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Acute central effects of corticotropin-releasing factor (CRF) on energy balance: Effects of age and gender
Accepted Manuscript Title: Acute central effects of corticotropin-releasing factor (CRF) on energy balance: effects of age and gender Authors: Judit Tenk Ildik´o Rost´as N´ora Furedi ¨ Alexandra Mik´o Szilvia So´os Margit Solym´ar Bal´azs Gaszner Mikl´os Sz´ekely Erika P´eterv´ari M´arta Balask´o M.D., Ph.D. PII: DOI: Reference:
S0196-9781(16)30182-6 http://dx.doi.org/doi:10.1016/j.peptides.2016.09.005 PEP 69671
To appear in:
Peptides
Received date: Revised date: Accepted date:
6-6-2016 12-9-2016 12-9-2016
Please cite this article as: Tenk Judit, Rost´as Ildik´o, Furedi ¨ N´ora, Mik´o Alexandra, So´os Szilvia, Solym´ar Margit, Gaszner Bal´azs, Sz´ekely Mikl´os, P´eterv´ari Erika, Balask´o M´arta.Acute central effects of corticotropin-releasing factor (CRF) on energy balance: effects of age and gender.Peptides http://dx.doi.org/10.1016/j.peptides.2016.09.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Acute central effects of corticotropin-releasing factor (CRF) on energy balance: effects of age and gender
Judit Tenk1, Ildikó Rostás1, Nóra Füredi1, Alexandra Mikó1, Szilvia Soós1, Margit Solymár1, Balázs Gaszner2, Miklós Székely1, Erika Pétervári1, Márta Balaskó1
From: 1
Institute for Translational Medicine, Medical School, University of Pécs, Hungary
2
Department of Anatomy, Medical School, University of Pécs, Hungary
Submitted to: PEPTIDES
Corresponding author: Márta Balaskó, M.D., Ph.D. Institute for Translational Medicine Medical School, University of Pécs 12 Szigeti str., Pécs H-7624, Hungary Phone: +36-72-536-246 Fax: +36-72-536-247 e-mail: [email protected]
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Highlights CRF-induced anorexia declines with aging in male, but not in female rats CRF-induced hypermetabolism declines strongly with aging in females, but not in males Maintained CRF-anorexia in all female rats may help prevent middle-aged obesity Age-related changes in the responsiveness of CRF1 and CRF2 receptors depend on gender
Abstract
Previously demonstrated age-related changes in the catabolic melanocortin system that may contribute to middle-aged obesity and aging anorexia, raise the question of the potential involvement of corticotropin-releasing factor (CRF) in these phenomena, as this catabolic hypothalamic mediator acts downstream to melanocortins. Catabolic effects of CRF were shown to be mediated by both CRF1 (hypermetabolism) and CRF2 (anorexia) receptors. To test the potential role of CRF in age-related obesity and aging anorexia, we investigated acute central effects of the peptide on energy balance in male and female rats during the course of aging. Effects of an intracerebroventricular CRF injection on food intake (FI), oxygen-consumption (VO2), core- and tail skin temperatures (Tc and Ts) were studied in male and female Wistar rats of five different age-groups (from 3- to 24-month). Anorexigenic responsiveness was tested during 180-min re-feeding (FeedScale) following 24-h fasting. Thermoregulatory analysis was performed by indirect calorimetry (Oxymax) complemented by thermocouples recording Tc and Ts (indicating heat loss). CRF suppressed FI in 3-month male and female animals. In males, CRF-induced anorexia declined with aging, whereas in females it was maintained in all groups. The peptide increased VO2 and Tc in all male age-groups, while the weaker hypermetabolic response characterizing 3-month females declined rapidly with aging. Thus, age-related alterations in acute central anorexigenic and hypermetabolic effects of CRF show different non-parallel patterns in males and females. Our findings underline the importance of gender differences. They also call the attention to the differential age-related changes in the CRF1 and CRF2 receptor systems.
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Abbreviations: BW, body weight; CRF, corticotropin- releasing factor; CRF1 receptor, corticotropin- releasing factor type 1 receptor; CRF2 receptor, corticotropin- releasing factor type 2 receptor; F, female; FI, food intake; HPA, hypothalamic-pituitary-adrenal; ICV, intracerebroventricular; IP, intraperitoneal; M, male; MSH, melanocyte- stimulating hormone; PFS, pyrogen-free saline; Tc, core body temperature; Ts, tail skin temperature; Ucn2, urocortin 2; Ucn3, urocortin 3; VO2, oxygen-consumption
Keywords: corticotropin-releasing factor (CRF), energy balance, food intake, metabolic rate, aging, gender
1. INTRODUCTION
Long-term regulation of body weight (BW) and body composition shows two different trends in mammals: obesity develops typically in the middle-aged, whereas old age is characterized by anorexia, weight loss and sarcopenia [15], [38], [55]. Both trends imply world-wide public health burdens [69], [70]. As most mammals also show similar trends in their long-term BW development [63], a dysregulation of energy homeostasis may also contribute to these phenomena. Therefore, the investigation of regulatory alterations in energy balance during the course of aging, are of outstanding importance. And indeed, earlier studies demonstrated the potential role of age-related shifts in the responsiveness to such centrally administered catabolic mediators as leptin [44] or endogenous melanocortin agonist alpha-melanocytestimulating hormone (alpha-MSH) in the development of the above mentioned BW trends [43], [52]. Based on these previous findings, the question arises, whether corticotropins (downstream to melanocortins and leptin) may also contribute to the metabolic dysregulation characterizing aging.
The first member of the corticotropin peptide family, corticotropin-releasing factor (CRF), a 41-aa neuropeptide was described by Vale and coworkers in 1981 [66]. This peptide is produced predominantly in the parvocellular neurons of the paraventricular nucleus of the hypothalamus [66], but expression of CRF mRNA has been detected, among other sites, in the cerebral cortex, amygdala and in the hippocampus [37], [68].
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Although the main functions of CRF are thought to be the activation of the hypothalamicpituitary-adrenal (HPA) axis [39], [49], the activation of the sympathetic tone [8] and coordination of the body’s responses to stress [32], this peptide also participates in a great variety of regulatory processes. It has been shown to induce fear, to participate in anxiety and in depressive disorders of animals [9] and humans [2]. In addition, CRF elicits coordinated catabolic effects via inducing anorexia [48] and hypermetabolism accompanied by increased brown-fat thermogenesis [8], [10], [33]. Moreover, in genetically obese rats (fa/fa) the peptide prevented weight gain [50].
Two subtypes of G-protein coupled receptors (CRF1 and CRF2) mediate the effects of corticotropins, CRF activates mainly CRF1 and to a lesser extent CRF2 receptor [35], [42], [67], while urocortin 1, another member of the corticotropin family shows similar affinity to both receptor types [35], [42], [67]. Widespread expression of CRF1 receptor was described in hypothalamic nuclei [45], [47], [65] and also in the anterior pituitary [65]. Additionally, they are found in the forebrain, in the septal region and amygdala [29], in the cerebral cortex and in the cerebellum [47], [65]. Anxiogenic actions, depressive behavior and hyperthermic/hypermetabolic effects have been attributed to the activation of this receptor type [18], [47], [65]. Its anorexigenic effects are thought to be based on emotional stress [28]. In contrast, CRF2 receptor expression appears to be more restricted in the brain, these receptors were mainly detected in the lateral septum, amygdala, hippocampus, also in hypothalamic nuclei (e.g. in the ventromedial hypothalamus) [65] or in the nucleus of the solitary tract [6]. Type 2 receptors mediate anorexia, anxiolytic and antidepressive effects [13], [47], [65]. This latter receptor type has been shown to mediate anorexigenic actions of centrally applied CRF or those of other members of the corticotropin peptide family, such as urocortin 2 (Ucn2) or urocortin 3 (Ucn3) [13], [40], [58]. The latter members of the corticotropin family show enhanced affinity to CRF2 receptors [13], [34], [40], [60].
During the course of aging, characteristic changes of the corticotropin system have been reported in humans and mammals [25]. Most studies found increased hypothalamic CRF expression in old age compensated by some CRF1 receptor downregulation [1], [11], [56], [64]. Nevertheless, a few studies described reduced or unchanged CRF expression in old agegroups [12], [31]. Post mortem investigations in men demonstrated increased activity of CRF, especially in depressed subjects or in deceased patients with Alzheimer's disease [4], [46].
5 Other investigators suggested a role for the hyperactive HPA axis in neuronal deterioration of aged humans and in old experimental animals [16], [54], [61]. An intriguing additional feature of the corticotropin system is that its functions show gender differences [22], [26], [36]. Therefore, age-related alterations of the hypothalamic corticotropin system may also show different patterns in males and females. Such differences may also provide some explanation for the gender differences in the long-term BW development of male and female rats: in contrast with males showing middle-aged obesity and a decline in BW in old age-groups, females maintain a stable low BW throughout life [17], [63].
Therefore, we aimed to investigate the potential involvement of acute central catabolic CRF effects (anorexia and hypermetabolism) in the development of age-related obesity and aging anorexia leading to weight loss in old male Wistar rats. These acute central catabolic effects of CRF were also analyzed in females during the course of aging to test their potential contribution to gender differences in long-term BW development in this strain.
2. METHODS
2.1. Animals Various age-groups of male (n = 79) and female (n = 75) Wistar rats (M and F, respectively) from the Colony of the Institute for Translational Medicine of the Medical School, University of Pécs, Hungary were used in the experiments of the present study: young adult (M = 14, F = 12), younger (M = 14, F = 16) and older middle-aged (M = 15, F = 13), aging (M = 16, F = 16) and old (M = 10, F = 18) (3-, 6- and 12-, 18- and 24-months old, respectively). (Maximal life-span of our Colony reaches 30 months, about 50% of rats survive 26 months, but after the age of 24 months surgical interventions involve high mortality). In this study altogether 7 rats failed to survive surgery. For thermoregulatory analysis and for tests of food intake separate groups of animals had to be used. Each animal in each age-group received CRF and its solvent in random order. Such tests were divided by 7 days. Following tests, animals were sacrificed; no repeated testing across age-groups was possible. After they have reached the appropriate age, rats were kept in individual plastic home-cages containing wood-chip bedding at an ambient temperature of 22–25 °C. Cages were equipped with a steel grid top with feeder and bottle container. This environment provided a thermoneutral temperature in the nest. Lights were on between 06.00 and 18.00 h. Standard
6 rat chow (11 kJ/g; CRLT/N rodent chow, Szindbád Kft., Gödöllő, Hungary) and tap water were available ad libitum, except for the 24-h fasting period when only water was provided for the appropriate groups. All animals were accustomed to regular handling. Spontaneous daily food intake (FI) and BW were measured every day at 09.00 h. Table 1 shows BW values of the different male and female age-groups. Initial BW-s of treated and those of control animals did not differ within age-groups, as shown by one-way ANOVA (p > 0.05 in all cases, Table 1).
2.2. Surgeries, drug administration A 22-gauge stainless-steel guide cannula was implanted into the right lateral cerebral ventricle of rats using a stereotaxic apparatus for the purpose of intracerebroventricular (ICV) injections. The tip of the guide cannula (fixed to the skull by dental cement) was positioned at A: -1.0 mm (to bregma), L: 1.5 mm (right lateral to bregma), V: 3.5 mm (ventral to dura) (coordinates according to the Rat Brain Atlas, [41]). A stylet closed the lumen of the guide cannula which was replaced during the experiments by a 28-gauge injection cannula outreaching the guide cannula by about 0.5 mm. Surgical interventions were performed under intraperitoneal (IP) ketamine-xylazine [78 mg/kg (Calypsol, Richter) + 13 mg/kg (Sedaxylan, Eurovet)] anesthesia. Gentamycin (2 mg IP) was used for the prevention of infections. In order to check the appropriate location of the guide cannula angiotensin II (Sigma, A9525, 20 ng/5 l) was injected through a pp10 polyethylene tube attachment around 3 days after the implantation. Appropriate location was assumed if at least 5 ml water was consumed within 30 min [43]. Experiments started 7 days after the cannula implantation.
After the experiments rats were sacrificed by an IP overdose of urethane (3-5 g/kg, Reanal). Post mortem check of the injection sites were performed by observing macroscopically the coronal sections of the removed brains that were fixed in 4% paraformaldehyde for 48 hours. Only rats with appropriate cannula location were included in the analysis. Because of incorrect location (judged by the in vivo or post mortem test), data of 9 of the 154 rats were excluded from the analysis.
When testing anorexigenic effects, the animals received CRF or pyrogen-free saline (PFS) after 24-h fasting. Corticotropin-releasing factor (0.3 g Bachem, H2435) dissolved in PFS in a volume of 5 l or 5 l PFS filled the proximal end of a 20-25 cm-long pp10 polyethylene
7 tube attachment of the injection cannula. A small bubble separated the substance prepared for ICV injection from the PFS that filled the distal part of the tube. In thermoregulatory tests an amount of 0.3, 1 and 3 g of the peptide was dissolved in PFS in a volume of 5 l and was injected similarly. Control rats received PFS in 5 l volume. In these studies CRF was injected at a thermal steady state (usually 60-90 min following the start of the experiment). All injections were given remotely, without causing any acute discomfort to the rats and they were administered around 09.00 h, early in the inactive phase of the day.
2.3 Assessment of food consumption At least 10-14 days before the experiments, rats were transferred to the automated FeedScale system (Columbus, OH) to get habituated to the environment and to the powdered form of rat chow. The system allowed continuous recording of the amount of consumed food. Data were registered every 10 minutes. On day 1 at 09.00 h food was removed for 24-h. Five minutes before the re-feeding started (on day 2 at 09.00) assigned rat groups received 0.3 g ICV CRF or PFS injection to test the inhibitory effect of the peptide on 3-h cumulative FI.
2.4 Assessment of thermoregulatory functions During thermoregulatory analysis oxygen consumption (VO2, representing metabolic rate), core temperature (Tc) and tail skin temperature (Ts, indicating heat loss) were recorded. The tests were performed between 09.00 h and 15.00 h on semi-restrained rats, singly enclosed in cylindrical wire-mesh confiners in separate tightly sealed plexiglass metabolic chambers continuously ventilated with room air (size: 20 x 30 x 18.5 cm). Four chambers were used simultaneously that were immersed into a thermostatically controlled water-bath. For thermoregulatory analysis of CRF a slightly subthermoneutral environment [ambient temperature (Ta) was 25 ºC] was maintained. As the animals were previously accustomed to the semi-restraining cages for at least a week (starting with a 120-min, followed by a 180-, a 240- and two 300-min sessions), we could minimize the stress during the experiments, as shown by the normal initial Tc values between 37.2 and 37.6 °C. Nevertheless, the gradual moderate decline in the Tc and VO2 values of control male (and to a lesser extent of female) PFS-treated rats indicates that some initial stress must have induced a slight increase in these values that declined later in the familiar environment. During tests the animals could not eat or drink.
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The injection cannula was attached to the proximal end of the 20-25 cm-long pp10 polyethylene tube. At the distal part of the tube, 5 μl PFS was slowly injected that resulted in the ICV administration of the 5 μl CRF solution or PFS via the injection cannula.
Following the ICV injection the VO2 was registered in 10-min intervals for 3 hours. For the recording of Tc and Ts copper-constantan thermocouples were applied. The colon thermocouple was inserted 10 cm beyond the anal sphincter and it was fixed by tape to the tail. The tail skin thermocouple was fixed on the dorsal skin of the distal part of the tail. One thermocouple recorded Ta of the chamber. The rate of heat loss (heat loss index, HLI) was calculated from the relationship of the monitored temperatures: HLI = (Ts-Ta) / (Tc-Ta) [51]. HLI near 0 (when the Ts value approaches Ta) suggests vasoconstriction as a heat conserving mechanism, HLI near 1 (when the Ts value approaches Tc) indicates vasodilation as indication of heat loss. In the semi-restraining cages the animals could not turn around or reach the thermocouples. The thermocouples and the extensions of the ICV cannula were pulled through a tightly sealed port of the metabolic chamber allowing the remote administration of substances. Oxygen consumption (ml O2/kg/min) and carbon-dioxide (CO2) production (ml/kg/min) were determined by indirect calorimetry (Oxymax, Equal Flow, Columbus, OH) from the air flowing through the chambers. Temperature data were collected by a Digi-Sense Benchtop Thermometer (Cole-Parmer) for electronic processing and evaluation.
2.5. Statistical analysis All age-groups contained at least 6-8 rats. SPSS 11.0 for Windows was used for the statistical analysis of the data with application of one-way, two-way (univariate or repeated-measures analysis) or repeated-measures ANOVA complemented by Tukey’s post hoc test, when more than two groups were compared. All results are presented as mean ± S.E.M. The level of significance was set at p < 0.05.
2.6. Ethical issues The general rules and those determined by the special permit of the University of Pécs Ethical Committee for the Protection of Animals in Research (BA 02/200-11/2011 valid for 5 years) were strictly observed in all experimental interventions and procedures. These rules are in accordance with the main directives of the National Ethical Council for Animal Research and
9 those of the European Communities Council (86/609/EEC, Directive 2010/63/EU of the European Parliament and of the Council).
3. RESULTS
Table 1 shows BW values of different male and female age-groups. Values of male Wistar rats (Table 1) were in accord with those observed in our previous studies [3], [44]: up to 18 months of age BW showed a rising tendency, then it declined in the oldest animals. Body weights (Table 1) of male rats exceeded those of corresponding females of the same agegroup.
Following 24-h fasting, weight loss of rats ranged from 7% to 9% of initial BW in males, and from 3% to 10% in females.
3.1 Anorexigenic CRF effects in male and female rats in the course of aging The small 0.3 g dose applied in these tests was chosen based on previous reports of the literature [57], [71]. In young adult (M3) and younger and older middle-aged (M6 and M12, respectively) male rats the ICV administered acute CRF injection caused a strong suppression of 3-h cumulative FI during re-feeding following 24-h fasting [repeated-measures ANOVA for the 3-h refeeding period from 10 to 180 min for M3: F(1,14) = 10.280, p = 0.006, for M6: F(1,12) = 17.339, p = 0.001 and for M12: F(1,14) = 16.700, p = 0.001, Fig. 1]. The peptide failed to induce a significant anorexigenic response in aging (M18) and old (M24) rats (Fig. 1). These results suggest that the anorexigenic effects of an acute central injection of CRF show a gradual decline with aging [F(4,63) = 3.410, p= 0.014, as indicated by two-way ANOVA repeated-measures analysis].
Cumulative 3-h re-feeding of control female rats reached similar values as those of corresponding age-groups of males (Figs 1, 2). In female rats, anorexigenic effects of an acute ICV CRF injection (0.3 g) were maintained at a significant level in all age-groups from F3 to F24 [repeated-measures ANOVA for the 3-h re-feeding period from 10 to 180 min for F3: F(1,10) = 21.365, p = 0.002, for F6: F(1,14) = 67.986, p < 0.001, for F18: F(1,10) = 10.210, p = 0.010, for F24: F(1,10) = 5.550, p = 0.040 and from 40 to 180 min for F12: F(1,10) = 5.749, p = 0.043, Fig. 2]. Therefore, in females no age-dependence of acute central CRF-anorexia
10 was found within the observed age-groups [F(4,54) = 1.287, p = 0.287, as indicated by twoway ANOVA repeated-measures analysis].
3.2. Hypermetabolic/hyperthermic CRF effects in male and female rats in the course of aging Initially three different doses of CRF (0.3, 1 and 3 g) were tested in the thermoregulatory experiments of young adult male rats and all doses elicited similar, significant hyperthermic responses. However, the 1 g dose elicited the largest increase in oxygen consumption (p < 0.05, comparison of different doses not shown). Therefore, the 1 g dose was applied in all subsequent thermoregulatory experiments.
The young adult male age-group showed a CRF-induced increase in metabolic rate [represented by the change in VO2 (VO2), at 25 °C Ta), with a consequent significant rise in Tc (shown as Tc in Fig. 3). The rise in oxygen consumption and Tc started directly upon the ICV injection. Fig. 3 demonstrates CRF-induced hypermetabolic/hyperthermic effects (1 g dose) at 25 °C in young adult rats [ANOVA repeated measures for VO2: F(1,12) = 21.335, p = 0.001; Tc: F(1,12) = 104.208, p <0.001]. No compensatory rise in heat loss was detectable, as indicated by HLI. Based on these data, CRF-induced hyperthermia appears to be coordinated, in which case both heat production and heat conservation (lack of vasodilation) promote the rise in Tc.
Regarding age-related variations of these reactions (Fig. 4), in addition to the a strong response of young adult rats to CRF concerning maximal Tc (p < 0.001, one-way ANOVA) and VO2 changes (p < 0.001, one-way ANOVA as compared with their respective controls), similar CRF-induced hyperthermic and hypermetabolic reactions were observed in older rats, as well (for Tcmax M6: p = 0.003, M12: p < 0.001, M18: p < 0.001 and M24: p= 0.004; for VO2max M6: p = 0.005, M12: p = 0.007, M18: p = 0.023 and M24: p = 0.040).
Although these responses proved to be significant in all age-groups, the maximal increase in Tc and VO2 (Fig. 4) declined with aging (two-way ANOVA univariate analysis for maximal rises in Tc or VO2: F(9,55) = 3.929, p = 0.007 or F(9,55) = 2.67, p = 0.041, respectively). Heat loss mechanisms did not show any age-related alteration.
11 Young adult female rats showed a weaker CRF-induced increase in metabolic rate and Tc (at 25 °C Ta) than the corresponding male age-group. The rise in VO2 and Tc started directly upon the ICV injection. Fig. 5 demonstrates CRF-induced hypermetabolic/hyperthermic effects (1 g dose) in young adult female rats [ANOVA repeated-measures for VO2 from 10 to 180 min: F(1,12) = 8.254, p = 0.014; for Tc from 10 to 180 min.: F(1,12) = 13.342, p = 0.003]. No vasodilation, indicated by HLI occurred in these female rats (Fig. 5).
The maximal increase in Tc and VO2 (Fig. 6) showed some variations with aging in female rats. The peptide induced hyperthermia (for Tcmax: F3: p < 0.001; F6: p=0,017; one-way ANOVA) and hypermetabolism (for VO2max: F3: p = 0.004; F6 p= 0,047; one-way ANOVA) in the young adult and younger middle-aged females. However, this response was weaker than in the corresponding male groups. Older female age-groups failed to show any significant change in Tc or VO2. Thus, the hyperthermic/hypermetabolic effects of CRF showed a gradual age-dependent decline in female rats [two-way ANOVA univariate analysis, F(9,59) = 3.309, p = 0.037 for Tcmax and F(9,59) = 2.731, p = 0.016 for VO2max].
4. DISCUSSION
The present study aimed to investigate age-related changes in the acute central catabolic effects of CRF in male and female Wistar rats with their different life-long body weight developments (Table 1) in view.
4.1 Anorexigenic CRF effects in male and female rats in the course of aging In males, acute ICV CRF administration-induced suppression of re-feeding showed agedependence, as it was significant in the young adult and also in the younger and older middleaged groups, whereas this effect failed to develop in older animals (Fig. 1). This age-related pattern of acute central CRF-anorexia distinctly differs from that of acute central alpha-MSHinduced suppression of re-feeding reported in male Wistar rats [43]. According to that study, male rats showed a strong decline of melanocortin-induced anorexia in the middle-aged group (as compared with that seen in young adult animals) but pronounced effects were observed again in the old rats [43]. Such an age-related pattern potentially contributes to the explanation of middle-aged obesity and aging anorexia. In contrast, the age-related pattern
12 derived from our experiments, characterizing acute central CRF-anorexia is unlikely to promote the development of either the middle-aged obesity or the aging anorexia in male Wistar rats. Regarding the potential involvement of the major CRF receptor subtypes in the above described age-related pattern, CRF2 receptors (rather than CRF1 receptors) may play a decisive role in it. Although CRF shows much higher affinity to CRF1 receptors, their contribution to CRF-induced anorexia is mainly attributed to emotional stress [28]. Moreover, these receptors activate the HPA axis [65] and therefore they are responsible for the dramatic increase in peripheral plasma corticosterone level upon an acute ICV CRF injection, described in previous studies [13]. A rise in peripheral corticosterone level or even an ICV administration of the hormone failed to change FI in mice [14], whereas a CRF-induced increase in peripheral cortisol has been shown to be coupled with increased FI in healthy humans [19]. In addition, the vast majority of the related literature also attributes anorexigenic CRF effects to CRF2 receptor mediation [13], [34], [40], [60]. Thus, our present findings may have implications for the potential lack of involvement of CRF2 receptors in aging-induced variations of BW in male rats.
In females, CRF-induced anorexia did not exhibit age-dependence within the studied agegroups, as it was maintained in all female animals. This continuous efficacy of acute ICV anorexigenic actions of CRF may have helped to prevent rapid weight gain in middle-aged and older female animals, but apparently failed to induce weight loss up to 24 months of age. However, it cannot be excluded that even older age-groups of female rats (e.g. 30-month), not studied in the present work, would show anorexia and weight loss as a result of this maintained efficacy. In summary, a gender difference emerges in the age-related patterns of CRF-induced anorexia and of CRF2 receptor responsiveness in Wistar rats: these CRF effects decline in males, but they are maintained in females during aging (until 24 months of age).
4.2. Hypermetabolic/hyperthermic CRF effects in male and female rats in the course of aging Concerning hypermetabolic/hyperthermic effects in young adult males, ICV CRF administration elicited a prompt rise in VO2 inducing a steady rise in Tc. This hyperthermia was accompanied by continuous heat conservation as indicated by the HLI. This thermoregulatory response appears to be coordinated, i.e. similar to fever-like coordinated hyperthermias, in which increased heat production is accompanied by suppressed heat loss
13 coordinated by thermoregulatory centers of the hypothalamus [3], [59], [62]. The role of CRF in thermoregulation is still controversial. Some studies suggested a role of CRF in the development of fever [27], [53]. However, other findings contradict the hypothesis of the participation of CRF in febrigenesis. Neither non-selective cyclooxygenase (COX) inhibitors, nor COX-2 antagonists suppressed CRF-induced hyperthermia [18], [53], although prostaglandin E2, a product of COX, is a key mediator of fever (for review see [7]). Antipyretic effects of centrally applied CRF were also demonstrated previously [5]. However, the present test also failed to detect compensatory vasodilation during the course of CRFhyperthermia arguing for a coordinated hyperthermic response. The mechanisms of this seemingly coordinated hyperthermia still remain unknown.
Regarding the age-related pattern of hypermetabolic/hyperthermic acute central CRF actions in males, these effects are maintained at a significant level across all age-groups, although a decline begins in the early middle-aged group continuing in the course of aging. Such a pattern may contribute to the development of middle-aged obesity. In addition, the low hypermetabolic responsiveness of old rats to CRF may contribute to the diminished capacity of old populations to develop fever. These findings may also have implications for the potential involvement of CRF1 receptors in aging-induced variations of BW, since acute central hyperthermic CRF effects were shown to be mediated predominantly by CRF1 receptors [18].
Young female rats exhibited a much weaker hypermetabolic/hyperthermic response upon acute central CRF administration (Fig.6) as compared with that of males. Even an elevated dose of CRF (3 g) failed to elicit an equivalent CRF-hyperthermia in females. This phenomenon cannot be explained by a diminished thermogenic capacity of females, as febrile responses of male and female rats to toxic agents did not differ in previous studies [20], [21]. Moreover, heat production capacity of the brown adipose tissue was even enhanced in female rats [30]. It may be concluded that the hypermetabolic responsiveness of female Wistar rats to acute central CRF administration is weaker than that of the males. The decline of the response during the course of aging was faster than in males: from 12 months of age no significant rise of VO2, or Tc was observed in older female animals.
Based on our results, we propose that acute central anorexigenic CRF effects (associated with CRF2 receptor activation) change in different, non-parallel, i.e. disparate ways in male and
14 female Wistar rats during the course of aging. Whereas males show declining anorexigenic and maintained hypermetabolic responsiveness to CRF during the course of aging, in females, the CRF-induced anorexia remains significant and the hypermetabolic response declines. The maintained anorexigenic efficacy of CRF in females may contribute to their lack of middle-aged obesity and may possibly promote later weight loss in older female animals. In males, no such association may be observed. Regarding hypermetabolic effects (associated with CRF1 receptor activation), despite the differences in the extent of CRF-hyperthermia, female and male rats appear to share the tendency for age-related decline of the responsiveness to acute central CRF administration. However, the high remaining level of CRF-induced hypermetabolism of old male rats, may contribute to their age-related weight loss. Our present findings underline the importance of gender differences in the age-related regulatory changes in the background of life-long BW development, at least regarding the contribution of the central catabolic corticotropin system. These results also emphasize the differential age-related changes in the corresponding receptor systems: CRF-induced hyperthermia mediated by CRF1 receptors in young rats [18] appear to be maintained at higher levels in males during the course aging, whereas CRF-induced anorexia mediated predominantly by CRF2 receptors in young animals [13] remain significant in all female agegroups until 24 months of age.
5. PERSPECTIVES
In the background of disparate age-related changes in acute central CRF effects, the differential involvement of CRF1 and CRF2 receptors and their subpopulations may be hypothesized. This problem may be further investigated by the application of specific agonists (e.g. Ucn2 for CRF2 receptors) and antagonists (e.g. antalarmin for CRF1 receptors, and antisauvagine-30 for CRF2 receptors) of the two major receptor types. In addition, differential age-related variations in the signal transduction pathways may also be assumed and investigated.
Gender differences raise the possibility of the involvement of sex hormones in long-term BW regulation and in CRF effects. Although a previous report [48] described similar baseline serum levels of plasma corticosterone in male and female Wistar rats and a similar increase in their respective serum values by the end of a 13-day CRF infusion, to date the effects of
15 estrogen or testosterone on CRF activity (from hypothalamic immunoreactivity to serum levels of the peptide) remain controversial [23], [24]. Ovariectomy in different age-groups of rats (carried out a month before the experiments) may help identify the special contribution of estrogen to these gender differences. However, the contribution of sex hormones would decrease during the course of aging, as their levels decline following menopause or by old age in males.
FUNDING This work was supported by grants from the University of Pécs (PTE 34039/KA-OTKA/1302, PTE ÁOK KA-2013/13-25, and PTE-AOK-KA-2015-14)
ACKNOWLEDGEMENT The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.
The excellent and expert technical help during this study provided by Ms. A. Jech-Mihálffy, Ms. M. Koncsecskó-Gáspár, Ms. A. Bóka-Kiss, and Ms. E. Sós is gratefully acknowledged.
DISCLOSURES All authors disclaim any form of conflicts of interest.
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24 FIGURE CAPTIONS
Fig. 1 Cumulative food intake (FI) values during 3-h re-feeding following 24-h fasting upon an intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection. M3, M6, M12, M18 and M24 indicate 3-month, 6-month, 12-month, 18month and 24-month male Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between 3-h re-feeding FI of CRF- vs. PFStreated male rats (repeated-measures ANOVA).
Fig. 2 Cumulative food intake (FI) values during 3-h re-feeding following 24-h fasting upon an intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection. F3, F6, F12, F18 and F24 indicate 3-month, 6-month, 12-month, 18-month and 24-month female Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between 3-h FI of CRF- vs. PFS-treated female rats (repeated-measures ANOVA).
Fig. 3 Changes of core temperature (Tc), oxygen consumption (Vand heat loss index (HLI) induced by a intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogenfree saline (PFS) injection in young adult male Wistar rats (M3). The groups consisted of minimum 6-8 animals. The mean value of resting initial Tc and VO2 were similar in all agegroups around 37.5 ºC and 24 ml/kg/min, in accord with our previous observations [52]. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. PFS-treated male rats (repeated-measures ANOVA).
Fig. 4 Maximal increases in core temperature (Tcmax) and oxygen consumption (VO2max) induced by intracerebroventricular corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection in male Wistar rats. M3, M6, M12, M18 and M24 indicate 3-month, 6month, 12-month, 18-month and 24-month male Wistar rats. The groups consisted of
25 minimum 6-8 animals. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. corresponding PFS-treated male rats (one-way ANOVA).
Fig. 5 Changes of core temperature (Tc), oxygen consumption (Vand heat loss index (HLI) induced by an intracerebroventricular (ICV) corticotropin-releasing factor (CRF) or pyrogenfree saline (PFS) injection in young adult female Wistar rats (F3). The groups consisted of minimum 6-8 animals. The mean value of resting initial Tc and VO2 were similar in all agegroups around 37.5 ºC and 24 ml/kg/min. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. PFS-treated female rats (repeated-measures ANOVA).
Fig. 6 Maximal increases in core temperature (Tcmax) and oxygen consumption (VO2max) induced by intracerebroventricular corticotropin-releasing factor (CRF) or pyrogen-free saline (PFS) injection in female Wistar rats. F3, F6, F12, F18 and F24 indicate 3-month, 6-month, 12-month, 18-month and 24-month female Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. corresponding PFS-treated female rats (one-way ANOVA).
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
26 Table 1: Body weight values (BW) of different age-groups of male (M) and female (F) Wistar rats group (age gender) M3
M6
initial and BW (g)
384.0±11.8
411.1±5.7
M12
506.5±8.5a,b
M18
543.4±15.4a,b
M24
514.0±14.1a,b
F3
220.3±6.9
F6
262.2±2.6a
F12
275.0±12.2a
F18
278.0±9.7a
F24
298.2±10.2a,b
group BW (g) (gender, age, before fasting treatment)
BW (g) before re-feeding
M3 PFS
387.4 ± 15.8
359.3 ± 13.4
M3 CRF
360.5 ± 11.5
346.5 ± 18.8
M6 PFS
416.5 ± 9.6
382.8 ± 10.1
M6 CRF
405.7 ± 6.3
375.1 ± 7.1
M12 PFS
507.3 ± 9.2
477.0 ± 8.9
M12 CRF
505.8 ± 15.0
474.8 ± 15.6
M18 PFS
532.8 ± 19.4
493.6 ± 17.7
M18 CRF
554.0 ± 5.1
511.8 ± 24.0
M24 PFS
526.3 ± 18.5
494.8 ± 18.0
M24 CRF
484.4 ± 11.2
477.6 ± 11.8
F3 PFS
233.2 ± 23.1
219.9 ± 5.6
F3 CRF
207.4 ± 10.2
195.8 ± 8.4
F6 PFS
260.5 ± 3.1
247.7 ± 2.7
F6 CRF
263.9 ± 4.3
255.2 ± 4.1
F12 PFS
278.0 ± 17.5
261.8 ± 18.2
F12 CRF
272.0 ± 18.8
258.4 ± 16.8
F18 PFS
276.5 ± 17.2
259.7 ± 15.2
F18 CRF
279.5 ± 10.8
252.6 ± 8.8
F24 PFS
304.4 ± 10.4
281.1 ± 6.5
F24 CRF
293.9 ± 16.3
273.7 ± 15.4
Groups are listed according to age and gender. Male and female rats were divided into five-five age-groups: young adult (3-month), younger and older middle aged (6- and 12-month) and aging or old (18-month or 24month). In addition to the initial BW values characterizing male and female age-groups, pre-fasting and pre-re-feeding BW-s of corticotropin-releasing factor (CRF)- or pyrogen-free saline (PFS)-treated animals are also shown. Values are expressed as mean ± S.E.M. for a minimum of six-eight rats in each group. Regarding initial BW-s, the following statistically significant differences were denoted: a 3-month male or female rats vs. other gender-matched age-groups (p<0.005), b 6-month male or female rats vs. other gender- matched age-groups (p<0.05). BW values of PFS- vs. CRF-treated groups of the same age and gender measured before or after a 24-h fasting did not differ.
S0196-9781(16)30182-6 http://dx.doi.org/doi:10.1016/j.peptides.2016.09.005 PEP 69671
To appear in:
Peptides
Received date: Revised date: Accepted date:
6-6-2016 12-9-2016 12-9-2016
Please cite this article as: Tenk Judit, Rost´as Ildik´o, Furedi ¨ N´ora, Mik´o Alexandra, So´os Szilvia, Solym´ar Margit, Gaszner Bal´azs, Sz´ekely Mikl´os, P´eterv´ari Erika, Balask´o M´arta.Acute central effects of corticotropin-releasing factor (CRF) on energy balance: effects of age and gender.Peptides http://dx.doi.org/10.1016/j.peptides.2016.09.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Acute central effects of corticotropin-releasing factor (CRF) on energy balance: effects of age and gender
Judit Tenk1, Ildikó Rostás1, Nóra Füredi1, Alexandra Mikó1, Szilvia Soós1, Margit Solymár1, Balázs Gaszner2, Miklós Székely1, Erika Pétervári1, Márta Balaskó1
From: 1
Institute for Translational Medicine, Medical School, University of Pécs, Hungary
2
Department of Anatomy, Medical School, University of Pécs, Hungary
Submitted to: PEPTIDES
Corresponding author: Márta Balaskó, M.D., Ph.D. Institute for Translational Medicine Medical School, University of Pécs 12 Szigeti str., Pécs H-7624, Hungary Phone: +36-72-536-246 Fax: +36-72-536-247 e-mail: [email protected]
2
Highlights CRF-induced anorexia declines with aging in male, but not in female rats CRF-induced hypermetabolism declines strongly with aging in females, but not in males Maintained CRF-anorexia in all female rats may help prevent middle-aged obesity Age-related changes in the responsiveness of CRF1 and CRF2 receptors depend on gender
Abstract
Previously demonstrated age-related changes in the catabolic melanocortin system that may contribute to middle-aged obesity and aging anorexia, raise the question of the potential involvement of corticotropin-releasing factor (CRF) in these phenomena, as this catabolic hypothalamic mediator acts downstream to melanocortins. Catabolic effects of CRF were shown to be mediated by both CRF1 (hypermetabolism) and CRF2 (anorexia) receptors. To test the potential role of CRF in age-related obesity and aging anorexia, we investigated acute central effects of the peptide on energy balance in male and female rats during the course of aging. Effects of an intracerebroventricular CRF injection on food intake (FI), oxygen-consumption (VO2), core- and tail skin temperatures (Tc and Ts) were studied in male and female Wistar rats of five different age-groups (from 3- to 24-month). Anorexigenic responsiveness was tested during 180-min re-feeding (FeedScale) following 24-h fasting. Thermoregulatory analysis was performed by indirect calorimetry (Oxymax) complemented by thermocouples recording Tc and Ts (indicating heat loss). CRF suppressed FI in 3-month male and female animals. In males, CRF-induced anorexia declined with aging, whereas in females it was maintained in all groups. The peptide increased VO2 and Tc in all male age-groups, while the weaker hypermetabolic response characterizing 3-month females declined rapidly with aging. Thus, age-related alterations in acute central anorexigenic and hypermetabolic effects of CRF show different non-parallel patterns in males and females. Our findings underline the importance of gender differences. They also call the attention to the differential age-related changes in the CRF1 and CRF2 receptor systems.
3
Abbreviations: BW, body weight; CRF, corticotropin- releasing factor; CRF1 receptor, corticotropin- releasing factor type 1 receptor; CRF2 receptor, corticotropin- releasing factor type 2 receptor; F, female; FI, food intake; HPA, hypothalamic-pituitary-adrenal; ICV, intracerebroventricular; IP, intraperitoneal; M, male; MSH, melanocyte- stimulating hormone; PFS, pyrogen-free saline; Tc, core body temperature; Ts, tail skin temperature; Ucn2, urocortin 2; Ucn3, urocortin 3; VO2, oxygen-consumption
Keywords: corticotropin-releasing factor (CRF), energy balance, food intake, metabolic rate, aging, gender
1. INTRODUCTION
Long-term regulation of body weight (BW) and body composition shows two different trends in mammals: obesity develops typically in the middle-aged, whereas old age is characterized by anorexia, weight loss and sarcopenia [15], [38], [55]. Both trends imply world-wide public health burdens [69], [70]. As most mammals also show similar trends in their long-term BW development [63], a dysregulation of energy homeostasis may also contribute to these phenomena. Therefore, the investigation of regulatory alterations in energy balance during the course of aging, are of outstanding importance. And indeed, earlier studies demonstrated the potential role of age-related shifts in the responsiveness to such centrally administered catabolic mediators as leptin [44] or endogenous melanocortin agonist alpha-melanocytestimulating hormone (alpha-MSH) in the development of the above mentioned BW trends [43], [52]. Based on these previous findings, the question arises, whether corticotropins (downstream to melanocortins and leptin) may also contribute to the metabolic dysregulation characterizing aging.
The first member of the corticotropin peptide family, corticotropin-releasing factor (CRF), a 41-aa neuropeptide was described by Vale and coworkers in 1981 [66]. This peptide is produced predominantly in the parvocellular neurons of the paraventricular nucleus of the hypothalamus [66], but expression of CRF mRNA has been detected, among other sites, in the cerebral cortex, amygdala and in the hippocampus [37], [68].
4
Although the main functions of CRF are thought to be the activation of the hypothalamicpituitary-adrenal (HPA) axis [39], [49], the activation of the sympathetic tone [8] and coordination of the body’s responses to stress [32], this peptide also participates in a great variety of regulatory processes. It has been shown to induce fear, to participate in anxiety and in depressive disorders of animals [9] and humans [2]. In addition, CRF elicits coordinated catabolic effects via inducing anorexia [48] and hypermetabolism accompanied by increased brown-fat thermogenesis [8], [10], [33]. Moreover, in genetically obese rats (fa/fa) the peptide prevented weight gain [50].
Two subtypes of G-protein coupled receptors (CRF1 and CRF2) mediate the effects of corticotropins, CRF activates mainly CRF1 and to a lesser extent CRF2 receptor [35], [42], [67], while urocortin 1, another member of the corticotropin family shows similar affinity to both receptor types [35], [42], [67]. Widespread expression of CRF1 receptor was described in hypothalamic nuclei [45], [47], [65] and also in the anterior pituitary [65]. Additionally, they are found in the forebrain, in the septal region and amygdala [29], in the cerebral cortex and in the cerebellum [47], [65]. Anxiogenic actions, depressive behavior and hyperthermic/hypermetabolic effects have been attributed to the activation of this receptor type [18], [47], [65]. Its anorexigenic effects are thought to be based on emotional stress [28]. In contrast, CRF2 receptor expression appears to be more restricted in the brain, these receptors were mainly detected in the lateral septum, amygdala, hippocampus, also in hypothalamic nuclei (e.g. in the ventromedial hypothalamus) [65] or in the nucleus of the solitary tract [6]. Type 2 receptors mediate anorexia, anxiolytic and antidepressive effects [13], [47], [65]. This latter receptor type has been shown to mediate anorexigenic actions of centrally applied CRF or those of other members of the corticotropin peptide family, such as urocortin 2 (Ucn2) or urocortin 3 (Ucn3) [13], [40], [58]. The latter members of the corticotropin family show enhanced affinity to CRF2 receptors [13], [34], [40], [60].
During the course of aging, characteristic changes of the corticotropin system have been reported in humans and mammals [25]. Most studies found increased hypothalamic CRF expression in old age compensated by some CRF1 receptor downregulation [1], [11], [56], [64]. Nevertheless, a few studies described reduced or unchanged CRF expression in old agegroups [12], [31]. Post mortem investigations in men demonstrated increased activity of CRF, especially in depressed subjects or in deceased patients with Alzheimer's disease [4], [46].
5 Other investigators suggested a role for the hyperactive HPA axis in neuronal deterioration of aged humans and in old experimental animals [16], [54], [61]. An intriguing additional feature of the corticotropin system is that its functions show gender differences [22], [26], [36]. Therefore, age-related alterations of the hypothalamic corticotropin system may also show different patterns in males and females. Such differences may also provide some explanation for the gender differences in the long-term BW development of male and female rats: in contrast with males showing middle-aged obesity and a decline in BW in old age-groups, females maintain a stable low BW throughout life [17], [63].
Therefore, we aimed to investigate the potential involvement of acute central catabolic CRF effects (anorexia and hypermetabolism) in the development of age-related obesity and aging anorexia leading to weight loss in old male Wistar rats. These acute central catabolic effects of CRF were also analyzed in females during the course of aging to test their potential contribution to gender differences in long-term BW development in this strain.
2. METHODS
2.1. Animals Various age-groups of male (n = 79) and female (n = 75) Wistar rats (M and F, respectively) from the Colony of the Institute for Translational Medicine of the Medical School, University of Pécs, Hungary were used in the experiments of the present study: young adult (M = 14, F = 12), younger (M = 14, F = 16) and older middle-aged (M = 15, F = 13), aging (M = 16, F = 16) and old (M = 10, F = 18) (3-, 6- and 12-, 18- and 24-months old, respectively). (Maximal life-span of our Colony reaches 30 months, about 50% of rats survive 26 months, but after the age of 24 months surgical interventions involve high mortality). In this study altogether 7 rats failed to survive surgery. For thermoregulatory analysis and for tests of food intake separate groups of animals had to be used. Each animal in each age-group received CRF and its solvent in random order. Such tests were divided by 7 days. Following tests, animals were sacrificed; no repeated testing across age-groups was possible. After they have reached the appropriate age, rats were kept in individual plastic home-cages containing wood-chip bedding at an ambient temperature of 22–25 °C. Cages were equipped with a steel grid top with feeder and bottle container. This environment provided a thermoneutral temperature in the nest. Lights were on between 06.00 and 18.00 h. Standard
6 rat chow (11 kJ/g; CRLT/N rodent chow, Szindbád Kft., Gödöllő, Hungary) and tap water were available ad libitum, except for the 24-h fasting period when only water was provided for the appropriate groups. All animals were accustomed to regular handling. Spontaneous daily food intake (FI) and BW were measured every day at 09.00 h. Table 1 shows BW values of the different male and female age-groups. Initial BW-s of treated and those of control animals did not differ within age-groups, as shown by one-way ANOVA (p > 0.05 in all cases, Table 1).
2.2. Surgeries, drug administration A 22-gauge stainless-steel guide cannula was implanted into the right lateral cerebral ventricle of rats using a stereotaxic apparatus for the purpose of intracerebroventricular (ICV) injections. The tip of the guide cannula (fixed to the skull by dental cement) was positioned at A: -1.0 mm (to bregma), L: 1.5 mm (right lateral to bregma), V: 3.5 mm (ventral to dura) (coordinates according to the Rat Brain Atlas, [41]). A stylet closed the lumen of the guide cannula which was replaced during the experiments by a 28-gauge injection cannula outreaching the guide cannula by about 0.5 mm. Surgical interventions were performed under intraperitoneal (IP) ketamine-xylazine [78 mg/kg (Calypsol, Richter) + 13 mg/kg (Sedaxylan, Eurovet)] anesthesia. Gentamycin (2 mg IP) was used for the prevention of infections. In order to check the appropriate location of the guide cannula angiotensin II (Sigma, A9525, 20 ng/5 l) was injected through a pp10 polyethylene tube attachment around 3 days after the implantation. Appropriate location was assumed if at least 5 ml water was consumed within 30 min [43]. Experiments started 7 days after the cannula implantation.
After the experiments rats were sacrificed by an IP overdose of urethane (3-5 g/kg, Reanal). Post mortem check of the injection sites were performed by observing macroscopically the coronal sections of the removed brains that were fixed in 4% paraformaldehyde for 48 hours. Only rats with appropriate cannula location were included in the analysis. Because of incorrect location (judged by the in vivo or post mortem test), data of 9 of the 154 rats were excluded from the analysis.
When testing anorexigenic effects, the animals received CRF or pyrogen-free saline (PFS) after 24-h fasting. Corticotropin-releasing factor (0.3 g Bachem, H2435) dissolved in PFS in a volume of 5 l or 5 l PFS filled the proximal end of a 20-25 cm-long pp10 polyethylene
7 tube attachment of the injection cannula. A small bubble separated the substance prepared for ICV injection from the PFS that filled the distal part of the tube. In thermoregulatory tests an amount of 0.3, 1 and 3 g of the peptide was dissolved in PFS in a volume of 5 l and was injected similarly. Control rats received PFS in 5 l volume. In these studies CRF was injected at a thermal steady state (usually 60-90 min following the start of the experiment). All injections were given remotely, without causing any acute discomfort to the rats and they were administered around 09.00 h, early in the inactive phase of the day.
2.3 Assessment of food consumption At least 10-14 days before the experiments, rats were transferred to the automated FeedScale system (Columbus, OH) to get habituated to the environment and to the powdered form of rat chow. The system allowed continuous recording of the amount of consumed food. Data were registered every 10 minutes. On day 1 at 09.00 h food was removed for 24-h. Five minutes before the re-feeding started (on day 2 at 09.00) assigned rat groups received 0.3 g ICV CRF or PFS injection to test the inhibitory effect of the peptide on 3-h cumulative FI.
2.4 Assessment of thermoregulatory functions During thermoregulatory analysis oxygen consumption (VO2, representing metabolic rate), core temperature (Tc) and tail skin temperature (Ts, indicating heat loss) were recorded. The tests were performed between 09.00 h and 15.00 h on semi-restrained rats, singly enclosed in cylindrical wire-mesh confiners in separate tightly sealed plexiglass metabolic chambers continuously ventilated with room air (size: 20 x 30 x 18.5 cm). Four chambers were used simultaneously that were immersed into a thermostatically controlled water-bath. For thermoregulatory analysis of CRF a slightly subthermoneutral environment [ambient temperature (Ta) was 25 ºC] was maintained. As the animals were previously accustomed to the semi-restraining cages for at least a week (starting with a 120-min, followed by a 180-, a 240- and two 300-min sessions), we could minimize the stress during the experiments, as shown by the normal initial Tc values between 37.2 and 37.6 °C. Nevertheless, the gradual moderate decline in the Tc and VO2 values of control male (and to a lesser extent of female) PFS-treated rats indicates that some initial stress must have induced a slight increase in these values that declined later in the familiar environment. During tests the animals could not eat or drink.
8
The injection cannula was attached to the proximal end of the 20-25 cm-long pp10 polyethylene tube. At the distal part of the tube, 5 μl PFS was slowly injected that resulted in the ICV administration of the 5 μl CRF solution or PFS via the injection cannula.
Following the ICV injection the VO2 was registered in 10-min intervals for 3 hours. For the recording of Tc and Ts copper-constantan thermocouples were applied. The colon thermocouple was inserted 10 cm beyond the anal sphincter and it was fixed by tape to the tail. The tail skin thermocouple was fixed on the dorsal skin of the distal part of the tail. One thermocouple recorded Ta of the chamber. The rate of heat loss (heat loss index, HLI) was calculated from the relationship of the monitored temperatures: HLI = (Ts-Ta) / (Tc-Ta) [51]. HLI near 0 (when the Ts value approaches Ta) suggests vasoconstriction as a heat conserving mechanism, HLI near 1 (when the Ts value approaches Tc) indicates vasodilation as indication of heat loss. In the semi-restraining cages the animals could not turn around or reach the thermocouples. The thermocouples and the extensions of the ICV cannula were pulled through a tightly sealed port of the metabolic chamber allowing the remote administration of substances. Oxygen consumption (ml O2/kg/min) and carbon-dioxide (CO2) production (ml/kg/min) were determined by indirect calorimetry (Oxymax, Equal Flow, Columbus, OH) from the air flowing through the chambers. Temperature data were collected by a Digi-Sense Benchtop Thermometer (Cole-Parmer) for electronic processing and evaluation.
2.5. Statistical analysis All age-groups contained at least 6-8 rats. SPSS 11.0 for Windows was used for the statistical analysis of the data with application of one-way, two-way (univariate or repeated-measures analysis) or repeated-measures ANOVA complemented by Tukey’s post hoc test, when more than two groups were compared. All results are presented as mean ± S.E.M. The level of significance was set at p < 0.05.
2.6. Ethical issues The general rules and those determined by the special permit of the University of Pécs Ethical Committee for the Protection of Animals in Research (BA 02/200-11/2011 valid for 5 years) were strictly observed in all experimental interventions and procedures. These rules are in accordance with the main directives of the National Ethical Council for Animal Research and
9 those of the European Communities Council (86/609/EEC, Directive 2010/63/EU of the European Parliament and of the Council).
3. RESULTS
Table 1 shows BW values of different male and female age-groups. Values of male Wistar rats (Table 1) were in accord with those observed in our previous studies [3], [44]: up to 18 months of age BW showed a rising tendency, then it declined in the oldest animals. Body weights (Table 1) of male rats exceeded those of corresponding females of the same agegroup.
Following 24-h fasting, weight loss of rats ranged from 7% to 9% of initial BW in males, and from 3% to 10% in females.
3.1 Anorexigenic CRF effects in male and female rats in the course of aging The small 0.3 g dose applied in these tests was chosen based on previous reports of the literature [57], [71]. In young adult (M3) and younger and older middle-aged (M6 and M12, respectively) male rats the ICV administered acute CRF injection caused a strong suppression of 3-h cumulative FI during re-feeding following 24-h fasting [repeated-measures ANOVA for the 3-h refeeding period from 10 to 180 min for M3: F(1,14) = 10.280, p = 0.006, for M6: F(1,12) = 17.339, p = 0.001 and for M12: F(1,14) = 16.700, p = 0.001, Fig. 1]. The peptide failed to induce a significant anorexigenic response in aging (M18) and old (M24) rats (Fig. 1). These results suggest that the anorexigenic effects of an acute central injection of CRF show a gradual decline with aging [F(4,63) = 3.410, p= 0.014, as indicated by two-way ANOVA repeated-measures analysis].
Cumulative 3-h re-feeding of control female rats reached similar values as those of corresponding age-groups of males (Figs 1, 2). In female rats, anorexigenic effects of an acute ICV CRF injection (0.3 g) were maintained at a significant level in all age-groups from F3 to F24 [repeated-measures ANOVA for the 3-h re-feeding period from 10 to 180 min for F3: F(1,10) = 21.365, p = 0.002, for F6: F(1,14) = 67.986, p < 0.001, for F18: F(1,10) = 10.210, p = 0.010, for F24: F(1,10) = 5.550, p = 0.040 and from 40 to 180 min for F12: F(1,10) = 5.749, p = 0.043, Fig. 2]. Therefore, in females no age-dependence of acute central CRF-anorexia
10 was found within the observed age-groups [F(4,54) = 1.287, p = 0.287, as indicated by twoway ANOVA repeated-measures analysis].
3.2. Hypermetabolic/hyperthermic CRF effects in male and female rats in the course of aging Initially three different doses of CRF (0.3, 1 and 3 g) were tested in the thermoregulatory experiments of young adult male rats and all doses elicited similar, significant hyperthermic responses. However, the 1 g dose elicited the largest increase in oxygen consumption (p < 0.05, comparison of different doses not shown). Therefore, the 1 g dose was applied in all subsequent thermoregulatory experiments.
The young adult male age-group showed a CRF-induced increase in metabolic rate [represented by the change in VO2 (VO2), at 25 °C Ta), with a consequent significant rise in Tc (shown as Tc in Fig. 3). The rise in oxygen consumption and Tc started directly upon the ICV injection. Fig. 3 demonstrates CRF-induced hypermetabolic/hyperthermic effects (1 g dose) at 25 °C in young adult rats [ANOVA repeated measures for VO2: F(1,12) = 21.335, p = 0.001; Tc: F(1,12) = 104.208, p <0.001]. No compensatory rise in heat loss was detectable, as indicated by HLI. Based on these data, CRF-induced hyperthermia appears to be coordinated, in which case both heat production and heat conservation (lack of vasodilation) promote the rise in Tc.
Regarding age-related variations of these reactions (Fig. 4), in addition to the a strong response of young adult rats to CRF concerning maximal Tc (p < 0.001, one-way ANOVA) and VO2 changes (p < 0.001, one-way ANOVA as compared with their respective controls), similar CRF-induced hyperthermic and hypermetabolic reactions were observed in older rats, as well (for Tcmax M6: p = 0.003, M12: p < 0.001, M18: p < 0.001 and M24: p= 0.004; for VO2max M6: p = 0.005, M12: p = 0.007, M18: p = 0.023 and M24: p = 0.040).
Although these responses proved to be significant in all age-groups, the maximal increase in Tc and VO2 (Fig. 4) declined with aging (two-way ANOVA univariate analysis for maximal rises in Tc or VO2: F(9,55) = 3.929, p = 0.007 or F(9,55) = 2.67, p = 0.041, respectively). Heat loss mechanisms did not show any age-related alteration.
11 Young adult female rats showed a weaker CRF-induced increase in metabolic rate and Tc (at 25 °C Ta) than the corresponding male age-group. The rise in VO2 and Tc started directly upon the ICV injection. Fig. 5 demonstrates CRF-induced hypermetabolic/hyperthermic effects (1 g dose) in young adult female rats [ANOVA repeated-measures for VO2 from 10 to 180 min: F(1,12) = 8.254, p = 0.014; for Tc from 10 to 180 min.: F(1,12) = 13.342, p = 0.003]. No vasodilation, indicated by HLI occurred in these female rats (Fig. 5).
The maximal increase in Tc and VO2 (Fig. 6) showed some variations with aging in female rats. The peptide induced hyperthermia (for Tcmax: F3: p < 0.001; F6: p=0,017; one-way ANOVA) and hypermetabolism (for VO2max: F3: p = 0.004; F6 p= 0,047; one-way ANOVA) in the young adult and younger middle-aged females. However, this response was weaker than in the corresponding male groups. Older female age-groups failed to show any significant change in Tc or VO2. Thus, the hyperthermic/hypermetabolic effects of CRF showed a gradual age-dependent decline in female rats [two-way ANOVA univariate analysis, F(9,59) = 3.309, p = 0.037 for Tcmax and F(9,59) = 2.731, p = 0.016 for VO2max].
4. DISCUSSION
The present study aimed to investigate age-related changes in the acute central catabolic effects of CRF in male and female Wistar rats with their different life-long body weight developments (Table 1) in view.
4.1 Anorexigenic CRF effects in male and female rats in the course of aging In males, acute ICV CRF administration-induced suppression of re-feeding showed agedependence, as it was significant in the young adult and also in the younger and older middleaged groups, whereas this effect failed to develop in older animals (Fig. 1). This age-related pattern of acute central CRF-anorexia distinctly differs from that of acute central alpha-MSHinduced suppression of re-feeding reported in male Wistar rats [43]. According to that study, male rats showed a strong decline of melanocortin-induced anorexia in the middle-aged group (as compared with that seen in young adult animals) but pronounced effects were observed again in the old rats [43]. Such an age-related pattern potentially contributes to the explanation of middle-aged obesity and aging anorexia. In contrast, the age-related pattern
12 derived from our experiments, characterizing acute central CRF-anorexia is unlikely to promote the development of either the middle-aged obesity or the aging anorexia in male Wistar rats. Regarding the potential involvement of the major CRF receptor subtypes in the above described age-related pattern, CRF2 receptors (rather than CRF1 receptors) may play a decisive role in it. Although CRF shows much higher affinity to CRF1 receptors, their contribution to CRF-induced anorexia is mainly attributed to emotional stress [28]. Moreover, these receptors activate the HPA axis [65] and therefore they are responsible for the dramatic increase in peripheral plasma corticosterone level upon an acute ICV CRF injection, described in previous studies [13]. A rise in peripheral corticosterone level or even an ICV administration of the hormone failed to change FI in mice [14], whereas a CRF-induced increase in peripheral cortisol has been shown to be coupled with increased FI in healthy humans [19]. In addition, the vast majority of the related literature also attributes anorexigenic CRF effects to CRF2 receptor mediation [13], [34], [40], [60]. Thus, our present findings may have implications for the potential lack of involvement of CRF2 receptors in aging-induced variations of BW in male rats.
In females, CRF-induced anorexia did not exhibit age-dependence within the studied agegroups, as it was maintained in all female animals. This continuous efficacy of acute ICV anorexigenic actions of CRF may have helped to prevent rapid weight gain in middle-aged and older female animals, but apparently failed to induce weight loss up to 24 months of age. However, it cannot be excluded that even older age-groups of female rats (e.g. 30-month), not studied in the present work, would show anorexia and weight loss as a result of this maintained efficacy. In summary, a gender difference emerges in the age-related patterns of CRF-induced anorexia and of CRF2 receptor responsiveness in Wistar rats: these CRF effects decline in males, but they are maintained in females during aging (until 24 months of age).
4.2. Hypermetabolic/hyperthermic CRF effects in male and female rats in the course of aging Concerning hypermetabolic/hyperthermic effects in young adult males, ICV CRF administration elicited a prompt rise in VO2 inducing a steady rise in Tc. This hyperthermia was accompanied by continuous heat conservation as indicated by the HLI. This thermoregulatory response appears to be coordinated, i.e. similar to fever-like coordinated hyperthermias, in which increased heat production is accompanied by suppressed heat loss
13 coordinated by thermoregulatory centers of the hypothalamus [3], [59], [62]. The role of CRF in thermoregulation is still controversial. Some studies suggested a role of CRF in the development of fever [27], [53]. However, other findings contradict the hypothesis of the participation of CRF in febrigenesis. Neither non-selective cyclooxygenase (COX) inhibitors, nor COX-2 antagonists suppressed CRF-induced hyperthermia [18], [53], although prostaglandin E2, a product of COX, is a key mediator of fever (for review see [7]). Antipyretic effects of centrally applied CRF were also demonstrated previously [5]. However, the present test also failed to detect compensatory vasodilation during the course of CRFhyperthermia arguing for a coordinated hyperthermic response. The mechanisms of this seemingly coordinated hyperthermia still remain unknown.
Regarding the age-related pattern of hypermetabolic/hyperthermic acute central CRF actions in males, these effects are maintained at a significant level across all age-groups, although a decline begins in the early middle-aged group continuing in the course of aging. Such a pattern may contribute to the development of middle-aged obesity. In addition, the low hypermetabolic responsiveness of old rats to CRF may contribute to the diminished capacity of old populations to develop fever. These findings may also have implications for the potential involvement of CRF1 receptors in aging-induced variations of BW, since acute central hyperthermic CRF effects were shown to be mediated predominantly by CRF1 receptors [18].
Young female rats exhibited a much weaker hypermetabolic/hyperthermic response upon acute central CRF administration (Fig.6) as compared with that of males. Even an elevated dose of CRF (3 g) failed to elicit an equivalent CRF-hyperthermia in females. This phenomenon cannot be explained by a diminished thermogenic capacity of females, as febrile responses of male and female rats to toxic agents did not differ in previous studies [20], [21]. Moreover, heat production capacity of the brown adipose tissue was even enhanced in female rats [30]. It may be concluded that the hypermetabolic responsiveness of female Wistar rats to acute central CRF administration is weaker than that of the males. The decline of the response during the course of aging was faster than in males: from 12 months of age no significant rise of VO2, or Tc was observed in older female animals.
Based on our results, we propose that acute central anorexigenic CRF effects (associated with CRF2 receptor activation) change in different, non-parallel, i.e. disparate ways in male and
14 female Wistar rats during the course of aging. Whereas males show declining anorexigenic and maintained hypermetabolic responsiveness to CRF during the course of aging, in females, the CRF-induced anorexia remains significant and the hypermetabolic response declines. The maintained anorexigenic efficacy of CRF in females may contribute to their lack of middle-aged obesity and may possibly promote later weight loss in older female animals. In males, no such association may be observed. Regarding hypermetabolic effects (associated with CRF1 receptor activation), despite the differences in the extent of CRF-hyperthermia, female and male rats appear to share the tendency for age-related decline of the responsiveness to acute central CRF administration. However, the high remaining level of CRF-induced hypermetabolism of old male rats, may contribute to their age-related weight loss. Our present findings underline the importance of gender differences in the age-related regulatory changes in the background of life-long BW development, at least regarding the contribution of the central catabolic corticotropin system. These results also emphasize the differential age-related changes in the corresponding receptor systems: CRF-induced hyperthermia mediated by CRF1 receptors in young rats [18] appear to be maintained at higher levels in males during the course aging, whereas CRF-induced anorexia mediated predominantly by CRF2 receptors in young animals [13] remain significant in all female agegroups until 24 months of age.
5. PERSPECTIVES
In the background of disparate age-related changes in acute central CRF effects, the differential involvement of CRF1 and CRF2 receptors and their subpopulations may be hypothesized. This problem may be further investigated by the application of specific agonists (e.g. Ucn2 for CRF2 receptors) and antagonists (e.g. antalarmin for CRF1 receptors, and antisauvagine-30 for CRF2 receptors) of the two major receptor types. In addition, differential age-related variations in the signal transduction pathways may also be assumed and investigated.
Gender differences raise the possibility of the involvement of sex hormones in long-term BW regulation and in CRF effects. Although a previous report [48] described similar baseline serum levels of plasma corticosterone in male and female Wistar rats and a similar increase in their respective serum values by the end of a 13-day CRF infusion, to date the effects of
15 estrogen or testosterone on CRF activity (from hypothalamic immunoreactivity to serum levels of the peptide) remain controversial [23], [24]. Ovariectomy in different age-groups of rats (carried out a month before the experiments) may help identify the special contribution of estrogen to these gender differences. However, the contribution of sex hormones would decrease during the course of aging, as their levels decline following menopause or by old age in males.
FUNDING This work was supported by grants from the University of Pécs (PTE 34039/KA-OTKA/1302, PTE ÁOK KA-2013/13-25, and PTE-AOK-KA-2015-14)
ACKNOWLEDGEMENT The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.
The excellent and expert technical help during this study provided by Ms. A. Jech-Mihálffy, Ms. M. Koncsecskó-Gáspár, Ms. A. Bóka-Kiss, and Ms. E. Sós is gratefully acknowledged.
DISCLOSURES All authors disclaim any form of conflicts of interest.
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24 FIGURE CAPTIONS
Fig. 1 Cumulative food intake (FI) values during 3-h re-feeding following 24-h fasting upon an intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection. M3, M6, M12, M18 and M24 indicate 3-month, 6-month, 12-month, 18month and 24-month male Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between 3-h re-feeding FI of CRF- vs. PFStreated male rats (repeated-measures ANOVA).
Fig. 2 Cumulative food intake (FI) values during 3-h re-feeding following 24-h fasting upon an intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection. F3, F6, F12, F18 and F24 indicate 3-month, 6-month, 12-month, 18-month and 24-month female Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between 3-h FI of CRF- vs. PFS-treated female rats (repeated-measures ANOVA).
Fig. 3 Changes of core temperature (Tc), oxygen consumption (Vand heat loss index (HLI) induced by a intracerebroventricular (ICV) corticotropin- releasing factor (CRF) or pyrogenfree saline (PFS) injection in young adult male Wistar rats (M3). The groups consisted of minimum 6-8 animals. The mean value of resting initial Tc and VO2 were similar in all agegroups around 37.5 ºC and 24 ml/kg/min, in accord with our previous observations [52]. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. PFS-treated male rats (repeated-measures ANOVA).
Fig. 4 Maximal increases in core temperature (Tcmax) and oxygen consumption (VO2max) induced by intracerebroventricular corticotropin- releasing factor (CRF) or pyrogen- free saline (PFS) injection in male Wistar rats. M3, M6, M12, M18 and M24 indicate 3-month, 6month, 12-month, 18-month and 24-month male Wistar rats. The groups consisted of
25 minimum 6-8 animals. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. corresponding PFS-treated male rats (one-way ANOVA).
Fig. 5 Changes of core temperature (Tc), oxygen consumption (Vand heat loss index (HLI) induced by an intracerebroventricular (ICV) corticotropin-releasing factor (CRF) or pyrogenfree saline (PFS) injection in young adult female Wistar rats (F3). The groups consisted of minimum 6-8 animals. The mean value of resting initial Tc and VO2 were similar in all agegroups around 37.5 ºC and 24 ml/kg/min. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. PFS-treated female rats (repeated-measures ANOVA).
Fig. 6 Maximal increases in core temperature (Tcmax) and oxygen consumption (VO2max) induced by intracerebroventricular corticotropin-releasing factor (CRF) or pyrogen-free saline (PFS) injection in female Wistar rats. F3, F6, F12, F18 and F24 indicate 3-month, 6-month, 12-month, 18-month and 24-month female Wistar rats. The groups consisted of minimum 6-8 animals. Asterisks (*) indicate significant differences between Tc and Vof CRF- vs. corresponding PFS-treated female rats (one-way ANOVA).
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
26 Table 1: Body weight values (BW) of different age-groups of male (M) and female (F) Wistar rats group (age gender) M3
M6
initial and BW (g)
384.0±11.8
411.1±5.7
M12
506.5±8.5a,b
M18
543.4±15.4a,b
M24
514.0±14.1a,b
F3
220.3±6.9
F6
262.2±2.6a
F12
275.0±12.2a
F18
278.0±9.7a
F24
298.2±10.2a,b
group BW (g) (gender, age, before fasting treatment)
BW (g) before re-feeding
M3 PFS
387.4 ± 15.8
359.3 ± 13.4
M3 CRF
360.5 ± 11.5
346.5 ± 18.8
M6 PFS
416.5 ± 9.6
382.8 ± 10.1
M6 CRF
405.7 ± 6.3
375.1 ± 7.1
M12 PFS
507.3 ± 9.2
477.0 ± 8.9
M12 CRF
505.8 ± 15.0
474.8 ± 15.6
M18 PFS
532.8 ± 19.4
493.6 ± 17.7
M18 CRF
554.0 ± 5.1
511.8 ± 24.0
M24 PFS
526.3 ± 18.5
494.8 ± 18.0
M24 CRF
484.4 ± 11.2
477.6 ± 11.8
F3 PFS
233.2 ± 23.1
219.9 ± 5.6
F3 CRF
207.4 ± 10.2
195.8 ± 8.4
F6 PFS
260.5 ± 3.1
247.7 ± 2.7
F6 CRF
263.9 ± 4.3
255.2 ± 4.1
F12 PFS
278.0 ± 17.5
261.8 ± 18.2
F12 CRF
272.0 ± 18.8
258.4 ± 16.8
F18 PFS
276.5 ± 17.2
259.7 ± 15.2
F18 CRF
279.5 ± 10.8
252.6 ± 8.8
F24 PFS
304.4 ± 10.4
281.1 ± 6.5
F24 CRF
293.9 ± 16.3
273.7 ± 15.4
Groups are listed according to age and gender. Male and female rats were divided into five-five age-groups: young adult (3-month), younger and older middle aged (6- and 12-month) and aging or old (18-month or 24month). In addition to the initial BW values characterizing male and female age-groups, pre-fasting and pre-re-feeding BW-s of corticotropin-releasing factor (CRF)- or pyrogen-free saline (PFS)-treated animals are also shown. Values are expressed as mean ± S.E.M. for a minimum of six-eight rats in each group. Regarding initial BW-s, the following statistically significant differences were denoted: a 3-month male or female rats vs. other gender-matched age-groups (p<0.005), b 6-month male or female rats vs. other gender- matched age-groups (p<0.05). BW values of PFS- vs. CRF-treated groups of the same age and gender measured before or after a 24-h fasting did not differ.