Metabolomic approaches are used to detect and analyze changes in the abundance of low molecular weight metabolites in biological samples [11]. Here we used GC–MS and multivariate statistical analysis to examine the metabolic profile of mice following CR with the aim of identifying metabolites that are associated with the protective effects of CR and may reveal the underlying physiologic mechanisms. CR improved markers of general health and body weight and reduced plasma lipid, fasting glucose (Figure 1). The GC–MS analysis revealed 35 metabolites showing differential abundance between the CR and control groups; several AAs, FAs, and metabolites related to the tricarboxylic acid cycle and urea cycle were present significant change in CR mice (Table 1). AAs participate in many metabolic pathways as well as protein synthesis and are an important energy source. The AAs that showed differential abundance between the 2 groups were glycine, l-isoleucine, l-proline, l-aspartic acid, l-serine, l-hydroxyproline, l-alanine, l-valine, l-threonine, l-glutamic acid, and phenylalanine. Consistent with these results, the KEGG functional pathways enriched under CR included Alanine, aspartate, and glutamate metabolism; Phenylalanine, tyrosine, and tryptophan biosynthesis; Arginine biosynthesis.
In this study, the main metabolite change is amino acids, which implied dietary protein may be the key determinant in CR on metabolism. CR diet with high protein cannot extend lifespan, mice fed a low protein diet lived longer than high protein diet [12]. Individuals in a high protein diet have a higher incidence of developing diabetes and cardiovascular mortality than a low protein diet [13, 14]. Randomized controlled trials indicated that humans fed a protein restriction diet can improve metabolic health, decrease cancer incidence and mortality rates in individuals under 65 [15]. CR delays aging through protein restriction act on cellular pathways, including AMPK, mTOR, and GH/insulin/IGF-1 pathways [16–19]. Dietary protein and AA can influence the level of insulin, IGF-1 and activity of mTOR [12, 20, 21]. Protein restriction acts on these pathways reduces anabolic responses, increases mitochondrial function and autophagy, and thus delays aging [22]. Mammalian target of rapamycin (mTOR) is a master regulator in cell metabolism that senses cellular energy status and integrates signals from nutrients, growth factors, and stress factors to promote cell growth [23]. Restriction of dietary protein protects the important organ and promotes longevity through inhibition of mTOR pathway, improves the stress resistance after hepatic injury and protects from renal ischemia reperfusion injury [24, 25]. Study found that reduced dietary protein inhibits tumor growth and also inhibits mTOR activity in tumor [26]. Chronic overnutrition increases mTOR signaling, which is associated with the development of insulin resistance (IR) [27]. CR in humans is able to increase insulin sensitivity, which is beneficial to health and longevity [28]. We found that the level of all most AA were decreased in the CR group in our study. In fasting condition, the utilization of energy diverted from cellular growth to protection, protection against oxidative stress by the repression of translation [29, 30]. mTOR regulates translation of mRNA by phosphorylating the translation inhibitors, promotes translation initiation of mRNA, thus decreasing mRNA translation by mTOR inhibition that slows aging [31]. Therefore, in our study the reduction of AA content may be associated with decreased mRNA translation.
We observed the level of l-isoleucine and l-valine were decreased compared to the control group in our study. Branched chain amino acids (BCAAs) may be an important role in AA regulation of metabolic health. Restricting the level of BCAAs in low protein diet can improve the mice metabolic health as same as low protein diet [20]. Amino acids can regulate IR, and reduction in fasting AA concentrations could improve insulin sensitivity effectively. Study showed that IR is associated with elevated concentrations of BCAAs and related metabolites [32]. The level of BCAAs was found to be increased in obesity, insulin resistance (IR), T2DM, coronary disease (CAD) and metabolic syndrome [33–36]. Alternation of the level in BCAAs could serve as biomarker for the metabolic syndrome. BCAAs was also found to be able to activate mTOR activity in cell culture, rodents and humans [37–40]. BCAAs restriction lower mTOR activity to improve metabolic health and longevity [15]. So, reduction of BCAAs in diet can improve IR and metabolic health by restricting mTOR activity. Moreover, BCAAs could exacerbate mitochondrial dysfunction by increasing acylcarnitine accumulation in muscle [41]. In heart failure, the accumulation of BCAA and its metabolites may cause oxidative stress and impair mitochondrial function [42, 43]. Thus, CR protects tissues against oxidative stress damage by reduction of BCAAs.
In addition, aromatic amino acids, phenylalanine and tryptophan are elevated in individuals with obesity and IR [36, 44]. Our study showed that the level of phenylalanine is significantly decreased in the CR group, which may be used as a biomarker for T2DM developing. Glutamic acid is used to synthesize glutamine, and the l-glutamine content regulates the mTOR pathway, preventing cellular uptake of l-glutamine leads to inhibition of mTOR signaling and activation of autophagy [45].
CR also provides a protective effect against obesity. Randomized clinical trial showed that non-obese adults had a significant weight loss thorough decreased visceral fat under CR diet [46]. Adipose tissue is the primary site for the synthesis of FAs, which are the main energy source under fasting conditions, TG stored in adipocytes hydrolyzed to fatty acids and release ATP after oxidation. In our study, 4 FA metabolites were significantly decreased in white adipose tissue of the CR group compared to control mice including palmitic acid, 1-monopalmitin (MG [16:0/0:0/0:0]), glycerol monostearate (MG [0:0/18:0/0:0]), and cholesterol. The levels of docosahexaenoic acid, 16-octadecenoic acid, oleic acid, stearic acid, and hexanoic acid were also altered by CR, which was associated with changes in glycerolipid metabolism and primary bile acid biosynthesis.
Elevated cholesterol is a main risk factor that induces the development of atherosclerosis, long-term CR results in beneficial effects on cardiovascular disease by decreasing cholesterol [47]. CR has been shown to increase the expression of genes involved in FA oxidation and decrease that of genes related to FA synthesis [48–51]. In obesity and T2DM, enhanced of adipose lipolysis cause the increasing of glycerol and fatty acids, and inflammatory cytokines, and lead to IR [52, 53]. CR exerts a potent anti-inflammatory effect, reduce inflammation and improve human health [54]. Furthermore, excessive FA could impair mitochondrial function of muscle and cause incomplete β-oxidation [55]. Our study showed that the levels of palmitic acid, 1-monopalmitin, and glycerol monostearate—which are markers of endogenous FA synthesis—were decreased in the adipose tissue of CR mice. Thus, the regulation of FA metabolism may be a health benefit of CR, which was supported by our finding that FA synthesis was decreased along with FA oxidation in different tissues of CR mice. CR may also reduce oxidative damage; FA oxidation increases the flavin adenine dinucleotide (FADH)/nicotinamide adenine dinucleotide (NADH) ratio, which suppresses reactive oxygen species production [56]. CR was shown to protect the heart by inhibiting cardiac remodeling and fibrosis and increasing contractility and energy generation via lipid β-oxidation in mice [57]. In a clinical trial, FA was more effectively mobilized and oxidized in the fasting state induced by CR [58]. The pathway of primary bile acid biosynthesis showed a significant change. Bile acids emulsify dietary fats, activate lipase, catalyze the hydrolysis of glycerol into fatty acids, and promote the absorption of these lipids [59]. Moreover, bile acids stimulate enterocyte secretion of gut hormones, glucagon-like peptide 1 (GLP-1), which augments insulin secretion to help maintain glucose homeostasis [60].