Both ghrelin deletion and unacylated ghrelin overexpression preserve muscles in aging mice

Changes of AG and UnAG level changes in WT mice during aging

Beside the already demonstrated age-related increase of AG [16], in WT mice, we also observed a rise in the circulating levels of UnAG (Figure 1A, 1B), a phenomenon that could represent a compensatory mechanism to age-induced ghrelin resistance [17]. It has been proposed that age-related ghrelin resistance could be due to a decline in ghrelin-elicited signaling or to changes in the levels of its receptor. However, the levels of AG receptor in skeletal muscle were no detectable neither in young nor in old WT animals (data not shown) and, being the receptor mediating the common AG/UnAG antiatrophic activity [7] still unknown, its expression cannot be assessed. Therefore, the relationship between AG/UnAG levels and their receptors still remains to be elucidated.

Figure 1. Increase of AG and UnAG plasmatic levels during aging in WT mice. Plasmatic levels of AG (A) and UnAG (B) in 3-, 12-, and 24-month-old mice determined by EIA; 3-month-old mice: N = 5, 12-month-old mice N = 8, 24-month-old mice N = 6. Data in bar graph are presented as mean ± SEM. *p<0.05 and **p<0.01 vs. 3-month-old mice.

Body weight, body composition, and food intake in young, adult, middle aged, and old WT, Tg, and Ghrl KO mice

To address whether ghrelin levels during aging affect sarcopenia development, we examined young (3-month-old), adult (6-month-old), middle-aged (12-month-old), and old (24-month-old) Myh6/Ghrl transgenic mice characterized by high levels of circulating UnAG, up to 100 times more than WT animals (Tg; [7, 13]), their WT littermates, and ghrelin knockout (Ghrl KO; [22]) mice, lacking all the gene-derived peptides.

We observed a general increase in body weight and BMI for all genotypes during aging up to 12 months, with a remarkable drop at 24 months of age (Figure 2A and Table 1). A similar trend was observed also for epididymal fat and skeletal muscles (Figure 2B, 2C and Table 1), suggesting the establishment of progressive sarcopenia starting from 12 months in all genotypes.

Figure 2. Body mass composition in young and old WT, Tg, and Ghrl KO mice. (A) Total body weight in 3-month old (young), 6-month old (adult), 12-month old (middle aged), and 24-month old (old) mice. (B) Epidydimal fat mass normalized to nose-to-anus length (NAL) and (C) gastrocnemius weight normalized to the tibial length (TL). Young mice: WT = 6, Tg = 7, Ghrl KO = 5; adult mice: WT = 7, Tg = 4, Ghrl KO = 5; middle aged: WT = 5, Tg = 6, Ghrl KO = 9; old mice: WT= 13, Tg= 14, Ghrl KO = 11. Data in bar graphs are presented as mean ± SEM. For each box plot, the lower and up boundaries denote the 25^th and the 75^th percentile of each data set, respectively, the horizontal line represents the median, and the whiskers represent the min and max of values. *p<0.05 and **p<0.01 vs. young mice; ^£p<0.05 and ^££p<0.01 vs. adult mice; ^#p<0.05 and ^##p<0.01 vs. middle aged mice; ^$p<0.05 and ^$$p<0.01 Tg and Ghrl KO vs. WT.

Other organs, such as the heart, liver, and spleen, on the contrary, steadily increased during aging (Table 1). Daily food intake increased in adult mice compared to young ones in all genotypes. Afterwards, in WT and Tg, daily food intake decreased with aging. On the contrary, Ghrl KO mice exhibited a higher food intake at 3 months of age that remained stable during aging (Table 1).

Muscle and cognitive performance in young and old WT, Tg, and Ghrl KO mice

To assess the effect of altered ghrelin levels during aging on muscle functionality, we measured the performances of young and old WT, Tg, and Ghrl KO mice using the hanging wire test. This test allows determining muscle function and coordination by counting the times a mouse falls or reaches one of the sides of the wire and the tension that the animal develops for maintaining itself on the wire, against gravity, by measuring the longest suspension time. In general, mice from all genotypes showed a decrease in muscle performance during aging. However, surprisingly, Ghrl KO mice outclassed both WT and Tg mice, at both ages, in the “falls and reach” score, as well in the holding impulse (Figure 3A, 3B). On the other hand, Tg mice performances were barely distinguishable from those of their WT littermates, both in young and old animals.

Figure 3. Muscle functionality and behavior in young and old WT, Tg, and Ghrl KO mice. (A) Average score trend in hanging wire test of 3-month old (young) and 24-month old (old) WT, Tg, and Ghrl KO mice and (B) average holding impulse in the same test. Young mice: WT = 21, Tg = 21, Ghrl KO = 27; old mice: WT = 9, Tg = 16, Ghrl KO = 7. *p<0.05 and **p<0.01 vs WT. (C) Memory was evaluated through the object recognition test and expressed as a discrimination index between a familiar and a novel object (seconds on novel – seconds on familiar)/(seconds on novel + seconds on familiar). (D) Anxiety was assessed as the time spent immobile during 6 minutes of tail suspension. Young mice: WT = 5, Tg = 5, Ghrl KO = 6; old mice: WT = 6, Tg = 7, Ghrl KO = 6. For each box plot, the lower and up boundaries denote the 25^th and the 75^th percentile of each data set, respectively, the horizontal line represents the median, and the whiskers represent the min and max of values. *p<0.05 and **p<0.01 old vs. young; ^$p<0.05 and ^$$p<0.01 Tg and Ghrl KO vs. WT.

Since the score on the hanging wire test could be affected by cognitive performance and stress susceptibility, we assessed if cognitive capability and anxiety/depression of mice were different among the strains. Young Ghrl KO mice undergoing the object recognition test obtained a higher discrimination index than young WT and Tg (Figure 3C). However, at 24 months of age, while no significant changes were observed in the discrimination index of WT and Tg mice, that of Ghrl KO mice dropped to a level similar to those of WT mice. The tail suspension test, with the time spent immobile representing a measure of anguish, showed no differences in the immobility time of young mice (Figure 3D). During aging, both WT and Tg mice had a propensity to develop depression-like behaviors, while Ghrl KO mice showed substantial preservation of the immobility time during aging.

Skeletal muscle atrophy during aging in WT, Tg, and Ghrl KO mice

Despite the lack of significant differences in the weight of muscles among the three genotypes and the general decrease in muscle mass in aged mice (Figure 2C and Table 1), a closer examination of muscles revealed differences in the expression of Atrogin-1 (Fbxo32), a marker and a mediator of muscle atrophy, during aging [23]. The age-dependent increase of Atrogin-1 mRNA expressions was remarkable in gastrocnemii of WT and Tg mice and was milder in Ghrl KO mice (Figure 4A), although it should be considered that Atrogin-1 mRNA levels were basally lower in young Ghrl KO mice compared to the other genotypes. This was also true in tibialis anterior (TA) muscles, where Atrogin-1 was significantly increased during aging only in Ghrl KO mice, reaching the values of young WT and Tg animals. Conversely, the increase of Atrogin-1 during aging was not significant in WT and absent in Tg animals. The mRNA expression of other markers of autophagic-mediated catabolism, such as cathepsin-L and Bnip-3, showed a general tendency to increase with aging in all genotypes (Figure 4B, 4C), although the increase in cathepsin-L was significant only in WT and Tg mice. In the aged groups, cathepsin-L gene expression in both Tg and Ghrl KO mice was reduced compared to the WT animals, while Bnip-3 levels significantly decreased only in Tg mice. On the contrary, the expression of the anabolic marker IGF1 showed the general tendency to decrease with aging in Tg and Ghrl KO mice, although the reduction was significant only in the former (Figure 4D). Nevertheless, in old animals, Ghrl KO mice displayed a statistically significant lower expression of IGF1 compared to WT mice. Analyzing the fiber cross-sectional area distributions of gastrocnemius (GAS) muscles from young adult and old WT mice, it was evident a clear shift toward smaller areas in the latter (Supplementary Figure 1). Since the muscles of young Tg and KO mice, in unstimulated conditions, do not differ from those of WT animals [13, 22], the shift towards larger areas in old Tg and Ghrl KO muscles compared to old WT muscles (Figure 4E) indicates that both high levels of circulating UnAG and the lack of ghrelin peptides protect against muscle mass loss during aging.

Figure 4. Ghrelin peptides effects on sarcopenia-dependent atrophy. (A) Atrogin-1 (Fbxo32) expression in gastrocnemius (GAS) and tibialis anterior (TA) of 3-month old (young) and 24-month old (old) mice determined by real-time RT-PCR. In GAS, young mice: WT = 4, Tg = 4, Ghrl KO = 4; old mice: WT = 4, Tg = 3, Ghrl KO = 4. In TA, young mice: WT = 4, Tg = 3, Ghrl KO = 4; old mice: WT = 8, Tg = 6, Ghrl KO = 5. (B) Cathepsin-L (Ctsl), (C) Bnip-3 (Bnip3), and (D) IGF-1 (Igf1) expression in gastrocnemius of 3-month old (young) and 24-month old (old) mice determined by real-time RT-PCR. Young mice: WT = 3, Tg = 3, Ghrl KO = 4; old mice: WT = 7, Tg = 9, Ghrl KO = 5. (E) Cross-sectional area (CSA) frequency distribution of myofibers in GAS of old WT, Tg, and Ghrl KO mice. WT= 4, Tg= 3, Ghrl KO= 5. The shadowed area of the graph represents the section of statistically significant differences among curves. Data are presented as mean ± SEM. *p<0.05 and **p<0.01 old vs. young; ^$p<0.05 and ^$$p<0.01 Tg and Ghrl KO vs. WT.

Muscle fiber types and metabolism in old WT, Tg, and Ghrl KO mice

Muscles are more susceptible/resistant to atrophy also depending on their fiber type composition, being glycolytic fibers more prone to undergo mass loss compared to the oxidative ones [24]; therefore, we assessed if high levels of UnAG or the lack of ghrelin peptides could affect muscle metabolism. Succinate-dehydrogenase (SDH) staining revealed that oxidative ability, i.e., the activity of complex II of the electron transport chain, decreases with aging both in WT and Ghrl KO mice, whereas it was maintained in Tg (Figure 5A), suggesting that UnAG stimulates an oxidative metabolism, counteracting the age-associated shift from oxidative to glycolytic metabolism.

Figure 5. Metabolic shift in TA muscles of WT, Tg, and Ghrl KO mice. (A) Representative images of succinate dehydrogenase (SDH) staining (scale bars: 200 μm) representing the oxidative capacity of TA muscles of 3-month old (young) and 24-month old (old) mice and quantification of SDH-positive fibers in TA muscle presented as the percentage of SDH-positive area above the total muscle surface. Young mice: WT= 5, Tg= 4, Ghrl KO= 5; old mice: WT= 4, Tg= 3, Ghrl KO= 5. (B) Representative Western blots images and protein densitometry quantification for mitochondrial transcription factor A (TFAM), ATP-synthase β-subunit (β-ATPase), succinate-dehydrogenase complex subunit-A (SDHA), peroxisome proliferator-activated receptor gamma (PPARγ), and PPARγ-coactivator-1-α (PGC1α) protein levels. Protein levels were probed in gastrocnemii of old mice, WT= 4, Tg= 5, Ghrl KO= 5. (C) Representative images of cytochrome c oxidase subunit 4 (COX4) immunofluorescence (scale bars: 200 μm) and quantification. Young mice: WT = 3, Tg = 3, Ghrl KO = 3; old mice: WT = 4, Tg = 4, Ghrl KO = 4. Data are presented as mean ± SEM. *p<0.05 and **p<0.01, old vs. young; ^$p<0.05 and ^$$p<0.01, Tg and Ghrl KO vs. WT.

Since aging muscles display mitochondrial dysfunction [25], we assessed the expression levels of mitochondria and oxidative metabolism markers such as mitochondrial transcription factor A (TFAM), ATP-synthase β-subunit (β-ATPase), SDH subunit-A (SDHA), peroxisome proliferator-activated receptor gamma (PPARγ), and PPARγ-coactivator-1-alpha (PGC1α). In line with the results reported in Figure 5A, we observed an up-regulation of SDHA in old Tg mice compared to WT (Figure 5B). In addition, old Tg mice showed an increase in PPARγ. On the other hand, β-ATPase and PGC1α, a master regulator of mitochondrial biogenesis, did not show different expression levels among the three genotypes. Yet, Ghrl KO mice showed an up-regulation of TFAM, another marker of mitochondrial biogenesis. Coherently, the mitochondrial content in muscles, assessed as the quantity of cytochrome c oxidase subunit 4 (COX4) in immunofluorescence, was higher in Ghrl KO mice than in WT, both in young and old animals (Figure 5C). Of note, also old Tg animals had significantly higher levels of COX4 compared to WT.

Inflammation markers in old WT, Tg, and Ghrl KO mice

With age, the spleen undergoes enlargement and disorganization of its architecture that can lead to age-related decline of immune function and chronic inflammation, that, in turn, could contribute to the decrease in muscle mass and functionality [26–28]. All three genotypes showed a significant increase of spleen weight with age, although to a lesser extent in Ghrl KO mice (Table 1). Adipose tissue as well contributes to the establishment of age-associated systemic inflammation, as it becomes dysfunctional with age, increasing the production of pro-inflammatory cytokines while decreasing the anti-inflammatory ones [29]. TNFα mRNA levels in epididymal fat increased during aging in all the genotypes, but to a lesser extent in Tg and Ghrl KO mice (Figure 6A), confirming the low-grade systemic inflammation in these mice. The impact of ghrelin peptide levels on age-related systemic inflammation was explored through an antibody-spotted array directed against several circulating markers of inflammation on pooled samples (Figure 6B and Supplementary Figure 2). As expected, old WT mice showed a general tendency to increase pro-inflammatory markers and to decrease the anti-inflammatory ones compared to young mice (reference line in Figure 6B). By contrast, a trend towards reduction could be observed in both old Tg and Ghrl KO mice compared to old WT for some pro-inflammatory markers, such as SDF1α, eotaxin-1 (CCL11), I-309 (CCL1), leptin, M-CSF, and IL-12p70, seemed to be lower. Conversely, the anti-inflammatory soluble TNF-R1 gave the impression of being increased in both Tg and Ghrl KO mice.

Figure 6. Inflammatory response in old WT, Tg, and Ghrl KO mice. (A) TNF-α (Tnf) expression in epididymal fat of 3-month old (young) and 24-month old (old) WT, Tg, and Ghrl KO mice, determined by real-time RT-PCR. Data in bar graph are presented as mean ± SEM. Young mice: WT= 3, Tg= 4, Ghrl KO = 4; old mice: WT= 5, Tg= 7, Ghrl KO = 5. (B) Serum biomarkers involved in systemic inflammation normalized on the levels in young WT mice (reference line). Young mice WT=4; old mice: WT= 4, Tg= 4, Ghrl KO = 4 pulled into two groups. Data in bar graph are presented as mean ± SD. *p<0.05 and **p<0.01, old vs. young; ^$p<0.05 Tg and Ghrl KO vs. WT.

UnAG pharmacological treatment improved the physical performance of old WT mice by inducing a metabolic switch in muscles

Old WT mice were treated with UnAG (100 μg/kg intraperitoneally once a day) for 28 days and compared to age- and weight-matched controls treated with saline. UnAG administration had no significant effect on total body weight (Figure 7A), daily food intake (Figure 7B), and epidydimal fat (Figure 7C). On the other hand, muscle weights showed a tendency towards higher values in UnAG-treated mice that were significant in gastrocnemii, quadriceps, an d EDL muscles (Figure 7D). UnAG progressively increased the physical performance of mice, seen as “falls and reach” score and holding impulse in the hanging wire test (Figure 7E, 7F). The difference in the holding impulse in UnAG-treated mice reached statistical significance at 28 days. However, at the end of the experimental period, there was no shift towards larger cross-sectional areas of gastrocnemius fibers, although the shape of UnAG-treated muscle distribution showed an increment of middle-sized fibers (Figure 7G). Despite the increase in muscle weight (Figure 7D), UnAG did not alter the expression of IGF-1 (Figure 7H). Furthermore, UnAG treatment was unable to decrease the expression of Atrogin-1 and cathepsin-L (Figure 7I, 7J). Since the better muscle performance could derive from a shift in muscle metabolism, we assessed SDH activity in gastrocnemii and observed that, indeed, UnAG induced a substantial increase in SDH staining in muscles (Figure 7K).

Figure 7. Exogenous administration of UnAG in old WT mice attenuates age-related muscle decline. UnAG 100 μg/Kg was injected daily i.p. for 28 days to 18-month-old mice (N = 6). Age- and weight-matched controls were injected with saline (N =6). (A) Total body weight; (B) daily food intake; (C) change in epidydimal fat mass normalized to nose-to-anus length (NAL) and in (D) muscle mass normalized to the tibial length (TL). GAS: gastrocnemius, SOL: soleus, QUAD: quadriceps, TA: tibialis anterior, EDL: extensor digitorum longus. (E) Average score trend in hanging wire test and (F) average holding impulse. (G) Cross-sectional area (CSA) frequency distribution of myofibers in GAS. The shadowed areas of the graph represent the sections of statistically significant differences among curves. (H) IGF-1 (Igf1), (I) Atrogin-1 (Fbxo32), and (J) Cathepsin-L (Ctsl) expression in gastrocnemius determined by real-time RT-PCR. (K) Quantification of SDH staining in TA muscle presented as the percentage of SDH-positive area above the total muscle surface. Data in bar graph are presented as mean ± SEM. For box plots, the lower and up boundaries denote the 25^th and the 75^th percentile of each data set, respectively, the horizontal line represents the median, and the whiskers represent the min and max of values. *p<0.05 and **p<0.01 vs. saline-treated controls.

Animals and treatments

Animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee at the University of Piemonte Orientale. C57BL/6 male mice, matched for age and weight, were used for all experiments. C57BL/6 Ghrl^-/- (Ghrl KO) and C57BL/6-Myh6/Ghrl transgenic (Tg) mice were generated as previously described [7, 22, 43]. WT mice were littermates of Tg animals. Ghrl KO strain was kept in homozygosity. Animals were housed in groups of 4/6 animals and fed ad libitum with unrestricted access to drinking water. For food intake analysis and behavioral tests, mice were single-caged for the acclimation plus experimental periods. The light/dark cycle in the room consisted of 12/12 h with artificial light. The numbers of mice ranged from 4 to 27 per experiment. BMI was calculated as animal weight/(nose-to-anus length)². AG and UnAG plasmatic levels were measured by EIA kits (SPIbio Bertin Pharma). In the experiments with i.p. injections of 100 μg/Kg UnAG (PolyPeptide Laboratories; Strasbourg, France), controls were age- and weight-matched saline-injected animals. Mice were killed through cervical dislocation, and tissues used for the study immediately excised, weighted, and processed or stored for further analysis.

Hanging wire test

A wire-hanging test was employed to assess whole-body muscle strength and endurance. The test was performed as previously described [13]. Briefly, mice were subjected to 180 sec hanging test, during which “falling” and “reaching” scores were recorded. When a mouse fell or reached one of the sides of the wire, the “falling” score or “reaching” score was diminished or increased by 1, respectively. A Kaplan-Meier-like curve was created afterward. Moreover, the longest time between two falls was taken as the latency-to-fall value and used to calculate the holding Impulse (i.e., the hanging time x body mass).

Object recognition test

Recognition memory was measured using various objects of different sizes and materials, as previously described [44]. Briefly, mice were separated in single cages 10 days before the test. The task started with a familiarization trial (day 1) in which two identical objects were presented to the animals, followed by the test trial (day 2), where one familiar object was substituted with a novel one. In detail, in day 1, mice were left with two identical objects (familiarization phase). Exploration was recorded for 10 min by an investigator blinded to the strain assessed. Sniffing, touching, and stretching the head toward the object at a distance ≤ 2 cm were scored as object investigation. In day 2 mice were again left in the same cage containing two objects: one identical to one of the objects presented during the familiarization phase (familiar object), and a new, different one (novel object), and the time spent exploring the two objects was recorded for 10 min. Memory was expressed as a discrimination index calculated as follows: (seconds on novel–seconds on familiar)/(seconds on novel + seconds on familiar). Animals with memory impairment spend lesser investigating time with the novel object, thus giving a lower discrimination index.

Tail suspension test

Mice anxiety/depression was measured as previously described [45]. Briefly, mice were individually suspended in the air by tape attached to a shelf (50 cm height) for 6 min. Trials were video-recorded, and a blinded experimenter scored the time during which mice remained immobile as a measure of depressive-like behavior. A small cylindrical tube was slipped over the mouse tail to prevent climbing motion and to escape.

Histological analysis

Muscles were excised, weighted, mounted in Killik embedding medium (Bio-optica), frozen in liquid-nitrogen-cooled isopentane, and stored at -80 °C. Transverse muscle sections (7 μm) were cryosectioned from the middle part of each muscle. Myofiber areas were determined by immunofluorescence with anti-laminin (1:200; Dako). Slices were fixed in 4% PFA for 20 min, washed, permeabilized with 0.2% Triton X-100 in 1% BSA for 15 min, and blocked with 4% BSA for 30 min. One hour of incubation with primary antibodies was followed by 45 min of secondary antibody (Alexa Fluor 488-anti-rabbit; Thermo Fisher Scientific) at RT. DAPI was incubated for 5 min at RT to visualize nuclei. SDH staining was performed to reveal the oxidative fibers within a muscle. Frozen transverse TA muscles were incubated in a working solution (SUCCINIC DEHYDROGENASE Stain Lyophilized, Bio-optica) for 45 minutes at 37°C. The sections were then rinsed in distilled water, fixed in 4% PFA for 10 minutes, placed in 15% ethanol for 10 minutes, and, finally, mounted with aqueous mounting medium. Images of whole muscle sections were acquired with the slide scanner Pannoramic Midi 1.14 (3D Histech) and cross-sectional areas (CSA) of fibers or SDH staining quantified with ImageJ software (v1.49o).

Immunofluorescence

Tissue sections were fixed in 4% PFA for 10 minutes, washed in PBS, permeabilized with 1% BSA 0,2% triton x-100 for 20 minutes. For blocking the unspecific binding sites, slices were incubated in 4% bovine serum albumin (BSA) for 2 hours at RT and then sections were stained with an anti-COX4 antibody (1:250; Cell Signaling technology, Danvers, MA, USA) or with an anti-laminin antibody (1:200; Dako, Agilent Technologies, Santa Clara, CA). After washing, sections were incubated with the appropriate Alexa Fluor Dyes-conjugated secondary antibody (488-anti-rabbit; Thermo Fisher Scientific) for 1 hour at RT. 40,6-diamidino-2-phenylindole (DAPI) was incubated for 5 minutes to visualize nuclei.

Images were acquired using the slide scanner Pannoramic Midi Scanner 1.14 (3D Histech) and quantified with ImageJ v1.49o software.

Gene expression analysis

Total RNA from muscles was extracted by RNAzol (Sigma-Aldrich). RNA was retro-transcribed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and real-time PCR was performed with the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using the following TaqMan assays: Mm00499518_m1 (Fbxo32, Atrogin-1/MAFbx), Mm00439560_m1 (Igf1), (Mm00515597_m1 (Ctsl), Mm01208835_m1 (Ppargc1a), Mm00616415_m1 (Ghsr), Mm01225600-g1 (Bnip3), Mm00443258_m1 (Tnf), and Mm00506384_m1 (Ppif).

Western blotting

Tissue samples from gastrocnemii were homogenized by using GentleMACS Dissociator (Miltenyi Biotec) and lysed in ice-cold RIPA Buffer (50 mM Tris/HCL, pH= 8.0; 1% Triton X-100; 150mM NaCl; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Sigma-Aldrich). A clear supernatant was obtained by centrifugation of lysates at 13,000 g for 20 min at 4°C. Protein concentration in the supernatant was determined by Bradford protein assay (Bio-Rad). Aliquots of total cell lysates (20 μg) were then separated by SDS-PAGE by using Miniprotean precast gels (BioRad), and proteins were transferred to nitrocellulose membranes (BioRad).

Membranes were blocked 1h at RT with 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 and then probed by using the following antibodies directed against PGC1α (AB3242; Millipore), β chain-ATP Synthase (MAB3494; Millipore), PPARγ (sc-7273; Santa Cruz Biotechnology), SDH (sc-377302; Santa Cruz Biotechnology), TFAM (sc-23588; Santa Cruz Biotechnology), GAPDH (G8795; Sigma-Aldrich).

The appropriate secondary horseradish peroxidase-conjugated antibodies from Jackson Immunoresearch were used in 5% not-fat- milk (blocking solution) for 1 h at room temperature. Immunoreactive bands were visualized by Clarity Western ECL Substrate (Biorad). Equal loading of samples was confirmed by GAPDH (G8795; Sigma-Aldrich) normalized and quantified by densitometry by ImageJ Software.

Mouse inflammation antibody array

Serum cytokines and biomarkers of systemic inflammation were detected using the Mouse Inflammation Antibody Array C1 (RayBiotech, Norcross, Georgia, USA) according to manufacturer instructions.

Statistical analysis

All data were expressed as mean ± SEM or mean ± SD, when appropriate, absolute values, or percentages. Outliers in the measurements were identified by mean of the interquartile range (IQR), as either below Q1 – 1.5 IQR or above Q3 + 1.5 IQR, and excluded from the analysis. The variation between groups was compared by using nonparametric Wilcoxon, Mann-Whitney U, Friedman, or χ2 tests, as appropriate. Statistical significance was assumed for p<0.05. The statistical analysis was performed with IBM SPSS Statistics for Windows version 25.0. The * symbol indicates a statistically significant difference between old and young animals, for any genotype group; the § symbol identifies a statistically significant difference between WT animals and either Tg or Ghrl KO within the same age group.