Abstract
The growth hormone secretagogue receptor (GHSR) mediates key properties of the gut hormone ghrelin on metabolism and behavior. Nevertheless, most recent observations also support that the GHSR is a constitutively active G protein-coupled receptor (GPCR) endowed with a sophisticated tuning involving a balance of endogenous ligands. Demonstrating the feasibility of shifting GHSR canonical signaling in vivo, we previously reported that a model with enhanced sensitivity to ghrelin (GhsrQ343X mutant rats) developed fat accumulation and glucose intolerance. Herein, we investigated the contribution of energy homeostasis to the onset of this phenotype, as well as behavioral responses to feeding or pharmacological challenges, by comparing GhsrM/M rats to WT littermate rats: (1) as freely behaving animals and (2) in feeding and locomotor paradigms. Herein, GhsrM/M rats showed enhanced locomotor response to a GHSR agonist while locomotor or anorexigenic responses to amphetamine or cabergoline (dopamine receptor 2 agonist), respectively, were preserved. Ad libitum fedGhsrM/M rats consumed and conditioned for sucrose similarly to littermate control rats . In calorie-restricted conditions, GhsrM/M rats retained food anticipatory activity and maintained better body weight and glycemia. Importantly, prior to fat accumulation, male GhsrM/M rats preferentially used carbohydrates as fuel substrate without alterations of energy intake, energy expenditure or physical activity and showed alterations of the GHSR system (i.e. enhanced ratio of GHSR hormones LEAP2: acyl-ghrelin and increased Ghsr expression in the hypothalamus). Overall, the present study provides proof for the concept that shifted GHSR signaling can specifically alter nutrient partitioning resulting in modified balance of carbohydrate/lipid utilization.
Introduction
The growth hormone secretagogue receptor (GHSR) (Howard et al. 1996) holds a unique interest as the target of the gut hormone ghrelin (Kojima et al. 1999), a hormone with key pharmacological properties such as growth hormone (GH) release (Kojima et al. 1999), enhanced fat storage and food intake, mediated by its action on brain energy homeostasis centers (Tschop et al. 2000, Nakazato et al. 2001, Theander-Carrillo et al. 2006) and hedonic circuits (Naleid et al. 2005, Abizaid et al. 2006, Jerlhag et al. 2007). While the ablation of the Ghsr gene (Ghsr−/−) or ghrelin-producing cells in mice reported mitigated results on energy homeostasis (Muller et al. 2015), Ghsr−/− mice failed to enhance locomotor activity similarly to control mice during scheduled feeding (Blum et al. 2009, LeSauter et al. 2009) and to preserve glycemic control during severe caloric restriction (Wang et al. 2014). The GHSR could therefore play key roles in situations of stress while its role in the fed state remains largely unknown (Mani & Zigman 2017).
The GHSR is a G protein-coupled receptor (GPCR) showing high constitutive activity as documented in cellular (Holst et al. 2003) and acellular systems (Damian et al. 2012). Recent observations identified the liver hormone LEAP2 as a novel GHSR ligand (Ge et al. 2018) with inverse agonist properties (M’Kadmi et al. 2019, Mani et al. 2019). Therefore, according to its expression pattern in the periphery and in several key brain regions, including the hypothalamus, ventral tegmental area (VTA) or hippocampus (Zigman et al. 2006), the GHSR could exert its functions according to a complex balance between GHSR ligands, whose accessibility in each GHSR expressing brain structure still needs to be delineated (Perello et al. 2019). Overall, these observations, that appear as a game changer (Andrews 2019), support an unprecedented refinement for an endocrine system, questioning the therapeutic potential of this GPCR target (Al-Massadi et al. 2018). We reasoned that a preclinical model with shifted canonical GHSR signaling could provide key insights for future drug discovery.
To this purpose, we used a rat model carrying the GhsrQ343X nonsense mutation that is predicted to cause the deletion of the last 22 amino acids of the GHSR, a domain containing most Ser/Thr phosphorylation sites (Bulbul et al. 2011). Although some studies initially documented altered responses when ghrelin was injected to homozygous mutant rats carrying this allele (GhsrM/M rats) (Bulbul et al. 2011, Clifford et al. 2012, MacKay et al. 2016), we proposed that the mutant receptor encoded by this mutated allele could enhance GHSR sensitivity to ligands (Chebani et al. 2016). First, in cellular systems, the GHSR-Q343X receptor shows altered β-arrestin recruitment and internalization to the benefit of G protein signaling in response to the agonist. Second, young adult GhsrM/M rats show increased GH release and chow intake at low doses of the agonist. Third, GhsrM/M rats develop fat accumulation and insulin resistance with age, an evolutive phenotype consistent with enhanced ghrelin effects (Chebani et al. 2016). However, the contribution of energy homeostasis to the onset of fat accumulation in GhsrM/M rats remained unexplored, as well as the physiological consequences of this mutation on food-related behaviors.
In this study, we assessed the consequence of shifting GHSR signaling onto: (1) feeding and locomotor response to pharmacological manipulations or feeding challenges; and (2) metabolic efficiency, nutrient partitioning by using an automated food and calorimetric recording system.
Material and methods
Animals
The mutant line of FHH-Ghsrm1Mcwi rats carrying the GhsrQ343X allele was obtained from the Medical College of Wisconsin (Milwaukee, WI, USA). The homozygous FHH-Ghsrm1Mcwi rats and WT littermates are referred to as GhsrM/M and GhsrWT/WT rats, respectively. Animals used in this study (187 rats from 7 litters) were obtained from crossing heterozygous rats. Animals were raised four by cages with free access to water and chow diet (A04, SAFE), in a room with controlled temperature (22–24°C) and illumination (12-h light:12-h darkness schedule with lights on at 7:00 am). The genotype of the rats at the Ghsr locus was determined as previously described (Chebani et al. 2016). Experiments were performed with male or female rats with ad libitum access to food and water, unless otherwise specified. The procedures involving rats were approved by the ethics committee of animal experimentation of the Université Paris-Descartes.
Analysis of metabolic efficiency
Calorimetry exploration was performed using 10-week-old male rats at the start of the experiment. Body composition was assessed at the start of the experiment, at the end of baseline, after 24 h fasting and after 48 h refeeding (end of the experiment) using an echo medical system (EchoMRI 100, Whole Body Composition Analyzer, EchoMRI, Houston, USA). Energy expenditure, oxygen consumption and carbon dioxide production, respiratory exchange ratio (RER), food intake and homecage activity were obtained using calorimetric chambers (Labmaster, TSE Systems GmbH, Bad Homburg, Germany) as previously depicted (Joly-Amado et al. 2012). Rats were individually housed, fed standard chow and acclimated to the chambers for 48 h before experimental measurements. A meal was defined as the consumption of >0.3 g of food, separated from the next feeding episode by at least 10 min. To assess the metabolic flexibility, all RER data were compiled to obtain relative cumulative frequency curves for GhsrM/M and GhsrWT/WT rats. Sigmoidal dose–response curves were fitted to determine EC50 and Hill slopes (indicative of metabolic rigidity) (Riachi et al. 2004).
Hormone measurements
Blood samples were collected from the same cohort of rats in a second experiment performed 3 weeks after the calorimetry exploration. Blood samples were obtained by tail bleeding in ad libitum fed and 24 h fasted conditions. Samples for LEAP2 measurements were collected on EDTA and centribuged, and plasma were stored at −80°C until measurements. Sample preparation for ghrelin measurements were performed as previously reported (Chebani et al. 2016). Rat acyl ghrelin and des-acyl ghrelin were measured with ELISA kits (Bertin Pharma) and LEAP2 was measured using an EIA kit (Phoenix Pharmaceuticals).
Statistics
Results are presented as mean ± s.e.m. Sample size (n) and P values are given in the figure captions. Statistical analyses were performed using GraphPad Prism® 5.01, SPSS statistics (IBM Inc.) and R software. Differences between two groups were determined using non-parametric Mann–Whitney or Wilcoxon tests, or parametric Student’s t-tests, as appropriate. Comparisons of multiple groups were performed using two-way ANOVA, repeated measure ANOVA or ANOVA on aligned rank transformed data (ARTool package) and P value of post hoc tests were adjusted with the Sidak correction. Covariance analyses (ANCOVA) used the Mouse Metabolic Phenotyping Center web page (http://www.mmpc.org/shared/regression.aspx). For all statistical analyses, a P value less than 0.05 was considered as significant.
Results
Pharmacological probing of central dopaminergic circuits in GhsrM/M rats reveals enhanced locomotor response to a GHSR agonist but preserved responses to amphetamine or to a DRD2 agonist
To examine central dopaminergic circuits in GhsrM/M rats, locomotion or refeeding response was examined in GhsrM/M and GhsrWT/WT rats in response to pharmacologic modulators of dopamine signaling or GHSR agonist. First, we assessed the locomotor response to amphetamine (AMPH), a psychoactive drug known to reverse dopamine transporter activity at post-synaptic target leading to enhanced dopamine (DA) release and action (Fig. 1A, B and C). Both experimental groups displayed similar response to the novel environment (Fig. 1A) and to AMPH-induced hyperlocomotion (Fig. 1B and C). Second, GHSR-DRD2 heteromers can produce behavioral response independent of ghrelin binding and correlated to DA function (Kern et al. 2012). GhsrM/M rat model offers a great platform to probe how possible interaction of GHSR and DRD2 receptor might contribute to alter the response to DRD2 agonist. For this purpose, the anorexigenic effect of the DRD2 agonist cabergoline was evaluated in GhsrM/M and GhsrWT/WT rats. Cabergoline mediated a potent anorexigenic response in the refeeding of prefasted GhsrM/M and GhsrWT/WT animals (Fig. 1D and E). No significant difference was found across the genotypes. Third, the pharmacological properties of the GHSR on locomotion were tested in GhsrM/M and GhsrWT/WT rats challenged with varying doses of the agonist hexarelin. Repeated measure ANOVA revealed a significant dose × genotype interaction (P < 0.01) suggestive of a differential dose–response effect between the two groups of rats (Fig. 1F). Indeed, post hoc analyses showed that the locomotor response to the highest tested dose of hexarelin was twice as high in GhsrM/M rats compared to GhsrWT/WT littermate rats, an observation further confirmed in the analyses of the response to the highest hexarelin dose (Fig. 1G and H). Overall, these observations, while supporting enhanced GHSR responsiveness in GhsrM/M rats, also rule out gross abnormalities in the dopaminergic system of these rats.
Pharmacological probing of central dopaminergic circuits in GhsrM/M rats. (A) Time course of the locomotor response to a novel environment and (B and C) to the injection of i.p. amphetamine (2 mg/kg) on 2 consecutive days in GhsrM/M (n = 11) and GhsrWT/WT rats (n =10), Ghsr heterozygous rats served as the saline control group (n = 7). Anorexigenic effects of a DRD2 agonist in a refeeding paradigm performed in fasted GhsrWT/WT (n = 9) (D) and GhsrM/M (n = 9) (E) rats injected s.c. with cabergoline (0.5 mg/kg) or with saline before refeeding. (F) Total locomotor response to increasing doses of the GHSR agonist hexarelin s.c. injected in male GhsrWT/WT (n = 13) and GhsrM/M (n = 13) rats, and (G and H) locomotor responses obtained with the highest injected hexarelin dose or saline across time. Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; ** P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Pharmacological probing of central dopaminergic circuits in GhsrM/M rats. (A) Time course of the locomotor response to a novel environment and (B and C) to the injection of i.p. amphetamine (2 mg/kg) on 2 consecutive days in GhsrM/M (n = 11) and GhsrWT/WT rats (n =10), Ghsr heterozygous rats served as the saline control group (n = 7). Anorexigenic effects of a DRD2 agonist in a refeeding paradigm performed in fasted GhsrWT/WT (n = 9) (D) and GhsrM/M (n = 9) (E) rats injected s.c. with cabergoline (0.5 mg/kg) or with saline before refeeding. (F) Total locomotor response to increasing doses of the GHSR agonist hexarelin s.c. injected in male GhsrWT/WT (n = 13) and GhsrM/M (n = 13) rats, and (G and H) locomotor responses obtained with the highest injected hexarelin dose or saline across time. Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; ** P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Pharmacological probing of central dopaminergic circuits in GhsrM/M rats. (A) Time course of the locomotor response to a novel environment and (B and C) to the injection of i.p. amphetamine (2 mg/kg) on 2 consecutive days in GhsrM/M (n = 11) and GhsrWT/WT rats (n =10), Ghsr heterozygous rats served as the saline control group (n = 7). Anorexigenic effects of a DRD2 agonist in a refeeding paradigm performed in fasted GhsrWT/WT (n = 9) (D) and GhsrM/M (n = 9) (E) rats injected s.c. with cabergoline (0.5 mg/kg) or with saline before refeeding. (F) Total locomotor response to increasing doses of the GHSR agonist hexarelin s.c. injected in male GhsrWT/WT (n = 13) and GhsrM/M (n = 13) rats, and (G and H) locomotor responses obtained with the highest injected hexarelin dose or saline across time. Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; ** P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M rats show unaltered spontaneous conditioning and motivation for sucrose but accelerated performance in operant system
The former results indicated that relative to their body weight, chow consumption was comparable between GhsrM/M and GhsrWT/WT rats, while showing enhanced sensitivity to endogenous ghrelin (Chebani et al. 2016). We hypothesized that these animals might show improved consumption and/or motivation for palatable food. To test this, we took the advantage of the spontaneous high preference for sucrose in the fawn hood strain (Tordoff et al. 2008) to investigate the spontaneous consumption and reinforcing properties of sucrose in GhsrM/M and GhsrWT/WT rats using free choice and instrumental conditioning paradigms. In a two-bottle choice between sucrose solution and water, both GhsrM/M and GhsrWT/WT male littermates displayed similar levels of consumption throughout the days, with a strong, sustained preference for the sucrose solution over water (Fig. 2A). For instrumental conditioning paradigms, we focused our interest on female rats who successfully achieved conditioning without requiring calorie restriction, a manipulation known to interfere with ghrelin endogenous tone. During conditioning sessions, ad libitum fed GhsrM/M and GhsrWT/WT rats achieved success criterion during the last two sessions of each fixed ratio (FR) schedule (Fig. 2B upper panel; repeated measure ANOVA; session effect; P < 0.001). Total responding levels were similar between GhsrM/M and GhsrWT/WT rats on the progressive ratio (PR) schedule, and for both the groups, nose-poking activity on the inactive, dummy target was close to zero, indicating specific, goal-oriented responding at the active nose-poking hole. Interestingly, at 5 min after the beginning of sessions, GhsrM/M rats had significantly achieved more responses than GhsrWT/WT rats, starting from the third FR3 session (Fig. 2B lower panel), although the total number of responses was comparable between GhsrM/M and GhsrWT/WT rats throughout the FR and PR schedules. This significant difference in performing speed was also seen 15 min after the beginning of the session (Fig. 2B lower panel). Overall, GhsrM/M rats fed ad libitum showed preference or consumption for palatable food similar to GhsrWT/WT rats, but performed faster to obtain rewarding food in an operant nose-poking task.
Consumption and motivation for sucrose in GhsrM/M rats. (A) Cumulative intake of sucrose solution (0.75%) (plain lines) and water (dotted line) during daily 1 h two-bottle choice in GhsrM/M (n = 17) and GhsrWT/WT (n = 13) ad libitum fed rats. (B) Operant nose-poking responses for sucrose pellets in GhsrM/M (n =16) and GhsrWT/WT (n =15) ad libitum fed rats during each session (upper panel) or at intermediate times of the session (lower panel). Data were analyzed by two-way repeated measure ANOVA.* P < 0.05; ** P < 0.01. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Consumption and motivation for sucrose in GhsrM/M rats. (A) Cumulative intake of sucrose solution (0.75%) (plain lines) and water (dotted line) during daily 1 h two-bottle choice in GhsrM/M (n = 17) and GhsrWT/WT (n = 13) ad libitum fed rats. (B) Operant nose-poking responses for sucrose pellets in GhsrM/M (n =16) and GhsrWT/WT (n =15) ad libitum fed rats during each session (upper panel) or at intermediate times of the session (lower panel). Data were analyzed by two-way repeated measure ANOVA.* P < 0.05; ** P < 0.01. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Consumption and motivation for sucrose in GhsrM/M rats. (A) Cumulative intake of sucrose solution (0.75%) (plain lines) and water (dotted line) during daily 1 h two-bottle choice in GhsrM/M (n = 17) and GhsrWT/WT (n = 13) ad libitum fed rats. (B) Operant nose-poking responses for sucrose pellets in GhsrM/M (n =16) and GhsrWT/WT (n =15) ad libitum fed rats during each session (upper panel) or at intermediate times of the session (lower panel). Data were analyzed by two-way repeated measure ANOVA.* P < 0.05; ** P < 0.01. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M display preserved food anticipatory activity in response to scheduled feeding
Previous investigations revealed that GhsrM/M rats preserved better body weight and glycemia in scheduled restricted feeding conditions (Chebani et al. 2016); however, whether this condition also affected other behavioral response related to food-seeking response remained unexplored. To test this, two paradigms differing on the kind of locomotor activity measured (wheel running or ambulatory locomotion) were used. In the first experiment, GhsrM/M and GhsrWT/WT littermate rats habituated to a single housing cage with free access to running wheels were put on a restricted feeding schedule. Unexpectedly, during thead libitum feeding period, GhsrM/M rats showed decreased running activity during the first hour of darkness phase (Fig. 3A) . At the beginning of caloric restriction, wheel running activity was increased in both groups of rats and the activity of GhsrM/M rats no longer differed from that of GhsrWT/WT rats (Fig. 3B). Running activity was further enhanced at the end of the caloric restriction and the activity pattern was re-distributed with maximal activity levels in anticipation to darkness phase and meal access (Fig. 3C). GhsrM/M and GhsrWT/WT rats displayed comparable levels of daily activity, as well as similar food anticipatory behavior (Fig. 3C). Besides, throughout caloric restriction GhsrM/M rats consumed similar amounts of food compared to GhsrWT/WT littermates (Fig. 3D), but were better able to maintain their body weight (Fig. 3E) and glycemia (Fig. 3F). To verify that food anticipatory activity was indeed unaltered in GhsrM/M rats while avoiding the potentially confounding effect of wheel running, which is rewarding by itself, we used an alternative paradigm in which another cohort of rats at same age were put on a 4 h restricted feeding schedule and recorded daily in actimetry cages during the 2 h preceding food access. In this setting, GhsrM/M and GhsrWT/WT rats developed food anticipatory activity to comparable levels (Fig. 3G), while chow intake and body weight were similar across genotypes (Fig. 3H and I). Altogether, GhsrM/M rats therefore show anticipatory activity and food consumption similar to GhsrWT/WT littermates on a restricted feeding schedule, suggesting that the GhsrQ343X mutation does not affect behavioral anticipation of food.
Food anticipatory activity is preserved in GhsrM/M rats. Wheel running activity in GhsrM/M (n = 7) and GhsrWT/WT (n = 8) rats averaged over 10 days of ad libitum feeding (A) or at beginning (B) or end of the restricted feeding schedule (C), daily food intake (D), body weight (E) and glycemia (F) of rats across the protocol. Travelled distance recorded in an open field in the 2 h preceding food access in GhsrM/M (n = 8) and GhsrWT/WT (n = 7) rats put on a 4 h restricted feeding schedule (initial: averaged on the first 4 days; final: averaged on the last 4 days) (G), daily food intake normalized to body weight (H) and body weight (I). Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Food anticipatory activity is preserved in GhsrM/M rats. Wheel running activity in GhsrM/M (n = 7) and GhsrWT/WT (n = 8) rats averaged over 10 days of ad libitum feeding (A) or at beginning (B) or end of the restricted feeding schedule (C), daily food intake (D), body weight (E) and glycemia (F) of rats across the protocol. Travelled distance recorded in an open field in the 2 h preceding food access in GhsrM/M (n = 8) and GhsrWT/WT (n = 7) rats put on a 4 h restricted feeding schedule (initial: averaged on the first 4 days; final: averaged on the last 4 days) (G), daily food intake normalized to body weight (H) and body weight (I). Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Food anticipatory activity is preserved in GhsrM/M rats. Wheel running activity in GhsrM/M (n = 7) and GhsrWT/WT (n = 8) rats averaged over 10 days of ad libitum feeding (A) or at beginning (B) or end of the restricted feeding schedule (C), daily food intake (D), body weight (E) and glycemia (F) of rats across the protocol. Travelled distance recorded in an open field in the 2 h preceding food access in GhsrM/M (n = 8) and GhsrWT/WT (n = 7) rats put on a 4 h restricted feeding schedule (initial: averaged on the first 4 days; final: averaged on the last 4 days) (G), daily food intake normalized to body weight (H) and body weight (I). Data were analyzed by two-way repeated measure ANOVA followed by Sidak’s post hoc tests. *P < 0.05; **P < 0.01; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M rats display increased body mass and similar body composition at 12 weeks of age
In order to determine whether alteration in energy homeostasis precede the development of insulin resistance in 6-month-old rats (Chebani et al. 2016), metabolic efficiency was assessed using indirect calorimetry in asymptomatic mutant rats in response to nutritional manipulation. GhsrM/M and GhsrWT/WT rats entered in metabolic cages at similar age (12.3 ± 0.2 and 12.0 ± 0.3-week-old, respectively). Upon entry, GhsrM/M rats were on average 7% heavier than GhsrWT/WT littermates, but proportions of fat and lean masses relatively to total body mass were comparable in GhsrM/M and GhsrWT/WT rats (Fig. 4A). Across the experiment, GhsrM/M and GhsrWT/WT animals similarly lost and regained total body mass, fat mass and lean mass upon fasting and refeeding, respectively (Fig. 4B). Thus, in young adult rats, the GhsrQ343X mutation resulted in higher body weight, with increases in both fat and lean masses in proportion to total body mass, suggesting an aggravation of the phenotype later with age, which is in line with similar blood glucose levels between genotypes in all feeding conditions (Supplementary Fig. 1, see section on supplementary materials given at the end of this article).
GhsrM/M rats show preserved circadian locomotor, feeding and energy balance rhythms across feeding conditions. (A) Body composition at the start of the calorimetric experiment in young adult (12-week-old) rats (n = 12/genotype). (B) Changes in total body weight across ad libitum feeding (4 days), fasting (24 h) and refeeding (48 h). (C) Daily fluctuations of homecage activity and (D) 24 h homecage activity. (E) Daily fluctuations of food intake and (F) 24 h food intake as a function of body mass in each rat. (G) Daily fluctuations of energy expenditure and (H) 24 h energy expenditure as a function of body mass in each rat. Values represent means ± s.e.m. Data were analyzed by Mann–Whitney test (A), by ANOVA on aligned rank transformed data (B, C, D, E and G) or by ANCOVA using body weight as a covariate (F and H). *P < 0.05; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M rats show preserved circadian locomotor, feeding and energy balance rhythms across feeding conditions. (A) Body composition at the start of the calorimetric experiment in young adult (12-week-old) rats (n = 12/genotype). (B) Changes in total body weight across ad libitum feeding (4 days), fasting (24 h) and refeeding (48 h). (C) Daily fluctuations of homecage activity and (D) 24 h homecage activity. (E) Daily fluctuations of food intake and (F) 24 h food intake as a function of body mass in each rat. (G) Daily fluctuations of energy expenditure and (H) 24 h energy expenditure as a function of body mass in each rat. Values represent means ± s.e.m. Data were analyzed by Mann–Whitney test (A), by ANOVA on aligned rank transformed data (B, C, D, E and G) or by ANCOVA using body weight as a covariate (F and H). *P < 0.05; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M rats show preserved circadian locomotor, feeding and energy balance rhythms across feeding conditions. (A) Body composition at the start of the calorimetric experiment in young adult (12-week-old) rats (n = 12/genotype). (B) Changes in total body weight across ad libitum feeding (4 days), fasting (24 h) and refeeding (48 h). (C) Daily fluctuations of homecage activity and (D) 24 h homecage activity. (E) Daily fluctuations of food intake and (F) 24 h food intake as a function of body mass in each rat. (G) Daily fluctuations of energy expenditure and (H) 24 h energy expenditure as a function of body mass in each rat. Values represent means ± s.e.m. Data were analyzed by Mann–Whitney test (A), by ANOVA on aligned rank transformed data (B, C, D, E and G) or by ANCOVA using body weight as a covariate (F and H). *P < 0.05; ***P < 0.001; ~non-significant trend (P < 0.1). Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
As shown Fig. 4C, GhsrM/M and GhsrWT/WT animals showed comparable 24 h patterns of activity across baseline, fasting and refeeding, both in light and darkness phases. In both groups of rats, fasting increased homecage activity, while refeeding returned it to basal level (Fig. 4D). As expected, the feeding pattern post-fast significantly differed from the ad libitum pre-fast condition for both groups of rats (Fig. 4E), with no differences across genotypes regarding 24 h intake (Fig. 4F). Furthermore, the diurnal and nocturnal meal parameters across feeding conditions were comparable between GhsrM/M and GhsrWT/WT rats, as measured by mean meal size, total time spent eating, number of meals, meal duration, inter-meal intervals and ingestion rate (Supplementary Fig. 2). Not surprisingly, both groups of rats showed similar diurnal and nocturnal levels of energy expenditure characterized by decreased energy expenditure during fasting, while there was a general tendency to return to its basal level upon refeeding (Fig. 4G). Analyses of 24 h energy expenditure revealed no differences between genotypes in the ad libitum feeding condition, but a trend to decreased energy expenditure was observed in GhsrM/M rats during fast and refeeding conditions (Fig. 4H). Estimated resting metabolic rate was also comparable for GhsrM/M rats and their GhsrWT/WT littermates (9.1 ± 0.4 kcal/h/lean mass and 9.4 ± 0.3 kcal/h/lean mass, respectively). Altogether, these results suggest that in young adult rats, the GhsrQ343X mutation does not alter daily locomotor activity, caloric intake, meal patterns or energy expenditure in conditions of ad libitum access to food as well as short-term food deprivation.
GhsrM/M rats exhibit metabolic fuel preference towards carbohydrates
As GhsrM/M rats showed increased body weight without any major changes in energy intake or expenditure compared to their GhsrWT/WT littermates, we then investigated if GhsrM/M and GhsrWT/WT rats displayed differences in utilization of metabolic substrates, by exploring RER in baseline, fasted and refed conditions (Fig. 5A, B and C). In all nutritional states, mean 24 h RER was higher in GhsrM/M rats compared to GhsrWT/WT littermates (Fig. 5D), indicating a slight but sustained increase in carbohydrate utilization as energy substrate, at the expense of fat, in GhsrM/M rats compared to GhsrWT/WT rats. However, qualitative RER variations across the experiment were similar in GhsrM/M and GhsrWT/WT animals, with RER decreasing during fasting compared to ad libitum basal feeding (Fig. 5A, B and D), and returning to baseline levels during the first 24 h of refeeding (Fig. 5C and D). Separate analyses of light and darkness phases indicated that, during light phase, GhsrM/M rats had in all conditions a higher RER than GhsrWT/WT rats (~2% in the fed state) (Fig. 5E) which is not the case during the darkness phase (Fig. 5F). Thus, GhsrM/M compared to GhsrWT/WT rats presented an overall decreased use of lipids and increased use of carbohydrates as energy substrates illustrated further by decreased cumulative fat oxidation across feeding conditions (Supplementary Fig. 3A, B, C and D). In order to explore metabolic flexibility, RER data were compiled to obtain relative cumulative frequency curves for GhsrM/M and GhsrWT/WT rats. Comparison of the fits revealed a right shift of the curve for GhsrM/M compared to GhsrWT/WT rats (Supplementary Fig. 3E), indicating that RER distribution of the GhsrM/M group is narrower and skewed toward higher values compared to the GhsrWT/WT group. Moreover, 1/Hill slope was lower for GhsrM/M rats compared with GhsrWT/WT rats indicative of decreased metabolic flexibility in GhsrM/M rats.
Increased respiratory exchange ratio (RER) in GhsrM/M rats across feeding conditions. Daily pattern of RER during ad libitum condition (4-day average) (A), 24 h fasting (B) and the first 24 h of refeeding (C) in 12-week-old male rats (n = 12/genotype). Averaged RER over 24 h (D), light phase (E) and darkness phase (F) for each feeding condition. Plasma concentrations of LEAP2 (G), acyl-ghrelin (H) and the calculated LEAP2:acyl-ghrelin molar ratio (I) in a rat subgroup during ad libitum feeding and fasting conditions. Data were analyzed by ANOVA on aligned rank transformed data (A, B, C, D, E, F and I) or two-way ANOVA (G and H). *P < 0.05; **P < 0.01; ***P < 0.001. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Increased respiratory exchange ratio (RER) in GhsrM/M rats across feeding conditions. Daily pattern of RER during ad libitum condition (4-day average) (A), 24 h fasting (B) and the first 24 h of refeeding (C) in 12-week-old male rats (n = 12/genotype). Averaged RER over 24 h (D), light phase (E) and darkness phase (F) for each feeding condition. Plasma concentrations of LEAP2 (G), acyl-ghrelin (H) and the calculated LEAP2:acyl-ghrelin molar ratio (I) in a rat subgroup during ad libitum feeding and fasting conditions. Data were analyzed by ANOVA on aligned rank transformed data (A, B, C, D, E, F and I) or two-way ANOVA (G and H). *P < 0.05; **P < 0.01; ***P < 0.001. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Increased respiratory exchange ratio (RER) in GhsrM/M rats across feeding conditions. Daily pattern of RER during ad libitum condition (4-day average) (A), 24 h fasting (B) and the first 24 h of refeeding (C) in 12-week-old male rats (n = 12/genotype). Averaged RER over 24 h (D), light phase (E) and darkness phase (F) for each feeding condition. Plasma concentrations of LEAP2 (G), acyl-ghrelin (H) and the calculated LEAP2:acyl-ghrelin molar ratio (I) in a rat subgroup during ad libitum feeding and fasting conditions. Data were analyzed by ANOVA on aligned rank transformed data (A, B, C, D, E, F and I) or two-way ANOVA (G and H). *P < 0.05; **P < 0.01; ***P < 0.001. Data represent mean ± s.e.m.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
GhsrM/M rats show enhanced LEAP2:acyl-ghrelin molar ratio but unaltered energy or glucose homeostasis markers centrally or in the periphery
Then, blood concentrations of GHSR hormones (i.e. LEAP2 and acyl-ghrelin) were assessed in satiated and fasted states using a subgroup of these same experimental rats (Fig. 5). The two-way ANOVA performed on circulating LEAP2 levels showed a significant genotype × feeding condition interaction (P < 0.01) revealing significantly higher LEAP2 concentrations in GhsrM/M compared to GhsrWT/WT littermates rats in the fed state only (Fig. 5G). The same analysis performed on circulating acyl ghrelin levels also showed a significant genotype × feeding condition interaction (P < 0.05) revealing this time lower circulating acyl ghrelin levels in GhsrM/M as compared to GhsrWT/WT rats only in the fasting state (Fig. 5H). Additionally, des-acyl ghrelin levels showed a pattern of response similar to acyl ghrelin (data not shown). When these same data were analyzed using a three-way ANOVA, a significant genotype × feeding condition × hormone interaction (P < 0.05) was observed, illustrating a biologically significant interplay between GHSR hormones across feeding conditions in GhsrM/M rats. Furthermore, Fig. 5I shows that this interplay benefits LEAP2 over acyl-ghrelin levels in GhsrM/M rats compared to GhsrWT/WT rats as suggested by increased LEAP2: acyl-ghrelin molar ratio in the former. Finally, to rule out energy or glucose homeostasis abnormalities at gene expression level in asymptomatic GhsrM/M rats, the expression of selected genes was compared in GhsrM/M and GhsrWT/WT rat tissues. These investigations essentially revealed no significant differences across the panel of selected genes between groups (Fig. 6). Nevertheless, the Ghsr gene showed enhanced expression in the hypothalamus of GhsrM/M compared to GhsrWT/WT rats, an observation that specifically matched with the Ghsr1a isoform (Fig. 6A). Despite the low expression level of Ghsr in the peripheral tissues tested, the enhanced sensitivity of the digital PCR technique disclosed similar Ghsr gene expression across groups in the soleus muscle (Fig. 6H). Overall, when still asymptomatic regarding body composition or disordered gene expression markers, GhsrM/M rats may nevertheless show altered GHSR markers (hormones and gene expression).
mRNA expression of known markers of energy homeostasis, adiposity and glucose homeostasis in GhsrM/M and GhsrWT/WT rats in the hypothalamus and in peripheral tissues. Quantitative RT-PCR (qRT-PCR) analyses in hypothalamus (A), epididymal white adipose tissue (B), inguinal white adipose tissue (C), brown adipose tissue (D), liver (E), tibialis muscle (F), soleus muscle (G) and ddPCR analysis in the soleus muscle (H). Data represent mean ± s.e.m. Data were analyzed by multiple Mann–Whitney tests using adjusted P values. **P < 0.01; ***P < 0.001; ND, not detectable.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
mRNA expression of known markers of energy homeostasis, adiposity and glucose homeostasis in GhsrM/M and GhsrWT/WT rats in the hypothalamus and in peripheral tissues. Quantitative RT-PCR (qRT-PCR) analyses in hypothalamus (A), epididymal white adipose tissue (B), inguinal white adipose tissue (C), brown adipose tissue (D), liver (E), tibialis muscle (F), soleus muscle (G) and ddPCR analysis in the soleus muscle (H). Data represent mean ± s.e.m. Data were analyzed by multiple Mann–Whitney tests using adjusted P values. **P < 0.01; ***P < 0.001; ND, not detectable.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
mRNA expression of known markers of energy homeostasis, adiposity and glucose homeostasis in GhsrM/M and GhsrWT/WT rats in the hypothalamus and in peripheral tissues. Quantitative RT-PCR (qRT-PCR) analyses in hypothalamus (A), epididymal white adipose tissue (B), inguinal white adipose tissue (C), brown adipose tissue (D), liver (E), tibialis muscle (F), soleus muscle (G) and ddPCR analysis in the soleus muscle (H). Data represent mean ± s.e.m. Data were analyzed by multiple Mann–Whitney tests using adjusted P values. **P < 0.01; ***P < 0.001; ND, not detectable.
Citation: Journal of Endocrinology 251, 3; 10.1530/JOE-20-0576
Discussion
Recent observations suggest that the GHSR is a constitutively active GPCR endowed with a sophisticated tuning involving a balance of endogenous ligands (Ge et al. 2018, Mani et al. 2019). Therefore, shifting GHSR canonical signaling could provide unique insights for future drug discovery. This is the case of functionally significant GhsrQ343X mutation, whose homozygous rat carriers display enhanced responsiveness to GHSR agonists, enhanced fat accumulation and insulin resistance (Chebani et al. 2016). The present study shows that GhsrM/M rats specifically display an enhanced locomotor response to a GHSR agonist while responses using dopaminergic drugs were seemingly unaltered. Similarly, spontaneous consumption and conditioning for sucrose appeared not to be impacted by the GhsrQ343X mutation, nor did food anticipatory activity. GhsrM/M rats, prior to fat accumulation and insulin resistance, show a shifted fuel preference toward carbohydrates at early adulthood. In contrast, GhsrM/M rats did not show obvious qualitative or quantitative alterations of energy intake, energy expenditure or locomotion. Altogether, the present study supports the feasibility of biasing GHSR signaling to the benefit of the storage of fat by acting preferentially on nutrient partitioning.
The present study provides novel insights into the relationship between GHSR signaling and metabolic fuel preference. Indeed, GhsrM/M rats showed a higher RER than GhsrWT/WT littermates, indicating a shift in metabolic preference toward decreased use of fat. It is noteworthy that increased RER in GhsrM/M rats was essentially observed during the light phase in the ad libitum condition (physiological fasting) and during early fasting and early refeeding. These observations are therefore in line with acute and chronic pharmacological studies showing that supraphysiological levels of acyl ghrelin elevates RER, indicating shifted metabolic fuel preference toward enhanced carbohydrate utilization, while energy expenditure and home cage activity are unaffected (Tschop et al. 2000, Currie et al. 2005, Theander-Carrillo et al. 2006). Overall, based on the assets of the GhsrQ343X model enhancing ghrelin responsiveness (Chebani et al. 2016), the present data support a physiological role for GHSR signaling in regular diet conditions to promote fat storage and preservation by acting on nutrient partitioning, a mechanism that might be, at least in part, centrally mediated (Joly-Amado et al. 2012).
Metabolic characterization of GhsrM/M and GhsrWT/WT littermates explored herein supports a specific alteration of substrate balance (decreased fat oxidation) associated with the GhsrQ343X mutation, rather than a modification of total energy intake or expenditure. Indeed, as measured in freely behaving conditions, GhsrM/M and GhsrWT/WT littermate rats showed similar chow intake, feeding patterns and energy expenditure. Although the latter did tend to decrease in GhsrM/M rats compared to GhsrWT/WT rats in situations of energy deficit, it did not differ in the fed condition, thus, excluding a key role of GHSR signaling in energy expenditure control. Interestingly, young adult GhsrM/M rats showed increased body weight compared to their GhsrWT/WT littermates, but displayed equivalent proportions of fat and lean masses relatively to total body mass, comparable fasting glycemia as well as similar expression of several molecular markers of energy and glucose homeostasis in the hypothalamus and several peripheral tissues. In comparison, as exemplified in our prior study (Chebani et al. 2016), older GhsrM/M rats showed enhanced body weight and adiposity and decreased glucose tolerance, suggesting that the phenotype of these rats worsen with age, which is consistent with studies in Ghsr−/− mice involving the GHSR in metabolic aging (Lin et al. 2011, Ma et al. 2011). Indeed, as they are aging, Ghsr−/− mice fed on a standard diet display reduced obesity and improved insulin sensitivity compared to Ghsr+/+ littermates, a phenotype mirroring that of adult GhsrM/M rats. In contrast to the mechanism described in GhsrM/M rats (lower fat oxidation associated with development of adiposity), while RER of 1-year-old mice was similar amongst genotypes, it was increased in 2-year-old Ghsr−/− mice compared to Ghsr+/+ littermates. It was suggested that increased RER in Ghsr−/− mice could be related to their lean phenotype and improved insulin sensitivity (Lin et al. 2011). Altogether, the phenotype of GhsrM/M rats and Ghsr−/− mice seems to implicate GHSR signaling in age-associated adiposity and insulin resistance. In addition, we speculate that enhanced acyl ghrelin sensitivity in GhsrQ343X rats illustrates how low fat oxidation may contribute to the occurrence of overweight (Galgani & Ravussin 2008).
The analysis of circulating levels of GHSR hormones performed herein provide a new perspective regarding the GhsrQ343X model. The present study confirmed our previous observation that acyl ghrelin levels are similar among groups, but increased less during fasted or negative energy balance states in GhsrM/M rats compared to their GhsrWT/WT littermates (Chebani et al. 2016). Second, the LEAP2 assay disclosed increased levels in GhsrM/M rats over GhsrWT/WT rats in the fed condition while these were similar in fasted rats. Altogether, the LEAP2:acyl-ghrelin molar ratio was significantly enhanced across feeding conditions in GhsrM/M rats compared to GhsrWT/WT rats, consistent with a concerted regulation of these two hormones across feeding conditions. This could be interpreted as an adaptive response to the enhanced GHSR sensitivity in GhsrM/M rats. Interestingly, enhanced LEAP2:acyl-ghrelin molar ratio was recently documented in obesity conditions in mice and human patients (Mani et al. 2019). In our case, however, the 12-week-old GhsrM/M rats did not show changes in fat mass or glycemia. Therefore, further studies are needed regarding the LEAP2:-acyl ghrelin balance in rat carriers of the GhsrQ343X allele. Finally, these observations may illustrate an evolutive biological state of so called ghrelin resistance in GhsrM/M rats, a condition documented in obesity (Briggs et al. 2010, Zigman et al. 2016). We speculate that this aggravating state could participate in the sometime contradictory pharmacological GHSR responses documented in adult GhsrM/M rats (Bulbul et al. 2011, Clifford et al. 2012, Chebani et al. 2016, MacKay et al. 2016).
The results obtained herein using pharmacological tools refine and further support previous observations on the mechanism of action of the GhsrQ343X mutation in rat. First, enhanced locomotor response to the GHSR agonist hexarelin in GhsrM/M rats may suggest increased dopaminergic responsiveness compared to GhsrWT/WT littermates. Former results using dose–responses of ghrelin or hexarelin already showed improved GH release and 4 h chow intake, observations indicative of enhanced GHSR responsivity in the hypothalamus (Chebani et al. 2016). Nevertheless, in contrast to these former results, obtaining a significant locomotor response required the highest dose of the injected agonist suggesting that this response may be non-physiological. In sum, these experiments are consistent with the hypothesis that the GhsrQ343X mutation, that results in G protein biased signaling in response to agonist in cellular systems (Chebani et al. 2016), could recapitulate a gain-of-function mutation in the GHSR. Additionally, and as disclosed herein, the enhanced expression of Ghsr1a in the hypothalamus but not in a peripheral tissue of young adult GhsrM/M rats, could also be considered as a possible contributing factor, although this requires further investigations. Second, pharmacological challenges designed to probe dopaminergic circuits using a DAT blocker (amphetamine) or a DRD2 agonist (cabergoline) revealed no difference across GhsrM/M and GhsrWT/WT rats. These observations, therefore, suggest that the GhsrQ343X allele has no obvious direct or indirect effect regarding both of these pharmacological responses, involving enhanced extracellular dopamine tone (amphetamine) or DRD2-GHSR heterodimers (cabergoline) (Kern et al. 2012). Interestingly, rare human missense or nonsense GHSR variants segregating with short stature or GH deficiency, presumed to be loss-of-function mutations on the basis of their in vitro mechanism of action, were documented (Pantel et al. 2006, 2009, Inoue et al. 2011, Pugliese-Pires et al. 2011), including the GHSRA204E mutation that specifically alters constitutive activity in vitro. Just recently, a knock-in mice model expressing this mutation showed altered GH release, food intake and glycemic control (Torz et al. 2020), therefore demonstrating that the GHSRA204E disease-causing mutation is related to a partial impairment of GHSR functioning. This kind of genetic defect mirrors the mechanism of the present GhsrQ343X mutation in rat, documenting enhanced GHSR function both in vitro and in vivo. Altogether, Ghsr mutant models appear as very relevant tools to probe the significance of GHSR signaling in vivo, more especially, since this constitutively active GPCR was recently established as the key target of both the agonist hormone ghrelin and LEAP2, an endogenous ligand with inverse agonist properties (M’Kadmi et al. 2019, Mani et al. 2019).
Using the GhsrQ343X model to probe food anticipatory activity, GhsrM/M and GhsrWT/WT rats were found to have similar running activity in anticipation of food, suggesting that the GhsrQ343X mutation does not impact the development nor expression of food anticipatory activity (FAA). Acyl ghrelin has been proposed to be a food-entrainable oscillator that participates in food anticipatory activity in rodents put on a restricted feeding schedule (LeSauter et al. 2009, Laermans et al. 2015). Furthermore, when mice are put on a restricted feeding schedule, Ghsr knock-out results in attenuated FAA (Blum et al. 2009, LeSauter et al. 2009, Davis et al. 2011) and reduced activation of several hypothalamic and midbrain nuclei prior to food access (Lamont et al. 2012), altogether supporting that GHSR signaling plays a role in food anticipation. FAA was suggested to have a 'go, no-go' property (LeSauter et al. 2009), therefore, while complete removal of the GHSR results in delayed onset of FAA, the presence of functional canonical acyl ghrelin-GHSR signaling in GhsrM/M rats, albeit enhanced (Chebani et al. 2016), might produce 'go' decisions with probabilities similar to the WT GHSR without affecting FAA onset. Of note, GhsrM/M rats were previously reported to have decreased FAA as assessed by total number of infrared bream breaks during the 2 h preceding food access (MacKay et al. 2016), a result that is not supported by present observations using two different paradigms (running wheels and actimetry cages). However, in this former study, FAA was not normalized to total 24 h activity levels, and the observed decrease in FAA is likely to be the result of the general reduction in activity levels related to the metabolic phenotype of GhsrM/M rats, rather than a specific attenuation of food-oriented anticipatory activity.
Taking advantage of the high preference for sucrose of the fawn hood strain (Tordoff et al. 2008) to probe spontaneous phenotypes in ad libitum fed GhsrM/M and GhsrWT/WT rats, the present study disclosed: (1) similar consumption of sucrose in a two-bottle choice paradigm, suggesting that the hedonic perception of sucrose is not altered; and (2) similar levels of total nose-poke responses for sucrose in an instrumental task, supporting similar motivation to obtain food rewards. Thus far, pharmacological studies support that acyl ghrelin-GHSR signaling modulates the appetitive properties of palatable food. Indeed, peripherally administered acyl ghrelin promotes, whereas GHSR antagonist JMV2959 reduces, consumption and motivation to obtain palatable food inad libitum chow fed rodents (Perello et al. 2010, Landgren et al. 2011, Skibicka et al. 2012), effects that are essentially reproduced by i.c.v., intra-VTA or intra-ventral hippocampus (vHPC) administration of acyl ghrelin or JMV2959 (Skibicka et al. 2011, 2012, Kanoski et al. 2013). However, the physiological significance of these effects is still unclear, as Ghsr−/−, Ghrl−/− and Goat−/− mouse models rarely showed results supporting altered hedonic feeding behaviors when animals are explored in the fed state (Disse et al. 2010, Davis et al. 2012, Lockie et al. 2015). In the physiological setting of the present study, it is interesting to note that GhsrM/M rats, considered with enhanced response to endogenous ghrelin, did not produce an increase in the number of responses over GhsrWT/WT rats, but had a subtler effect on the performing speed of the animals, which was increased. This observation may suggest enhanced impulsivity to obtain food rewards. Interestingly, ghrelin injected i.c.v. or into the VTA of rats was indeed recently shown to increase impulsive behavior to obtain palatable food (Anderberg et al. 2016). Overall, the observation supporting a possible effect of the GhsrQ343X mutation with qualitative rather than a quantitative modulation of spontaneous operant responding for sucrose, at least in female rats, is of particular interest and needs to be clarified further, keeping in mind possible confounding factor such as the current rat strain, as well as the metabolic phenotype of aged GhsrM/M rats.
The present study shows limits regarding putative sex differences associated with the GhsrQ343X mutation. First, the present calorimetry study only focused on male rats. While female GhsrM/M rats developed overweight with age with a timing similar to male rats (our former study), a study specifically investigating the energy intake, energy expenditure or RER parameters in female animals is mandatory in order to delineate possible gender differences. Indeed, female animals in mice or rat are known to gain less weight than males in HFD models, and these observations are known to be contributed, at least in part, to differences in sensitivity to energy homeostasis hormones such as leptin or ghrelin. More specifically, ghrelin sensitivity was shown to be modulated by estrogens (Clegg et al. 2007). Second, in the reward experiments using sucrose, while female and male GhsrM/M rats show similar responses in the two-bottle choice paradigm, only female rats succeeded the operant conditioning steps, precluding the analysis of any possible specific effect of the mutation in male GhsrM/M rats.
Altogether, the present study strengthens the observations that GhsrM/M rats may specifically show enhanced responsivity to GHSR agonist in vivo. In the fed or fasted conditions, young adult GhsrM/M rats did not show obvious feeding or locomotor alterations but showed shifted fuel preference toward carbohydrates, therefore providing a possible mechanism to the enhanced fat accumulation documented in these rats later with age. Finally, these data also support the feasibility of tricking GHSR signaling to the benefit of fat storage by acting preferentially on nutrient partitioning.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-20-0576.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by INSERM, by a grant of the FRM-Institut Danone (to J P), by the Université Sorbonne Paris Cité (USPC) for a fellowship to C M and by CNRS.
Author contribution statement
C M, P Z, R G P D, T L-L and J P conceptualized the research, C M, P Z, R G P D, R H, Y C, H D and J P conducted the experiments, C M, P Z, R G P D, T L-L and J P analyzed the data, G L P, D C, F N, S L and J P supervised the experiments, C M and J P wrote the initial draft of the manuscript. All authors edited and revised the manuscript.
Acknowledgements
The authors are grateful to the PhysGen program for providing the Ghsr FHH rats (Dr. Howard Jacob, Medical College of Wisconsin), to the animal core and PhenoBrain facilities of the Institute of Psychiatry and Neuroscience of Paris (INSERM UMR S-1266 | Université de Paris), the animal core facility of BioMedTech (INSERM US36 | CNRS UMS2009 | Université de Paris). The authors are also thankful to Corinne Canestrelli, Claire Dovergne, Racha Fayad and Adèle Guillard for technical support, to Gabriel Montaldo for help with activity wheel recording. We also acknowledge the technical platform metabolism of the Unit ‘Biologie Fonctionnelle et Adaptative’ (University de Paris, BFA UMR CNRS 8251) for metabolic analysis and the animal core facility ‘Buffon’ of the University Paris Diderot Paris 7/Institut Jacques Monod, Paris for animal housing and the transcriptomic facility of INSERM U1215 Neurocenter Magendie, funded by INSERM and LabEX BRAIN ANR-10-LABX-43.
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