Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages

The protocol for this study was approved by the Institutional Ethical Committee for Animal Experimentation (project number P051/2010). Male, 24-week old, C57BL/6J mice (Janvier SAS, Chassal, France) were anesthetized with an intraperitoneal (i.p.) injection of ketamine (3 mg, Univet, Heusden-Zolder, Belgium) and xylazine (0.4 mg, VMD, Brussels, Belgium) and submitted to dual-energy-X-ray-absorptiometry (DEXA) to identify body composition (Lunar PIXImus™, GE Medical-Lunar, Madison, WI, USA) (Figure 1). After a 48-hour recovery, animals were again anaesthetized by i.p. injection of ketamine and xylazine. Animals were placed on a heating pad maintained at 25°C. Spontaneously breathing mice were continuously inhaling 1 to 2% isoflurane-vet (Schering-Plough, Hull, UK) with oxygen (2L/min) through a facial mask. The left central jugular vein was dissected under microscopy and a sterilized Renathane catheter (model MRE025; Braintree Scientific, Braintree, MA, USA) was inserted. The catheter was tunneled to the back, attached to a single-channel-fluid-swivel (model 375/25; Instech Laboratories, Plymouth Meeting, PA, USA) allowing free movement and continuous perfusion according to the resuscitation scheme (Figure 1). Sepsis was produced by a 50% ligation of the cecum (at half the distance between the distal pole and the base of the cecum) with a 3.0 silk suture (Mersilk, Ethicon, Johnson & Johnson, St.-Stevens-Woluwe, Belgium), and a single puncture through-and-through with a 20-gauge needle. The cecum was gently squeezed to extrude feces and returned to its original position [13‐15]. Surgical wounds were closed with 7.0 silk sutures (Mersilk, Ethicon, Johnson & Johnson, St.-Stevens-Woluwe, Belgium). Post-operative analgesia was provided by local infiltration of the surgical wounds with ropivacaine (1%, AstraZeneca, Brussels, Belgium) and subcutaneous (sc) administration of buprenorphine (9 μg, Schering-Plough, UK). Mice received the broad-spectrum antibiotic Imipenem (0.25 mg sc, Meropenem, AstraZeneca, Brixham, UK) and continuous intravenous fluid-resuscitation (NaCl_0.45%/glucose_2.5% + 6% hydroxyethyl starch (Voluven, Fresenius Klabi, Schelle, Belgium)) in a 4:1 proportion at 0.5 ml/H for the first 18 hours [16]. Afterwards, animals were randomly allocated to the "restricted" or "fed" protocol. Animals in the 'CLP-restricted' protocol (n = 13) received progressive increments of NaCl_0.45%/glucose_50% (0.4 to 1.3 Kcal/day). Animals in the 'CLP-fed' protocol (n = 14) received increments of total parenteral nutrition (TPN) (consisting of 29% kcal from lipids, 14% kcal from amino acids, and 57% kcal from glucose) from 2.9 to 8.6 Kcal/day (Clinomel N7, Baxter, Braine-l'Alleud, Belgium). Healthy controls (n = 11) received 5 g of standard chow daily (13 kcal/24 h). A nutrient-restricted healthy group (pair-fed for the CLP-restricted group) was unfeasible as the restricted nutrient intake was lethal for healthy mice. Every 12 hours septic mice received sc 0.25 mg Imipenem and 9 to 12 μg buprenorphine. Buprenorphine was chosen because it shares clinical attributes of standard opioid analgesics but evokes less respiratory depression and minor immune suppression in mice models [17]. Post-operative pain was regularly assessed by the Mice Grimace Scale [18]. All mice were kept normoglycemic (80 to 120 mg/dl) with iv infusion of insulin (Actrapid Penfill, Novo Nordisk, Bagsvaerd, Denmark) using an in-house developed protocol. To avoid hypothermia, the cage temperature was kept at 27 ± 1°C with heating lamps. Animals that lost their catheter were excluded from the study. After five days, surviving animals were submitted to a second DEXA-scan, and sacrificed by decapitation. Due to technical problems, not all mice were scanned twice. Blood was collected from the trunk, and organs were removed. AT was removed from the inguinal, epididymal and peri-renal sites. All tissue samples were snap-frozen in liquid nitrogen and stored at -80°C until analysis. Due to the small size of AT biopsies, not all biopsies could be analyzed for both immunocytochemistry and RNA analysis.

Patients were enrolled in a large RCT (n = 4,640) to compare early versus late initiation of parenteral nutrition to supplement failing enteral feeding during the first week of CI. Details of the study were previously published [19]. The protocol and consent forms were approved by the Institutional Ethical Review Board (ML4190). During the randomized control trial, subcutaneous biopsies were collected from 188 patients. To avoid bias of prior-to-illness body mass index (BMI)-related changes in the AT, only patients with a normal BMI (between 18 to 25 kg/m²) were selected for this study (n = 72). From these 72 patients, 20 early PN and 20 late PN patients, matched for admission characteristics and illness severity (Table 1), were compared with 13 healthy demographically-matched volunteers. On Day 8 ± 1 of the ICU stay, a subcutaneous in vivo AT biopsy was collected in the quadriceps region with a 5-mm-Bergström muscle-biopsy cannula according to the protocol described earlier [6]. The biopsy specimens were snap frozen in liquid nitrogen and stored at -80°C.

Triglyceride content of tissue was determined with a colorimetric commercial assay (triglyceride quantification kit, Abcam, Cambridge, UK).

Paraformaldehyde-fixed paraffin sections were stained with F4/80Ab (general anti-macrophage antibody (Ab) for mice (Abcam)), CD163Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)-Ab (Abcam). For human samples, the general macrophage marker CD68 was used (Dako, Glostrup, Denmark). Macrophage accumulation in AT sections was evaluated in a blinded fashion by two observers on a Leica DM3000 Microscope (Wetzlar, Germany). Macrophage staining in liver and lungs, PPARγ staining in AT and adipocyte size were quantified on digital microscopical images with in-house designed algorithms developed in Matlab 7.4.0 and Image Processing Toolbox (Matworks, Natick, MA, USA) as described earlier [20]. The estimated proportion of adipocytes was calculated by dividing total end weight of body fat (second DEXA quantification) by mean adipocyte size. This calculation was then plotted proportionally to the mean estimated number of adipocytes of the healthy mice [21].

Messenger RNA was isolated and cDNA quantified in real time as previously described [22]. Data are expressed normalized to GAPDH or HPRT as fold-change of the mean of the control group. For the quantification of Proliferating Cell Nuclear Antigen (PCNA), CCAAT/enhancer binding protein-β (C/EBPβ), hormone sensitive lipase (HSL), tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), Arginase-1, interleukin 10 (IL-10), chemokine (C-C motif) ligand 2 (CCL2), and CD-163 messenger RNA, commercial gene expression assays (TaqMan, Life Technologies, Gent, Belgium) were used.

The beginning bodyweight of the animals was 29.3 ± 0.4 g (mean ± SE), of which 4.2 ± 0.1 g were fat. Changes in body composition were quantified in eight animals per group. CLP-fed animals lost weight, total body fat mass and total body fat-free mass as compared with healthy animals (Figure 2A-C). The loss of fat mass in CLP-fed mice corresponded to a 26% reduction in total body fat mass (from mean 4.1 ± 0.1 g to 3.4 ± 0.2g). CLP-fed and CLP-restricted animals showed a comparable reduction in lost weight, fat mass and fat-free mass.

Part of the fat was lost from ectopic sites, evidenced by lowered triglyceride content in liver and skeletal muscle in CLP-fed compared to healthy mice. Reduction of triglyceride content from liver and skeletal muscle was further accentuated in CLP-restricted compared to CLP-fed animals (Figure 2D). Total body fat mass correlated with triglyceride content in liver and muscle (R = 0.625, P = 0.01).

Adipocytes of CLP-fed mice were smaller than in healthy controls in subcutaneous (46% smaller), epididymal (62% smaller) and renal AT (51% smaller) (Figure 3A). CLP-restricted mice showed a comparable reduction in adipocyte size as CLP-fed mice. Because smaller adipocytes could theoretically be explained by more newly formed small adipocytes (adipogenesis), or by adipocytes releasing stored triglycerides (lipolysis), we quantified markers for both processes. To assess lipolysis, gene expression of HSL, the rate-limiting enzyme in the release of stored triglycerides, was quantified. HSL gene expression was lower in CLP-fed than in healthy animals in subcutaneous AT (median (IQR) 1.3 (0.8 to 1.5) to 0.4 (0.4 to 0.5) Arbitrary Units (AU), P = 0.05) and renal AT (1.0 (0.9 to 1.2) to 0.6 (0.6 to 0.7) AU, P = 0.005). In the epididymal AT, HSL gene expression tended (P = 0.1) to be lower in CLP-fed than in healthy mice. There was a significant further reduction of HSL gene expression in visceral (P = 0.004), but not in subcutaneous AT (P = 0.7) of CLP-restricted compared to CLP-fed mice.

To assess adipogenesis, the number of adipocytes was estimated by dividing total body fat mass by adipocyte size: CLP-fed mice displayed 50% higher number of adipocytes compared to healthy mice (Figure 3B). CLP-fed and CLP-restricted mice showed a comparable increased number of adipocytes. Gene expression of the proliferation marker PCNA was up-regulated in CLP-fed mice compared to healthy controls in subcutaneous and renal AT (not in epidydimal AT). CLP-fed and CLP-restricted mice showed a comparable PCNA gene expression (Figure 3C). Immunostaining of the adipogenic transcription factor PPARγ was significantly higher in CLP-fed compared to healthy mice in all AT depots. CLP-fed and CLP-restricted mice showed comparable PPARγ immunostaining (Figure 3D). C/EBPβ expression was higher in CLP-fed than in healthy animals in all AT depots. In subcutaneous AT, CLP-restricted mice showed a further increase in C/EBPβ gene expression when compared to CLP-fed mice (Figure 3E). In visceral AT, CLP-fed and CLP-restricted mice showed comparable increase in C/EBPβ gene expression.

Immunostaining revealed that macrophages accumulated in all AT depots of CLP-fed animals compared with healthy animals. There was comparable accumulation of macrophages in CLP-fed and CLP-restricted animals (Table 2). Macrophages can polarize to either an M1- or an M2-phenotype, each expressing distinct markers. We quantified gene expression of TNFα and iNOS as M1-markers; and arginase-1, IL10 and CD163 as M2-markers. In subcutaneous AT of CLP-fed mice, gene expression of TNFα and iNOS was similar to healthy mice (Figure 4A, B). In contrast, the M2-markers arginase-1, IL-10 and CD163 were markedly up-regulated in subcutaneous AT of CLP-fed mice compared to healthy mice. In visceral AT, TNFα (renal and epididymal) and iNOS (renal) gene expressions were increased up to two-fold in CLP-fed compared to healthy mice (Figure 4A, B). In contrast, gene expression of the M2-marker arginase-1 increased more than 10-fold in CLP-fed compared to healthy mice.

Expression of M1 and M2 markers was comparable in CLP-fed and CLP-restricted mice in all AT depots (Figure 4). Only in subcutaneous and epididymal AT, iNOS was reduced and in renal AT, arginase expression was further increased in CLP-restricted compared to CLP-fed mice. Subcutaneous and visceral AT sections were immunostained for CD163. Positive M2 macrophage staining was present in all AT depots of CLP-fed and CLP-restricted mice.

Immuno-staining revealed that macrophages also accumulated in the liver and lungs of CLP-fed compared with healthy mice (Figure 5). This increased staining corresponded in the liver and lungs with an increased expression of both M1-marker TNFα and M2-markers arginase-1 and CD163. In the liver, macrophage immuno-staining correlated significantly with CD163 gene expression (R = 0.541, P = 0.002) and with TNFα gene expression (R = 0.405, P = 0.003). There was equal accumulation of macrophages in the liver and lungs of CLP-fed and CLP-restricted mice. Also, expression of M1-marker TNFα and M2-marker arginase-1 was comparable in CLP-fed and CLP-restricted mice. In contrast, expression of M2-marker CD163 was further increased in CLP-restricted when compared to CLP-fed mice (Figure 5).

As a possible trigger for the macrophage accumulation, we quantified gene expression of the chemo-attractant factor CCL2. The gene expression of CCL2 was higher in all AT depots, lungs and liver of CLP-fed when compared to healthy mice (Figures 4F and 5). The gene expression of CCL2 in CLP-fed and CLP-restricted mice was comparable in all AT depots, lungs and liver. CCL2 expression correlated positively with arginase expression (R = 0.566, P = 0.007 in inguinal AT, R = 0.912, P < 0.0001 in epidydimal AT, R = 0.598, P = 0.009 in renal AT). CCL2 gene expression correlated positively with CD163 gene expression in the liver (R = 0.424, P = 0.01) and lungs (R = 0.730, P < 0.0001).

To assess the clinical relevance of our animal study observations, we additionally studied a matched set of 40 human AT biopsies collected in vivo during the EPaNIC trial [12]. Small needle biopsies were collected at Day 8, and compared with 13 biopsies from healthy volunteers. Total caloric intake was significantly higher in early-PN patients (n = 20) compared to late-PN (n = 20) patients during the first seven days in the ICU (Figure 6A).

Adipocyte size decreased in early PN 'fed' critically-ill patients compared to healthy controls (Figure 6B). Medium adipocyte size was equally reduced in early PN 'fed' patients and late PN 'restricted' patients. Immuno-staining revealed accumulation of macrophages in all biopsies of critically-ill patients (Figure 6C). Gene expression of TNFα was unaffected by illness, while arginase-1 gene expression was higher in early PN 'fed' critically-ill patients than in controls (Figure 6D, E). Gene expression of TNFα was equally up-regulated in early PN 'fed' and late PN 'restricted' patients.

It is known from the literature that glucocorticoids can both stimulate adipogenesis [23] and promote M2 polarization [24]. Nineteen of the 40 patients received corticosteroids during the study period. However, arginase expression (P = 0.25), TNFα expression (P = 0.47) and adipocyte size (P = 0.6) were not different between patients receiving or not receiving steroids. In contrast, patients receiving steroids during the study period, had a higher percentage of CD68 staining then patients not receiving steroids (P = 0.03).