Untargeted metabolomics reveals effects of calorie restriction on skeletal muscle in older female and male rats

Abstract

This study used untargeted metabolomics to investigate how calorie restriction (CR) affects skeletal muscle in older female and male rats. Fischer-344 Brown Norway rats of both sexes (22-23-month-old) were randomly assigned to an ad libitum-fed (AL) group or a CR group, which received 65% of the daily food intake of their sex-matched AL counterparts for eight weeks. Epitrochlearis muscles were collected and analyzed using untargeted metabolomics and immunoblotting. CR altered the abundance of 554 out of the 999 detected muscle metabolites. Additionally, 521 metabolites differed between females and males. CR raised the levels of metabolites related to bile acids, with significantly greater CR-induced elevation of secondary bile acids in females, suggesting possible sex-dependent interactions involving the gut, liver, and muscle. Furthermore, CR increased the levels of five glycolytic metabolites. These changes occurred without increases in the abundance of phosphofructokinase (a rate-limiting glycolytic enzyme) or in key allosteric activators (AMP and ADP). There was no evidence for a CR-induced increase in NAD⁺/NADH ratio: NAD⁺ levels decreased and NADH levels increased for CR versus AL rats. Approximately 78% of dipeptides were lower in CR rats compared to AL rats, and 38% of dipeptides were greater for females versus males. Levels for 5-methyl-2’-deoxycytidine (a methylated DNA breakdown product and potential marker for changes in DNA remodeling) were significantly reduced by CR only in females. Together, these findings offer important insights for understanding both the shared and sex-specific mechanisms through which CR influences muscle metabolism, health, and function.

Introduction

Moderate calorie restriction (CR), a 20%-40% reduction in calorie intake compared to eating freely (ad libitum [AL]), has multiple effects on physiological function and health with advancing age. CR provides many metabolic benefits, including better insulin sensitivity and glucose tolerance, improved blood lipid profiles, and reduced oxidative stress.¹ Skeletal muscle is a key tissue involved in a number of these metabolic processes and is highly responsive to the effects of CR.^2–5

For decades, researchers have examined the metabolic effects of CR using conventional approaches focused on relatively small numbers of targeted metabolites. More recently, the use of untargeted metabolomics has broadened our understanding of CR-associated metabolic changes in mammals.^6–20 These untargeted metabolomics studies reveal complex metabolic responses that are not captured by conventional analyses focused on a small set of metabolites. Their results often include changes in lipid metabolism,^{6–13,16–18,20} and sometimes detect shifts in energy and redox pathways linked to mitochondrial function.^8,9,12 Several reports also indicate effects on bile acid-related metabolites.^7,11,19,20 Publications using untargeted metabolomics with mammalian CR models include a study conducted in rhesus macaques,⁶ but the other studies focused on mice. Extending CR research using untargeted metabolomics to other species, including rats, would provide additional insights.²¹ Prior studies often analyzed only plasma, serum, and/or urine.^{6–8,16,17,19} Other studies have analyzed various tissues, including liver, kidney, white adipose tissue, heart, hypothalamus, cerebellum, cortex, hippocampus, lung, or skeletal muscle.^9–15,18,20 Few studies included both sexes,^9,10,14 and even fewer rigorously evaluated sex-related differences by direct, statistical comparison.⁹

We sought to address several of the gaps in prior CR metabolomics publications by testing whether CR effects differ by sex in older rats. Older male and female rats were studied (24-25 months at study completion) after 8 weeks of CR, during which they received 65% of the food consumed by AL controls. We collected epitrochlearis muscles and performed untargeted metabolomics using a platform with broad pathway coverage, a comprehensive reference library, and high accuracy and reproducibility in metabolite identification. We focused on the epitrochlearis because its fiber-type composition closely reflects the overall rat skeletal muscle profile^22,23 and because earlier work established this muscle as a well-characterized model for studying metabolic effects of CR and/or aging.^24–27 To quantify sex and diet effects, we used two-way ANOVA (main effects: sex and diet) and tested sex × diet interactions to identify sex-specific CR responses. We hypothesized that most CR-induced metabolite changes would be shared between sexes, with a subset showing sex-selective responses. Guided by the metabolomics findings, we conducted follow-up protein analyses by immunoblotting. Overall, the results provide new insights into CR-induced metabolic remodeling in older muscle, including both shared and sex-specific effects.

Method

Animal treatment

Animal care procedures were approved by the University of Michigan Committee on Use and Care of Animals. Female (n = 18) and male (n = 18) Fischer-344 Brown Norway rats were obtained from the National Institute of Aging (NIA) at 22-23-month-old. Animals were individually housed in specific pathogen-free conditions, maintained on a 12:12 h light-dark cycle (lights out at 1700 h). Rats had AL access to food (Laboratory Diet no. 5L0D; Lab Diet, St. Louis, MO, USA) and water for a one-week acclimation period, with food intake determined daily. After this acclimation period, rats were randomly assigned to AL (n = 9 per sex) or CR (n = 9 per sex) groups. AL rats had unlimited access to the rodent chow. CR rats were restricted to 65% of the mean daily intake of the sex-matched AL group for 8-weeks. Rats received their daily food allocation between 1600 h and 1700 h. Intake and body mass were measured each week. Rats were euthanized at 1000 h to 1100 h when they were 24-25 months old. At the time of anesthesia, CR rats had already consumed their food allocation from the previous evening. AL rats had continuous access to food until anesthesia.

Tissue dissection

When rats were deeply anesthetized (intraperitoneal injection of 50 mg/kg ketamine; 5 mg/kg xylazine), their epitrochlearis muscles were dissected out and freeze-clamped using aluminum tongs cooled to the temperature of liquid nitrogen. A retroperitoneal fat pad was dissected out and weighed.

Muscle sample preparation for metabolomics analysis and immunoblotting analysis

A portion of the frozen muscles was prepared using the automated MicroLab STAR^® system from Hamilton Company. Each sample was extracted equivalently by weight using methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000), followed by centrifugation. The resulting extract was divided into multiple fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, while the remaining fractions were reserved for backup. Samples were placed briefly on a TurboVap^® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis. Another portion of the frozen muscles was homogenized for immunoblotting as described.²⁸

Metabolomics analysis

Untargeted metabolomics analysis was performed by Metabolon, Inc. (Morrisville, NC) using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS). All methods used a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35 000 mass resolution. Dried sample extracts were reconstituted in solvents compatible with each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds (PosEarly). In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds (PosLate). In this method, the extract was gradient eluted from the same aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column (Neg). The basic extracts were gradient eluted from the column using methanol and water, however, with 6.5 mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1 × 150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10 mM Ammonium Formate, pH 10.8 (HILIC).

Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Authenticated standards with known retention time/index (RI), mass to charge ratio (m/z), and fragmentation data for all molecules were in the library. Biochemical identifications were based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/− 10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores were based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum.

Immunoblotting analysis

Chemicals used for immunoblotting were from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Hanover Park, IL, USA). The reagents and apparatus for SDS-PAGE and nonfat dry milk (no. 170-6404) were purchased from Bio-Rad (Hercules, CA, USA). Pierce MemCode Reversible Protein Stain Kit (no. 24585), bicinchoninic acid protein assay (no. 23225), and Tissue Protein Extraction Reagent (T-PER; no. 78510) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-carnitine palmitoyltransferase 1B (CPT1B, no. 22170-1-AP), anti-carnosine dipeptidase 2 (CNDP2, no. 14925-1-AP), anti-phosphofructokinase, muscle type (PFKM, no. 55028-1-AP), anti-Poly (ADP-ribose) polymerase 1 (PARP1, no. 13371-1-AP), and anti-Sirtuin 1 (SIRT1, no. 13161-1-AP) were purchased from ProteinTech (Rosemont, USA). Anti-rabbit IgG horseradish peroxidase conjugate (no. 7074) was from Cell Signaling Technology. Enhanced chemiluminescence Luminata Forte Western HRP Substrate (no. WBLUF0100) was purchased from EMD Millipore (Billerica, MA, USA).

Immunoblotting procedures were performed as described.²⁸ Equal amounts of protein from each muscle lysate were mixed with 6x Laemmli buffer, boiled for 5 min, and then separated by SDS-PAGE. The protein was then transferred to polyvinylidene difluoride membranes. Equal loading was confirmed with MemCode protein stain.²⁹ Membranes were blocked in TBST (Tris-buffered saline, pH 7.5 plus 0.1% Tween-20) that was supplemented with 5% nonfat milk for 1 h at room temperature. After blocking, the membranes were incubated with the appropriate primary and secondary antibodies, subjected to enhanced chemiluminescence, and quantified by densitometry (AlphaView; ProteinSimple, San Jose, CA, USA). Results were expressed relative to the normalized average of all the samples on the blot.

Statistical analysis

Body mass, fat pad mass, epitrochlearis mass, and food intake

Body mass, fat pad mass, epitrochlearis mass, and food intake of AL and CR groups were compared for each sex using Student’s t-test with SigmaPlot 16.0 (San Jose, CA). Values are presented as means ±SD. p-values ≤ .05 were considered statistically significant.

Metabolomics data

Following imputation of missing values with the minimum observed value for each compound, and log transformation, two-way ANOVA (main effects of Diet, AL or CR, and Sex, female or male) was performed. To adjust for false discovery rate with multiple comparisons, q-values ≤ .05 were considered statistically significant. The q-values were calculated based on estimating the proportion of true null hypotheses for each list of p-values using an R script as previously described.³⁰

Immunoblotting data

Two-way analysis of variance (ANOVA) with SigmaPlot 16.0 was used to assess the main effects of sex (female or male) and diet (AL or CR), and the sex × diet interaction on metabolomics and immunoblotting results. Two-way repeated measures ANOVA was used to determine the effects of diet and time on body mass during the 8-week dietary intervention. Tukey post hoc tests were used to identify the source of significant variance. Final values for body mass, fat pad mass, and epitrochlearis mass, and mean values for food intake in AL and CR groups within each sex were compared using a Student’s t-test. Values are presented as means ±SD. p-values ≤ .05 were considered statistically significant.

Results

Body mass, fat pad mass, epitrochlearis mass, and food intake

As intended, the food intake of CR rats was ∼65% of the value for AL rats of each sex (Figure 1A). Initial body masses were not significantly different between AL and CR diet groups in either females (AL = 263.2 ± 20.5 g; CR = 272.8 ± 15.9 g) or males (AL = 539.4 ± 20.1 g; CR = 539.2 ± 41.8 g). Body mass of the CR rats declined ∼4%-7% per week during the initial 2-3 weeks of the CR protocol (Figure S1). Thereafter, the rate of loss progressively decreased to ∼1%-1.5% per week during the final 2-3 weeks of the intervention. Body mass values for CR rats approached a near plateau, with no significant differences within the CR rats at week-7 versus week-8. The retroperitoneal fat pad mass, epitrochlearis mass, and body mass after 8-weeks of CR were lower for CR versus AL animals of each sex (Figure 1A).

Figure 1.

A: Body mass, retroperitoneal fat pad mass, epitrochlearis mass, and mean daily food intake in female and male rats. *p ≤ .05, **p < .001 for CR versus AL in both sexes. Values are shown as mean ± SD. B: Principal component analysis (PCA) of rat skeletal muscle samples (circles represent AL; squares represent CR; pink represents females; blue represents males). C: Summary of the Two-Way ANOVA. D: Summary of the main effects for sex and diet and sex × diet interactions for each of the super pathways.

Metabolomics and immunoblotting

Metabolomics analysis detected a total of 999 biochemicals, and Principal Component Analysis (PCA) showed clear separation of samples based on both diet and sex (Figure 1B). The first principal component (PC1), which explained 20% of the variance, was associated with diet. The second principal component (PC2) explained 15.9% of the variance and was associated with sex. Diet had a somewhat greater effect on the variation in biochemical profiles than sex. Two-way ANOVA revealed significant main effects of both sex and diet as well as interactions between sex and diet (Figure 1C).

The biochemicals were grouped into nine Super Pathways (Figure 1D) with the following proportions: Lipids (39%), Amino Acids (19%), Peptides (15%), Unnamed (8%), Xenobiotics (6%), Nucleotides (5%), Carbohydrates (4%), Cofactors and Vitamins (3%), and Energy (1%). The Unnamed Super Pathway includes compounds with known chemical identities that could not be confidently assigned to any established Super Pathway because of limited or ambiguous information.

Two-Way ANOVA showed significant main effects of sex and diet, and significant sex × diet interactions (Figure 1D). Approximately 48% of the 999 metabolites were not significantly different between females and males. Significantly greater values for females compared to males were detected for ∼39% of the metabolites, with the remainder (∼14%) significantly lower for females. About 45% of the metabolites were not significantly different between the diet groups. Significantly lower values were detected for ∼43% of the compounds in CR compared to AL groups, with significantly greater values for CR animals in ∼13% of the compounds. Significant sex × diet interactions were identified for 15 metabolites (Figure 1D, Supplementary Table, Figure S2A to S2O).

Lipids

Approximately 39% of the 392 detected lipids showed no difference between sexes (Figure 1D). About 47% of the lipids were greater for female versus male rats, and ∼14% of the lipids were lower for females than males. Approximately 43% of the lipids were lower for CR versus AL rats, and about 17% of CR values exceeded AL values of lipids.

Several metabolites involved in lipid oxidation for energy production, including 3-hydroxybutyrate and 3-hydroxyhexanoylcarnitine, were higher in CR rats compared to AL rats (Figure 2A). This finding aligns with previous studies showing increased in vivo fat oxidation in both female³¹ and male^32,33 rats that were studied several hours following their daily food allotment under CR protocols similar to the current study. Muscle abundance of CPT1B, a rate-limiting enzyme for long-chain fatty acid oxidation, was greater for male versus female rats, but it was not altered by diet (Figure 2B).

Figure 2.

A: Sex and diet main effects and sex × diet interactions on 3-Hydroxybutyrate (BHBA) and 3-Hydroxyhexanolycarnitine values in skeletal muscles. The shaded boxes represent the Middle 50% of the data, and the whiskers represent the range of the data. The solid bar across the box represents the median value. The plus sign (+) represents the mean. Data are scaled such that the median value measured across all samples is set to 1.0. Outliers are shown as circles. Sex and diet main effects and sex × diet interactions, *q ≤ 0.05. B: Carnitine palmitoyltransferase-1B (CPT1B) abundance in skeletal muscles determined by immunoblotting. *p ≤ .05, main effect of sex. Values are shown as mean ± SD along with a representative blot. C: Volcano plots depicting significant (q ≤ 0.05) sex and diet effects for polyunsaturated acyl carnitines in skeletal muscle. D: Volcano plots depicting significant (q ≤ 0.05) sex and diet effects for long-chain polyunsaturated acyl fatty acids (n-3 and n-6) in skeletal muscle. E: Sex and diet effects on primary and secondary bile acid metabolites in skeletal muscle. Red represents significant (q ≤ 0.05) positive and green represents significant negative differences, with the fold of change values indicated. The figure of the bile acids pathway was created with BioRender.com. F: Sex and diet main effects and sex × diet interactions on ursodeoxycholate, isoursodeoxycholate, hyocholate, and 7-ketodeoxycholate values in skeletal muscles, *q ≤ 0.05.

Many polyunsaturated lipids consistently responded to CR. All eight members of the polyunsaturated acyl carnitine subpathway were lower in CR rats compared to AL rats (Figure 2C). Additionally, 94% of the lipids in the long-chain polyunsaturated fatty acid (n-3 and n-6) subpathway were reduced in CR rats compared to their respective AL counterparts (Figure 2D). Earlier research has demonstrated reductions in levels of these polyunsaturated fatty acids, including docosapentaenoate (n3 DPA; 22:5n3) and docosahexaenoate (DHA; 22:6n3), in cell membranes.³⁴

Pronounced effects of sex and diet were seen in bile acid levels (Figure 2E). Females compared to males had greater values for 5 of 8 (63%) primary bile acids (produced in the liver) and 5 of 7 (71%) secondary bile acids (modified by the gut microbiome and reabsorbed into circulation). CR versus AL rats had higher levels of 4 (50%) primary bile acids and 6 (86%) secondary bile acids. A significant sex × diet interaction was identified for 4 (57%) secondary bile acids with a greater CR-related increase for females, but not males (Figure 2F).

Carbohydrates

Most (62%) of the 42 detected carbohydrates did not differ between the sexes (Figure 1D). Approximately 19% of the carbohydrates were greater for females versus males, with the remaining 19% were lower in females. Most (64%) of the carbohydrates were not significantly altered by diet. Values were significantly greater for ∼21% of the carbohydrates of AL compared to CR rats, with the remainder (∼14%) greater for CR animals.

The glycolytic pathway was highly responsive to diet. Although muscle glucose concentration was lower for CR compared to AL rats (Figure 3A), CR significantly increased the levels of five glycolytic metabolites (Figure 3A). Muscle glucose and pyruvate levels were lower in females versus males (Figure 3A). There was a significant sex × diet interaction for pyruvate levels, with a significant CR-related increase in pyruvate in females, but not in males. In the absence of hyperglycemia, CR typically reduces circulating insulin without causing large decrements in glycemia, and prior research suggests that in vivo muscle glucose uptake is similar or somewhat greater for the CR versus AL rats.²⁶ There was no CR effect on the abundance of PFKM, a rate-limiting enzyme for glycolysis (Figure 3B).

Figure 3.

A: Sex and diet main effects and sex × diet interactions on glycolytic metabolites in skeletal muscle. Red represents significant (q ≤ 0.05) positive and green represents significant negative differences, with the fold of change values indicated. The metabolites and enzymes measured in this study are shown in boxes in the glycolysis pathway diagram. The figure of the glycolysis pathway was created with BioRender.com. B: Phosphofructokinase, muscle type (PFKM) abundance in skeletal muscles determined by immunoblotting. Values are shown as mean ± SD along with a representative blot. C: Sex and diet main effects and sex × diet interactions on N6-carboxymethyllysine values in skeletal muscle. *q ≤ 0.05. D: Sex and diet main effects and sex × diet interactions on TCA cycle metabolites in skeletal muscle. Red represents significant (q ≤ .05) positive and green represents significant negative differences, with the fold of change values indicated.

Advanced glycation end-products (AGEs) are formed by non-enzymatic glycation of proteins, lipids, and nucleic acids. Levels of N6-carboxymethyllysine (CML), a widely studied AGE, were greater for female versus male rats and lower for CR compared to AL rats (Figure 3C). Earlier work demonstrated that CR resulted in lower CML in heart mitochondria from 6-month-old male rats after 4-months of CR (consuming 60% of AL intake).³⁵

Energy

The tricarboxylic acid (TCA) cycle intermediates citrate, aconitate, and malate levels were lower in the CR group compared to the AL group (Figure 3D). However, the levels of other TCA cycle intermediates that were detected (α-ketoglutarate, succinate, and fumarate) were unaffected by diet.

Amino acids

Of the 186 compounds in the Amino Acid Super Pathway, ∼49% did not differ between the sexes (Figure 1D). Values for ∼36% were greater for females versus males. About 50% of amino acid metabolites were not altered by diet, and ∼34% were lower for muscles from CR versus AL rats. Significant sex × diet interactions were detected for four members of this pathway (Figure 1D). Significant diet effects were detected only in females for threonine (CR > AL) and N, N-dimethylalanine (CR < AL). A significant diet effect was detected only in males for aspartate (CR > AL). The magnitude of the diet effect on dimethylglycine (CR < AL) was significantly greater for males versus females.

Glutathione is a key regulator of cellular redox balance and exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG values were greater for males compared to females (Figure 4A). CR led to lower GSSG levels, while GSH levels had a non-significant trend (q = 0.056) to be greater for CR versus AL animals. These results are consistent with a lower GSSG/GSH ratio and an improved oxidative state for CR versus AL rats.

Figure 4.

A: Sex and diet main effects and sex × diet interactions on glutathione, reduced (GSH) and glutathione, oxidized (GSSG) values in skeletal muscle, *q ≤ 0.05. B-C: Volcano plots depicting significant (q ≤ 0.05) sex and diet effects for dipeptides in skeletal muscle.

Peptides

Over 90% of the compounds in the Peptides Super Pathway (136 out of 149) were dipeptides (Supplementary Table). Values for 38% of dipeptides were greater for females versus males, and only a single dipeptide was lower for females versus males. Notably, 78% of dipeptides decreased with CR compared to AL, while only ∼1.5% increased; the remaining ∼20.5% were unchanged (Figure 4B-C). A significant sex × diet interaction was detected for glycylisoleucine, which was significantly altered by diet (CR < AL) only in males. A recent study also reported that CR reduced the levels of numerous dipeptides, along with only a few dipeptides increasing in the gastrocnemius muscle of mice.¹⁴

Cofactors and vitamins

Fifty percent of the metabolites in this Super Pathway showed no differences between sexes (Figure 1D). About 37% of these metabolites were greater for females versus males, and ∼13% were greater for males versus females. Most (∼53%) of the compounds in this Super Pathway were lower for CR compared to AL, with 40% not significantly different between diets. Significant sex × diet interactions were identified for a vitamin C metabolite (2-O-methylascoribic acid, CR < AL only in males), and a vitamin B6 metabolite (pyridoxamine phosphate, CR < AL only in females).

Nicotinamide adenine dinucleotide exists in two forms: oxidized (NAD⁺) and reduced (NADH). There were significant main effects of diet for both NAD⁺ and NADH. CR led to lower NAD⁺ levels and higher NADH levels compared to the AL group (Figure 5A). Although there were no significant main effects of sex or sex × diet interactions for either compound, there was a non-significant trend (q-value = 0.09) for an interaction in NADH. Neither sex nor diet altered the abundance of SIRT1 or PARP1, which are enzymes that can lead to the degradation of NAD⁺ (Figure 5B and C).

Figure 5.

A: Sex and diet main effects and sex × diet interactions on oxidized (NAD⁺) and reduced (NADH) nicotinamide adenine dinucleotide values in skeletal muscle *q ≤ 0.05. B-C: Sirtuin1 (SIRT1) and poly (ADP-ribose) polymerase 1 (PARP1) abundance in skeletal muscles determined by immunoblotting. Values are shown as mean ± SD along with a representative blot. D: Sex and diet main effects and sex × diet interactions on ADP and AMP values in skeletal muscle *q ≤ 0.05. E: Sex and diet main effects and sex × diet interactions on 5-Methyl-2’-deoxycytidine values in skeletal muscle. *q ≤ 0.05.

Nucleotides

About 46% of the 48 metabolites in this Super Pathway were unaltered by sex ( Figure 1D). A third (33.3%) of the compounds in the Super Pathway were greater for females versus males. Most (∼60%) of the compounds in this category were not responsive to diet, and ∼27% were greater for AL compared to CR.

The level of ADP was greater in female versus male rats (Figure 5D). There was a significant main effect of diet on AMP, with significantly lower values in CR compared to AL. Our previous study, which evaluated only male rats, reported that CR did not alter muscle AMP.²⁷ To our knowledge, this is the first report of CR effects on ADP and AMP levels in skeletal muscle from both male and female rats.

There was a significant sex × diet interaction for muscle levels of 5-methyl-2’-deoxycytidine. CR versus AL values were significantly lower only in females, with a non-significant trend to be greater for CR versus AL males (q-value = 0.0553) (Figure 5E). This compound is a methylated DNA breakdown product that likely reflects changes in DNA methylation patterns due to DNA remodeling. It is unclear whether these findings indicate sex-specific epigenetic remodeling in response to CR. However, CR has been reported to induce changes in DNA methylation in mice.³⁶

Xenobiotics

The majority (60%) of compounds in this Super Pathway did not differ between sexes (Figure 1D). Similarly, most compounds in this category (53%) did not show any response to diet, while ∼40% were lower in CR rats compared to AL rats.

Discussion

This study used untargeted metabolomics to explore the metabolic effects of CR on skeletal muscle in both female and male rats. The findings demonstrated that CR had widespread impacts, altering the levels of approximately 55% of metabolites detected across diverse metabolic pathways. While many CR-responsive metabolites overlapped between the sexes, several metabolites and pathways showed distinct, sex-specific changes. These results uncovered several previously unidentified effects of CR that may have important functional implications.

The increase in glycolytic intermediates with CR raises questions about the underlying mechanism. CR did not increase the levels of either ADP or AMP, both of which are allosteric activators of PFK. This does not support the idea that CR increases allosteric activation of PFK. We also found that CR rats had lower citrate levels. Since citrate inhibits PFK, this decrease could potentially favor greater PFK activity. However, citrate’s inhibitory effect is enhanced when ATP levels are low. Although we did not measure ATP in this study, previous research²⁷ showed that CR did not alter muscle ATP levels in male rats. Earlier research has not reported whether CR alters muscle ATP in female rats. Overall, we found little evidence for increased allosteric activation of PFK, and PFK protein abundance was not higher in CR compared to AL rats. Therefore, other regulatory mechanisms are likely to be important for the increase in glycolytic intermediates observed in CR animals. It has been reported that locomotor activity is greater for CR versus AL rats.³⁷ Greater muscle recruitment could lead to greater activation of glycolysis.

Another notable finding was that CR led to reduced concentrations of many dipeptides. Dipeptides are formed either by proteolysis or by enzymes linking two amino acids together by creating a dipeptide bond. Interestingly, while CR had a dramatic effect on dipeptide levels, there was no consistent relationship between the influence of CR effects on the levels of the specific amino acids that comprised CR-responsive dipeptides. It is possible that CR reduces dipeptide levels by decreasing proteolysis. Although previous research³⁸ indicates that CR does not affect the overall rate of protein synthesis in skeletal muscle, it is unclear whether CR alters the rate of muscle proteolysis.

Carnosine, an abundant dipeptide in skeletal muscle that is formed by linking L-histidine and β-alanine, has several well-documented functions, including pH buffering and antioxidant activity.³⁹ Carnosine levels were lower for CR versus AL rats. Therefore, the beneficial roles of carnosine may be diminished by the CR-induced reduction of this dipeptide in muscle.

A recent study examined how five different lifespan-extending interventions (CR or treatment with rapamycin, canagliflozin, acarbose, or 17-α-estradiol) affected metabolites, including dipeptides, in several tissues.¹⁴ They found that lower skeletal muscle dipeptide levels were associated with increased lifespan with the interventions. These findings highlight the need for further research to understand how CR’s effects on skeletal muscle dipeptides may influence muscle function and health.

The sex-specific effects of CR on bile acids were observed in secondary bile acids, suggesting that CR may cause sex-dependent changes in gut microbiome activity. Although skeletal muscle does not produce bile acids, these molecules can still influence muscle metabolism through signaling pathways involving the membrane receptor TGR5 and the nuclear receptor FXR.⁴⁰

Prior studies indicate that CR can increase circulating bile acids in mice, although the magnitude may vary with age, duration of CR, and sex. In adult (7-8-month-old) male mice, 5 months of CR increased bile acid levels in both plasma¹⁹ and liver.¹¹ In younger (12-week-old) male mice, a shorter intervention (2 weeks) also elevated serum bile acids and was accompanied by increased hepatic expression of multiple bile acid synthetic enzymes, including higher abundance of CYP7A1, the rate-limiting enzyme in primary bile acid synthesis.⁴¹

Sex also influences baseline bile acid profiles and the response to CR. With AL feeding in 11-week-old mice, females had higher serum levels of several bile acids than males.⁴² After three weeks of CR, these sex differences narrowed largely because bile acid levels rose in males. CR also produced sex-specific changes in hepatic expression of bile acid synthetic enzymes. Liver Cyp7a1 mRNA was increased by CR in females but was unchanged in males. Cyp39a1 mRNA was greater in the livers from CR mice of both sexes. Liver mRNA values for Cyp7b1 were decreased only in males. Hepatic expression of Cyp8b1 was decreased in both sexes. While these findings with brief CR in young mice are informative, most of the results are limited to mRNA expression rather than protein abundance or enzyme activity. Furthermore, comparable work has not yet been reported for longer duration CR in older female and male rats.

It is well established that CR can alter the gut microbiota.⁴³ For example, a recent study showed that lithocholic acid (LCA), a secondary bile acid responsive to CR, can reproduce many of the health benefits typically seen with CR.⁴⁴ Although LCA was not detected among the secondary bile acids measured in the current study, CR increased the levels of 6-oxolithocholate, a bile acid derived from LCA, in both sexes. Additionally, research in obese women and men showed that four months of CR led to sexually dimorphic changes in gut microbiota composition.⁴⁵ Together, these findings indicate that CR can have significant, and sometimes sex-specific, effects on both bile acids and the gut microbiome.

We found evidence that CR increased fat oxidation in muscle, as indicated by higher concentrations of 3-hydroxybutyrate and 3-hydroxyhexanoylcarnitine. These findings align with earlier studies showing increased in vivo fat oxidation with CR.^31–33 Increased fat oxidation was not attributable to higher muscle CPT1 abundance induced by CR. Another mechanism for greater fat oxidation would be elevated availability of lipid substrates, but previous research has shown that CR causes circulating non-esterified fatty acid (NEFA) concentrations in rats to either stay the same or decrease.²⁶ Stearate (18:0) and palmitate (16:0) are the most abundant NEFAs in rat plasma.⁴⁶ CR reduced muscle levels of both lipids. These results provide evidence that CR effects on fat oxidation are not because of greater availability of lipid substrates in either the plasma or muscle.

It has been suggested that an increased NAD⁺/NADH ratio is an expected outcome of CR.^47,48 Consistent with this idea, CR decreased NADH levels while NAD⁺ remained unchanged, leading to a higher ratio in yeast.⁴⁹ However, there is limited data on how CR affects NAD⁺ and NADH levels in rodent skeletal muscle. One study found that CR increased NAD⁺ levels in the skeletal muscle of mice (sex not specified) but did not affect NADH.⁵⁰ Another study reported that CR raised NAD⁺ levels in skeletal muscle from male mice but did not provide data on NADH.⁵¹ To our knowledge, the current study is the first to measure the effects of CR on both NAD⁺ and NADH in skeletal muscle from both male and female rodents. Our findings do not support the idea that CR uniformly increases the NAD⁺/NADH ratio in mammalian tissues.

What factors contribute to the sex-specific effects of CR in older animals? In young adults, most sex differences are attributable to the influence of sex hormones, such as estrogen and testosterone, together with the influence of genetic differences associated with the sex chromosomes (XX and XY).⁵² As animals age and sex hormone levels decline,^52,53 differences inherent to the sex chromosomes may become increasingly important in shaping the sex-specific effects of CR in older animals.

The effects of CR on metabolism are influenced by several key aspects of the experimental protocol, including the timing of food access in relation to the light: dark cycle and the schedule of metabolic measurements. Previous studies have compared various protocols by measuring metabolic outcomes, such as respiratory quotient, oxygen consumption, plasma glucose and insulin levels, glucose and insulin tolerance, plasma metabolites and proteins, phosphorylation of signaling proteins in the liver and muscle, muscle triglycerides, and glucose uptake in different tissues.^{26,31,32,54–57} However, the impact of varying the timing of food access for CR rats and/or the timing of tissue sampling on a comprehensive profile of skeletal muscle metabolites has not yet been explored.

In this study, CR rats were provided with their daily allotment one hour before the onset of the dark phase. Under a 12:12-hour light: dark cycle, AL rats consume a large amount of food during the period between the last hour of the light phase through the early hours of darkness. Skeletal muscle was sampled the following day, and food was not removed from AL animals in the hours preceding muscle sampling. This approach was also used in several earlier studies that assessed skeletal muscle metabolic outcomes in CR and AL animals without imposing a pre-sampling fast on the AL group.^25,27,58,59 The intent is to capture metabolic conditions that reflect typical feeding behavior, rather than adding an additional experimental variable (an enforced fast) for AL animals. A caveat of this design is that AL and CR groups differed in both the timing and the amount of food consumed. Therefore, group differences in metabolite profiles potentially represent the combined effects of intake timing and caloric intake, and this should be considered when interpreting the results. It should also be recognized that other CR protocols that remove food from AL animals several hours before sampling do not perfectly align the AL group’s temporal pattern of food intake with that of the CR cohort. Previous metabolomics studies that evaluated CR in mammals have numerous differences in their feeding protocols. Almost half of these studies subjected AL animals to several hours of fasting before tissues were sampled.^{6,11,13,17–20} The other studies either did not specify if AL animals were fasted or explicitly stated that AL animals were not fasted.^{7–10,12,14–16} Differences in experimental design across studies should be considered when interpreting their results.

When CR was initiated at a young age (14 weeks), maximal lifespan increased in both female and male Fischer 344 Brown Norway rats.⁶⁰ In contrast, initiating CR later in life (at 18 or 26 months) did not significantly extend longevity in male Fischer 344 Brown Norway rats.⁶¹ The longevity effects of late-life CR have not been reported for female Fischer 344 Brown Norway rats. Importantly, late-life CR can improve health and physiological function even when it does not extend lifespan. For example, CR initiated at 22.5 months in male Fischer 344 Brown Norway rats increased insulin-stimulated glucose uptake in skeletal muscle.⁶² Similarly, female Fischer 344 rats that began CR at 24 months showed improved insulin-stimulated skeletal muscle glucose uptake.⁶³ These findings suggest that CR-induced improvements in insulin sensitivity may be accompanied by, and potentially linked to, changes in skeletal muscle metabolite profiles.

A limitation of this study is that we did not perform necropsies or systematically evaluate each rat’s pathological status. As a result, we cannot rule out the possibility that undetected differences in pathology among animals influenced the outcomes that we report.

In conclusion, CR significantly improves metabolic health in both sexes. However, previous studies have shown that the mechanisms underlying the benefits of various interventions are often not identical between males and females.^64–67 Our results show that while most effects of CR on muscle metabolites are similar in both sexes, a subset of effects differ between females and males in older rats. These findings offer important insights for understanding both the shared and sex-specific mechanisms through which CR influences skeletal muscle metabolism, health, and function.

Supplementary material

Supplementary material is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online.

Funding

This work was supported by the National Institutes of Health (R21AG084931).

Conflicts of interest

None declared.

Data availability

Data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Author contributions

Conceptualization: Gregory D. Cartee and Haiyan Wang; Project Administration: Haiyan Wang; Investigation: Haiyan Wang and Jiwei Hao; Writing—Original Draft: Gregory D. Cartee and Haiyan Wang; Writing—Critical Review and Editing: Gregory D. Cartee, Haiyan Wang, and Jiwei Hao; Data Curation: Haiyan Wang; Formal Analysis: Haiyan Wang; Visualization: Haiyan Wang; Funding Acquisition: Gregory D. Cartee; Resources and Supervision: Gregory D. Cartee. All authors reviewed and approved the final version of the manuscript.