The greatest significant influence on human life span and health is inevitable ageing. Most individuals on earth are middle-aged or older, and in high-income nations, the proportion of those over 60 is increasing faster than that of any other age group (Beard et al., 2016). The number of Americans 65 years old and above was anticipated to be around 43.1 million in 2012, and by the year 2050, it is expected to increase to 83.7 million (Ortman et al., 2014). By 2050, 1.6 billion people over 65 are expected to live on the planet (Roberts et al., 2018). This demographic transformation is one of the most significant socio-economic issues of the current decade because ageing is linked to several comorbidities that lead to fast-rising health care expenses (Gasior et al., 2012). One of the distinguishing characteristics of ageing is the gradual decrease of muscle mass and physical function, often known as sarcopenia (Roubenoff, 2000). Sarcopenia typically begins in the fifth decade of life (Miljkovic et al., 2015; Cruz-Jentoft et al., 2010; Clark and Manini, 2012). It can affect mobility and increase the risk of falling, thus increasing the cost of healthcare and hospitalisation, which increases the economic burden within the society (Chung et al., 2018). Since sarcopenia is now recognised as a significant clinical phenomenon, interdisciplinary efforts are being made to recognise, comprehend, prevent and treat this disorder (Cooper et al., 2013; Jean-Yves Reginster, 2016).

Insight into the molecular mechanisms that cause ageing has advanced remarkably. Sarcopenia may result from various age-related molecular and cellular damage, also known as the hallmarks or pillars of ageing. These include loss of proteostasis, dysregulated nutrient sensing, stem cell exhaustion/dysfunction, cellular senescence, genomic instability, mitochondrial dysfunction and epigenetic alterations (López-Otín et al., 2013) (Kennedy et al., 2014). One of the most significant roles of skeletal muscle is its exceptional ability to produce force and power. Strength, or the maximum capacity of skeletal muscle to generate force, is a function of the muscle’s cross-sectional area and the nervous system’s ability to activate the corresponding motor fully. According to cross-sectional research, older adults strongly correlate muscle size and maximum strength. However, a more nuanced association has been found in studies that tracked changes in muscle strength and mass over time (Goodpaster et al., 2006) (Lauretani et al., 2003).

The simultaneous decrease of skeletal muscle mass and strength with age affects independence and quality of life by reducing locomotor performance and jeopardising metabolic health. This is shown in the solid epidemiological links between sarcopenia and dynapenia, risk of non-communicable diseases (such as diabetes, chronic obstructive pulmonary disease and cancer), and ensuing morbidity and mortality (Nichols et al., 2019; Newman et al., 2006). Developing methods for stratification, identifying targets for treatment, and triaging those in need of early intervention could all be accomplished by identifying molecular signals linked to muscle ageing (Sood et al., 2015; Xia et al., 2013).

In recent years, utilising omics technologies (such as transcriptomics, proteomics and metabolomics) has allowed researchers to better understand the underlying causes of various illness states and create biomarkers for either diagnosis or prognosis (Bhattacharya et al., 2014; Zierer et al., 2015). For instance, metabolomic methods have already provided insight into alterations in the muscle metabolome with ageing. A recent study by Fazelzadeh and others into age-related changes in muscle metabolome revealed that metabolites linked to mitochondrial function, fibre type and tissue turnover varied between age groups (Fazelzadeh et al., 2016). It is anticipated that local levels of metabolites will reflect the age-related changes in gene expression levels in muscle. According to a recent animal study, age impacts muscle glucose and fatty acid metabolism (Houtkooper et al., 2011). In another study, it was found that ageing causes changes in lipid and glucose metabolism that are specific to certain muscle groups and are consistent with mitochondrial dysfunction (Garvey et al., 2014).

Intriguingly, baseline levels of metabolites from the tricarboxylic acid cycle were reported to be lower in healthy older adults than in younger people in muscle biopsies. These differences were accompanied by lower concentrations of ATP, ADP, branched-chain amino acids, and acylcarnitine in older, healthy subjects, suggesting impaired mitochondrial function or a lower number of mitochondria in the older subjects’ muscle, which in turn may be a result of the older subjects’ lower habitual physical activity (Fazelzadeh et al., 2016). Biochemical features of mammals’ skeletal muscle have been shown to change in the course of ageing, mainly within the energy metabolism system (Ermini 1976). This study looks into untargeted metabolomics whereby all the low molecular weight compounds or metabolites that are present and participate in biochemical reactions at a particular time are captured and quantified (Horgan and Kenny 2011). Utilising a metabolomic-based approach could provide insight into the overall molecular mechanisms of disease progressions and how a therapeutic agent works at a metabolic level.

The metabolic basis of sarcopenia is not very much known, and there have been no reports on how tocotrienol can affect the metabolic profiling of skeletal muscle in ageing and sarcopenia. Sarcopenia has been shown to decrease metabolic activities in mitochondria, muscle, kidney, and methylation compared with the declined metabolic activities for antioxidation in frailty (Kameda et al., 2020). There has been growing evidence that tocotrienol, which is a vitamin E isomer, can prevent the onset of age-related chronic illnesses and metabolic disturbances, like obesity (Zhao et al., 2016; Fukui 2019; Pang and Chin 2019), cardiovascular disease (Ramanathan et al., 2018), type-2 diabetes mellitus (Chung et al., 2020; Mahjabeen et al., 2021), arthritis (Haleagrahara et al., 2014; Radhakrishnan et al., 2014; Chin et al., 2019), osteoporosis (Shen et al., 2017) and sarcopenia (Khor et al., 2014; Khor et al., 2016; Chung et al., 2018; Lim et al., 2019).

Vitamin E comprises two subgroups: tocopherols and tocotrienols (Schneider 2005). It has been established that tocotrienols have superior anti-inflammatory and antioxidant activities compared to α-tocopherols (Peh et al., 2016). Palm oil is the most abundant source of tocotrienols, referred to as tocotrienol-rich fraction (TRF). Using palm oil as a lipid carrier appears to enhance the bioavailability and solubility of the treatment, improving the permeability and effectiveness of medications, stabilising the emulsification of formulation between emulsifier and surfactant, and extending the shelf life of the therapeutic agent. Palm oil has demonstrated beneficial behaviour in giving variety in medicine design, shape and delivery (Goon et al., 2019). The long-chain triglycerides in palm oil are enzymatically processed before being taken up by enterocytes and absorbed into the bloodstream (Goon et al., 2019).

The skeletal muscle ageing process is typically accompanied by impaired muscle metabolism, including mitochondrial dysfunction and insulin resistance (Distefano and Goodpaster, 2017). However, strategies to slow ageing have mostly failed to achieve their desired response. As a result, the current study aimed to identify any potential impacts of TRF on several metabolic pathways in the ageing Sprague Dawley rat’s muscle tissue.

Sustaining the health of one’s muscles requires a sufficient intake of vitamin E. In a previous study using animal models, severe tocopherol deficiency has been observed to impair muscle performance even though vitamin E deficiency is rarely reported in humans (Nier et al., 2006; Rafique et al., 2004). Decrease in muscle mass and function in sarcopenia has been reported to be correlated with low vitamin E levels, as shown by the physical performance test. Invecchiare in Chianti study reported that the level of vitamin E daily intake positively correlates with knee extension strength and total the physical performance (Cesari et al., 2004) indicating the requirement of vitamin E in maintaining muscle strength and function.

In this study, physical performance was observed by conducting the weight-loaded performance swim test and the grid hang test. The results of our study showed that muscle strength was reduced with ageing. Treatment with TRF for 3 months however, increased the exhaustion time in weight-loaded performance swim test of young rats eventhough no similar effects were observed in adult and old rats treated with TRF. This might occur because the young TRF-treated rats have more significant muscle regeneration potential than the adult or old TRF-treated rats. The ability of muscles to regenerate relied entirely on the renewal of satellite cells, which eventually declined with age (Dumont et al., 2015). It has been previously reported that ageing causes the number of satellite cells to decrease (Miljkovic et al., 2015). According to a study by Khor et al., 2016, senescent myoblasts have low proliferative capacities, which showed downregulation of myogenic differentiation genes but increased expression of oxidative damage-associated genes. This study may explain the reduced ability of adults and old TRF-treated rats to regenerate muscle in this study.

This study also demonstrated that supplementation of TRF to young, adult and old rats for 3 months could reduce the levels of DNA damage indicating the capacity of TRF to reduce damaged DNA due to excessive ROS generation. This finding was in line with a previous study which reported that tocotrienols are rich vitamin E derivatives that aid in preventing DNA damage brought on by ageing (Chin et al., 2019; Georgousopoulou et al., 2017; Pathak et al., 2021; Taridi et al., 2014). A previous randomised clinical trial involving middle-aged and older adults supplemented with tocotrienol for 6 months showed a significant reduction in DNA damage by 3 months, which is a benefit that persisted through the 6 months of the study (Chin et al., 2019).

In this study, the effect of TRF supplementation on young, adult and old Sprague Dawley rats and its molecular mechanism was elucidated using an untargeted metabolomic approach. The animal model used in this study has been reported to be appropriate for studying sarcopenia (Shavlakadze and Grounds, 2006) (Chai et al., 2011; Schiaffino and Mammucari, 2011; Pötsch et al., 2014; Kob et al., 2015; Tarantini et al., 2019). Aged rat models are the most natural ageing model and have morbidities and mechanisms reasonably similar to those found in humans with sarcopenia (Baek et al., 2020). Rats grow quickly and at around 6 weeks old, they are sexually mature, and after five or 6 months, they are socially mature. An adult animal’s month is roughly similar to 2.5 years in a human (Andreollo et al., 2012). The decline in physical activity that occurs with ageing causes metabolic changes and worsens metabolic abnormalities (Pataky et al., 2021).

In this study, the metabolites profile of the control groups was compared to provide insight into the metabolites regulated by ageing. Distinctive characteristics of ageing skeletal muscle metabolomes were summarised in Figure 8. Our results showed the reduction of alanine, phenylalanine, tyrosine and glycine as well as the branched-chain amino acids (BCAAs)—isoleucine, leucine and valine in the old control group indicating an imbalance between protein synthesis and breakdown during ageing. Interestingly, none of these amino acid alterations was present in the young or adult groups, indicating that ageing affects dysregulated amino acid metabolism. A biomarker of muscle protein degradation, 3-methylhistidine, was noticeably downregulated in the muscle tissue of the elderly control group. This metabolite is not recycled but quantitatively excreted in the urine and has been proposed as an index of muscle protein turnover.

One of the most striking metabolite changes with age is in the level of biochemical markers of glucose metabolism. Levels of glycolytic intermediates decreased significantly in the muscle of adults and old rats. 3-Phosphoglycerate and phosphoenol pyruvate were each detected with decreased fold change in the adult and old control groups besides downregulation of nicotinamide adenine dinucleotide (NAD), a co-enzyme central to metabolism. Glycolysis is the primary pathway of glucose metabolism for energy production (Xiang et al., 2021). A decrease in glycolytic intermediate means that there is an impairment in energy production. It is often believed that ageing causes glucose intolerance, and skeletal muscle is the primary site of glucose uptake (Kohrt and Holloszy, 1995). Previous research reported that resting skeletal muscle glycogen stores were 60% lower in old than young individuals (Meredith et al., 1989). This suggests an age-related impairment in the glycogen synthase pathways (Consitt et al., 2019). Other studies have reported that skeletal muscle glycogen synthase activity is diminished in older adults compared to younger individuals (Pehleman et al., 2005; Poulsen et al., 2005; Biensø et al., 2015).

Carnitine is an amino acid derivative that functions as a transporter of long-chain fatty acids from the cytosol to the mitochondrial matrix for fatty acid β-oxidation (Gnoni et al., 2020). Acylcarnitine is a metabolite of fatty acid transportation whereby fatty acyl CoA will be released from acylcarnitine for fatty acid oxidation. Decanoyl carnitine and L-palmitoyl carnitine are medium-chain acylcarnitine, an intermediate of fatty acid β-oxidation by-products (Ramos-Roman et al., 2012). It is also a marker for incomplete fatty acid oxidation (Mai et al., 2013), and acylcarnitine accumulation is a marker for mitochondrial dysfunction (Al-Bakheit et al., 2016). This study shows an increase in decanoyl carnitine and L-palmitoyl carnitine levels in ageing rats, which is in line with recent reports that suggest the rise of acylcarnitine levels may be caused by the impairment of lipid oxidation or a natural response to an excess lipid supply (Ramos-Roman et al., 2012). It is known that lipid oxidation indeed decreases in elderly individuals (Calles-Escandón and Poehlman, 1997).

Phosphoenolpyruvate is an intermediate for both glycolysis and gluconeogenesis. It can be converted to pyruvate by pyruvate kinase to produce energy (Hamasaki et al., 1978). Recent studies have reported that the ageing of C. elegans is highlighted by a progressive decline in cytosolic phosphoenolpyruvate carboxykinase, which is the enzyme responsible for converting pyruvate to phosphoenolpyruvate in gluconeogenesis (Yuan et al., 2016). This study shows a decrease in phosphoenolpyruvate in the adult control compared to the young control group indicating inefficient glycolysis and gluconeogenesis. Thus, changes in phosphoenolpyruvate with age are likely involved in energy metabolism that acutely affects the physiology of ageing organisms, thus impacting the ageing process (Feng et al., 2016).

In this study, TRF supplemented group was compared with the RBD palm olein supplemented group as a control to provide insight into the different metabolites being regulated by TRF supplementation in young (3 months old), adult (9 months old) and old (21 months old) rats. The RBD palm olein was used in this study as it is the carrier for TRF. It is the liquid form of palm oil that is edible and is commonly used for cooking, which is easily accessible. TRF used in this study was extracted from the Malaysian palm tree Elaeis guineensis. Palm-based TRF is easily accessible and has been studied for its therapeutic properties, mainly in Malaysia. On the other hand, the Westerns are more inclined toward annatto oil or rice bran oil-based TRF. What differs between these oils is the amount of vitamin E homologs in them. Annatto and rice bran oil-based TRF encompasses a higher concentration of δ-tocotrienol and a lower amount of tocopherol (Frega et al., 1998; Burdeos et al., 2012; Allen et al., 2017). Contrarily, palm oil is one of the most abundant natural sources of tocotrienols, which contains up to 800 mg/kg weight of the α- and γ-tocotrienol isotypes in crude palm oil (also known as tocotrienol-rich fraction). In palm oil, vitamin E is distributed as 30% tocopherols and 70% tocotrienols (Sen et al., 2010). Therefore, utilising palm-based TRF in this study may produce robust knowledge in the metabolomic analysis as both tocotrienol and tocopherol would affect multiple genes and metabolic pathways (Pang and Chin, 2019).

Rats supplemented with TRF for 3 months showed an increase in spermine in the adult treated group. The same metabolite was downregulated in the adult control rats when compared to young control group. Spermine, which is oxidised by spermine oxidase, is an essential regulator of muscle gene expressions and fibre size. Studies have shown that a repressed level of spermine leads to muscle atrophy (Ceci et al., 2017). The decrease in spermine with ageing observed in this study was counter-regulated by TRF as shown by the increase of spermine with TRF treatment indicating that supplementation of TRF can reverse the repression of spermine due to ageing. Another metabolite that was found to be upregulated with TRF treatment in adult rats was nicotinamide adenine dinucleotide (NAD). Previous research has reported a decline in NAD⁺ concentrations with ageing in a tissue-specific manner (Braidy et al., 2011; Massudi et al., 2012). NAD⁺ is a coenzyme that facilitates redox reactions in all living cells mainly for energy production (Obrador et al., 2021). The upregulation of NAD⁺ in this study after 3 months of TRF supplementation in the adult treated group coincides with the TRF antioxidant properties that may have reversed the ageing-decline of NAD⁺ and promoted energy production.

Another metabolite highly downregulated with TRF treatment in the adult group was citric acid, which shows a 17.30-fold downregulation in the treated group compared to the control group. Citric acid is an intermediate of the tricarboxylic acid (TCA) cycle (Baldwin and Krebs 1981), which is involved in a metabolic pathway converting carbohydrates, proteins and fats into carbon dioxide and water to produce energy. Evidence demonstrates that energy production progressively decreases with age, mainly due to the decline in mitochondrial function (Bratic and Trifunovic 2010). A decrease in the level of citric acid shows usage of the metabolite in the TCA cycle, which in this study is shown in the TRF-treated group. Thus, showing that TRF supplementation reverses the age-associated decrease in energy production.

In this study, glycerol 3-phosphate was highly upregulated with ageing. Glycerol 3-phosphate is produced from the phosphorylation of glycerol by a glycerol kinase. The source of glycerol can be from the breakdown of triacylglycerol or triglycerides via lipolysis. Glycerol 3-phosphate is converted to dihydroxyacetone phosphate (DHAP), an intermediate of glycolysis and gluconeogenesis by glycerol 3-phosphate dehydrogenase (mGDPH) utilising NAD⁺ via a reversible reaction. Studies have shown that loss of mGDPH attenuates muscle regeneration, while overexpression of it ameliorated dystrophic pathology in mice (Liu et al., 2018). Muscle regeneration decreases with age as ageing causes satellite cells to malfunction, influencing their regenerative and self-renewal capacities (Sousa-Victor et al., 2014). Other studies have also shown how age reduces glycerol 3-phosphate (Nemutlu et al., 2015). The findings of this study showed that there was an accumulation of glycerol 3-phosphate in old rats which was alleviated by TRF treatment. This agrees with a previous study, which reported that TRF increases the proliferation capacity of senescent myoblasts, thus increasing muscle regeneration (Khor et al., 2016). The accumulation of glycerol 3-phosphate was linked with increased NAD⁺, because less NAD⁺ is converted to NADH due to less conversion of glycerol 3-phosphate to DHAP. In this study, an upregulation of NAD⁺ was seen after 3 months of TRF supplementation in the adult group.

As for the muscle metabolomes expression in the old group of rats, several metabolites particularly the amino acids were downregulated. TRF treatment however, increased the expression of aspartic acid, phenylalanine, tryptophan and histidine besides upregulating N6-Me-adenosine, cytidine, xanthine, indoleacrylic acid and N-acetylneuraminic acid in old rats. Aspartic acid is important in glutamate flux through the TCA cycle and glutamate transamination (Birsoy et al., 2015). A previous study showed that aspartic acid levels decreased in old age (Lo et al., 2020) which was similar to the findings of this current study. However, with TRF treatment, the aspartic acid level was upregulated by 8.98-fold in old group of rats. This shows that TRF supplementation reverses the age-declining cause of aspartic acid involvement in biochemical pathways of TCA cycle activity (Lancha et al., 2009).

The metabolites profiled also detected an upregulation of N6-Me-Adenosine. Researchers in Bonn, Germany, have reported that adenosine, a metabolite and signaling molecule, can enhance muscle function and brown fat during ageing and obesity. N6-methyladenosine is a methylation product which occurs in the N6-position of adenosine, the most prevalent internal modification of eukaryotic mRNA. Many reports have suggested that N6-methyladenosine modulates gene expression, which regulates cellular processes, including cell self-renewal, differentiation, invasion and apoptosis (He et al., 2019). Recent studies have shown that N6-methyladenosine plays an essential function in skeletal muscle development by regulating myoblast proliferation and differentiation during myogenesis and is the key to biological processes and regulatory pathways during skeletal muscle development (Zhang et al., 2020). N6-methyladenosine can accurately regulate stem cell differentiation and reprogram it during stem cell development by modulating the gene expression involved in the corresponding processes (Wang et al., 2019). Similar to the function of glycerol 3-phosphate, which plays a role in muscle regeneration, our results showed that N6-methyl adenosine was downregulated in old rats when compared to the young and adult control groups. However, after 3 months of TRF supplementation, the concentration of N6-methyl adenosine has increased in both adult and old groups of rats, showing that TRF supplementation might be involved in facilitating the muscle regeneration process. Significant metabolic changes between the control and treated group were summarised in Figure 9.

The muscle performance finding from this study was limited as there were logistic issues due to the Covid-19 pandemic and the lack of proper software to observe the behaviour of the rats during the muscle performance tests. Therefore, further study is needed to explain the effect of TRF on muscle performance. However, the molecular analysis of the mechanism of TRF in reducing the level of oxidative stress in muscle supported by the modulation of the metabolic pathways observed in the present study strengthens the properties of TRF as a potential agent to prevent age-related muscle degeneration and sarcopenia.

Our data indicate that TRF improves muscular function, and delays muscle degeneration due to ageing in Sprague Dawley rats. Our findings also showed the principal variations in muscle metabolomes due to ageing as shown in the old and young groups of rats which were related to tissue turnover, fibre size and mitochondrial function. The effect of tocotrienol-rich fraction on reducing several metabolites such as glycerol 3-phosphate and upregulating several amino acids and N6-Me-adenosine are likely to ameliorate muscle regeneration and cell self-renewal in the old group of rats. The mechanisms underlying the modulation in skeletal muscle metabolomes expression following TRF supplementation positively impact the potential treatment of sarcopenia in aged rats.