Abstract Appearance of middle-aged obesity and aging anorexia both in humans and rodents suggests a role for regulatory alterations. Hypothalamic melanocortin agonist, α-melanocyte-stimulating hormone (α-MSH) produced in the arcuate nucleus (ARC), reduces body weight via inducing hypermetabolism and anorexia mainly through melanocortin 4 receptors (MC4Rs) in the paraventricular nucleus (PVN). Orexigenic ARC-derived agouti-related protein (AgRP) is an inverse agonist on MC4R in the PVN. Previously, we demonstrated that characteristic age-related shifts in the catabolic effects of α-MSH may contribute both to middle-aged obesity and aging anorexia. Responsiveness to α-MSH decreases in middle-aged rats compared with young adults, whereas in old age it rises again significantly. We hypothesized corresponding age-related dynamics of endogenous melanocortins. Therefore, we quantified mRNA gene expression and peptide or protein level of α-MSH, AgRP, and MC4R in the ARC and PVN of male Wistar rats of five age groups (from young to old). Immunofluorescence and quantitative reverse transcriptase polymerase chain reaction were applied. α-MSH and MC4R immunoreactivities in the ARC and PVN declined in middle-aged and increased together with their expressions in aging rats. AgRP gene expression but not its immunoreactivity increased in aging rats. Our results demonstrate that age-dependent changes of endogenous melanocortins contribute to middle-aged obesity and aging anorexia. Obesity, Aging anorexia, Brain aging, Hormones, Metabolism Obesity, a common risk factor for numerous diseases, became a major healthcare challenge in the last decades (1). Although middle-aged populations tend to become obese (2), old age is rather characterized by anorexia and consequent loss of active tissues leading to sarcopenia (3,4). As both trends are also observed in other mammals (5), common endogenous regulatory alterations may contribute to their development (6). However, our knowledge on their possible neurobiological background is still insufficient. In the regulation of energy homeostasis, the hypothalamic melanocortin (MC) system plays a dominant catabolic role via suppression of food intake (FI) and increase of energy expenditure leading to loss of body weight (BW) (7,8). Melanocortins (including α-melanocyte-stimulating hormone [α-MSH]) are cleaved from a precursor polypeptide encoded by the pro-opiomelanocortin (POMC) gene. Among the five different G-protein-coupled receptors (MC1R–MC5R), the melanocortin 3 receptor (MC3R) and MC4R are the important central receptor types concerning the hypothalamic regulation of energy homeostasis (9). Because MC4R knockout mice display the more severe obese phenotype (10,11), this receptor subtype appears to be more important than MC3R. Its abnormalities were also detected in human obesity (12,13). α-MSH, the main endogenous agonist of the MC system, is produced in the arcuate nucleus (ARC) of the hypothalamus, and it acts mainly through MC4Rs of second-order neurons in the paraventricular nucleus (PVN) of the hypothalamus (14). Moreover, recently, an additional local self-up-regulatory action of α-MSH via MC4R on POMC neurons in the ARC was demonstrated (15). The endogenous inverse MC4R/MC3R agonist agouti-related peptide (AgRP) is also produced in the ARC, eliciting an orexigenic effect via second-order neurons of the PVN (16). Additionally, AgRP producing GABAergic neurons have an inhibitory effect on POMC neurons in the ARC (17). As the MC system plays a key role in the control of BW and body composition (18), the final control signal on energy homeostasis may strongly depend on the balance of melanocortins possessing physiologically opposing agonistic (ie α-MSH) and antagonistic (ie AgRP) effects (9). We hypothesized that age-related shifts in this balance play an important role in long-term trends of BW during the course of aging. In our previous in vivo studies, the catabolic effects of intracerebroventricularly (ICV) injected α-MSH were shown to be age-dependent: the effects of the peptide were strong in young and again in old rats, but they were weak in the middle-aged groups (19,20). A similar pattern was observed in case of the anorexigenic actions of a 7-day α-MSH infusion as well (21). These latter findings were in accord with the robust MC infusion-induced response of old rats reported by Zhang and co-workers (22). However, other previous in vitro studies failed to reveal an unequivocal age-related pattern in the endogenous activity of the hypothalamic MC system as indicated by gene expression of POMC: either decreased (23–28) or unchanged MC activity was described in old rodents (22,29,30). The aim of the present study was to investigate the age-related dynamics of the endogenous MC system including those of α-MSH, AgRP, and MC4R in the ARC and PVN using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and a semiquantitative immunohistochemical approach. Dynamics of these age-related changes were then systematically compared with those of the in vivo results. In order to carry out this analysis, responsiveness to ICV administered α-MSH had to be tested in all corresponding age groups complementing our previous experiments. Materials and Methods Animals Young (Y), middle-aged (M), and old (O) age groups of male Wistar rats from the Colony of the Institute for Translational Medicine of the Medical School, University of Pecs, Hungary were used in our study: 3, 6 or 12, 18, and 24 months of age (Y3, M6 or M12, O18, and O24), corresponding to human young adult, younger or older middle-aged, aging, and old populations, respectively. Two cohorts of rats were subjected to ICV cannula implantation surgery (see below) for investigation of α-MSH effects on (a) FI (cohort a: n = 6–14/age group) and (b) metabolic measurements (cohort b: n = 6–16/age group). Further two cohorts of intact rats were used for (c) immunohistochemistry (cohort c: n = 5–8/age group) and (d) qRT-PCR (cohort d: n = 5–9/age group). Animals were housed in standard plastic cages with woodchip bedding at 23–26°C ambient temperature. Lights were on between 06:00 and 18:00 hours. Standard rat chow (CRLT/N rodent chow, Szindbád Kft., Gödöllő, Hungary, 11 kJ/g) and tap water were available ad libitum. Animals were weighed once a week. Intact rats were housed three per cage, whereas following cannula implantation, animals were housed singly. All our protocols and procedures were approved by the National Ethical Council for Animal Research (Permit number: BA 02/200–11/2011 valid for 5 years). They were also in accord with the directives of the European Communities Council on the protection of animals used for scientific purposes (86/609/EEC, Directive 2010/63/EU of the European Parliament and of the Council). Surgeries and Drug Administration Upon reaching the appropriate age, animals were operated for the purpose of implanting an ICV cannula into the right lateral cerebral ventricle under intraperitoneal ketamine-xylazine [78 (Calypsol, Richter) + 13 mg/kg (Sedaxylan, Eurovet)] general anesthesia. To prevent postoperative infections, intramuscular gentamycin (2 mg/kg) was also given. The implantation was performed using a stereotaxic apparatus as described earlier (19), coordinates were determined according to the rat brain atlas of Paxinos and Watson (A: −1.0 [posterior to bregma], L: 1.5 [to midline], V: 3.5 mm [ventral to dura]) (31). Experiments started 7 days after the cannula implantation. During the tests, a single 5 µL ICV injection of α-MSH (Bachem AG Switzerland, 5 µg dissolved in pyrogen-free saline [PFS]) or PFS as control was given in random order as described earlier (19). This dose was chosen based on earlier observations (19,32). After 7 days, the substances were switched and the measurements were repeated. After the experiments, rats were sacrificed by an intraperitoneal overdose of urethane (3–5 g/kg, Reanal). Post-mortem check of the injection sites was performed by observing macroscopically the coronal sections of the removed brains. Only rats with appropriate cannula location were included in the analysis. Assessment of Anorexigenic Effects of α-MSH Two weeks before the tests, rats (cohort a) were transferred individually to chambers of the automated FeedScale system (Columbus, OH). Thus, they were habituated to the environment and to the powdered rat chow. This system allowed continuous recording of their FI. Data were registered automatically every 10 minutes as published earlier (19,33,34). One day before the ICV injection, at 09:00 hours, food was removed for 24 hours. Five minutes before the re-feeding started (at 09:00 hours) assigned rat groups received 5 µg ICV α-MSH or PFS to test the inhibitory effect of the peptide on 2 hours cumulative FI. Assessment of Hypermetabolic Effects of α-MSH Oxygen consumption (mL O2/kg/min, VO2 representing metabolic rate) was determined by indirect calorimetry (Oxymax, Equal Flow, Columbus, OH). Tests were performed between 09:00 and 15:00 hours on semirestrained rats (cohort b), singly enclosed in cylindrical wire-mesh confiners in separate metabolic chambers at 25ºC where the animals had no access to food or water for 6 hours. These confiners were necessary to allow recording changes in resting metabolic rate upon a remote administration of α-MSH or PFS (via an extension of the ICV cannula without disturbance of the animals), as described earlier (20). To minimize the restraint stress, animals were carefully accustomed to the confiners and the metabolic chambers for at least 2 weeks prior to the tests (involving 2 × 30 minutes, 1, 2, and 8 × 4 hours sessions) (35). Following the ICV injection, VO2 was registered in 10 minutes intervals for 3 hours. Maximal increase in VO2 was usually observed about 20 minutes following the injection; therefore, these values were used for the analysis. Tissue Sampling for Immunohistochemistry Intact Wistar rats of all age groups (cohort c) were deeply anesthetized by an intraperitoneal overdose of urethane (3–5 g/kg, Reanal). After their breathing slowed down, their chest cavity was opened, and they were transcardially perfused with ice-cold 50 mL of 0.1M phosphate buffered saline (PBS, pH 7.5) followed by 300 mL chilled 4% paraformaldehyde in 0.1M Millonig buffer for 20 minutes. Consecutively, brains were carefully removed and post fixed in the same fixative for 7 days at 4°C. Coronal sections (30 µm) were cut using a Leica VT1000 S vibratome (Leica, Wetzlar, Germany). For this study, two series of sections were collected from a hypothalamic tissue block between −1.5 and −2.5 mm to the bregma, each interspaced by 60 µm containing the PVN and ARC. Sections were stored at −20°C in antifreeze solution until further use (36). Double Labeling Immunofluorescence for α-MSH and MC4R The first series of sections was washed in PBS for 4 × 15 minutes, in order to remove antifreeze solution and fixative. Then, a treatment with 0.5% Triton X-100 (diluted in PBS) for 30 minutes was applied to permeabilize the cell membranes and to enhance antibody penetration. Subsequently, sections were treated with 2% normal donkey serum (NDS, Jackson Immunoresearch Europe, Suffolk, UK) diluted in PBS for 30 minutes, to reduce the background signal. Next, sections were incubated for 2 days in the cocktail of rabbit anti-α-MSH antibody diluted to 1:5000 in PBS (Peninsula Laboratories, CA, USA) and in goat anti-MC4R antibody, diluted to 1:100 in PBS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. On the third day, samples were rinsed with PBS for 2 × 15 minutes. Then, samples were incubated for 24 hours at 4°C in the mixture of the following secondary antibodies: Cy2 conjugated antirabbit (1:250 in PBS; Jackson), SP-biotin conjugated donkey antigoat (1:2000 in PBS; Jackson). After 2 × 15 minutes, PBS-washed samples were incubated for 3 hours at room temperature with Cy3-conjugated streptavidin (1:1000 in PBS; Jackson). After PBS washes, sections were mounted to gelatin-coated slides, and they were air-dried and covered with glycerol/PBS (1:1) solution. Single Immunofluorescence for AgRP The second series of sections, as defined above, was used to investigate the AgRP immunoreactivity. Sections, containing PVN and ARC, were selected and washed in PBS for 4 × 15 minutes, and then samples were subjected to heat-induced epitope retrieval at 90°C in Na-citrate buffer (pH 6.0) for 10 minutes. After cooling for 20 minutes in the same solution, samples were transferred into 0.5% Triton X-100 for 30 minutes. Subsequently, sections were treated with 2% NDS diluted in PBS (Jackson) for 30 minutes. The primary antibody (rabbit anti-AgRP; Phoenix Pharmaceuticals, 1:6000) diluted in PBS with 2% NDS was applied for 2 days at 4°C. After washed in PBS, sections were incubated for 24 hours at 4°C in SP-biotin conjugated donkey antigoat serum (Jackson, 1:1500). Subsequently, sections were washed with PBS for 2 × 15 minutes and incubated for 3 hours at room temperature in Cy3-conjugated streptavidin (1:1000 in PBS; Jackson). Samples were washed in PBS, mounted to gelatin-coated slides, air-dried, and covered with glycerol/PBS (1:1) solution. Microscopy and Morphometry Sections were digitalized using a confocal laser scanning microscope (Olympus Fluoview FV1000) (Olympus MicroImaging, Japan). To perform the semiquantitation of fluorescent signal, the photon count mode was preferred with the following settings: confocal aperture: 105 μm, optical sectioning by 5 μm step size, 20× lens with a numeric aperture of 0.75, in a resolution of 1024 × 1024 pixels with 10 μs excitation time per pixel. Fluorophores (ie Cy2 and Cy3) were excited by 100% intensity 542 and 550 nm laser beams, respectively. Images of both channels were saved and automatically superimposed. Manual cell counting and densitometry were carried out on nonedited images. The intensity of the immunofluorescence was determined in 10 perikarya per section for α-MSH in the ARC and for MC4R both in the PVN and ARC. The immunosignals on 10 nerve fibers per section were measured for α-MSH in the PVN. The AgRP fiber density was evaluated both in the PVN and ARC. To determine the immunosignal, the Image J software (version 1.37, NIH, Bethesda, MD) was used. Data were corrected for the background density outside the ARC/PVN, yielding the specific signal density (SSD) per neuron or nerve fiber, which was expressed in arbitrary units (a.u.). Antiserum Characterization and Immunohistochemistry Controls The rabbit α-MSH antiserum was generated against whole α-MSH. The high serum specificities were confirmed by preabsorption experiments on rat brain samples with the respective synthetic peptides to which they had been raised (α-MSH, Bachem). The AgRP antibody (Phoenix Pharmaceuticals, Inc., USA) was produced in rabbit against the AgRP (83–131) amide. Preabsorption of this antiserum with the synthetic AgRP (Phoenix) prevented the immunosignal. MC4R was raised against the C-terminus of MC4R of rat origin. The blocking peptide (SC-6880-P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) abolished staining in all specificity controls. In addition, primary serum omission or replacement by nonimmune goat or rabbit serum at the dilution of the respective primary antiserum completely prevented immunoreaction. Sampling for qRT-PCR Intact ad-libitum fed Wistar rats of all age groups (cohort d) were removed from their home cages and decapitated. To avoid the stress effects of anesthetic injection potentially biasing mRNA expression profiles, no anesthesia was applied. The procedure was performed at 08:00 hours in all age groups. Brains were immediately dissected and quickly frozen in liquid nitrogen and stored at −70°C until further use. PVN and ARC samples were punched from 1 mm thick slices (−2 to −3 mm from the bregma (31)) of the brains cut on a brain matrix (Ted Pella, CA, USA) by two razor blades. Sections were placed on an ice-chilled mat. From the mediobasal hypothalamic area, punches of (a) the PVN and (b) ARC were microdissected by a 1 mm diameter Harris punching needle (Sigma-Aldrich, Budapest, Hungary). The total amount of RNA was isolated with the Pure Link RNA Mini Kit (Life Sciences, Carlsbad CA, USA) according to the protocol suggested by the manufacturer. High Capacity cDNA kit was applied (Applied Biosystems, Foster City, CA, USA) to perform cDNA synthesis, using 1 µg of the total RNA sample according to the official protocol. SensiFast SYBR Green reagent (BioLine) was used to perform qRT-PCR for gene expression analysis. Amplifications were run on an ABI StepOnePlus system. StepOne software was used to analyze gene expressions that were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. The appropriate housekeeping gene was selected based on our recent study (34). The primer sequences are shown in Supplementary Table 1. PCR conditions were set as follows: one cycle at 95°C for 2 minutes, 40 cycles at 95°C for 5 seconds, and 60°C for 30 seconds. The amplification of PCR products was calculated according to the 2-ΔΔCt method. Statistical Analysis All results were presented as mean ± standard error of mean (S.E.M.). After confirmation of the normal data distribution, one-way analysis of variance (ANOVA) was applied followed by Fisher’s post hoc analysis. SPSS 11.0 for Windows and Statistica 8.0 for Windows softwares were used. The level of significance was set at p < .05. Results In Vivo Results: Decreased Catabolic Efficacy of α-MSH Precedes Middle-Aged Weight Gain, and Later on, an Increased Efficacy Precedes Weight Loss in Old Rats The BW development of male Wistar rats of our colony shows a marked continuous age-related rise until 18 months of age: a period of rapid growth to 6 months is followed by a more moderately rising slope reaching the peak of the growth curve at 18 months (Figure 1A). Thereafter, a pronounced decline is observed (Figure 1A). Both hypermetabolic and anorexigenic components of the catabolic effects of ICV α-MSH-injection show characteristic age-related changes. The hypermetabolic effect (based on VO2) remained significant in all age groups (as compared with their age-matched controls treated with PFS without any hypermetabolism, not shown) except for older middle-aged (M12) rats. Accordingly, this effect reached its nadir in the M12-group followed by an increase in older animals (Figure 1B). Similarly, the anorexigenic effect (based on suppression of fasting-induced refeeding) was also significant in all age groups (as compared with their age-matched controls, not shown) except for older M12 rats. Accordingly, this effect of the peptide also reached its minimum in the M12-group followed by an increased efficacy in older animals (Figure 1C). Thus, the age-related drop in the hypermetabolic and anorexigenic responsiveness to α-MSH occurs (12 months) before the marked weight gain observed by 18 months of age, whereas the strong responsiveness of the O18-group precedes the appearance of weight loss by 24 months of age. These results suggest that the observed age-related shifts in catabolic α-MSH-effects may contribute to the development of middle-aged obesity and later to that of weight loss of old age. Figure 1. View largeDownload slide Age-related changes in BW (A) of male Wistar rats, and their hypermetabolic (B) and anorexigenic (C) responsiveness to ICV injected α-MSH, 5 µg. (A) BW development curve constructed from mean ± S.E.M. values of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats. #p < .001 3 months vs. all other age groups, *p < .001 18 months vs. all other age groups. (B) α-MSH-induced increase in oxygen consumption (ΔVO2, expressed in % of the initial value) at 20 minutes following α-MSH injection in different age groups of rats. The initial VO2-values did not differ (21.7 ± 1.2, 21.4 ± 1.7, 23.5 ± 1.7, 22.0 ± 1.5, and 19.1 ± 1.9 mL/kg/min for 3-, 6-, 12-, 18-, and 24-month-old groups, respectively).*p < .05 concerning the difference between 12-month-old and 3- or 24-month-old groups. (C) α-MSH-induced suppression of 2 hours cumulative refeeding FI following 24 hours fasting in various age groups of rats. Anorexigenic responsiveness is represented by the difference between cumulative FI values of the treated and the age-matched control groups (injected ICV with physiological saline) expressed as % of the control cumulative values (control refeeding FI values: 21.7 ± 1.2, 21.4 ± 1.7, 23.5 ± 1.7, 22.0 ± 1.5, and 19.1 ± 1.9 g for 3-, 6-, 12-, 18-, and 24-month-old groups, respectively). *p < .001 12 months vs. other age groups; #p < .02 18 months vs. younger age groups. All data (A–C) are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA with Fisher’s post hoc test. Figure 1. View largeDownload slide Age-related changes in BW (A) of male Wistar rats, and their hypermetabolic (B) and anorexigenic (C) responsiveness to ICV injected α-MSH, 5 µg. (A) BW development curve constructed from mean ± S.E.M. values of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats. #p < .001 3 months vs. all other age groups, *p < .001 18 months vs. all other age groups. (B) α-MSH-induced increase in oxygen consumption (ΔVO2, expressed in % of the initial value) at 20 minutes following α-MSH injection in different age groups of rats. The initial VO2-values did not differ (21.7 ± 1.2, 21.4 ± 1.7, 23.5 ± 1.7, 22.0 ± 1.5, and 19.1 ± 1.9 mL/kg/min for 3-, 6-, 12-, 18-, and 24-month-old groups, respectively).*p < .05 concerning the difference between 12-month-old and 3- or 24-month-old groups. (C) α-MSH-induced suppression of 2 hours cumulative refeeding FI following 24 hours fasting in various age groups of rats. Anorexigenic responsiveness is represented by the difference between cumulative FI values of the treated and the age-matched control groups (injected ICV with physiological saline) expressed as % of the control cumulative values (control refeeding FI values: 21.7 ± 1.2, 21.4 ± 1.7, 23.5 ± 1.7, 22.0 ± 1.5, and 19.1 ± 1.9 g for 3-, 6-, 12-, 18-, and 24-month-old groups, respectively). *p < .001 12 months vs. other age groups; #p < .02 18 months vs. younger age groups. All data (A–C) are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA with Fisher’s post hoc test. In Vitro Results: α-MSH Immunoreactivity in the ARC and PVN Declines in Middle-Aged and Increases Together With POMC Gene Expression in Aging Rats To assess the age dependence of the main factors of the endogenous MC system at mRNA level in the ARC and PVN, qRT-PCR measurements were conducted (Figure 2). Regarding protein levels, immunofluorescence method was applied (Figures 3 and 4). Figure 2. View largeDownload slide Relative mRNA expressions of POMC, AgRP, and MC4R in the ARC (A–C, respectively) and in the PVN (D–F, respectively) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats. All data (A–F) are expressed as mean ± S.E.M. Lettering on top of the columns represents significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). Figure 2. View largeDownload slide Relative mRNA expressions of POMC, AgRP, and MC4R in the ARC (A–C, respectively) and in the PVN (D–F, respectively) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats. All data (A–F) are expressed as mean ± S.E.M. Lettering on top of the columns represents significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). Figure 3. View largeDownload slide Immunohistochemical detection of α-MSH and MC4R in the ARC of male Wistar rats revealed age-related differences using α-MSH-MC4R double labeling. Representative confocal immunofluorescent images show α-MSH (red; A–C) and MC4R (green; D–F) immunoreactive neuronal cell bodies in 6-, 12-, and 18-month-old male Wistar rats, respectively. Immunoreactivity in the middle-aged 12-month-old group is decreased as compared with the younger or older age group. Scale bar: 100 µm. Specific signal density (in arbitrary units, a.u.) of α-MSH (G) and MC4R (H) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats demonstrates a characteristic age-related pattern. All data (G and H) are expressed as mean ± S.E.M. Lettering on top of the columns indicates significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). Figure 3. View largeDownload slide Immunohistochemical detection of α-MSH and MC4R in the ARC of male Wistar rats revealed age-related differences using α-MSH-MC4R double labeling. Representative confocal immunofluorescent images show α-MSH (red; A–C) and MC4R (green; D–F) immunoreactive neuronal cell bodies in 6-, 12-, and 18-month-old male Wistar rats, respectively. Immunoreactivity in the middle-aged 12-month-old group is decreased as compared with the younger or older age group. Scale bar: 100 µm. Specific signal density (in arbitrary units, a.u.) of α-MSH (G) and MC4R (H) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats demonstrates a characteristic age-related pattern. All data (G and H) are expressed as mean ± S.E.M. Lettering on top of the columns indicates significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). Figure 4. View largeDownload slide Immunohistochemical detection of α-MSH and MC4R in the PVN of male Wistar rats revealed age-related differences using α-MSH-MC4R double labeling. Representative confocal immunofluorescent images show α-MSH (red; A–C) immunoreactive nerve fibers and MC4R (green; D–F) immunoreactive neuronal cell bodies in 6-, 12-, and 18-month-old male Wistar rats, respectively. Immunoreactivity in the middle-aged 12-month-old group is decreased as compared with the younger or older age group. Areas marked by white boxes are also depicted as higher magnification insets in the right bottom corner of the respective panel. Scale bar: 100 µm. Specific signal density (in arbitrary units, a.u.) of α-MSH (G) and MC4R (H) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats demonstrates a characteristic age-related pattern. All data (G and H) are expressed as mean ± S.E.M. Lettering on top of the columns indicates significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). Figure 4. View largeDownload slide Immunohistochemical detection of α-MSH and MC4R in the PVN of male Wistar rats revealed age-related differences using α-MSH-MC4R double labeling. Representative confocal immunofluorescent images show α-MSH (red; A–C) immunoreactive nerve fibers and MC4R (green; D–F) immunoreactive neuronal cell bodies in 6-, 12-, and 18-month-old male Wistar rats, respectively. Immunoreactivity in the middle-aged 12-month-old group is decreased as compared with the younger or older age group. Areas marked by white boxes are also depicted as higher magnification insets in the right bottom corner of the respective panel. Scale bar: 100 µm. Specific signal density (in arbitrary units, a.u.) of α-MSH (G) and MC4R (H) of 3-, 6-, 12-, 18-, and 24-month-old male Wistar rats demonstrates a characteristic age-related pattern. All data (G and H) are expressed as mean ± S.E.M. Lettering on top of the columns indicates significant differences between pairs of age groups according to Fisher’s post hoc test (p < .05). The relative expression of POMC mRNA in the ARC was a function of age (Figure 2A, ANOVA: F = 5.097; p < .005). Based on the post hoc comparison, gene expression increased until 18 months of age with a transient nonsignificant 30% decline between 6 and 12 months. In the oldest O24 rats, the gene expression became low again. Regarding immunohistochemical results, we found clearly recognizable neuronal cell bodies labeled by α-MSH antibodies in the ARC using α-MSH-MC4R double labeling (Figure 3A–C). The SSD measurement of α-MSH immunoreactivity (Figure 3G) revealed that the peptide content of ARC α-MSH neurons was also a function of age (ANOVA: F =3.761; p < .02). Here, resembling the results concerning the POMC gene expression (cp. Figure 2A), a somewhat similar age-related pattern emerged: compared with the similarly high SSD values of Y3 and M6 rats, the M12 animals showed a significant decline followed by a marked increase in the aging O18 animals. In the oldest O24 group, the SSD dropped significantly. Thus, the peak was found once again in the O18 rats (Figure 3G). At the site of release of α-MSH (ie in the PVN), relatively low POMC expression was found in all age groups with the exception of the aging O18 rats (Figure 2D, ANOVA: F = 6.062; p < .002). Their high value was followed by a nonsignificant decline in O24 rats (p = .19). Interestingly, the relative POMC expression of the oldest rats was still higher than that of the Y3-group (p < .001). At the PVN, we saw a dense network of α-MSH immunoreactive nerve fibers (Figure 4A–C). No α-MSH immunoreactive cell bodies were found here. Statistical analysis revealed an overall effect of age on α-MSH peptide immunosignal (Figure 4G, F = 4.804, p < .01). The age-related pattern was similar to that seen in the ARC (cp. Figure 3G): M12 animals showed a significant decline compared with the SSD values of M6 rats, followed by a marked increase in the aging O18 group. The decline in the oldest animals did not reach statistical significance (p = .06; Figure 4G). In Vitro Results: AgRP Gene Expression, But Not Its Immunoreactivity Increases in the ARC and PVN of Aging Rats AgRP produced in the ARC also binds to MC4R as an endogenous inverse agonist in the PVN. Results of the quantitation of AgRP mRNA expression (Figure 2B and E) were somewhat surprising since relative mRNA expression values exceeded those of POMC or MC4R (cp. Figure 2A, C, D, and F). In the ARC, the AgRP mRNA expression changed with aging (Figure 2B, ANOVA: F = 22.324; p < .001). It was low in Y3-, M6-, and M12-groups followed by a dramatic increase in O18 animals (a 30-fold increase was observed compared with the Y3 rats). In the oldest animals, AgRP mRNA expression decreased again. In the PVN, a similar age-related pattern of AgRP mRNA expression was seen (Figure 2E, ANOVA: F = 122.890; p < .001): uniformly low values in Y3-, M6-, and M12-groups were followed by a highly significant 50-fold rise in the O18 and O24 rats. In contrast with the ARC (cp. Figure 2B), the two oldest groups here did not differ significantly (Figure 5E). Figure 5. View largeDownload slide Schematic representation of the directions of age-related changes in relative mRNS expressions of POMC, AgRP, and MC4R and in immunosignals of α-MSH, AgRP, and MC4R in the ARC and PVN of the hypothalamus. Thin arrows represent directions of trends in mRNS expression between age groups following one another. Bold arrows demonstrate similar directions of trends in immunosignals. Dashed arrows indicate nonsignificant trends. The age of the animal groups are given in months (mo) under the corresponding boxes. Figure 5. View largeDownload slide Schematic representation of the directions of age-related changes in relative mRNS expressions of POMC, AgRP, and MC4R and in immunosignals of α-MSH, AgRP, and MC4R in the ARC and PVN of the hypothalamus. Thin arrows represent directions of trends in mRNS expression between age groups following one another. Bold arrows demonstrate similar directions of trends in immunosignals. Dashed arrows indicate nonsignificant trends. The age of the animal groups are given in months (mo) under the corresponding boxes. The AgRP staining was performed both in the ARC and PVN. In both locations, the signal was clear in nerve fibers. In contrast to AgRP mRNA expression, the high level of nerve fiber SSD for AgRP failed to show any age-dependence either in the ARC or in the PVN (Supplementary Figure 1A and B; for ARC: F = 1.103, p = .380, for PVN: F = 0.801, p = .539). In Vitro Results: MC4R Immunoreactivity in the ARC and PVN Declines in Middle-Aged and Increases in Aging Rats, Whereas Gene Expression Increases Only in the ARC of Aging Rats The presence of MC4Rs was detected not only in the PVN but also in the ARC. The qRT-PCR measurements of MC4R mRNA in the ARC (Figure 2C) showed that O18 rats expressed approximately four times more MC4R transcripts than other age groups (ANOVA: F = 50.560, p < .0001). Regarding immunohistochemical findings, α-MSH-MC4R double labeling revealed that α-MSH neurons in the ARC co-express MC4R (Figure 3D–F). The SSD of MC4R in the ARC showed age-dependence (Figure 3H, ANOVA: F = 5.447, p < .005). Post hoc comparison revealed that the MC4R immunosignal in M6- and O18-groups was approximately 90% higher than that of Y3, M12, or O24 rats. This age-related pattern showed similarities with that of α-MSH SSD in the ARC (cp. Figure 3G): the decline in the M12 animals was followed by an increase in the O18 aging group, and then again a decrease was observed in the oldest rats. The rise in the aging rats and the drop in the oldest ones were in accord with the mRNA expression data in the ARC (cp. Figure 2C). In the PVN, there was no difference in MC4R mRNA expression between the age-groups (Figure 2F, ANOVA: F = 1.625, p = .199). However, the age-related pattern of MC4R SSD values (immunofluorescence, Figure 4D–F and H) resembled those of α-MSH SSD in the ARC and PVN (cp. Figures 3A–C and G, and 4A–C and G). This general pattern involves a decline in the middle-aged followed by a rise in aging O18 and a final drop in the oldest animals. These data suggest corresponding age-related fluctuations in the activity of the endogenous MC system. On the other hand, these shifts in MC4R in the PVN may contribute to the explanation of the changes in the responsiveness to exogenously administered α-MSH (Figure 1B and C). Discussion The MC system plays a pivotal role in the control of energy homeostasis, with deep impact on BW and body composition (9,18). Based on earlier observations (19–22), we hypothesized that age-related changes in the intrinsic activity of the hypothalamic MC system contribute to the development of middle-aged obesity and aging anorexia/cachexia seen in humans and in rodents. To test this hypothesis, we investigated changes in the reactivity and also in the endogenous activity of the hypothalamic MC system during the course of aging. Thus, on the one hand, we analyzed complex acute catabolic (hypermetabolic and anorexigenic) effects of an exogenous MC agonist in five age groups of male Wistar rats from young adult to old age with special regard to the BW development curve. On the other hand, we also aimed to clarify the endogenous mechanisms of these in vivo phenomena at the level of hypothalamic gene expression and protein content in all five age groups. Our in vivo and in vitro results support our hypothesis as discussed below. Our in vivo results across five age groups completed and confirmed previously suggested age-related patterns (19,20,22). Both the decreased catabolic responsiveness of middle-aged animals and the enhanced responsiveness of aging rats appeared before characteristic alterations in the slope of the BW development curve. These dynamics suggest a regulatory role of the MC system in age-related changes of BW. These BW changes imply disadvantageous consequences regarding body composition: weight gain in middle-aged rats is usually a result of visceral fat accumulation (obesity), whereas weight loss in old animals affects mainly muscles (sarcopenia) (19,37,38). Therefore, investigation of their mechanisms using in vitro techniques is of major importance. Our results regarding immunofluorescent labeling of α-MSH neurons were in accord with earlier studies reporting α-MSH-immunopositive perikarya (39) in the ARC and α-MSH-immunoreactive nerve fibers projecting from the ARC into the PVN (40). Our α-MSH-MC4R double labeling did not only confirm the presence of MC4R in the PVN but also on α-MSH-immunopositive perikarya of the ARC (41–43). Detection of MC4R in the ARC has proven to be elusive so far; only a handful of studies reported its low density in addition to the moderate density of MC3R (41,44,45). The significance of MC4R expression in α-MSH immunopositive neurons was recently assessed in elegant studies by groups of Smith (46) in vitro and do Carmo (15) in vivo. These authors suggested a self–up-regulatory action of α-MSH on POMC neurons via their own MC4R that could amplify MC peptide release. In contrast to α-MSH, AgRP binding to MC4R of POMC neurons in the ARC would inhibit the above-mentioned positive feedback loop, through inverse agonism. Our immunofluorescent labeling of AgRP clearly showed positive nerve fibers both in the ARC and PVN (site of activation of second-order neurons), indicating active interactions with other neurons described earlier in both nuclei (14,47). We could not detect AgRP perikarya in the ARC, since the visualization of AgRP neurons by immunohistochemistry would have absolutely required a high-dose colchicine pretreatment (47), which would have biased the results in this experimental setup. With regard to gene expressions, our RT-PCR results revealed the presence of not only MC4R mRNA, but also that of POMC and AgRP in both nuclei. A discrepancy appears between the relatively high α-MSH and AgRP immunoreactivity and the low POMC and AgRP mRNA expression in the ARC of the Y3 group. This phenomenon awaits further studies. One could speculate that the gene transcription and the peptide release are slower in this age group, which leads to a relative accumulation of the immunoreactivity. Concerning age-related alterations, we analyzed our in vitro findings (depicted in a schematic way in Figure 5) in view of our in vivo observations. As compared with the responsiveness of Y3 animals, the decline in efficacy of α-MSH reached statistical significance by M12 (Figure 1B and C). This decline may be explained by the drop in MC4R immunoreactivity that we found in the PVN and even in the ARC of M12 (Figure 5). Interestingly, this drop was not associated with a parallel decrease in MC4R gene expression, suggesting an increased receptor turnover or decreased efficacy of synthesis. Moreover, the drop of MC4R immunoreactivity in the ARC could lead to a diminished positive feedback affecting POMC neurons that manifested in parallel drop of α-MSH immunoreactivity in the ARC and PVN, suggesting a suppressed endogenous activity of α-MSH (peptide content). Again, POMC gene expression was maintained in both nuclei; therefore, the decreased α-MSH peptide content could be explained by decreased efficacy of synthesis or increased elimination. At the same time, neither AgRP gene expression nor immunoreactivity decreased until M12 implying a maintained negative feedback affecting POMC neurons in the ARC and inverse agonist action in second-order neurons of the PVN. Accordingly, in M12, a relative AgRP dominance over α-MSH would explain the observed tendency for weight gain that precedes the appearance of BW peak by O18 (Figure 1A). This O18 age group with the maximal BW (associated with the peak fat mass (19,38)) shows a marked change of the MC system in the opposite direction. As compared with M12 animals, the responsiveness rises once again in O18 (Figure 1B and C). This rise may be explained by the significant increase in MC4R immunoreactivity that we found in the PVN and even in the ARC of O18 (Figure 5). Interestingly, the rise in the ARC but not in the PVN was associated with a parallel increase in MC4R gene expression, suggesting an increased synthesis of the receptor. In the PVN, a more efficient synthesis or decreased turnover could explain the higher MC4R immunoreactivity. Moreover, the activation of positive feedback affecting the POMC neurons of the ARC is indicated by the parallel rise of POMC gene expression and α-MSH immunoreactivity. In the PVN, similar increases were detectable. Interestingly, AgRP gene expression increased in the same age group without any change in AgRP immunoreactivity in either nucleus. Accordingly, in O18, a relative MC dominance over AgRP could explain the observed tendency for weight loss occurring between O18 and O24 (Figure 1A). These especially synchronous age-related changes may represent an adaptive reaction upon reaching a critical BW or adiposity. In the oldest animals, the synchronous decline in MC4R and α-MSH immunoreactivity characterizing both nuclei with parallel suppression of gene expression in the ARC suggests a coordinated decline in the endogenous activity of the MC system. (AgRP immunoreactivity did not change at this age, whereas gene expression declined only in the ARC.) These O24 animals appear to react to the previously occurring marked weight loss (leading to sarcopenia) with an adaptive counter-regulatory reaction also suggested previously by other authors (28). In summary, our in vitro findings revealed potential mechanisms of the age-related in vivo trends in BW regulation in male Wistar rats. In middle-aged rats, peptide contents of α-MSH and MC4R decreased both in the ARC and PVN (without matching changes in gene expression), whereas in aging animals, the increase in α-MSH and MC4R peptide contents was associated with parallel increases in POMC and MC4R mRNA expressions. Although AgRP mRNA expression in both nuclei showed some increase in aging animals, its protein content remained unchanged across all age groups. In addition to the MC system, other hypothalamic mediators may also contribute to the explanation of the age-related trends in BW regulation. Orexigenic neuropeptide Y (NPY), the other major regulator of the ARC has been reported to decline with aging (48). Thus, it may also contribute to aging anorexia, but not to middle-aged obesity. However, to date, no detailed analysis involving more than three age groups has been carried out. Thus, future studies are needed to clarify the role of NPY sufficiently. Our study is the first to analyze age-related shifts in the endogenous tone of the hypothalamic MC system across five age groups with parallel analysis of gene expression and immunohistochemistry. Previous studies have usually compared POMC and/or AgRP gene expressions of only a young and an old group (22,24–26,28); only a few of them included a third (middle-aged) group in the comparison (23,27,29,30). They showed controversial results: some studies reported an age-related decline (23–28), whereas others found unchanged MC activity in old rodents (22,29,30). The use of different rat and mouse strains, gender differences, or diverse methodologies (RT-PCR, in situ hybridization, analysis of the whole hypothalamus, or that of the ARC) may explain these contradictory findings. Our experimental setting allowed the detailed follow-up of age-related shifts within the hypothalamic MC system that could provide explanation for the long-term changes in BW development. Concluding Remarks and Perspectives To the best of our knowledge, this study is the first describing an age-related fluctuation of α-MSH immunoreactivity both in the ARC and PVN across five age groups. Our results prove that the α-MSH and MC4R peptide immunoreactivity along with the POMC and MC4R mRNA expression are affected by age in such a way that it may contribute to the explanation of aging anorexia. Moreover, the earlier occurring decline in α-MSH and MC4R peptide immunoreactivity may contribute to the explanation of the weight gain and obesity of middle-aged animals. Finally, our results suggest that reaching a critical BW or fat mass may provide the trigger for the activation of the MC system that in turn aggravates aging anorexia. Further studies are required to uncover why mRNA and protein expressions of the MC system do not overlap in certain age groups at certain sites. The authors predict the existence of age-related changes in the translation efficacy of mRNA or fluctuations in the protein turnover. Other studies are to be conducted to identify the significance of AgRP mRNA upregulation in aging rats. Supplementary Material Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online. Funding This work was supported by the University of Pecs, Hungary (PTE ÁOK-KA No: 2017/12 to E.P., PTE ÁOK-KA No: 2017/13 to M.B., PTE ÁOK-KA No: 2017/01 to B.G., and PTE-AOK-KA-2015-14 to M.S.). Acknowledgments The authors are grateful for the expert and excellent technical assistance of Ms. I. Orbán, Ms. A. Jech-Mihálffy, Ms. A. Bóka-Kiss, Ms. É. Sós, and Ms. M. Koncsecskó-Gáspár. 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The Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences – Oxford University Press
Published: Apr 1, 2018
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