Autocrine/Paracrine Role of Inflammation-Mediated Calcitonin Gene-Related Peptide and Adrenomedullin Expression in Human Adipose Tissue
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内分泌学杂志 2005年第6期
Departments of Research (P.L., D.S., H.Z., U.K., B.M.) and Endocrinology, Diabetology, and Clinical Nutrition (H.Z., U.K., B.M.), University Hospital, CH-4031 Basel, Switzerland
Address all correspondence and requests for reprints to: Philippe Linscheid, Ph.D., Department of Research, University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail: philippe.linscheid@unibas.ch.
Abstract
Human adipose tissue is a contributor to inflammation- and sepsis-induced elevation of serum procalcitonin (ProCT). Several calcitonin (CT) peptides, including ProCT, CT gene-related peptide (CGRP), and adrenomedullin (ADM) are suspected mediators in human inflammatory diseases. Therefore, we aimed to explore the expression, interactions, and potential roles of adipocyte-derived CT peptide production. Expression of CT peptide-specific transcripts was analyzed by RT-PCR and quantitative real-time PCR in human adipose tissue biopsies and three different inflammation-challenged human adipocyte models. ProCT, CGRP, and ADM secretions were assessed by immunological methods. Adipocyte transcriptional activity, glycerol release, and insulin-mediated glucose transport were studied after exogenous CGRP and ADM exposure. With the exception of amylin, CT peptides were expressed in adipose tissue biopsies from septic patients, inflammation-activated mature explanted adipocytes, and macrophage-activated preadipocyte-derived adipocytes. ProCT and CGRP productions were significantly augmented in IL-1? and lipopolysaccharide-challenged mesenchymal stem cell-derived adipocytes but not in undifferentiated mesenchymal stem cells. In contrast, ADM expression occurred before and after adipogenic differentiation. Interferon- coadministration inhibited IL-1?-mediated ProCT and CGRP secretion by 78 and 34%, respectively but augmented IL-1?-mediated ADM secretion by 50%. Exogenous CGRP and ADM administration induced CT, CGRP I, and CGRP II mRNAs and dose-dependently (10–10 and 10–6 M) enhanced glycerol release. In contrast, no CGRP- and ADM-mediated effects were noted on ADM, TNF, and IL-1? mRNA abundances. In summary, CGRP and ADM are two differentially regulated novel adipose tissue secretion factors exerting autocrine/paracrine roles. Their lipolytic effect (glycerol release) suggests a metabolic role in adipocytes during inflammation.
Introduction
THE CALCITONIN (CT) gene family presumably evolved from a common ancestor gene because calcitonin peptides share amino acid homologies and display related receptor binding and biological activities (1). The calcitonin I (CALC I) gene gives rise to two distinct peptides by tissue-specific alternative splicing of the pre-mRNA: CT encoded in exon 4 in thyroidal C-cells and calcitonin gene-related peptide (CGRP) I encoded in exons 5 and 6 in sensory nerves (2). CALC II produces CGRP II but not CT because exon 4 contains a stop codon (1). CALC III is a pseudogene (3). Adrenomedullin (ADM) is encoded by CALC IV and amylin by CALC V genes (1).
CT was named after its postulated role in calcium homeostasis (4). CGRP I and II are almost identical neurotransmitters with potent vasodilatory properties (5). ADM, another vasoactive peptide, was originally discovered in human pheochromocytoma (6). Amylin is a 37-amino acid peptide hormone that is cosecreted with insulin by the pancreatic ?-cells in response to a nutrient stimulus (7). CT-, CGRP-, ADM-, and amylin-mediated signaling occurs via calcitonin receptor (CTR) and CR-like receptor (CRLR) with receptor activity-modifying proteins (RAMPs) 1–3 determining specific binding (8).
Serum concentrations of CT peptides, including procalcitonin (ProCT), CGRP, and ADM are markedly elevated in severe inflammation, systemic infections, sepsis, and sepsis-like conditions (9, 10, 11). Importantly, recent data suggest deleterious sepsis-related effects of ProCT, CGRP, and ADM (12, 13, 14, 15, 16). In contrast to the classic neuroendocrine paradigm, CT and CGRP I mRNA are ubiquitously expressed in septic hamsters and baboons (17, 18, 19). In humans, we recently demonstrated nonneuroendocrine CALC I expression and ProCT secretion in adipose tissue of sepsis patients and cytokine-challenged primary adipocytes (20, 21). Herein we sought to further elucidate the dynamics and metabolic effects of inflammation- and sepsis-related CGRP and ADM production in human adipose tissue. Because primary adipocyte availability is often scarce, mesenchymal stem cell (MSC)-derived adipocytes were also used as a model of functional adipocytes (22). Specifically, we aimed to clarify whether different CT peptides are differentially expressed and secreted on inflammatory stimulation and whether their production is dependent on the differentiation status. In addition, we studied transcriptional activity, glycerol release, and insulin-mediated glucose transport in adipocytes after exposure to exogenous CGRP and ADM.
Materials and Methods
Adipocyte cultures
Human mature explanted adipocytes and preadipocyte-derived adipocytes were obtained as previously described (20).
Human MSCs between passages 4 and 10 (Cambrex Bio Science Verviers, S.p.r.l., Verviers, Belgium) were seeded in 6-well plates and grown in MSC basal medium (Cambrex) until confluent. Adipogenic differentiation was induced by incubating the cells in DMEM/F12 (Invitrogen, Basel, Switzerland) containing 3% fetal bovine serum (FBS, Invitrogen) and the following supplements: 250 μM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 0.2 nM 3,3,5-triiodo-L-thyronine, 5 μM transferrin (all from Sigma, Buchs, Switzerland), 100 nM insulin (Novo Nordisk, Küsnacht, Switzerland), 1 μM rosiglitazone (GlaxoSmithKline, Worthing, UK). After 15–18 d, supplements were removed by washing three times with warm PBS, and experiments were started 2–4 d after completing differentiation. MSC-derived adipocytes had visible lipid droplets and expressed adipocyte-specific mRNAs, e.g. peroxisomal proliferator-activated receptor (PPAR)2, leptin, adiponectin, or GLUT4. IL-1? and lipopolysaccharide (LPS) administration induced glycerol release and glucose uptake was stimulated by insulin addition.
Adipocytes were subjected to treatments using the following agents: 100 U/ml interferon (IFN)-, 10 ng/ml TNF, 20 U/ml IL-1? (all human-specific cytokines from PeproTech, London, UK). CGRP, CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), ADM, and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) were purchased from Phoenix Europe GmbH (Karlsruhe, Germany), LPS (Escherichia coli 026:B6), and cycloheximide (ready made) were from Sigma.
Viability of adipocytes after stimulation was assessed with trypan blue staining: viable cells excluding trypan blue; dead cells staining blue.
Adipocytes and macrophages in coculture
White blood cells were isolated by Ficoll-Paque PLUS (Amersham, Uppsala, Sweden) and used for adipocyte macrophages coculture experiments as previously described (21).
RT-PCR
Total RNA from homogenized tissues or adipocyte cultures was extracted by the single-step guanidinium-isothiocyanate method with a commercial reagent (Tri Reagent; Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically at 260 nm (BioPhotometer; Vaudaux-Eppendorf AG, Sch?nenbuch, Switzerland). Ratio of extinction at 260 and 280 nm was between 1.5 and 2.0, and the quality was assessed by gel electrophoresis. One microgram total RNA was subjected to reverse transcription (Omniscript reverse transcription kit; QIAGEN, Basel, Switzerland). PCR was performed on a conventional thermal cycler (Tgradient; Biometra, G?ttingen, Germany) using PCR Taq core kit (QIAGEN). Human gene-specific primers and conditions were as indicated in Table 1. Amplification products were separated and visualized on 2% agarose gels containing 0.5 μg/ml ethidium bromide. A 100-bp molecular ruler (Bio-Rad Laboratories, Reinach, Switzerland) was run as reference marker. PCR product identity was confirmed by direct nucleotide sequencing of the PCR products by dye deoxy terminator cycle sequencing.
TABLE 1. List of human gene-specific primers in RT-PCR analysis
Quantitative analysis of CT, CGRP, and ADM mRNA expression
cDNA obtained as described above was subjected to quantitative real-time PCR analysis using the ABI 7000 sequence detection system (PerkinElmer, Norwalk, CT). Specific primers yielding short PCR products suitable for Sybr-Green detection were designed using Primer Express software (version 1.0; Applied Biosystems, Rotkreuz, Switzerland). Sequences of primers were as indicated in Table 1. Reaction volume was 22 μl, and the conditions were set as suggested by the manufacturer. For quantitative mRNA estimations, results were expressed as the ratio of the respective CT mRNA, CGRP mRNA, and ADM mRNA, respectively, to hypoxanthine-guanine phosphoribosyltransferase mRNA threshold values. Product identity was confirmed by sequence analysis and electrophoresis on a 2.5% agarose gel containing ethidium bromide.
Protein measurements
MSC-derived adipocytes were incubated for 48 h with treatments. Supernatant aliquots were kept at –70 C until analyzed.
ProCT concentrations were determined in supernatants by an ultrasensitive chemiluminometric assay with a functional sensitivity of 6 pg/ml (ProCa-S assay; B.R.A.H.M.S. GmbH, Hennigsdorf-Berlin, Germany).
ADM and CGRP concentrations were determined in supernatants by human-specific RIAs (Phoenix Europe). The CGRP kit (IC50 10–20 pg/tube) cross-reacts 0.09% with amylin and 0.004% with ADM. The ADM kit (IC50 6–20 pg/tube) cross-reacts 0.032% with CGRP I as indicated by the manufacturer.
Glycerol release into culture medium
On d 1 differentiation medium was removed from MSC-derived adipocytes. Cells were washed three times in warm PBS and kept in phenol red-free DMEM:F12 containing 3% FBS. Medium was changed on d 2 and supplements were added. On d 3, 800-μl supernatant aliquots were collected and kept frozen at –20 C until used for glycerol measurement (glycerol UV-method, Roche Molecular Biochemicals/R-Biopharm, Darmstadt, Germany).
Insulin-mediated glucose uptake
On d 1 differentiation medium was removed from MSC-derived adipocytes. Cells were washed three times in warm PBS and kept in DMEM/F12 containing 5 mM glucose and 3% FBS. Supplements were added on d 2. On d 3 at t = 0 min, 100 nM insulin were added to some wells. At t = 20 min, 1 μC deoxy-D-glucose, 2-[3H(G)] [PerkinElmer (Schweiz) AG, Schwerzenbach, Switzerland] was added to all wells. After 15 min cells were washed three times in ice-cold PBS and lysed in 0.1% sodium dodecyl sulfate. Radioactivity was measured in a scintillation counter.
Patients
Adipose tissue biopsies were obtained from healthy patients undergoing abdominal plastic surgery. Adipose tissue samples were also obtained from four infected patients, as documented with elevated serum ProCT (23), requiring laparotomy (mean age 44 yr, range 19–65 yr). The septicemia were due to peritonitis because of perforated sigmoid diverticulitis, perforated appendicitis, ischemic colitis of the sigmoid colon, and necrotizing proctocolitis with perforation of the rectum and descending colon. In addition, adipose tissue was collected from noninfected patients requiring elective surgery (mean age 53 yr, range 29–71 yr). Informed consent was obtained. Harvested tissues were immediately incubated 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.
All patients gave written informed consent to participate in the study. The study protocol was reviewed and approved by the Human Ethics Committee of Basel.
Statistical analysis
Results are presented as means ± SEM. Groups of experiments were compared statistically using Student’s t tests. In addition, two group comparisons corrected for multiple testing, i.e. one-way ANOVA with post hoc analysis for least-square differences, were performed.
Results
Sepsis-induced expressions of CT mRNAs in adipose tissue
CGRP I, CGRP II, and ADM mRNAs were detected in adipose tissue biopsies obtained from four septic patients with elevated serum ProCT (Fig. 1A). In contrast, the respective transcripts were not detected in normal, noninfected subjects (Fig. 1B). Amylin mRNA was found in neither control nor septic adipose tissue biopsies.
FIG. 1. CGRP I, CGRP II, and ADM mRNA expressions in adipose tissue biopsies of septic patients. Omental (o) and sc (s) adipose tissue biopsies were obtained from four septic patients and subjected to RT-PCR analysis for CT peptide transcripts (A). Serum ProCT level of each donor is indicated on top. Samples from noninfected donors were used as controls (B). n.d., Not detected; rt–, no reverse transcriptase controls to exclude genomic DNA contamination; c+, positive control.
Inflammation-induced expressions of CT mRNAs in primary adipocytes
Expressions of CGRP I, CGRP II, and ADM mRNAs were analyzed by RT-PCR in explanted mature adipocytes kept in ceiling cultures (20). Endothelial cells represent an important fraction of nonadipose cells in adipose tissue (24). Previously published PCR primers for endothelial nitric oxide synthase mRNA (25) showed expression in whole adipose tissue but not adipocytes (not shown). This verified the absence of contaminating stromal cells in explanted mature adipocyte preparations. Compared with basal conditions, CGRP I and ADM mRNA expression were markedly induced after 24 h of exposure to TNF, IL-1?, and LPS treatments, respectively (Fig. 2). In some experiments a weak inflammatory CGRP II mRNA induction was also observed. In contrast, amylin mRNA was never detected in mature adipocytes.
FIG. 2. Inflammatory CGRP I, CGRP II, and ADM mRNA inductions in human primary adipocytes. Human explanted adipocytes were subjected to 24 h TNF, IL-1?, and LPS treatments, respectively. Preadipocyte-derived adipocytes were kept in coculture with activated PBMC-derived macrophages for 24 h. Total RNA was separately isolated and induction of CT peptide transcripts was analyzed by RT-PCR analysis. mac, Macrophages. Shown results are representatives of three independent experiments.
Next, we studied CT peptide gene induction in preadipocyte-derived adipocytes cocultured with peripheral blood mononuclear cell (PBMC)-derived macrophages. In some experiments coincubation with unstimulated macrophages resulted in weak induction of CGRP I, CGRP II, and ADM mRNAs in adipocytes. Conversely, adipocyte CGRP I, CGRP II, and ADM mRNA expressions were markedly increased when macrophages were activated with live E. coli before coculture with adipocytes. ADM mRNA was detected in macrophages but not CGRP I and CGRP II mRNAs. Amylin mRNA was detected in neither adipocytes nor macrophages.
Inflammatory CT peptide expression and secretion in MSC-derived adipocytes
Production of CT peptides was analyzed in MSCs before and after adipogenic differentiation. PPAR2 mRNA, an adipocyte-specific marker, was present in MSC-derived adipocytes but not in undifferentiated MSCs (Fig. 3). ADM mRNA was induced by IL-1?, LPS, or combined IL-1?/TNF/LPS treatments in undifferentiated MSCs but not the mRNAs of CT, CGRP I, and CGRP II. In contrast, cytokine treatment of MSC-derived adipocytes induced CT, CGRP I, and CGRP II as well as ADM mRNA expression. Amylin mRNA was detected in neither MSCs nor MSC-derived adipocytes.
FIG. 3. CT peptide mRNA inductions in MSCs and MSC-derived adipocytes. Total RNA was isolated from MSCs and MSC-derived adipocytes on 24-h inflammatory treatments as indicated and reverse-transcribed. cDNA was subjected to RT-PCR analysis. Shown results are representatives of four independent experiments. RT–, Negative control lacking reverse transcriptase; C+, positive control.
In addition to conventional PCR, abundance of CT, CGRP I, and ADM mRNAs on inflammatory treatments was analyzed in MSC-derived adipocytes by quantitative real-time PCR. Compared with controls, CT mRNA was increased 79-fold (P < 0.05) on IL-1? treatment, 111-fold (P < 0.05) on LPS treatment, and 61-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment (Fig. 4A). CGRP I mRNA was increased 1.4-fold on IL-1? treatment, 2.4-fold (P < 0.05) on LPS treatment, and 3.2-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment. ADM mRNA was increased 8.4-fold on IL-1?-treatment, 18-fold on LPS treatment, and 80-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment.
FIG. 4. Quantitative analysis of inflammation-induced ProCT, CGRP, and ADM productions. MSC-derived adipocytes were subjected to treatments as indicated. Abundance of CT, CGRP I, and ADM mRNAs was analyzed after 48 h by quantitative real-time PCR using primers specifically designed for sybr green detection (A). Release of corresponding peptides was determined by supersensitive chemiluminometric assay (ProCT) or RIA (CGRP and ADM) (B). CT peptide mRNA inductions by combined IL-1?/TNF/LPS were analyzed by RT-PCR after short incubations up to 8 h. Shown results are means ± SEM from at least three independent experiments (A, B) or representatives from four independent observations (C). *, P < 0.05, compared with controls; , P < 0.05 compared with IL-1? alone.
Concentrations of ProCT, CGRP, and ADM in basal culture supernatants after 48 h were 9.7 ± 1.4, 11.1 ± 0.8, and 221 ± 31.8 pg/ml, respectively (Fig. 4B). ProCT secretion was increased 4.8-fold to 47.4 ± 8.0 pg/ml (P < 0.05) on IL-1? treatment, 5.0-fold to 48.2 ± 3.3 pg/ml (P < 0.05) on LPS treatment, and 4.5-fold to 43.9 ± 2.6 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment. CGRP protein secretion was increased 2.5-fold to 28.7 ± 1.8 pg/ml (P < 0.05) on IL-1? treatment, 3.2-fold to 35.3 ± 2.2 pg/ml (P < 0.05) on LPS treatment, and 4.1-fold 45 ± 1.0 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment. ADM protein secretion was increased 3.1-fold to 683 ± 40.9 pg/ml (P < 0.05) on IL-1? treatment, 3.7-fold to 815 ± 50.1 pg/ml (P < 0.05) on LPS treatment, and 6.8-fold to 1495 ± 75.1 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment.
IFN administration alone had no influence on CT peptide expression and secretion (Figs. 3 and 4). IL-1?-induced CT, CGRP, and CGRP II mRNAs were potently blocked when IFN was coadministered. Accordingly, IL-1?-mediated ProCT and CGRP release was inhibited by 78% to 10.1 ± 1.5 pg/ml (P < 0.05) and 34% to 18.6 ± 4.2 pg/ml, respectively, by IFN coadministration (Fig. 4B). In contrast, combined IFN and IL-1? administration resulted in 4.0-fold increased ADM mRNA abundance, compared with IL-1? alone. Accordingly, IL-1?-mediated ADM secretion into culture supernatants was increased 2.0-fold to 1347 ± 64.2 pg/ml (P < 0.05) in the presence of IFN.
Combined IL-1?/TNF/LPS treatment evoked maximal CGRP I and ADM induction and was therefore chosen for short-term induction analysis. CT, CGRP I, and CGRP II mRNAs were present after 2-h incubations (Fig. 4C). Low-level ADM mRNA was markedly augmented after 1 h.
Distinct CGRP- and ADM-mediated autoregulatory and metabolic effects in adipocytes
mRNAs of CTR, CRLR, RAMP1, RAMP2, and RAMP3 as well as neutral endopeptidase (NEP) were detected by RT-PCR in basal MSC-derived adipocytes (Fig. 5A). Exogenous CGRP and ADM were administered to MSC-derived adipocytes in concentrations ranging from 10–10 to 10–6 M. After 24 h, induction of CT, CGRP I, and CGRP II mRNA was observed, even at low concentrations (Fig. 5B). In contrast, the same mRNAs were not induced in adipocytes exposed to truncated peptides CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). One-nanomolar ADM-mediated mRNA inductions were blocked by 1 h preadministration of 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52), alone or in combination (Fig. 5C). One-micromolar ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) did not antagonize mRNA inductions when 10 nM ADM was added instead of 1 nM (not shown). Ten-nanomolar CGRP-mediated mRNA inductions were blocked by 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) but not by 1 μM ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). IL-6 mRNA was induced by ADM at high concentrations but not by CGRP (Fig. 5B). mRNAs of ADM, IL-1?, and TNF mRNAs were induced by 1 ng/ml LPS but not by 10–6 M CGRP or ADM (Fig. 5D).
FIG. 5. CGRP- and ADM-mediated effects in MSC-derived adipocytes. MSC-derived adipocytes were subjected to RT-PCR analysis of genes known to mediate CT peptide-signaling (A). CT peptide mRNAs were analyzed by RT-PCR in MSC-derived adipocytes after 24 h exposure to indicated concentrations of exogenous CGRP and ADM (B). To test specificity of observations, truncated peptides CGRP(8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) and ADM(22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 ) were administered alone (B) or in combination with ADM and CGRP (C). Effect of high CGRP and ADM concentrations, respectively, were analyzed on ADM, TNF, and IL-1? mRNAs (D). One nanogram per milliliter LPS was administered alone as a positive stimulation control. Protein synthesis was blocked by cycloheximide (CH) administration in CGRP-, ADM-, and IL-1?-treated cells, and RT-PCR analysis was performed after 6-h incubations (E). Shown results are representative of at least three separate experiments.
In preliminary experiments, 6 h had been determined as the minimal incubation time needed for detecting CT peptide mRNA induction in MSC-derived adipocytes treated with IL-1? alone. Therefore, possible effects of cycloheximide-mediated protein synthesis inhibition on CT peptide mRNA induction were studied after 6-h incubation periods. Five micromalar cycloheximide alone had no effect on CT mRNA induction (Fig. 5E). When coadministered to 100 nM ADM, 100 nM CGRP, or 20 U/ml IL-1?, a potentiating effect was noted on CT, CGRP I, CGRP II, and ADM mRNAs.
Lipolytic activity was assessed by measuring glycerol release into culture medium. Addition of CGRP and ADM between 1 nM and 1 μM evoked a dose-dependent activation of lipolysis. In contrast, addition of truncated peptides CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) or ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) did not induce lipolysis (Fig. 6). However, preadministration of 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) or ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) alone or in combination did not antagonize ADM and CGRP effects on lipolysis.
FIG. 6. CGRP- and ADM-mediated lipolysis induction in MSC-derived adipocytes. MSC-derived adipocytes were subjected to exogenous CGRP and ADM exposure between 1 nM and 1 μM for 24 h. Release of glycerol into supernatant was measured as an indicator of lipolytic activity. Results are presented as means ± SEM. n, Number of separate wells.
Potential effects of CGRP and ADM on insulin sensitivity were studied by comparing basal and insulin-mediated glucose uptake after their administration. Adipocytes were relatively insulin resistant on 24 h TNF exposure (Fig. 7). Coadministered CGRP or ADM did not further impair insulin action. In some experiments, presence of CGRP and ADM alone resulted in reduced insulin-mediated glucose uptake. However, statistical significance was not reached.
FIG. 7. Insulin-mediated glucose uptake. MSC-derived adipocytes were subjected to indicated treatments for 24 h. One hundred nanomoles insulin were applied to cells for 20 min. Then 3H-deoxy-glucose was added and uptake was monitored after 15 min. Insulin stimulation indices were expressed as the ratio of basal and insulin-mediated 3H-deoxy-glucose uptake. Results are expressed as means ± SEM. n, Number of independent triplicate experiments. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.
Discussion
Herein we demonstrate the sepsis-related production and potential functions of the two novel human adipose tissue secretion products CGRP and ADM. Lethal outcome of sepsis correlates with high serum levels of CT peptides including ProCT, CGRP, and ADM (15, 16, 23). Septic shock, a major cause of mortality and morbidity in intensive care units, is mainly characterized by hypotension and multiple organ failure, and the underlying cellular and molecular mechanisms are still poorly understood (26). Recent in vivo data suggest that high ProCT concentrations mediate deleterious effects: ProCT immunoneutralization increased survival rate in septic hamsters and pigs, whereas exogenous ProCT administration worsened the outcome (12, 13, 14). Immunomodulating properties of ProCT might be partially responsible for these observations (27, 28). Further activities to be unveiled might include some of the numerous properties found for other CT peptides with which ProCT shares a common structure. CGRP and ADM are potent vasodilators and modulators of nitric oxide production (29, 30, 31, 32). In addition, pharmacological CGRP and amylin doses induce insulin resistance in rat model in vivo and in vitro (33, 34).
We recently identified human adipose tissue depots as major sepsis-related nonneuroendocrine CT mRNA expression sites leading to increased serum ProCT (20, 21). By using adipose tissue biopsies from septic patients and three different human adipocyte models, we extend our previous findings on CGRP and ADM production in response to inflammatory stimuli. To assure that the observed cytokine-mediated CT peptide mRNA expressions do indeed occur in adipocytes, experiments were successfully performed in adipocyte ceiling cultures obtained by negative buoyancy selection of lipid-laden cells. The limited availability of primary adipose cell cultures was overcome by using bone marrow-derived MSCs as adipocyte precursors (22). In accordance with our first report on in vitro ProCT expression in human primary adipocytes (20), IL-1? and LPS potently induced CALC I gene expression in MSC-derived adipocytes. Similar kinetics of CT peptide mRNA induction was observed in both adipocyte models: IL-1?-induced CT mRNA was detectable after at least 6-h incubations and long lasting (t > 48 h). Furthermore, the previously shown potent inhibition by IFN on ProCT production was herein confirmed in MSC-derived adipocytes (20). In addition, the present data extend IFN-mediated gene repression to CGRP I and CGRP II.
Undifferentiated MSCs as well as preadipocytes do not respond with ProCT and CGRP production on any inflammatory treatment. This observation indicates that sepsis- related ProCT and CGRP production in adipose tissue depends on final differentiated adipocytes. We previously reported tissue-wide CT and CGRP mRNA expression (17, 18) in septic hamsters. The precise cellular origin and mechanisms of ProCT and CGRP production in nonadipose tissues will need to be addressed in future studies, but based on our findings, it is likely that the widespread up-regulation of CT and CGRP mRNA is limited to differentiated cells.
The positive effect of IFN on CALC IV gene-derived ADM mRNA expression and ADM protein secretion indicates major differences in gene expression in comparison with CALC I and CALC II. In contrast to ProCT and CGRP, inflammation-induced ADM expression was reported in numerous tissues and in vitro models, including vascular endothelial cells, vascular smooth muscle cells, fibroblasts, neurons, macrophages, TNF-treated 3T3-L1 murine adipocytes, and very recently basal rat adipocytes (6, 35, 36, 37, 38). Accordingly, besides differentiated adipocytes, we found ADM mRNA induction in undifferentiated MSCs and PBMC-derived macrophages. Thus, in contrast to CALC I and CALC II products ProCT and CGRP, sepsis- and inflammation-related ADM production appears to occur ubiquitously in all cell types independently of the differentiation state. Therefore, nonadipose cells possibly contribute in cytokine-mediated ADM mRNA expression and ADM peptide secretion shown herein. An approximately 10-fold induction in ADM mRNA abundance was recently shown in rat adipose tissues from obese high-fat-fed rats (38). Obesity is related to increased presence of macrophages in mice adipose tissue (39, 40). In coculture experiments we herein demonstrate ADM and CGRP mRNA induction by macrophage-secreted factors. Thus, a potential role of inflammation-mediated CT peptide expression in human obesity will need to be addressed.
The presence of CTR, CRLR, RAMP1, RAMP2, and RAMP3 mRNAs in MSC-derived adipocytes is in accordance with recent data from rat adipocytes (38). This indicates possible expression of functional CT peptide receptors, which may be responsible for the herein reported CGRP and ADM effects (8). NEP mRNA expression in MSC-derived adipocytes is in line with a previous report on active NEP found in human adipocytes (41). NEP actively degrades CGRP and ADM (42, 43). Furthermore, ADM and NEP are inversely correlated in septic rats (44). Therefore, adipocyte NEP functions might include clearance of CT peptides in the vicinity of adipocytes.
Induction of CALC I and CALC II gene transcription was observed on exposure of adipocytes to exogenous CGRP and ADM in concentrations ranging from 1 nM to 1 μM. This induction was selective because the mRNAs of ADM, TNF, and IL-1? were not affected by high CGRP and ADM concentrations. In contrast, the mentioned transcripts were induced by 1 ng/ml LPS administration in the present adipocyte model. This excludes that CGRP and ADM effects on CT, CGRP I, and CGRP II mRNAs are endotoxin-mediated artifacts from the recombinant peptide preparation. The CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)- and ADM(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)-mediated antagonism on mRNA inductions is a further indicator of specific CGRP and ADM effects.
When maximally stimulated with combined IL-1?/TNF/LPS treatment, CT, CGRP I, and CGRP II mRNA appeared already after 2 h. Cycloheximide-mediated protein synthesis inhibition had positive additive effects on ADM-, CGRP-, and IL-1?-mediated CT peptide mRNA inductions, respectively. Thus, inflammatory CALC gene mRNA inductions appear not to depend on intermediate gene expression. Moreover, based on the presented data, the existence of a CALC gene transcription repressing factor with a high turnover rate may be hypothesized.
In addition to transcriptional activation, a positive dose-dependent effect of CGRP and ADM was observed on glycerol release. Thus, in analogy to proinflammatory cytokines (e.g. TNF), CGRP and ADM possibly enhance lipolytic activity (45). Control experiments with ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) or CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) remained negative on glycerol release. However, in contrast to mRNA inductions, preadministration of truncated peptides did not antagonize ADM and CGRP effects on lipolysis. This might be explained by a very sensitive and/or an alternative signaling pathway leading to enhanced lipolysis (46). Future studies will need to address this issue.
Intensive insulin therapy reduces mortality in intensive care unit patients, but the mechanisms responsible for sepsis-related insulin resistance induction are still poorly understood (47). Herein we assessed adipocyte insulin sensitivity by monitoring insulin-dependent glucose transport. The demonstrated TNF-induced insulin resistance is in accordance with currently recognized concepts and expands the knowledge on MSC-derived adipocyte in vitro metabolic functionality (22, 48). Previous data suggested glucose transport-related antiinsulin effect by CGRP (33, 34). Herein attenuation of insulin-mediated glucose uptake by CGRP and ADM, however, was quite variable, indicating that other factors that are not yet well understood may play an important role. Therefore, a final conclusion cannot be drawn based on our data.
The process of adipocyte differentiation is characterized by increasing IL-6 production and nuclear factor-B-related inflammation (49, 50). Cultured adipocytes are known to express inflammatory genes (51, 52). Accordingly, in MSC-derived adipocytes, we found low-level basal IL-6, TNF, and IL-1? mRNA expression. CGRP and ADM are known to potentate inflammatory events in several cells and tissues (29, 53). Therefore, the herein shown CGRP- and ADM- mediated effects in basal adipocytes might be interpreted as modulatory events, rather than culprit inductions. Sepsis-related adipocyte CT peptide expression in vivo occurs in the presence of numerous proinflammatory factors, which might be significantly influenced by local CT peptide production. The precise mechanisms of inflammatory CT peptide expression and action have to be addressed in future experiments.
Acknowledgments
We are grateful to Igor Langer for providing adipose tissue biopsies. We thank Kaethi Dembinski and Susanne Vosmeer for excellent technical assistance. The ultrasensitive assays for calcitonin precursors were kindly provided by B.R.A.H.M.S. GmbH.
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Address all correspondence and requests for reprints to: Philippe Linscheid, Ph.D., Department of Research, University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail: philippe.linscheid@unibas.ch.
Abstract
Human adipose tissue is a contributor to inflammation- and sepsis-induced elevation of serum procalcitonin (ProCT). Several calcitonin (CT) peptides, including ProCT, CT gene-related peptide (CGRP), and adrenomedullin (ADM) are suspected mediators in human inflammatory diseases. Therefore, we aimed to explore the expression, interactions, and potential roles of adipocyte-derived CT peptide production. Expression of CT peptide-specific transcripts was analyzed by RT-PCR and quantitative real-time PCR in human adipose tissue biopsies and three different inflammation-challenged human adipocyte models. ProCT, CGRP, and ADM secretions were assessed by immunological methods. Adipocyte transcriptional activity, glycerol release, and insulin-mediated glucose transport were studied after exogenous CGRP and ADM exposure. With the exception of amylin, CT peptides were expressed in adipose tissue biopsies from septic patients, inflammation-activated mature explanted adipocytes, and macrophage-activated preadipocyte-derived adipocytes. ProCT and CGRP productions were significantly augmented in IL-1? and lipopolysaccharide-challenged mesenchymal stem cell-derived adipocytes but not in undifferentiated mesenchymal stem cells. In contrast, ADM expression occurred before and after adipogenic differentiation. Interferon- coadministration inhibited IL-1?-mediated ProCT and CGRP secretion by 78 and 34%, respectively but augmented IL-1?-mediated ADM secretion by 50%. Exogenous CGRP and ADM administration induced CT, CGRP I, and CGRP II mRNAs and dose-dependently (10–10 and 10–6 M) enhanced glycerol release. In contrast, no CGRP- and ADM-mediated effects were noted on ADM, TNF, and IL-1? mRNA abundances. In summary, CGRP and ADM are two differentially regulated novel adipose tissue secretion factors exerting autocrine/paracrine roles. Their lipolytic effect (glycerol release) suggests a metabolic role in adipocytes during inflammation.
Introduction
THE CALCITONIN (CT) gene family presumably evolved from a common ancestor gene because calcitonin peptides share amino acid homologies and display related receptor binding and biological activities (1). The calcitonin I (CALC I) gene gives rise to two distinct peptides by tissue-specific alternative splicing of the pre-mRNA: CT encoded in exon 4 in thyroidal C-cells and calcitonin gene-related peptide (CGRP) I encoded in exons 5 and 6 in sensory nerves (2). CALC II produces CGRP II but not CT because exon 4 contains a stop codon (1). CALC III is a pseudogene (3). Adrenomedullin (ADM) is encoded by CALC IV and amylin by CALC V genes (1).
CT was named after its postulated role in calcium homeostasis (4). CGRP I and II are almost identical neurotransmitters with potent vasodilatory properties (5). ADM, another vasoactive peptide, was originally discovered in human pheochromocytoma (6). Amylin is a 37-amino acid peptide hormone that is cosecreted with insulin by the pancreatic ?-cells in response to a nutrient stimulus (7). CT-, CGRP-, ADM-, and amylin-mediated signaling occurs via calcitonin receptor (CTR) and CR-like receptor (CRLR) with receptor activity-modifying proteins (RAMPs) 1–3 determining specific binding (8).
Serum concentrations of CT peptides, including procalcitonin (ProCT), CGRP, and ADM are markedly elevated in severe inflammation, systemic infections, sepsis, and sepsis-like conditions (9, 10, 11). Importantly, recent data suggest deleterious sepsis-related effects of ProCT, CGRP, and ADM (12, 13, 14, 15, 16). In contrast to the classic neuroendocrine paradigm, CT and CGRP I mRNA are ubiquitously expressed in septic hamsters and baboons (17, 18, 19). In humans, we recently demonstrated nonneuroendocrine CALC I expression and ProCT secretion in adipose tissue of sepsis patients and cytokine-challenged primary adipocytes (20, 21). Herein we sought to further elucidate the dynamics and metabolic effects of inflammation- and sepsis-related CGRP and ADM production in human adipose tissue. Because primary adipocyte availability is often scarce, mesenchymal stem cell (MSC)-derived adipocytes were also used as a model of functional adipocytes (22). Specifically, we aimed to clarify whether different CT peptides are differentially expressed and secreted on inflammatory stimulation and whether their production is dependent on the differentiation status. In addition, we studied transcriptional activity, glycerol release, and insulin-mediated glucose transport in adipocytes after exposure to exogenous CGRP and ADM.
Materials and Methods
Adipocyte cultures
Human mature explanted adipocytes and preadipocyte-derived adipocytes were obtained as previously described (20).
Human MSCs between passages 4 and 10 (Cambrex Bio Science Verviers, S.p.r.l., Verviers, Belgium) were seeded in 6-well plates and grown in MSC basal medium (Cambrex) until confluent. Adipogenic differentiation was induced by incubating the cells in DMEM/F12 (Invitrogen, Basel, Switzerland) containing 3% fetal bovine serum (FBS, Invitrogen) and the following supplements: 250 μM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 0.2 nM 3,3,5-triiodo-L-thyronine, 5 μM transferrin (all from Sigma, Buchs, Switzerland), 100 nM insulin (Novo Nordisk, Küsnacht, Switzerland), 1 μM rosiglitazone (GlaxoSmithKline, Worthing, UK). After 15–18 d, supplements were removed by washing three times with warm PBS, and experiments were started 2–4 d after completing differentiation. MSC-derived adipocytes had visible lipid droplets and expressed adipocyte-specific mRNAs, e.g. peroxisomal proliferator-activated receptor (PPAR)2, leptin, adiponectin, or GLUT4. IL-1? and lipopolysaccharide (LPS) administration induced glycerol release and glucose uptake was stimulated by insulin addition.
Adipocytes were subjected to treatments using the following agents: 100 U/ml interferon (IFN)-, 10 ng/ml TNF, 20 U/ml IL-1? (all human-specific cytokines from PeproTech, London, UK). CGRP, CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), ADM, and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) were purchased from Phoenix Europe GmbH (Karlsruhe, Germany), LPS (Escherichia coli 026:B6), and cycloheximide (ready made) were from Sigma.
Viability of adipocytes after stimulation was assessed with trypan blue staining: viable cells excluding trypan blue; dead cells staining blue.
Adipocytes and macrophages in coculture
White blood cells were isolated by Ficoll-Paque PLUS (Amersham, Uppsala, Sweden) and used for adipocyte macrophages coculture experiments as previously described (21).
RT-PCR
Total RNA from homogenized tissues or adipocyte cultures was extracted by the single-step guanidinium-isothiocyanate method with a commercial reagent (Tri Reagent; Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically at 260 nm (BioPhotometer; Vaudaux-Eppendorf AG, Sch?nenbuch, Switzerland). Ratio of extinction at 260 and 280 nm was between 1.5 and 2.0, and the quality was assessed by gel electrophoresis. One microgram total RNA was subjected to reverse transcription (Omniscript reverse transcription kit; QIAGEN, Basel, Switzerland). PCR was performed on a conventional thermal cycler (Tgradient; Biometra, G?ttingen, Germany) using PCR Taq core kit (QIAGEN). Human gene-specific primers and conditions were as indicated in Table 1. Amplification products were separated and visualized on 2% agarose gels containing 0.5 μg/ml ethidium bromide. A 100-bp molecular ruler (Bio-Rad Laboratories, Reinach, Switzerland) was run as reference marker. PCR product identity was confirmed by direct nucleotide sequencing of the PCR products by dye deoxy terminator cycle sequencing.
TABLE 1. List of human gene-specific primers in RT-PCR analysis
Quantitative analysis of CT, CGRP, and ADM mRNA expression
cDNA obtained as described above was subjected to quantitative real-time PCR analysis using the ABI 7000 sequence detection system (PerkinElmer, Norwalk, CT). Specific primers yielding short PCR products suitable for Sybr-Green detection were designed using Primer Express software (version 1.0; Applied Biosystems, Rotkreuz, Switzerland). Sequences of primers were as indicated in Table 1. Reaction volume was 22 μl, and the conditions were set as suggested by the manufacturer. For quantitative mRNA estimations, results were expressed as the ratio of the respective CT mRNA, CGRP mRNA, and ADM mRNA, respectively, to hypoxanthine-guanine phosphoribosyltransferase mRNA threshold values. Product identity was confirmed by sequence analysis and electrophoresis on a 2.5% agarose gel containing ethidium bromide.
Protein measurements
MSC-derived adipocytes were incubated for 48 h with treatments. Supernatant aliquots were kept at –70 C until analyzed.
ProCT concentrations were determined in supernatants by an ultrasensitive chemiluminometric assay with a functional sensitivity of 6 pg/ml (ProCa-S assay; B.R.A.H.M.S. GmbH, Hennigsdorf-Berlin, Germany).
ADM and CGRP concentrations were determined in supernatants by human-specific RIAs (Phoenix Europe). The CGRP kit (IC50 10–20 pg/tube) cross-reacts 0.09% with amylin and 0.004% with ADM. The ADM kit (IC50 6–20 pg/tube) cross-reacts 0.032% with CGRP I as indicated by the manufacturer.
Glycerol release into culture medium
On d 1 differentiation medium was removed from MSC-derived adipocytes. Cells were washed three times in warm PBS and kept in phenol red-free DMEM:F12 containing 3% FBS. Medium was changed on d 2 and supplements were added. On d 3, 800-μl supernatant aliquots were collected and kept frozen at –20 C until used for glycerol measurement (glycerol UV-method, Roche Molecular Biochemicals/R-Biopharm, Darmstadt, Germany).
Insulin-mediated glucose uptake
On d 1 differentiation medium was removed from MSC-derived adipocytes. Cells were washed three times in warm PBS and kept in DMEM/F12 containing 5 mM glucose and 3% FBS. Supplements were added on d 2. On d 3 at t = 0 min, 100 nM insulin were added to some wells. At t = 20 min, 1 μC deoxy-D-glucose, 2-[3H(G)] [PerkinElmer (Schweiz) AG, Schwerzenbach, Switzerland] was added to all wells. After 15 min cells were washed three times in ice-cold PBS and lysed in 0.1% sodium dodecyl sulfate. Radioactivity was measured in a scintillation counter.
Patients
Adipose tissue biopsies were obtained from healthy patients undergoing abdominal plastic surgery. Adipose tissue samples were also obtained from four infected patients, as documented with elevated serum ProCT (23), requiring laparotomy (mean age 44 yr, range 19–65 yr). The septicemia were due to peritonitis because of perforated sigmoid diverticulitis, perforated appendicitis, ischemic colitis of the sigmoid colon, and necrotizing proctocolitis with perforation of the rectum and descending colon. In addition, adipose tissue was collected from noninfected patients requiring elective surgery (mean age 53 yr, range 29–71 yr). Informed consent was obtained. Harvested tissues were immediately incubated 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.
All patients gave written informed consent to participate in the study. The study protocol was reviewed and approved by the Human Ethics Committee of Basel.
Statistical analysis
Results are presented as means ± SEM. Groups of experiments were compared statistically using Student’s t tests. In addition, two group comparisons corrected for multiple testing, i.e. one-way ANOVA with post hoc analysis for least-square differences, were performed.
Results
Sepsis-induced expressions of CT mRNAs in adipose tissue
CGRP I, CGRP II, and ADM mRNAs were detected in adipose tissue biopsies obtained from four septic patients with elevated serum ProCT (Fig. 1A). In contrast, the respective transcripts were not detected in normal, noninfected subjects (Fig. 1B). Amylin mRNA was found in neither control nor septic adipose tissue biopsies.
FIG. 1. CGRP I, CGRP II, and ADM mRNA expressions in adipose tissue biopsies of septic patients. Omental (o) and sc (s) adipose tissue biopsies were obtained from four septic patients and subjected to RT-PCR analysis for CT peptide transcripts (A). Serum ProCT level of each donor is indicated on top. Samples from noninfected donors were used as controls (B). n.d., Not detected; rt–, no reverse transcriptase controls to exclude genomic DNA contamination; c+, positive control.
Inflammation-induced expressions of CT mRNAs in primary adipocytes
Expressions of CGRP I, CGRP II, and ADM mRNAs were analyzed by RT-PCR in explanted mature adipocytes kept in ceiling cultures (20). Endothelial cells represent an important fraction of nonadipose cells in adipose tissue (24). Previously published PCR primers for endothelial nitric oxide synthase mRNA (25) showed expression in whole adipose tissue but not adipocytes (not shown). This verified the absence of contaminating stromal cells in explanted mature adipocyte preparations. Compared with basal conditions, CGRP I and ADM mRNA expression were markedly induced after 24 h of exposure to TNF, IL-1?, and LPS treatments, respectively (Fig. 2). In some experiments a weak inflammatory CGRP II mRNA induction was also observed. In contrast, amylin mRNA was never detected in mature adipocytes.
FIG. 2. Inflammatory CGRP I, CGRP II, and ADM mRNA inductions in human primary adipocytes. Human explanted adipocytes were subjected to 24 h TNF, IL-1?, and LPS treatments, respectively. Preadipocyte-derived adipocytes were kept in coculture with activated PBMC-derived macrophages for 24 h. Total RNA was separately isolated and induction of CT peptide transcripts was analyzed by RT-PCR analysis. mac, Macrophages. Shown results are representatives of three independent experiments.
Next, we studied CT peptide gene induction in preadipocyte-derived adipocytes cocultured with peripheral blood mononuclear cell (PBMC)-derived macrophages. In some experiments coincubation with unstimulated macrophages resulted in weak induction of CGRP I, CGRP II, and ADM mRNAs in adipocytes. Conversely, adipocyte CGRP I, CGRP II, and ADM mRNA expressions were markedly increased when macrophages were activated with live E. coli before coculture with adipocytes. ADM mRNA was detected in macrophages but not CGRP I and CGRP II mRNAs. Amylin mRNA was detected in neither adipocytes nor macrophages.
Inflammatory CT peptide expression and secretion in MSC-derived adipocytes
Production of CT peptides was analyzed in MSCs before and after adipogenic differentiation. PPAR2 mRNA, an adipocyte-specific marker, was present in MSC-derived adipocytes but not in undifferentiated MSCs (Fig. 3). ADM mRNA was induced by IL-1?, LPS, or combined IL-1?/TNF/LPS treatments in undifferentiated MSCs but not the mRNAs of CT, CGRP I, and CGRP II. In contrast, cytokine treatment of MSC-derived adipocytes induced CT, CGRP I, and CGRP II as well as ADM mRNA expression. Amylin mRNA was detected in neither MSCs nor MSC-derived adipocytes.
FIG. 3. CT peptide mRNA inductions in MSCs and MSC-derived adipocytes. Total RNA was isolated from MSCs and MSC-derived adipocytes on 24-h inflammatory treatments as indicated and reverse-transcribed. cDNA was subjected to RT-PCR analysis. Shown results are representatives of four independent experiments. RT–, Negative control lacking reverse transcriptase; C+, positive control.
In addition to conventional PCR, abundance of CT, CGRP I, and ADM mRNAs on inflammatory treatments was analyzed in MSC-derived adipocytes by quantitative real-time PCR. Compared with controls, CT mRNA was increased 79-fold (P < 0.05) on IL-1? treatment, 111-fold (P < 0.05) on LPS treatment, and 61-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment (Fig. 4A). CGRP I mRNA was increased 1.4-fold on IL-1? treatment, 2.4-fold (P < 0.05) on LPS treatment, and 3.2-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment. ADM mRNA was increased 8.4-fold on IL-1?-treatment, 18-fold on LPS treatment, and 80-fold (P < 0.05) on combined IL-1?/TNF/LPS treatment.
FIG. 4. Quantitative analysis of inflammation-induced ProCT, CGRP, and ADM productions. MSC-derived adipocytes were subjected to treatments as indicated. Abundance of CT, CGRP I, and ADM mRNAs was analyzed after 48 h by quantitative real-time PCR using primers specifically designed for sybr green detection (A). Release of corresponding peptides was determined by supersensitive chemiluminometric assay (ProCT) or RIA (CGRP and ADM) (B). CT peptide mRNA inductions by combined IL-1?/TNF/LPS were analyzed by RT-PCR after short incubations up to 8 h. Shown results are means ± SEM from at least three independent experiments (A, B) or representatives from four independent observations (C). *, P < 0.05, compared with controls; , P < 0.05 compared with IL-1? alone.
Concentrations of ProCT, CGRP, and ADM in basal culture supernatants after 48 h were 9.7 ± 1.4, 11.1 ± 0.8, and 221 ± 31.8 pg/ml, respectively (Fig. 4B). ProCT secretion was increased 4.8-fold to 47.4 ± 8.0 pg/ml (P < 0.05) on IL-1? treatment, 5.0-fold to 48.2 ± 3.3 pg/ml (P < 0.05) on LPS treatment, and 4.5-fold to 43.9 ± 2.6 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment. CGRP protein secretion was increased 2.5-fold to 28.7 ± 1.8 pg/ml (P < 0.05) on IL-1? treatment, 3.2-fold to 35.3 ± 2.2 pg/ml (P < 0.05) on LPS treatment, and 4.1-fold 45 ± 1.0 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment. ADM protein secretion was increased 3.1-fold to 683 ± 40.9 pg/ml (P < 0.05) on IL-1? treatment, 3.7-fold to 815 ± 50.1 pg/ml (P < 0.05) on LPS treatment, and 6.8-fold to 1495 ± 75.1 pg/ml (P < 0.05) on combined IL-1?/TNF/LPS treatment.
IFN administration alone had no influence on CT peptide expression and secretion (Figs. 3 and 4). IL-1?-induced CT, CGRP, and CGRP II mRNAs were potently blocked when IFN was coadministered. Accordingly, IL-1?-mediated ProCT and CGRP release was inhibited by 78% to 10.1 ± 1.5 pg/ml (P < 0.05) and 34% to 18.6 ± 4.2 pg/ml, respectively, by IFN coadministration (Fig. 4B). In contrast, combined IFN and IL-1? administration resulted in 4.0-fold increased ADM mRNA abundance, compared with IL-1? alone. Accordingly, IL-1?-mediated ADM secretion into culture supernatants was increased 2.0-fold to 1347 ± 64.2 pg/ml (P < 0.05) in the presence of IFN.
Combined IL-1?/TNF/LPS treatment evoked maximal CGRP I and ADM induction and was therefore chosen for short-term induction analysis. CT, CGRP I, and CGRP II mRNAs were present after 2-h incubations (Fig. 4C). Low-level ADM mRNA was markedly augmented after 1 h.
Distinct CGRP- and ADM-mediated autoregulatory and metabolic effects in adipocytes
mRNAs of CTR, CRLR, RAMP1, RAMP2, and RAMP3 as well as neutral endopeptidase (NEP) were detected by RT-PCR in basal MSC-derived adipocytes (Fig. 5A). Exogenous CGRP and ADM were administered to MSC-derived adipocytes in concentrations ranging from 10–10 to 10–6 M. After 24 h, induction of CT, CGRP I, and CGRP II mRNA was observed, even at low concentrations (Fig. 5B). In contrast, the same mRNAs were not induced in adipocytes exposed to truncated peptides CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). One-nanomolar ADM-mediated mRNA inductions were blocked by 1 h preadministration of 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52), alone or in combination (Fig. 5C). One-micromolar ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) did not antagonize mRNA inductions when 10 nM ADM was added instead of 1 nM (not shown). Ten-nanomolar CGRP-mediated mRNA inductions were blocked by 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) but not by 1 μM ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). IL-6 mRNA was induced by ADM at high concentrations but not by CGRP (Fig. 5B). mRNAs of ADM, IL-1?, and TNF mRNAs were induced by 1 ng/ml LPS but not by 10–6 M CGRP or ADM (Fig. 5D).
FIG. 5. CGRP- and ADM-mediated effects in MSC-derived adipocytes. MSC-derived adipocytes were subjected to RT-PCR analysis of genes known to mediate CT peptide-signaling (A). CT peptide mRNAs were analyzed by RT-PCR in MSC-derived adipocytes after 24 h exposure to indicated concentrations of exogenous CGRP and ADM (B). To test specificity of observations, truncated peptides CGRP(8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) and ADM(22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 ) were administered alone (B) or in combination with ADM and CGRP (C). Effect of high CGRP and ADM concentrations, respectively, were analyzed on ADM, TNF, and IL-1? mRNAs (D). One nanogram per milliliter LPS was administered alone as a positive stimulation control. Protein synthesis was blocked by cycloheximide (CH) administration in CGRP-, ADM-, and IL-1?-treated cells, and RT-PCR analysis was performed after 6-h incubations (E). Shown results are representative of at least three separate experiments.
In preliminary experiments, 6 h had been determined as the minimal incubation time needed for detecting CT peptide mRNA induction in MSC-derived adipocytes treated with IL-1? alone. Therefore, possible effects of cycloheximide-mediated protein synthesis inhibition on CT peptide mRNA induction were studied after 6-h incubation periods. Five micromalar cycloheximide alone had no effect on CT mRNA induction (Fig. 5E). When coadministered to 100 nM ADM, 100 nM CGRP, or 20 U/ml IL-1?, a potentiating effect was noted on CT, CGRP I, CGRP II, and ADM mRNAs.
Lipolytic activity was assessed by measuring glycerol release into culture medium. Addition of CGRP and ADM between 1 nM and 1 μM evoked a dose-dependent activation of lipolysis. In contrast, addition of truncated peptides CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) or ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) did not induce lipolysis (Fig. 6). However, preadministration of 1 μM CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) or ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) alone or in combination did not antagonize ADM and CGRP effects on lipolysis.
FIG. 6. CGRP- and ADM-mediated lipolysis induction in MSC-derived adipocytes. MSC-derived adipocytes were subjected to exogenous CGRP and ADM exposure between 1 nM and 1 μM for 24 h. Release of glycerol into supernatant was measured as an indicator of lipolytic activity. Results are presented as means ± SEM. n, Number of separate wells.
Potential effects of CGRP and ADM on insulin sensitivity were studied by comparing basal and insulin-mediated glucose uptake after their administration. Adipocytes were relatively insulin resistant on 24 h TNF exposure (Fig. 7). Coadministered CGRP or ADM did not further impair insulin action. In some experiments, presence of CGRP and ADM alone resulted in reduced insulin-mediated glucose uptake. However, statistical significance was not reached.
FIG. 7. Insulin-mediated glucose uptake. MSC-derived adipocytes were subjected to indicated treatments for 24 h. One hundred nanomoles insulin were applied to cells for 20 min. Then 3H-deoxy-glucose was added and uptake was monitored after 15 min. Insulin stimulation indices were expressed as the ratio of basal and insulin-mediated 3H-deoxy-glucose uptake. Results are expressed as means ± SEM. n, Number of independent triplicate experiments. *, P < 0.05, compared with controls; **, P < 0.01, compared with controls.
Discussion
Herein we demonstrate the sepsis-related production and potential functions of the two novel human adipose tissue secretion products CGRP and ADM. Lethal outcome of sepsis correlates with high serum levels of CT peptides including ProCT, CGRP, and ADM (15, 16, 23). Septic shock, a major cause of mortality and morbidity in intensive care units, is mainly characterized by hypotension and multiple organ failure, and the underlying cellular and molecular mechanisms are still poorly understood (26). Recent in vivo data suggest that high ProCT concentrations mediate deleterious effects: ProCT immunoneutralization increased survival rate in septic hamsters and pigs, whereas exogenous ProCT administration worsened the outcome (12, 13, 14). Immunomodulating properties of ProCT might be partially responsible for these observations (27, 28). Further activities to be unveiled might include some of the numerous properties found for other CT peptides with which ProCT shares a common structure. CGRP and ADM are potent vasodilators and modulators of nitric oxide production (29, 30, 31, 32). In addition, pharmacological CGRP and amylin doses induce insulin resistance in rat model in vivo and in vitro (33, 34).
We recently identified human adipose tissue depots as major sepsis-related nonneuroendocrine CT mRNA expression sites leading to increased serum ProCT (20, 21). By using adipose tissue biopsies from septic patients and three different human adipocyte models, we extend our previous findings on CGRP and ADM production in response to inflammatory stimuli. To assure that the observed cytokine-mediated CT peptide mRNA expressions do indeed occur in adipocytes, experiments were successfully performed in adipocyte ceiling cultures obtained by negative buoyancy selection of lipid-laden cells. The limited availability of primary adipose cell cultures was overcome by using bone marrow-derived MSCs as adipocyte precursors (22). In accordance with our first report on in vitro ProCT expression in human primary adipocytes (20), IL-1? and LPS potently induced CALC I gene expression in MSC-derived adipocytes. Similar kinetics of CT peptide mRNA induction was observed in both adipocyte models: IL-1?-induced CT mRNA was detectable after at least 6-h incubations and long lasting (t > 48 h). Furthermore, the previously shown potent inhibition by IFN on ProCT production was herein confirmed in MSC-derived adipocytes (20). In addition, the present data extend IFN-mediated gene repression to CGRP I and CGRP II.
Undifferentiated MSCs as well as preadipocytes do not respond with ProCT and CGRP production on any inflammatory treatment. This observation indicates that sepsis- related ProCT and CGRP production in adipose tissue depends on final differentiated adipocytes. We previously reported tissue-wide CT and CGRP mRNA expression (17, 18) in septic hamsters. The precise cellular origin and mechanisms of ProCT and CGRP production in nonadipose tissues will need to be addressed in future studies, but based on our findings, it is likely that the widespread up-regulation of CT and CGRP mRNA is limited to differentiated cells.
The positive effect of IFN on CALC IV gene-derived ADM mRNA expression and ADM protein secretion indicates major differences in gene expression in comparison with CALC I and CALC II. In contrast to ProCT and CGRP, inflammation-induced ADM expression was reported in numerous tissues and in vitro models, including vascular endothelial cells, vascular smooth muscle cells, fibroblasts, neurons, macrophages, TNF-treated 3T3-L1 murine adipocytes, and very recently basal rat adipocytes (6, 35, 36, 37, 38). Accordingly, besides differentiated adipocytes, we found ADM mRNA induction in undifferentiated MSCs and PBMC-derived macrophages. Thus, in contrast to CALC I and CALC II products ProCT and CGRP, sepsis- and inflammation-related ADM production appears to occur ubiquitously in all cell types independently of the differentiation state. Therefore, nonadipose cells possibly contribute in cytokine-mediated ADM mRNA expression and ADM peptide secretion shown herein. An approximately 10-fold induction in ADM mRNA abundance was recently shown in rat adipose tissues from obese high-fat-fed rats (38). Obesity is related to increased presence of macrophages in mice adipose tissue (39, 40). In coculture experiments we herein demonstrate ADM and CGRP mRNA induction by macrophage-secreted factors. Thus, a potential role of inflammation-mediated CT peptide expression in human obesity will need to be addressed.
The presence of CTR, CRLR, RAMP1, RAMP2, and RAMP3 mRNAs in MSC-derived adipocytes is in accordance with recent data from rat adipocytes (38). This indicates possible expression of functional CT peptide receptors, which may be responsible for the herein reported CGRP and ADM effects (8). NEP mRNA expression in MSC-derived adipocytes is in line with a previous report on active NEP found in human adipocytes (41). NEP actively degrades CGRP and ADM (42, 43). Furthermore, ADM and NEP are inversely correlated in septic rats (44). Therefore, adipocyte NEP functions might include clearance of CT peptides in the vicinity of adipocytes.
Induction of CALC I and CALC II gene transcription was observed on exposure of adipocytes to exogenous CGRP and ADM in concentrations ranging from 1 nM to 1 μM. This induction was selective because the mRNAs of ADM, TNF, and IL-1? were not affected by high CGRP and ADM concentrations. In contrast, the mentioned transcripts were induced by 1 ng/ml LPS administration in the present adipocyte model. This excludes that CGRP and ADM effects on CT, CGRP I, and CGRP II mRNAs are endotoxin-mediated artifacts from the recombinant peptide preparation. The CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)- and ADM(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)-mediated antagonism on mRNA inductions is a further indicator of specific CGRP and ADM effects.
When maximally stimulated with combined IL-1?/TNF/LPS treatment, CT, CGRP I, and CGRP II mRNA appeared already after 2 h. Cycloheximide-mediated protein synthesis inhibition had positive additive effects on ADM-, CGRP-, and IL-1?-mediated CT peptide mRNA inductions, respectively. Thus, inflammatory CALC gene mRNA inductions appear not to depend on intermediate gene expression. Moreover, based on the presented data, the existence of a CALC gene transcription repressing factor with a high turnover rate may be hypothesized.
In addition to transcriptional activation, a positive dose-dependent effect of CGRP and ADM was observed on glycerol release. Thus, in analogy to proinflammatory cytokines (e.g. TNF), CGRP and ADM possibly enhance lipolytic activity (45). Control experiments with ADM(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) or CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) remained negative on glycerol release. However, in contrast to mRNA inductions, preadministration of truncated peptides did not antagonize ADM and CGRP effects on lipolysis. This might be explained by a very sensitive and/or an alternative signaling pathway leading to enhanced lipolysis (46). Future studies will need to address this issue.
Intensive insulin therapy reduces mortality in intensive care unit patients, but the mechanisms responsible for sepsis-related insulin resistance induction are still poorly understood (47). Herein we assessed adipocyte insulin sensitivity by monitoring insulin-dependent glucose transport. The demonstrated TNF-induced insulin resistance is in accordance with currently recognized concepts and expands the knowledge on MSC-derived adipocyte in vitro metabolic functionality (22, 48). Previous data suggested glucose transport-related antiinsulin effect by CGRP (33, 34). Herein attenuation of insulin-mediated glucose uptake by CGRP and ADM, however, was quite variable, indicating that other factors that are not yet well understood may play an important role. Therefore, a final conclusion cannot be drawn based on our data.
The process of adipocyte differentiation is characterized by increasing IL-6 production and nuclear factor-B-related inflammation (49, 50). Cultured adipocytes are known to express inflammatory genes (51, 52). Accordingly, in MSC-derived adipocytes, we found low-level basal IL-6, TNF, and IL-1? mRNA expression. CGRP and ADM are known to potentate inflammatory events in several cells and tissues (29, 53). Therefore, the herein shown CGRP- and ADM- mediated effects in basal adipocytes might be interpreted as modulatory events, rather than culprit inductions. Sepsis-related adipocyte CT peptide expression in vivo occurs in the presence of numerous proinflammatory factors, which might be significantly influenced by local CT peptide production. The precise mechanisms of inflammatory CT peptide expression and action have to be addressed in future experiments.
Acknowledgments
We are grateful to Igor Langer for providing adipose tissue biopsies. We thank Kaethi Dembinski and Susanne Vosmeer for excellent technical assistance. The ultrasensitive assays for calcitonin precursors were kindly provided by B.R.A.H.M.S. GmbH.
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