA expression has been described in murine macrophage cell lines after cytokine stimulation (8). Accordingly, increased plasma SRIF levels were measured in jugular and portal veins in endotoxin-injected sheep and in septic pigs (9, 10).

Five distinct receptors mediating SRIF activity are widely expressed in many tissues. As a neurotransmitter, SRIF inhibits the release of GH, dopamine, norepinephrine, TRH, and CRH. Among others, additional modulatory roles have been ascribed to SRIF in inflammatory conditions and lymphocyte function (11, 12, 13). Adipose tissue emerged as a major endocrine organ in humans. Numerous peptide hormones released by adipocytes affect energy homeostasis, glucose and lipid metabolism, immune response, and reproduction (14). In addition, adipose tissue participates in the release of mediators of low-grade inflammation, which cause insulin resistance, relative impairment of insulin secretion, and, ultimately, hyperglycemia (15, 16). Knowledge of mechanisms underlying these processes is scarce. In view of the variety of endocrine factors involved in these processes, we investigated the expression of SRIF as a potential modulator in adipose tissue exposed to inflammatory cytokines. Here, we present in vivo and in vitro evidence for SRIF expression in adipose tissue in response to inflammation.


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

Permission was obtained from the local ethics committee for all experiments with patients. Adipose tissue samples were obtained from seven septic patients requiring laparotomy (mean age, 56 yr; range, 19–75 yr), after giving informed consent. Septicemias were due to peritonitis because of perforated sigmoid diverticulitis, perforated sigmoid carcinoma, perforated appendicitis, ischemic colitis of the sigmoid colon, and necrotisizing proctocolitis with perforation of the rectum and descending colon, respectively. In addition, adipose tissue was collected from noninfected patients undergoing elective surgery (mean age, 53 yr; range, 29–71 yr). Informed consent was obtained. Harvested tissues were incubated immediately in RNA-later (Ambion, Inc., Austin, TX) to prevent RNA degradation. The samples were snap-frozen and stored at –70 C. Tissues were powdered under liquid nitrogen before RNA extraction using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Alternatively, tissues were stored in 4% formaldehyde for immunohistochemical analyses. Circulating levels of SRIF were assessed in six septic patients (mean age, 54.7 yr; range, 36.1–73.3 yr; body mass index, 28.1 ± 6.1 kg/m2). The underlying diagnosis of these patients was sepsis due to pneumonia (n = 5) and staphylococcal infection (n = 1), respectively. In addition, we investigated SRIF plasma concentrations in four healthy subjects (mean age, 49.5 yr; range, 38–59 yr; body mass index, 24.4 ± 2.4 kg/m2), all staff members.

Adipocyte cultures and isolation of preadipocytes

After informed consent, 50- to 500-g sc adipose tissue samples were obtained from noninfected obese patients undergoing plastic surgery. Primary cultures of human adipocytes were performed as described previously (17, 18), with modifications. Briefly, adipose tissue was minced, digested in 1 mg/ml collagenase 2 (Worthington Biochemical Corp., Freehold, NJ), filtered (150-µm nylon mesh), and centrifuged at 200 x g. The cell pellet was resuspended twice in erythrocyte lysis buffer, washed, and seeded in six-well plates. After a 24-h incubation in DMEM/F12 with 10% fetal calf serum (FCS; Life Technologies, Inc., Basel, Switzerland) allowing attachment, cells were washed in PBS and cultured in serum-free medium supplemented with agents [isobutyl-methyl-xanthine, dexamethasone (DEX), insulin, transferrin, rosiglitazone, and triiodothyronine] that induce differentiation of preadipocytes to adipocytes. Triglyceride-storing adipocytes, representing 40–80% of cultured cells, are visible within 5–10 d. Differentiation was confirmed by RT-PCR analysis for adipocyte-specific peroxisome proliferator-activated receptor {gamma}2 expression (19). Adipocytes were maintained for an additional 4 d in DMEM/F12 with 10% FCS before experiments, without supplementation with the above listed agents, namely rosiglitazone. In addition, floating mature adipocytes obtained after the centrifugation step were washed twice in medium (DMEM/F12 with 10% FCS). Packed adipocytes (0.5 ml) were inoculated into 50-ml flasks (Becton Dickinson Labware, Rutherford, NJ) completely filled with medium and allowed to attach to the top surface for 72 h at 37 C (20, 21). Flasks were subsequently turned around, and non-adherent cells, representing the bulk of initially inoculated cells, were removed. The adherent, triglyceride-storing mature adipocytes were cultured in 5 ml medium for 2 additional days before experiments.

Adipocytes were stimulated for 6 or 24 h with 1 µg/ml lipopolysaccharide (LPS) and 20 U/ml IL-1ß, respectively. In selected experiments, cells were pretreated for 2 h with 1 µM DEX before IL-1ß or LPS addition. Human-specific IL-1ß was purchased from PeproTech (London, UK). LPS (Escherichia coli 026:B6) and DEX were from Sigma (Buchs, Switzerland).

The viability of adipocytes after stimulations was assessed via trypan blue staining, in which dead cells stain blue.

Human mesenchymal stem cells (MSCs)

Human MSCs were purchased from BioWhittaker Europe, S.p.r.l. (Verviers, Belgium). Differentiation into adipogenic lineage was performed as recommended by the manufacturer’s protocol. Differentiated cells were exposed to LPS, IL-1ß, and DEX as described above.

Adipocytes and macrophages in cocultures

Peripheral blood mononuclear cells were isolated by Ficoll-Plaque PLUS (Amersham, Uppsala, Sweden) and washed four times with Hank’s balanced salt solution (Invitrogen, Basel, Switzerland) supplemented with 0.5% human albumin (Blutspendedienst SRK, Bern, Switzerland). Cells were resuspended in Iscove’s modified Dulbecco’s medium with 20% human serum and seeded in cell culture inserts with 0.4-µm pore size (Becton Dickinson Labware, Dietikon, Switzerland). After 1 h, non-adherent cells were removed by thorough washing with Hank’s balanced salt solution and 0.5% human albumin. Adherent monocytes were cultured for 5 d. The obtained monocyte-derived macrophages were activated with live E. coli or combined 1 µg/ml LPS, 20 U/ml IL-1ß, 10 ng/ml TNF{alpha} administration. After 2 h, stimulants were removed by repeated washing, and the inserts containing activated macrophages were added to ex vivo differentiated adipocytes kept in six-well plates for an additional 22 h. Then, adipocytes and macrophages were separately subjected to SRIF-mRNA analysis as described below.

RT-PCR

Total RNA from homogenized tissues, adipocyte cultures, or MSCs, respectively, was extracted by the single-step guanidinium-isothiocyanate method with a commercial reagent (TRIzol reagent; Life Technologies, Inc.) according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically, and the quality was assessed by gel electrophoresis. Equal amounts of RNA per tissue or in vitro treatment were subjected to RT (Omniscript RT kit; Qiagen, Basel, Switzerland). PCR was performed on a conventional thermal cycler (TGradient; Biometra, Göttingen, Germany) using the PCR Taq core kit (Qiagen) and the following intron border-spanning oligonucleotides (22): SSTR1 (318-bp PCR product; GenBank accession no. BC035618), 5'-ATGGTGGCCCTCAAGGCCGG-3' (sense) and 5'-CGCGGTGGCGTAATAGTCAA-3' (antisense); SSTR2 (318-bp PCR product; GenBank accession no. AY236542), 5'-TCCTCTGGAATCCGAGTGGG-3' (sense) and 5'-TTGTCCTGCTTACTGTCACT-3' (antisense); SSTR3 (332-bp PCR product; GenBank accession no. AY277678), 5'-TGCCACCCTGGGCAACGTGT-3' (sense) and 5'-CAGGCAGAATATGCTGGTGA-3' (antisense); SSTR4 (323-bp PCR product; GenBank accession no. NM_ 001052), 5'-GCGCGCGGCGACCTACCGGC-3' (sense) and 5'-GCCTGGTGATTTTCTTCTCC-3' (antisense); SSTR5 (222-bp PCR product; GenBank NM_ 001053), 5'-CGTCTTCATCATCTACACGG-3' (sense) and 5'-GGCCAGGTTGACGATGTTGA-3' (antisense); SRIF (Ref. 23 ; 356-bp PCR product; GenBank accession no. BC032625), 5'-GATGCTGTCCTGCCGCCTCCAG-3' (sense) and 5'-ACAGGATGTGAAAGTCTTCCA-3' (antisense); ß-actin (198-bp product; GenBank accession no. AF076191), 5'- TTCTGACCCATGCCCACCAT-3' (sense) and 5'-ATGGATGATGATATCGCCGCGCTC-3' (antisense).

The annealing temperature was 62 C for SSTR1–4, 58 C for SSTR5, 60 C for SRIF, and 65 C for ß-actin. Thirty-five cycles of PCR were used for detection. Cycles were reduced to 28 for ß-actin, to stop the reaction in the linear phase of amplification. ß-Actin was used to verify equal quantities of RNA loading in each reaction. PCR products were separated and visualized on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide. PCR product identity was confirmed by direct nucleotide sequencing of the PCR products by dye deoxy terminator cycle sequencing. The absence of genomic DNA contamination was confirmed by RT-negative control reactions for all RNA preparations.

Quantitative analyses of SRIF-mRNA expression

cDNA, obtained as described above, was subjected to quantitative real-time PCR analysis using the ABI 7000 Sequence detection system (PerkinElmer Life Sciences, Emeryville, CA). Specific primers yielding short PCR products suitable for Sybr-Green detection were designed using Primer Express software (version 1.0; PE Applied Biosystems, Foster City, CA). Sequences of primers were as follows: SRIF (82-bp product; GenBank accession no. BC032625), 3'-GATGCCCTGGAACCTGAAGA-5' (sense) and 3'-CCGGGTTTGAGTTAGCAGATC-5' (antisense); hypoxanthine-guanine phosphoribosyltransferase (HPRT; 85-bp product; GenBank accession no. M26434), 3'-TCAGGCAGTATAATCCAAAGATGGT-5' (sense) and 3'-AGTCTGGCTTATATCCAACACTTCG-5' (antisense). The reaction volume was 22 µl, and the conditions were set as suggested by the manufacturer. Each cDNA sample tested for quantitative SRIF-mRNA expression was also subjected to HPRT-mRNA analysis. Results were expressed as the ratio of the respective SRIF-mRNA and HPRT-mRNA threshold values. The product identity was confirmed by sequence analysis and electrophoresis on a 2.5% agarose gel containing ethidium bromide.

Peptide measurement

SRIF concentrations were determined in EDTA plasma and supernatants using a commercially available SRIF RIA kit (functional assay sensitivity, 5 pg/ml; Phoenix Pharmaceuticals Inc., Belmont, CA).

Procalcitonin (ProCT) concentrations were determined in sera by an ultra-sensitive chemiluminometric assay with a functional sensitivity of 5 pg/ml (ProCa-S assay; B.R.A.H.M.S. GmbH, Hennigsdorf-Berlin, Germany).

Immunohistochemistry

Adipose tissue biopsies were formalin fixed and paraffin wax embedded. The slides were dewaxed in xylene and rehydrated through graded ethanols. Heat-induced epitope retrieval was performed by immersing the slides in citrate buffer (10 mM, pH 6) and microwaving at 98 C for 30 min before cooling and rinsing with PBS. Immunohistochemical staining was performed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) as recommended by the manufacturer’s protocol. SRIF rabbit polyclonal antibody was purchased from Novocastra Laboratories Ltd. (Newcastle, UK).

Density measurements

Density measurements of PCR amplification products were performed using Scion Image 4.02 Beta for Windows (Scion Corp., Frederick, MD).

Statistical analysis

All data are presented as means ± SEM. Unpaired t tests (two-sided) or Mann-Whitney U tests in case of nonparametric distributions were used to identify differences among the groups. For multigroup comparisons, one-way ANOVA with post hoc analysis for least-square differences was performed. Data were analyzed using Statistica for Windows (version 6; StatSoft Inc., Tulsa, OK).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
SRIF-mRNA induction in vitro and in vivo

In ex vivo differentiated adipocytes and in mature explanted adipocytes obtained from noninfected patients undergoing plastic surgery, SRIF-mRNA expression was almost not detectable by conventional RT-PCR analysis using 35 amplification cycles (Fig. 1Go). SRIF-mRNA expression was found after a 6-h exposure to LPS or the proinflammatory cytokine IL-1ß in ex vivo differentiated adipocytes, adipogenic differentiated MSCs, and mature adipocytes. Induction of SRIF-mRNA expression was repressed after preincubation of ex vivo differentiated adipocytes and MSCs with DEX for 2 h (Fig. 1Go). Stimulation with 0.004% dimethylsulfoxide, the carrier for DEX, alone had no effect on the SRIF induction (data not shown). SRIF-mRNA induction was also observed in adipocytes kept in coculture with E. coli-activated or combined cytokine/LPS-activated macrophages (Fig. 2Go). In contrast, no SRIF-mRNA induction was observed in macrophages per se stimulated with E. coli or with combined cytokines/LPS, respectively.



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  		FIG. 1. Induction of SRIF-mRNA expression in cultured adipocytes and MSCs. Explanted mature adipocytes kept in so-called "ceiling cultures," ex vivo differentiated adipocytes, and MSCs differentiated into the adipogenic lineage were subjected to 6-h IL-1ß (20 U/ml) and LPS (1 µg/ml) treatments, respectively. Ex vivo differentiated adipocytes and MSCs differentiated into the adipogenic lineage were preincubated with DEX for 2 h. SRIF-mRNA expression was analyzed by RT-PCR. Data are one of at least four independent experiments.

 


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  		FIG. 2. Quantitative analysis of SRIF-mRNA expression in ex vivo differentiated adipocytes kept in coculture with macrophages. Peripheral blood mononuclear cell-derived macrophages kept in cell culture inserts with 0.4-µm pores were activated for 2 h with live E. coli. After repeated washing with PBS, inserts were added to ex vivo differentiated adipocytes in six-well plates for an additional 22 h. Total RNA from macrophages and adipocytes were separately isolated and subjected to quantitative real-time PCR analysis using specifically designed primers yielding an 82-bp product suitable for Sybr-Green detection. SRIF-mRNA threshold values were normalized with HPRT-mRNA values. In addition, conventional PCR was performed and visualized on a 2.5% agarose gel. Results are means ± SEM (n = 4) or one of four independent experiments.

 
In several patients with severe bacterial infection, mirrored in markedly elevated serum ProCT levels (24), we found SRIF-mRNA expression in visceral fat depots but not in sc biopsies (Fig. 3AGo). The SRIF gene was not expressed in adipose tissue samples from noninfected control patients, neither of visceral nor of sc origin (Fig. 3Go, B and C).



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  		FIG. 3. SRIF-mRNA expression in adipose tissue obtained from septic and nonseptic humans. A, Subcutaneous (s) and visceral (v) adipose tissue biopsies were obtained intraoperatively from patients with markedly increased circulating ProCT levels indicating bacterial infection. B, Noninfected tissues were obtained from patients with nondetectable levels of serum ProCT as controls. SRIF-mRNA analysis was assessed by RT-PCR. Amplification products were visualized on agarose gels. Verification of mRNA as the source of amplification template was obtained by omitting the RT in reactions for pooled infected samples (rt-), resulting in no bands after PCR. RNA extracted from human islet cells was taken as a positive control (c+). C, Quantitative real-time PCR analysis using specifically designed primers yielding an 82-bp product suitable for Sybr-Green detection was performed. SRIF-mRNA threshold values were normalized with HPRT-mRNA values and are presented as means ± SEM (n = 4). Shown are data from four infected and four control patients, respectively. n.d., Not detected.

 
SRIF secretion in vitro and in vivo

In supernatants of ex vivo differentiated unstimulated adipocytes, the SRIF protein concentration was below or at the detection limit of 5 pg/ml after a 24-h incubation (Fig. 4Go). In contrast, SRIF protein secretion was increased to 31 ± 5.0 pg/ml in supernatants of LPS-treated cells. The administration of LPS, IL-1ß, and TNF{alpha} combined led to an average SRIF protein concentration of 24.3 ± 3.9 pg/ml (n = 6 for each treatment; ANOVA for least-square differences, P < 0.001). The viability of adipocytes after a 24-h exposure to LPS, IL-1ß, and TNF{alpha} was unchanged as assessed by trypan blue staining (data not shown). No significant differences in peripheral blood levels of SRIF were found between infected and noninfected patients. Infected patients had a plasma SRIF level of 89.55 ± 24.1 pg/ml, and noninfected individuals had a level of 158 ± 73.73 pg/ml (P = 0.21).



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  		FIG. 4. SRIF release by ex vivo differentiated adipocytes. Ex vivo differentiated adipocytes were subjected to inflammatory treatment (20 U/ml IL-1ß, 10 ng/ml TNF{alpha}, 1 µg/ml LPS) for 2 h in the presence of 10% fetal bovine serum. Thereafter, medium was replaced by serum-free medium containing the same inflammatory agents and incubated for an additional 22 h. SRIF protein content was analyzed by RIA. Random values between 0 and 5 were generated for measurements below the detection limit of 5 pg/ml. Data are means ± SEM (n = 6). *, P < 0.01, comparison of LPS alone and combined IL-1ß/ TNF{alpha}/LPS vs. control, respectively.

 
Immunohistochemistry for the SRIF protein was negative in adipose tissue biopsies obtained from noninfected control patients (Fig. 5AGo) with nondetectable circulating ProCT levels. In contrast, the SRIF protein was detected in visceral adipose tissue biopsies from healthy individuals after an overnight incubation in LPS- and IL-1ß-supplemented culture medium (Fig. 5BGo). Similarly, in visceral adipose tissue biopsies obtained from septic patients documented by increased circulating ProCT levels, the SRIF protein was detected by immunohistochemistry (Fig. 5DGo). No protein expression was seen in biopsies of sc origin from the same subjects (Fig. 5CGo).



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  		FIG. 5. SRIF immunohistochemical staining in human adipose tissue. Adipose tissue sections were immunohistochemically stained to determine the presence of SRIF. A, Subcutaneous adipose tissue biopsies obtained from noninfected individuals were taken as control tissue. B, Subcutaneous adipose tissue biopsies obtained from noninfected individuals were stimulated with LPS and IL-1ß overnight in cell culture medium. Subcutaneous (C) and visceral (D) adipose tissue biopsies were obtained intraoperatively from a septic patient. All magnifications, x400.

 
SSTR1–5 gene expression in vitro

SSTR-mRNA expression was assessed using RT-PCR. In unstimulated human adipocytes, SSTR subtypes 1 and 2 were expressed, in contrast to subtypes 3, 4, and 5. SSTR1 down-regulation was observed in adipocytes kept in coculture with E. coli-activated macrophages (Fig. 6Go). SSTR3 and SSTR4 were induced in the same experiments. SSTR2 remained unchanged, and SSTR5 was never detected.



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  		FIG. 6. Semiquantitative analysis of SSTR1–5-mRNA expression in ex vivo differentiated adipocytes kept in coculture with macrophages. Peripheral blood mononuclear cell-derived macrophages kept in cell culture inserts with 0.4-µm pores were activated for 2 h with live E. coli. After repeated washing with PBS, inserts were added to ex vivo differentiated adipocytes in six-well plates for an additional 22 h. Total RNA from adipocytes was separately isolated and subjected to RT-PCR analysis using specifically designed primers for each SSTR subtype. Amplification products were visualized on agarose gels. Semiquantitative analysis was performed by density measurements of the bands on the agarose gels. Results are presented as means ± SEM (n = 4) or one of four independent experiments. *, P < 0.05, comparison of stimulated (S) vs. control (C), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The present study shows that SRIF is expressed and secreted in human adipose tissue in response to inflammation. Thus, adipose tissue represents a novel site of SRIF expression besides the well-described neuroendocrine cells in the brain and the gastrointestinal tract.

Ex vivo differentiated preadipocytes could be contaminated by other adipose tissue-derived cell types (e.g. macrophages, endothelial cells), which could provide a non-adipocyte source of SRIF expression. Therefore, we repeated the experiments with MSC-derived adipocytes, with virtually identical results. However, due to the very nature of developing MSCs, contribution of other not yet finally differentiated cells cannot be excluded. In a third model, SRIF expression in adipocytes clearly validated the initial findings using mature and purified adipocytes cultured in adipocyte-selecting "ceiling cultures" (20, 21). Accordingly, SRIF-mRNA was detectable in visceral adipose tissue of septic patients but not in noninfected subjects.

In confirmation of the transcriptional data, immunoreactive SRIF was also detected in supernatants of cytokine-stimulated adipocytes, indicating that SRIF is transcribed, processed, and secreted in fat cells. In addition, using immunohistochemistry, SRIF-positive cells were found in the visceral fat of septic patients as well as in cytokine-stimulated explants of adipose tissue.

In a coculture system, activated macrophages induced adipocyte SRIF gene expression in a similar way as did IL-1ß and LPS, suggesting macrophage-derived stimulants are key components for this induction. Interestingly, we were not able to confirm the previously reported SRIF-mRNA expression by macrophages after inflammatory activation. This may be due to species differences between rodent and human macrophages and macrophage-like cell lines (25).

SRIF was found in visceral, but not sc, adipose tissue of patients with septic disease. It could be argued that SRIF expression in visceral fat was related to the proximity of visceral fat to the site of bacterial infection in our patients (bowel infections). In this respect, secretion of cytokines in response to septic disease was sufficient to induce ProCT expression in both visceral and sc tissue in the same patients (26). Therefore, we hypothesized another modulatory factor that suppressed SRIF expression in sc adipose tissue in vivo despite cytokine stimulation (e.g. innervations).

Venous blood from visceral fat is drained via the hepatic portal vein, where SSTRs have been characterized at the nerve endings of afferent fibers of the vagal nerve (27). These receptors translate humoral SRIF signals into neural signals in the central nervous system that may contribute to the responses of the brain to sepsis and inflammation. Hence, SRIF released from visceral fat after inflammatory stimulation during infections could modulate hepato-pancreatic perfusion and/or metabolic function, thereby augmenting the relative insulin deficit and/or resistance observed in septic patients (16). Furthermore, in obese patients, enhanced inflammation-related adipose tissue SRIF production might participate in the obesity-related complications reported in patients in the intensive care unit (28). SRIF has a short plasma half-life of approximately 2–3 min and shows a diurnal rhythm (2). This may explain the present observation that there was no difference in peripheral-venous plasma SRIF levels between infected and noninfected subjects. These findings make other systemic endocrine effects of adipose tissue-derived SRIF less likely but do not rule them out because only single samples were obtained. In rats, SRIF administration lowers inflammatory markers and mediators (29). Furthermore, endogenous SRIF may participate in antiinflammatory actions of glucocorticoids (11). In this respect, the negative effect of DEX on SRIF expression in adipocytes is noteworthy. It is tempting to speculate that sepsis-induced SRIF expression described here exerts feedback effects on inflammation.

Interestingly, mRNAs for SSTR1, 3, and 4 are differentially expressed in the presence or absence of inflammatory mediators in human adipocytes. In isolated rat adipocytes, SRIF surface binding and lipolytic action has been reported (30). Hence, adipocyte-derived, inflammation-induced SRIF may exert multiple effects in an autocrine or paracrine manner, e.g. down-regulation of leptin secretion because plasma leptin levels decreased in human subjects after SRIF administration (31). A lipolytic effect of SRIF was also demonstrated in chicken adipocytes after prolonged exposure to the peptide (32).

Assuming that lower grades of inflammation also result in preferential SRIF expression in visceral adipose tissue, these observations may explain several previous human data on reduced GH secretion in association with visceral adiposity. Indeed, there is a highly significant negative correlation between the visceral fat mass and 24-h GH secretion in human subjects; this association was stronger than the influence of age, gender, and total body fat on GH secretion (33). In addition, GH concentrations were reduced in parallel with visceral adiposity among patients with HIV-lipodystrophy, and an increased SRIF tone in these patients was postulated (34, 35).

Visceral adipose tissue is more closely associated with the insulin resistance syndrome than sc fat. In comparison, visceral adipocytes have higher lipolytic rates, are under distinct sympathetic nervous system regulation, secrete larger amounts of IL-6 and plasminogen activator inhibitor-1, and secrete smaller amounts of adiponectin and leptin (36).

In conclusion, SRIF is secreted by human adipocytes, both ex vivo after stimulation with activated macrophages, LPS, and IL-1ß and in vivo in visceral adipose tissue harvested from septic patients. Human adipose tissue expresses several SSTRs that are differentially regulated by inflammatory stimuli. It remains to be elucidated whether in sepsis visceral adipose tissue-derived SRIF plays a role as a classical hormone, thereby suppressing insulin or GH secretion, or alternatively, interferes by paracrine or autocrine action with the release of other adipocytokines.


   Acknowledgments
 
We are grateful to Prof. G. Pierer for providing adipose tissue, to Prof. M. Oberholzer for SRIF immunohistochemical staining, to Kaethi Dembinski and Susanne Vosmeer for excellent technical assistance, and to Isabelle Widmer and Caroline Koenig for providing EDTA plasma samples.


   Footnotes
 
This work was supported by grants from the Swiss National Science Foundation (32-59012.99 and 32-068209.02), the "Sonderprogramm zur Förderung des akademischen Nachwuchses der Universität Basel," the Nora van Meeuwen-Häfliger Foundation, and the Krokus Foundation and by unconditional research grants from Novartis AG.

D.S. and P.L. contributed equally to this work.

Abbreviations: DEX, Dexamethasone; FCS, fetal calf serum; HPRT, hypoxanthine-guanine phosphoribosyltransferase; LPS, lipopolysaccharide; MSC, mesenchymal stem cell; ProCT, procalcitonin; SRIF, somatostatin.

Received February 12, 2004.

Accepted June 30, 2004.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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