Septic states are commonly associated with an acute anorexia leading to a severe malnutrition and cachexia that negatively affect patient's survival (1, 2). In the central nervous system (CNS), food intake and energy homeostasis are controlled by neuronal populations mainly located in the hypothalamic nuclei (3). In particular, neurons of the arcuate nucleus (AN) are the first targets for peripheral signals and express orexigenic neuropeptides including the neuropeptide Y (NPY) or anorexigenic peptides such as the α-melanocyte stimulating hormone (α-MSH), a cleavage product of the pro-opiomelanocortin (POMC) (4). The mechanisms involved in septic anorexia are not yet completely understood but there is evidence that acute systemic inflammation is a crucial factor in the pathogenesis of cachexia. Central administration of interleukin-1-β (IL-1β) or tumor necrosis factor-α (TNF-α) induces the activation of hypothalamic anorexigenic neurons in rat (5, 6). Moreover, it has been shown that systemic inflammation could interact with CNS, including hypothalamus (7).
Numerous data indicate that hypothalamic glial cells are sensitive to peripheral signals involved in the central regulation of food behavior (8, 9). It is now well established that astrocytes are not only essential for neuronal metabolism, but are also an important component of the tripartite synapse by releasing compounds acting on neurons and named gliotransmitters (10). Endozepines including the octadecaneuropeptide ODN derive from the diazepam-binding inhibitor DBI, a 86 amino acid polypeptide (11). They are widely distributed within the CNS and have been recognized as typical gliotransmitters (12, 13). They originally designate a family of neuropeptides considered endogenous ligands of benzodiazepine receptors in the rat brain (14). In the hypothalamus, high levels of endozepines have been found in astrocytes, ependymocytes bordering the third ventricle and tanycytes of the median eminence (12). Previous data indicate that the endozepine ODN modulates the activity of various hypothalamic neurons, such as NPY, POMC, and corticotropin releasing hormone (CRH) neurons (15, 16). It is now clearly demonstrated that ODN is a potent anorexigenic factor involving activation of the POMC/α-MSH hypothalamic pathway (13, 17).
We have recently demonstrated that plasmatic ODN level is increased during sepsis in both rat and human (18). In addition, a positive correlation was observed between TNF-α levels and ODN levels in patients with a systemic inflammatory response syndrome (18). Nevertheless, there is no report that describes interaction between inflammatory process and expression of endozepines in hypothalamic astroglial cells. However, in vivo and in vitro studies have shown that rat astrocytes express a number of pro-inflammatory cytokine receptors, and that TNF-α and IL-1β modulate the expression of several genes encoding chemokines and growth factors, suggesting that pro-inflammatory cytokines are important regulators of glial cell activity (19, 20).
In this context, the aim of the present study was to investigate the temporal variations of hypothalamic endozepines after a sepsis, using the animal model of cecal ligation and puncture (CLP), and the effects of the pro-inflammatory cytokines TNF-α and IL-1β on DBI gene expression in cultured rat astrocytes.
MATERIALS AND METHODS
Dulbecco's modified Eagle's medium (DMEM), F12 culture medium, D(+)-glucose, bovine serum albumin, trifluoroacetic acid, and Tri reagent were purchased from Sigma (St. Louis, MO). L-glutamine, N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid (HEPES), and the antibiotic-antimycotic solution were obtained from Biowhittaker (Lonza group, Basel, Switzerland). Fetal bovine serum (FBS) was from Dutscher (Brumath, France). Trypsin-EDTA was from Invitrogen (Life Technologies, Carlsbad, CA). Acetonitrile was from Prolabo (Fontenay-sous-Bois, France). [Tyr0]-ODN was from Neosystem (Strasbourg, France). TNF-α and IL-1β were from Eurobio (Les Ulis, France). All other reagents were of A grade purity.
Secondary cultures of rat cortical astrocytes were prepared as previously described (21). Briefly, cerebral hemispheres from newborn Wistar rats were collected in DMEM/F12 (2:1; v/v) culture medium supplemented with 2 mM glutamine, 1% insulin, 5 mM HEPES, 0.4% glucose, and 1% of the antibiotic-antimycotic solution. The tissues were dissociated mechanically with a syringe equipped with a 1-mm gauge needle, and filtered through a 100-μm sieve (Falcon, Franklin Lakes, NJ). Dissociated cells were resuspended in culture medium supplemented with 10% fetal bovine serum and seeded at a density of 0.6 × 106 cells/mL in 150-cm2 flasks (Techno Plastic Products, Trasandingen, Switzeland). When cultures were confluent, astrocytes were isolated from mixed glial cultures by shaking overnight the flasks with an orbital agitator (KS 15, Bühler, Germany). Adhesive cells were detached by trypsination and preplated for 2 min to discard contaminating oligodendrocytes and microglial cells. Then, the nonadhering astrocytes were harvested and plated on 35-mm Petri dishes (Dutscher, Brumath, France) at a density of 0.2 × 106 cells/mL. The cells were incubated at 37°C in a humid atmosphere (5% CO2). After 4 days of culture, more than 99% of the cells were labeled with antibodies against glial fibrillary acidic protein, a specific marker of astrocytes. All experiments were performed on 5- to 7-day-old secondary cultures.
Reverse transcription-polymerase chain reaction analysis
Cultured astrocytes were incubated at 37°C with fresh serum-free culture medium in the absence or the presence of test substances. At the end of the incubation, culture media were removed and the cells were rinsed twice with Rnase-free phosphate buffered saline (0.1 M, pH 7.4; Invitrogen, Life Technologies). Total RNA was extracted by the guanidine thiocyanate-phenol-chloroform method using Tri reagent. Approximately 1 μg of total RNA was reverse transcribed by ImPROM-IITM reverse transcriptase (50 U/μL; Invitrogen, Life Technologies) using random hexanucleotides as primers. Real-time polymerase chain reaction (RT-PCR) was performed on 15 ng of total cDNA with 1X SYBR Green universal PCR Master mix (Applied Biosystem, Life Technologies) containing dNTPs, MgCl2, AmpliTaq Gold DNA polymerase, forward (5’-TGCTCCCGCGCTTTCA-3’), and reverse (5’-CTGAGTCTTGAGGCGCTTCAC-3’) DBI primers (300 nM, each; Proligo, Sigma-Aldrich, Saint-Louis, MO). DBI cDNA was first subjected to 50°C for 2 min and 95°C for 10 min, followed by 40 reaction cycles of 95°C for 15 s, 60°C for 1 min, using the ABI Prism 7000 sequence detection system (Applied Biosystem, Life Technologies). The amount of DBI cDNA in each sample was calculated by the comparative threshold cycle (Ct) method and expressed as 2exp(–ΔΔCt) using glyceraldehyde 3-phosphate dehydrogenase as an internal control (22).
Cecal ligation and puncture experiments
Sprague-Dawley male rats (Charles River Inc, Elbeuf, France), weighing 200 g to 250 g at the beginning of the experiments, were housed under constant temperature (21°C) and a 14/10 h light/dark cycle (lights on 06:00 h). The animals had free access to standard rat chow and drinking tap water. The experiments were performed in accordance with the national and international policies and the protocol was approved by the North-West Regional Ethic Committee on Animal Experimentation (France, referral number: ceean0406-01, approval number: 01-04-06/03).
Sepsis was induced by cecal ligation and puncture (CLP). Rats were anaesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). A 2-cm midline incision was made, the cecum was isolated and ligated below the ileocecal valve as not to cause bowel obstruction, punctured twice on the antimesenteric side with an 18-gauge needle and gently squeezed to ensure that the cecum was perforated. The cecum was then placed back in the abdomen and the incision was closed with sutures. For sham-operated controls, the cecum was exposed in an identical manner, but was not ligated or punctured. Rats were killed by decapitation 1, 2, 3, 4, 6, 12 and 24 h after surgery (five animals per group for each time slot) and the hypothalamus was rapidly dissected. Hypothalamic tissue samples were stored at −80°C until endozepine extraction and radioimmunoassay. For in situ hybridization experiments, rats were deeply anaesthetized with ketamine-xylazine and rapidly perfused transcardially with 4% paraformaldehyde in 0.2 M phosphate buffer. The brains were removed, post-fixed in the same fixative at 4°C overnight, and placed in 15% sucrose in 0.1 M phosphate buffer at 4°C overnight. Thereafter, the tissues were frozen on dry ice in embedding medium (OCT, Bayer Corp, Elkhart, IN).
In situ hybridization
Frontal sections through an area extending from the medial preoptic area (MPOA) to the anterior hypothalamus were serially cut at 10 μm in a cryostat. The brain sections were then mounted on Superfrost/PLUS microscope slides (Fisher Scientific, Montreal, Canada) and maintained at −80°C until use.
In situ hybridization was performed as previously described (15). The pGEM4Z plasmid containing a 1.2-kb EcoR1 fragment of rat CRH cDNA (Dr K. Mayo), generously provided by Dr S. Rivest, was linearized with HindIII while the pGEM3Z plasmid containing a 287-pb (XBAI-Sal1) fragment of rat NPY cDNA (Dr D. Larhammar), generously provided by Dr S. Rivest, was linearized with EcoR1. Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl2, 40 mM Tris (pH 7.9), 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 0.2 mM ATP/GTP/CTP, 200 mCi [35S]UTP (Dupont NEN, Boston, MA), 40 U RNAsin (Promega, Madison, WI), and 20 U of either T7 (NPY probe) or SP6 (CRH probe) polymerase for 90 min at 37°C. The sense probes were prepared using 20 U of SP6 or T7 polymerase for the NPY and CRH probes, respectively. To reduce nonspecific labeling, unincorporated nucleotides were removed by using a Biospin column (Bio-Rad, Hercules, CA) 15 min after the addition of 1 mL of Dnase 1 μg/1 mL (Pharmacia Biotech Inc, Montreal, Canada) at 37°C. The [35S]dATP-labeled 30-bases of the rat POMC cDNA probe (bases 297–324) was prepared as previously described [Compère 2003]. The sequence corresponding to the probe is: 5’-CTTGCCCCAGCGGAAGTGCTCGGAGTA-3’. The vector used for production of the cRNA probe was constructed by insertion into a pCR-BluntII-TOPO (Invitrogen, Life Technologies) of a cDNA fragment of 266 pb for DBI (Genebank no. NM-007830). The cDNA fragments, located at position 215-461 downstream from the ATG start codon for DBI, were obtained by amplification using the polymerase chain reaction. After hybridation, the sections were dehydrated and coated with liquid photographic emulsion (Kodak NTB-2). Slides were exposed for 5 days for NPY, 3 days for CRH, 12 days for DBI, and 13 days for POMC. Semiquantitative analysis of the hybridization signal was carried out on nuclear emulsion-dipped slides over ependymal cells bordering the third ventricle of the hypothalamus using a Zeiss Optical System coupled to a Macintosh computer (Power PC 7500/100) and the Image software (version 1.60 non-FPU, W. Rasband, NIH, Bethesda, MD). The optical density (OD) of the hybridization signal was measured under dark-field illumination at ×10 magnification. Sections from the CLP and sham animals were matched for rostrocaudal level. The hypothalamus was digitized and subjected to densitometric analysis, yielding measurements of integrated OD. The OD of each specific region was then corrected for the average background signal, which was determined by sampling cells immediately outside the cell group of interest. Quantitative data (mean ± SEM) were calculated from measurements obtained from 12 to 18 sections per rat (five rats per group) in the same region of the nucleus.
Radioimmunoassay of ODN-related peptides
Endozepines were extracted from hypothalamus by homogenization of the samples in 1 mL of 2 M acetic acid, and heated at 90°C for 10 min. After centrifugation (20,000 g, 20 min) supernatants were dried by vacuum centrifugation (Speed Vac concentrator, Savant, Hicksville, NY). The dry samples were resuspended in phosphate buffer (0.1 M, pH 8) containing 0.1% Triton X-100 and the concentrations of ODN-like immunoreactivity (ODN-LI) were quantified by radioimmunoassay using an antiserum raised against synthetic rat ODN (12). [Tyr0]-ODN was iodinated using the chloramines-T procedure and purified on a Sep-Pak C18 cartridge (22). The final dilution of the ODN antiserum was 1:30,000 and the total amount of tracer was 6000 cpm/tube. After a 2-day incubation at 4°C, the antibody-bound ODN fraction was precipitated by adding bovine γ-globulins (1%, w/v; 100 μL) and polyethylene glycol 8000 (20%, w/v; 2 mL). After centrifugation, the pellet containing the bound fraction was counted in a gamma counter (LKB Wallac, Rockville, MI).
All values presented in the figures are mean ± SEM. Mann and Whitney, and Kruskal and Wallis followed by Dunn test were applied to determine statistical differences between values with an α risk of 5% and a β risk of 20%.
Effect of CLP on DBI mRNA expression and ODN like-immunoreactivity in rat hypothalamus
To examine whether the septic inflammation regulates the expression of DBI in the rat hypothalamus, the rat model of CLP was used. As illustrated by light microscope dark- and bright-field micrographs, DBI mRNA was primarily observed in tanycytes of the median eminence and in ependymal cells bordering the third ventricle of the hypothalamus (Fig. 1A–F). Semiquantitative analysis of the signal detected in tanycytes (median eminence area, ME) and ependymal cells (EA) indicated that CLP significantly increased DBI mRNA levels after 3 and 24 h in these two populations of cells (Fig. 1G and H). In parallel, time-course experiment over a period of 24 h revealed that CLP provoked a time-dependent increase of ODN-LI in hypothalamic extracts (Fig. 1I). A significant effect was observed within 4 h (+24%) and a maximum at 6 h (+39%). The increase in hypothalamic ODN-LI was maintained at least for 24 h.
Effects of TNF-α and IL-1β on DBI mRNA level in cultured rat astrocytes
To determine the capacity of inflammatory cytokines to modulate endozepine expression in astroglial cells, the effects of TNF-α and IL-1β were tested on cultured rat astrocytes. Time-course experiments showed that TNF-α (100 ng/mL) provoked a significant increase in DBI mRNA level that reached a maximum after 4 h of incubation (Fig. 2A). Thereafter, the action of TNF-α gradually declined and DBI mRNA concentration returned to basal level within 12 h after the onset of cytokine administration. Incubation of cells with graded concentrations of TNF-α (30 pg/mL to 300 ng/mL) for 4 h induced a dose-dependent increase of DBI mRNA levels with a maximum effect at a concentration of 3 ng/mL (Fig. 2B). In contrast, the pro-inflammatory cytokine IL-1β had no effect on DBI mRNA level, even at high concentrations (Fig. 2C and D).
Effect of CLP on POMC, NPY, and CRH mRNA level in the rat hypothalamus
We next tested the effect of CLP on the levels of mRNAs encoding POMC, the precursor of the anorexigenic peptide α-MSH, and the orexigenic peptide NPY in the AN. In situ hybridization with the [35S]POMC cDNA probe showed the occurrence of POMC-expressing neurons limited to the AN of the hypothalamus (Fig. 3A, B, and E). CLP induced a significant increase in POMC mRNA levels after 3 and 24 h (+108% and +53%, respectively; Fig. 3F). In situ hybridization with the NPY mRNA probe produced a staining of neurons of the AN which was not significantly affected by CLP (Fig. 3C, D, G, and H). To confirm that CLP likely activates the hypothalamic anorexigenic pathway, we have examined the effect of sepsis on one of the targets of POMC neurons, the CRH neurons of the paraventricular nucleus (PVN) of the hypothalamus. As illustrated by light microscope dark- and bright-field micrographs, neurons expressing CRH mRNA were observed throughout the medial parvocellular portion of the PVN (Fig. 4A–E). A significant increase in CRH mRNA expression was observed in septic animals, 3 and 24 h after CLP (+112% and +140%, respectively; Fig. 4F).
This study shows for the first time that CLP-induced sepsis increases DBI mRNA level in hypothalamic glial cells and increases endozepine protein synthesis (ODN-LI) in hypothalamic area. In addition, it is shown that pro-inflammatory cytokine TNF-α stimulates DBI mRNA level in cultured rat astrocytes.
We have first examined the effect of sepsis on the level of DBI mRNA in the rat hypothalamus, using the CLP method. Murine CLP is a well-established model of sepsis which mimics the clinical features of peritonitis, including polymicrobial infection from gastrointestinal tract origin. The present study showed that CLP induced a rapid and sustained increase of DBI mRNA level in the rat hypothalamus. It also indicates that CLP-induced DBI gene expression in rat was associated with a delayed increase of endozepine synthesis (ODN-LI) in the hypothalamus. Previous data have shown that plasma endozepine level is increased too during systemic inflammation in human and rodents (18).
The early phase of CLP is characterized by an over-expression of TNF-α and a modest increase in IL-1β (23). Thus, astroglial cells of the hypothalamus could be one the initial target for pro-inflammatory cytokines. This hypothesis is supported by the kinetic of the expression of the signal transducer and activator of transcription 3 (STAT3), a marker of cytokine-activated brain cells, in lipopolysaccharide (LPS)-treated animals (24). It was indeed shown that peripheral injection of LPS induced the translocation of STAT3 into the nucleus of, first ependymal cells (within 2 h), and later into the cells located in the parenchyma of the hypothalamus, neurons and glial cells (within 4 h) (24). The present study demonstrates that DBI mRNA level was significantly increased 3 h after CLP suggesting that stimulation of DBI gene could be one of the first responses of hypothalamus to sepsis. The hypothesis that sepsis-induced TNF-α synthesis may be responsible, at least in part, for the over-expression of the DBI gene in the hypothalamus, is in agreement with data indicating that nanomolar concentrations of TNF-α markedly and rapidly increased DBI mRNA level in cultured rat astrocytes (the present study) and that there is a positive correlation between endozepine and TNF-α levels in human serum during systemic inflammation (18). This is also consistent with the presence of functional TNF-α receptors in astrocytes (25). In contrast, while rodent astroglial cells are equipped with Il-1β receptors, this pro-inflammatory cytokine was unable to modify DBI mRNA level (26).
The mechanism of action of TNF-α on DBI gene expression is currently unknown. Although the DBI gene exhibits the hallmarks of a typical house keeping gene, several studies have shown that the DBI promoter encompasses consensus sequences for regulatory elements (27, 28). In addition, several neuropeptides, such as somatostatin or pituitary adenylate cyclase-activating polypeptide, are able to modulate DBI mRNA level in astroglial cells (22, 29). It has been shown that TNF-α increases activator protein-1 transcriptional activity in MCF7 cells (30). The fact that DBI promoter contains putative sequence for this regulatory suggests that TNF-α could, maybe, directly modulate DBI gene transcription in glial cells (27, 28). Nevertheless, it cannot be excluded that other peripheral signals could also modulate DBI expression. In particular, adrenal steroid hormones, which are increased during sepsis, could affect hypothalamic endozepine level as previously shown in non-septic rats (9, 31). Finally, there is a lack of data in our work to prove a direct and continuous interaction between TNF-α, in vivo DBI mRNA expression, ODN-like immunoreactivity, and anorexia.
The significance of DBI gene overexpression in the hypothalamus during sepsis is currently a matter of speculation. Septic states are commonly associated with anorexia/cachexia whose severity is associated with both quality of life and eventual mortality of the patient (1, 2). We previously demonstrated that glial cells of the hypothalamus produce the DBI-processing product ODN, a potent anorexigenic factor acting through the POMC pathway (13, 15). Thus, given the data of the present work, we hypothesize now that the increase of hypothalamic endozepine level could be responsive, at least in part, for anorexia observed in septic patients. The AN of the hypothalamus contains two populations of neurons, the POMC and the NPY neurons, which exert opposite effects on energy balance (32). In agreement with a previous study, the present data indicate that sepsis increases the level of mRNA encoding POMC (33). In contrast, CLP did not modify the expression of NPY, data which are consistent with a previous study indicating that intraperitoneal administration of LPS did not affect NPY mRNA level in the AN (33). Thus, the idea of the activation of the hypothalamic anorexigenic pathway by sepsis is strengthened by the delayed stimulation of CRH neurons of the NPV, one of the targets of POMC neurons (34). Nevertheless, the data found in our work can lead us to make a hypothesis concerning a potential role of endozepines in sepsis-induced anorexia through the POMC/CRH pathway, but this new hypothesis does not exclude a direct effect of TNF-α and/or IL-1β on CRH neurons as previously observed (5). Thus, an activation of CRH synthesis mediated by endozepines could be an additional mechanism involved in the anorectic effect of this neuropeptide. As ODN anorexigenic effect involves activation of the POMC/α-MSH hypothalamic pathway, and, as in patients with septic shock, the response of the pituitary to CRH stimulation in terms of release of α-MSH is impaired in non-survivors compared with survivors, it also seems interesting to consider the study of endozepine expression levels in survivors versus non-survivor during septic shock (13, 17, 35).
In conclusion, we have demonstrated that CLP stimulates DBI expression in rat hypothalamus and we suggest that this stimulation could involve TNF-α. On the basis of the potent anorexigenic activity of the DBI processing product ODN, we assume that endozepines, acting through the POMC/CRH anorexigenic pathway, could be in part responsive for sepsis-induced anorexia. There is a need of a future in vivo work to explore the role of TNF-α in hypothalamic endozepine expression and to look for a potential correlation between DBI mRNA expression, endozepine levels, and food intake in a preclinical model of sepsis.
The authors thank Ms Dorthe Cartier, Louise Désy, and Johanne Ouellet for expert technical assistance.
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Keywords:© 2016 by the Shock Society
Alpha-melanocyte-stimulating hormone; astroglial cells; corticotropin-releasing hormone; cytokines; diazepam binding inhibitor; endozepines; neuropeptide Y; pro-opiomelanocortin