Small peptides: could they have a big role in metabolism and the response to exercise?

Exercise is a powerful non‐pharmacological intervention for the treatment and prevention of numerous chronic diseases. Contracting skeletal muscles provoke widespread perturbations in numerous cells, tissues and organs, which stimulate multiple integrated adaptations that ultimately contribute to the many health benefits associated with regular exercise. Despite much research, the molecular mechanisms driving such changes are not completely resolved. Technological advancements beginning in the early 1960s have opened new avenues to explore the mechanisms responsible for the many beneficial adaptations to exercise. This has led to increased research into the role of small peptides (<100 amino acids) and mitochondrially derived peptides in metabolism and disease, including those coded within small open reading frames (sORFs; coding sequences that encode small peptides). Recently, it has been hypothesized that sORF‐encoded mitochondrially derived peptides and other small peptides play significant roles as exercise‐sensitive peptides in exercise‐induced physiological adaptation. In this review, we highlight the discovery of mitochondrially derived peptides and newly discovered small peptides involved in metabolism, with a specific emphasis on their functions in exercise‐induced adaptations and the prevention of metabolic diseases. In light of the few studies available, we also present data on how both single exercise sessions and exercise training affect expression of sORF‐encoded mitochondrially derived peptides. Finally, we outline numerous research questions that await investigation regarding the roles of mitochondrially derived peptides in metabolism and prevention of various diseases, in addition to their roles in exercise‐induced physiological adaptations, for future studies.


Introduction
Compelling evidence shows that exercise is a cornerstone therapy for the primary prevention of ≥35 chronic conditions, including metabolic syndrome, obesity, type 2 diabetes (T2D), coronary heart disease and hypertension (Booth et al., 2012;Pedersen & Saltin, 2015).As with other therapies, there is evidence for a dose-response relationship, with a recent meta-analysis indicating that higher levels of physical activity are associated with a lower risk of mortality, irrespective of weight status (Tarp et al., 2022).Note that in this review, 'exercise' indicates a single session of structured physical activity, whereas 'exercise training' or 'training' signifies repeated exercise sessions.
Many of the beneficial effects of exercise have been attributed to adaptations to skeletal muscle (Thyfault & Bergouignan, 2020).Seminal works published in the 1960s were the first to provide mechanistic insights into how training-induced changes to skeletal muscle might contribute to improved health (Bergström et al., 1967;Hermansen et al., 1967).Much of this work has focused on training-induced changes to mitochondria (the powerhouses of the cells).For example, the eminent scientist John Holloszy reported that exercise training increased total mitochondrial protein content and the activity of mitochondrial enzymes in the gastrocnemius muscle of rats (Holloszy, 1967).This was followed by research showing training-induced mitochondrial adaptations in humans, including elevated abundance of mitochondrial proteins, increased mitochondrial content and greater mitochondrial respiratory function (Daussin et al., 2008;Granata et al., 2016a, b;Montero & Lundby, 2017; for a comprehensive review, please see Granata et al., 2018).Although much research has investigated these and other skeletal muscle adaptations contributing to the health benefits of exercise, the underlying molecular mechanisms that drive these changes remain largely unclear (Bishop et al., 2023).
The flow of genetic information within a biological system is described by the central dogma of molecular biology (i.e.'DNA makes RNA, and RNA makes protein'; Crick, 1958).In the context of exercise, Williams and Neufer were the first to hypothesize that it is the cumulative effect of transient changes in mRNA levels in response to each individual exercise session that determines subsequent adaptive responses (e.g.altered steady-state protein abundance and concomitant physiological adaptations) (Williams & Neufer, 1996).Most research in the field of the molecular biology of exercise has subsequently focused on understanding changes in mRNA and protein levels after a single exercise session and exercise training (Bishop et al., 2019;Bishop & Hawley, 2022;Egan & Sharples, 2023;Granata et al., 2018Granata et al., , 2021;;Menshikova et al., 2006;Tarnopolsky et al., 2007; for comprehensive reviews, please see Bishop et al., 2019;Egan & Sharples 2023;Granata et al., 2018).
In the last decade, however, there has been increased research into the roles of non-coding RNAs (ncRNAs), i.e.RNA transcripts not translated into proteins (Couso & Patraquim, 2017).Although these were initially misannotated as 'transcriptional noise' , some have subsequently been observed to have regulatory functions (Yao et al., 2019).Recent research has also revealed that several ncRNAs contain open reading frames (ORFs), including small open reading frames (sORFs), which are coding sequences that encode small peptides (<100 amino acids) that can be translated to produce polypeptides (Anderson et al., 2015;Bi et al., 2017;Bonilauri & Dallagiovanna, 2021;Magny et al., 2013).However, owing to the difficulty of identifying sORF-encoded peptides, the biological roles and response to exercise of many sORF-encoded peptides remain to be discovered.
Recent research supports the notion that many sORF-encoded peptides are likely to play a pivotal role in biological functions in all organisms, including humans (Guerra-Almeida & Nunes-da-Fonseca, 2020;Miller et al., 2022;Schlesinger & Elsässer, 2022;van Heesch et al., 2019).Emerging evidence indicates that some sORF-encoded peptides have significant roles in the mitochondrion, an organelle involved in reactive oxygen species signalling, ATP production through aerobic respiration, and Ca 2+ cycling (Johannsen & Ravussin, 2009).The nuclear genome encodes some of these polypeptides, which are subsequently translocated to the mitochondria.There are also ncRNAs containing sORFs within the mitochondrial DNA (mtDNA) that encode small polypeptides (Fig. 1).Of these mitochondrially derived peptides (MDPs), the discovery of humanin (HN), mitochondrial sORF of the 12S ribosomal ribonucleic acid (rRNA)-c (MOTS-c) and small humanin-like peptides (SHLPs) 1−6 has ushered in a new interest in mitochondrial biology (Cobb et al., 2016;Hashimoto, Niikura, Tajima, et al.,  2001; Kim et al., 2017;Lee et al., 2015).Given that exercise training increases mitochondrial biogenesis, it is tempting to speculate that sORF-encoded MDPs and some newly discovered nuclear-encoded small peptides might function as exercise-sensitive peptides that help to regulate exercise-induced physiological adaptations.In Table 1, we provide gene names, protein names, genomic regions, synonyms, localization and universal protein resource knowledgebase identifiers for MDPs and small peptides.
In this review, we highlight the discovery of MDPs and newly discovered small peptides that have been proposed to influence metabolism, with a specific emphasis on their potential roles in preventing metabolic diseases and regulating exercise-induced adaptations.Although few studies are available, we also critically analyse evidence for a role of exercise and exercise training in altering sORF-encoded MDPs.

Mitochondrially derived peptides
Humanin.Humanin was the first MDP to be described.It is a 24-amino-acid peptide encoded by a 75 bp sORF within the 16S ribosomal RNA (rRNA) of mtDNA and was identified in 2001 using a complementary DNA library from brain sections of people with Alzheimer's disease (Hashimoto, Niikura, Ito, et al., 2001;Yang et al., 2019).Humanin is present in other tissues, including the colon, testes, kidney, pancreas, heart and skeletal muscle, in both rodents and humans (Charununtakorn et al., 2016;Liu et al., 2019).In addition to its tissue distribution, HN is also present in cerebrospinal fluid and seminal fluid (Muzumdar et al., 2009).In response to physiological and pathophysiological stressors, including exercise and metabolic diseases, HN can be produced in the above-mentioned tissues and secreted into the circulation (Liu et al., 2019;von Walden et al., 2021).Given its wide distribution in the body, HN might be a key factor in coordinating biological functions between multiple tissues.

Mechanisms of action.
Humanin is believed to function via its binding to two types of specific receptors: formyl peptide receptor-like 1 (FPRL1) and a trimeric receptor complex, ciliary neurotrophic factor receptor alpha/WSX-1/gp130 (Lee et al., 2013).The binding of HN to these two receptors activates downstream stress kinases linked to multiple metabolic processes, including extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (PKB, also known as Akt) and signal transducer and activator of transcription 3 (STAT3) (Hashimoto et al., 2009;Kim et al., 2016).In addition, in mouse pancreatic β-cells treated with HN, there was an increase in the phosphorylation of AMP-activated protein kinase (AMPK, a master regulator of cellular energy metabolism and mitochondrial biogenesis) and the mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A, which encodes the transcription coactivator PGC-1α) and other genes associated with mitochondrial biogenesis (Qin et al., 2018).

Role in metabolism.
The present evidence suggests that HN and its analogues induce similar metabolic actions to those observed with AMPK induction, such as increased cellular ATP production in a cultured murine β-cell line (βTC3) in vitro (Kuliawat et al., 2013) and enhanced activation of glucose consumption and glucose transporter type 4 (GLUT4) in the membrane fraction in COV434 cells (Wang, Zeng, et al., 2021).Palmitate-induced lipid accumulation and insulin resistance are attenuated by HN treatment through AMPK phosphorylation in human primary hepatocytes (Kwon et al., 2020).Moreover, small interfering RNA-mediated knockdown of AMPK blunts the protective effect of HN on lipid accumulation and insulin resistance in human primary hepatocytes (Kwon et al., 2020).Increased mitochondrial mass was also reported in HN-treated cells, and this change was inhibited by compound c, which is a potent AMPK inhibitor (Qin et al., 2018).These findings suggest that increased mitochondrial biogenesis is mediated by the HN-AMPK signalling axis and that AMPK substantially contributes to the effects of HN.
Another possible downstream target of HN is Akt, with HNG, a potent HN analogue with a glycine substitution, increasing phosphorylated Akt (p-Akt) levels in SH-SY5Y human neuroblastoma cells and in the brain of mice (Kim et al., 2016;Xu et al., 2008).Humanin was also reported to improve insulin sensitivity by increasing p-Akt levels in the skeletal muscle of rats (Muzumdar et al., 2009).In particular, given that HN is involved in the activation of Akt signalling and that loss of Akt signalling causes the development of obesity and T2D (Brozinick et al., 2003;Huang et al., 2018;Krook et al., 1998), HN might play a key role in metabolic health via insulin signalling.
Both in vitro and in vivo studies have been performed to investigate the metabolic actions of HN and its analogues further.For example, murine β-cell lines, derived from islets isolated from wild-type (WT) and db/db diabetic mice and treated with different doses of an HN analogue, HNGF6A, increased insulin secretion in a dose-dependent manner (Kuliawat et al., 2013).Treatment with HN has also been reported to reduce weight gain, visceral fat and fasting blood glucose and to increase energy expenditure in mice fed a high-fat diet (Gong et al., 2015), and fasting glucose and inflammatory cytokines (tumor necrosis factor alpha and interleukin-6) in both the heart and plasma were decreased in HN-treated mice with diabetes mellitus (Jiang et al., 2022;Zhang et al., 2021).Also, Hoang et al. (2010) documented that a 6-week of HN treatment enhanced glucose tolerance in non-obese diabetic mice.Collectively, research suggests that HN and its analogues with single amino acid substitutions might have the potential to improve metabolic functions by activating cellular pathways associated with energy metabolism and modulating insulin and glucose metabolism, probably owing to their insulin-sensitizing properties.
Role in disease.Humanin was first denoted as a rescue factor, with a role in restoring cognitive functions because of its neuroprotective and anti-apoptotic functions (Hashimoto, Ito, et al., 2001;Hashimoto, J Physiol 602.4 Niikura, Ito, et al., 2001).Owing to these specific functions, HN is considered a potential treatment to improve/cure symptoms of Alzheimer's disease (Rochette et al., 2020).Research has also shown that exogenous treatment with HN and its analogues can improve the prognosis of metabolic diseases, such as insulin resistance, obesity, diabetes and polycystic ovary syndrome (PCOS), in multiple rodent models (Burtscher et al., 2023;Gong et al., 2015;Muzumdar et al., 2009;Wang, Li, et al., 2021;Wang, Zeng, et al., 2021).Although promising effects of HN have been reported for in vivo and in vitro experiments, further studies in humans are necessary to understand comprehensively the specific metabolic changes that occur with HN supplementation.
Researchers have investigated HN protein levels in the blood and tissue of humans with different diseases to shed light on the potential impact of HN for health.However, the findings reported by these researchers have been inconsistent and contradictory.For instance, circulating HN protein levels were significantly decreased in patients with impaired fasting blood glucose levels, T2D, gestational diabetes mellitus, endothelial dysfunction and ischaemic heart diseases, when compared with control patients (Ma et al., 2018;Ramanjaneya, Bettahi, et al., 2019;Voigt & Jelinek, 2016;Widmer et al., 2013;Zhloba et al., 2018).Serum HN protein levels were also reported to be negatively correlated with age, haemoglobin A 1c and blood glucose and positively correlated with preserved endothelial function (Di Francescomarino et al., 2009;Ramanjaneya, Bettahi, et al., 2019;Widmer et al., 2013).Also, a recent study has reported that the expression of HN was considerably reduced in the ovaries of patients with PCOS (a disease also associated with insulin resistance) compared with the control group (Wang, Li, et al., 2021).However, when rat models of PCOS were given exogenous HNG supplementation, there was a significant decrease in body weight gain, improvement in ovarian morphological abnormalities and a reduction in endocrinological disorders (Wang, Li, et al., 2021).These findings suggest that supplementation with HNG could potentially serve as a new therapeutic approach for insulin resistance present in patients with PCOS.In contrast, RNA sequencing of skeletal muscle showed that the content of HN mRNA was increased in individuals with obesity compared with lean individuals (Kwon et al., 2020).Likewise, patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), characterized by mutations in mtDNA, have increased expression of HN protein in skeletal muscle (Kariya et al., 2005a).That is possibly a tissue-specific response to overcome an energy-deficient status, because in vitro HN treatment increased ATP production (Kariya et al., 2005a, b).However, more work is needed to unravel the discrepancies observed in previous studies.

Humanin and exercise.
There is also some evidence (Table 2) that HN protein levels change in response to exercise and exercise training, and this depends on the type (resistance or endurance), intensity (high or moderate) and frequency of exercise.For example, it was reported that there was a significant increase (∼35%) in skeletal muscle HN protein content in prediabetic men after 12 weeks of resistance training, but no such change in a Nordic walking group, a form of physical activity and exercise that involves walking with the use of specially designed poles (Gidlund et al., 2016).However, it is worth noting that basal HN protein levels were higher in the Nordic walking group compared with the resistance training group.These higher baseline protein levels of HN could potentially have attenuated the exercise-induced expression in skeletal muscle and complicate the conclusion of exercise-specific effects on this peptide.A recent study investigated the effects of a single session of moderate-intensity cycling [45 min at 70% of maximal oxygen uptake ( VO 2 max )] compared with a single session of resistance exercise (four sets of seven maximum-weight repetitions of leg-press and knee-extension exercise) in healthy, active participants (von Walden et al., 2021).In this study, it was reported that there was a significant increase in plasma HN protein levels at 30 min and 3 h postexercise only after the cycling exercise (von Walden et al., 2021), showing that endurance exercise provides a more potent stimulus compared with resistance exercise in increasing circulating HN protein levels at the specified sampling times.It is probable that this elevation in plasma HN levels was reflected by changes in the muscle, given that a previous study reported parallel increases in plasma HN protein concentration and muscle protein levels of HN in young, healthy, untrained men after a single session of high-intensity interval exercise (HIIE) on a cycle ergometer (10 × 1 min efforts at the power output associated with peak oxygen uptake and separated by 75 s of rest) (Woodhead et al., 2020).
Summary.Humanin, an exercise-inducible MDP, shows promising effects in the management of various metabolic diseases in both rodents and humans.The role of this potential therapeutic peptide in exercise-induced adaptations and its ability to prevent metabolic diseases have yet to be explored fully, underscoring the necessity for further in-depth mechanistic studies.

Mitochondrial open reading frame of the 12S rRNA-c.
In 2015, a 16-amino-acid mitochondrial-encoded peptide named MOTS-c was discovered by Lee et al. (2015).MOTS-c is considered a mitokine, i.e. a soluble molecule produced and secreted into the circulation in response to mitochondrial stress (Wan et al., 2023).In the presence of metabolic stress (e.g.glucose restriction), MOTS-c translocates to the nucleus to regulate gene expression (Kim et al., 2018).It has a significant role in the stress response, as indicated by the analysis of HeLa human cervical cancer cells and HEK293 human embryonic kidney cells (Kim et al., 2018).MOTS-c has been detected in tissues with high mitochondrial content, such as skeletal muscle, brain, liver and kidney (Lee et al., 2015).Despite its recent discovery (Lee et al., 2015), there has been considerable scientific interest in the role of MOTS-c in the body.A search on PubMed yields >130 research articles containing 'MOTS-c' or 'mitochondrial open reading frame of the 12S rRNA-c' (as of 24 November 2023).

Mechanisms of action.
Compelling evidence suggests that MOTS-c acts in an AMPK-and sirtuin (SIRT) 1-dependent manner (Lee et al., 2015;Zarse & Ristow, 2015).Research shows that treatment with and overexpression of MOTS-c results in an enhancement of AMPK activation and increased mRNA levels within downstream pathways in mouse skeletal muscle, with a concomitant increase in the protein level of the downstream glucose transporter GLUT4 (Lee et al., 2015).
The knockdown of AMPK leads to a significant decline (ranging from 16 to 40%) in the glycolytic rate stimulated by MOTS-c in vitro, using HEK293 cells (Lee et al., 2015).To test the potential role of SIRT1 in mediating the glycolytic actions of MOTS-c, (Lee et al. (2015) used small interfering RNA and EX527, a SIRT1 inhibitor, in HEK293 cells stably overexpressing MOTS-c to knock down SIRT1.They reported that there was a 40% decrease in glucose-stimulated glycolytic rate and that the administration of EX527 resulted in a 45% decrease in MOTS-c-expressing cells compared with their control (Lee et al., 2015).Additionally, MOTS-c has been reported to increase time-and dose-dependent phosphorylation of Akt (Ser473) in HEK293 cells (Lee et al., 2015), supporting the notion that the enhanced effectiveness of infused insulin in MOTS-c-treated mice hinges predominantly on the activation of skeletal muscle Akt, along with a concomitant elevation in MOTS-c protein levels (Lee et al., 2015).These findings substantiate the role of AMPK, Akt and SIRT1 in mediating the actions of MOTS-c, thus unveiling important mechanistic insights into the biological effects of this newly discovered peptide.

Role in metabolism.
There is evidence supporting a role of MOTS-c in both lipid and glucose metabolism.
For example, MOTS-c treatment in mice was reported to reduce ovariectomy-induced lipid accumulation in white adipose tissue and high-fat diet-induced gains in visceral fat and body mass, and to increase β-oxidation (Lu et al., 2019).Furthermore, the role of MOTS-c in the regulation of adipocyte lipid metabolism was totally blocked by the addition of an AMPK inhibitor (Lu et al., 2019).The role of MOTS-c in glucose metabolism is supported by increased glucose uptake in cultured cells after MOTS-c overexpression (Lee et al., 2015), in addition to the prevention of ovariectomy-induced insulin resistance, and improved insulin sensitivity and glucose tolerance in mice (Kim, Miller, Mehta, et al., 2019;Lee et al., 2015;Lu et al., 2019;Reynolds et al., 2021;Zempo et al., 2021).Moreover, systemic treatment with MOTS-c also reduced non-fasting glucose levels, significantly improved the response to glucose tolerance tests in mice (Lee et al., 2015) and suppressed autoimmune diabetes in non-obese diabetic mice (Kong et al., 2021).Considering that a significant proportion (70-85%) of insulin-stimulated glucose disposal occurs in skeletal muscle, it is highly plausible that MOTS-c could improve insulin sensitivity and glucose homeostasis primarily through its actions within this tissue, thereby promoting efficient glucose management and contributing to overall metabolic health.In addition, research has also reported that MOTS-c can enhance mitochondrial function and mitochondrial homeostasis in aged human placenta-derived mesenchymal stem cells (Yu et al., 2021).Furthermore, the translocation of MOTS-c to the nucleus in response to metabolic stress generated by glucose restriction, serum deprivation or oxidative stress in vitro indicates that MOTS-c plays a role in the adaptive response to different cellular stressors.In summary, MOTS-c exerts profound systemic effects on skeletal muscle (Li & Laher, 2015;Zarse & Ristow, 2015).To mediate its effects, MOTS-c interacts with AMPK, Akt and SIRT1 to improve lipid oxidation, glucose metabolism and insulin sensitivity; this makes MOTS-c a promising therapy for the chronic treatment of metabolic dysfunction.However, research on the metabolic role of MOTS-c in humans remains limited.

Role in disease.
In support of the above roles, MOTS-c has been linked to many metabolic diseases.The present evidence shows a possible role of MOTS-c in diseases associated with altered metabolism, supported by the results of studies in which mice were injected with MOTS-c (Kong et al., 2021;Lee et al., 2015;Lu et al., 2019;Yang et al., 2021).Additionally, metabolic responses to MOTS-c treatment in cultured cells and animals might act to improve fitness and health span (Reynolds et al., 2021).Fuku et al. (2015) suggested that m.1382A>C polymorphism located in the MOTS-c-encoding mtDNA, which is frequent among Northeast Asians, might be one of the key biological mechanisms contributing to the exceptional longevity of the Japanese population.However, further mechanistic studies are required to replicate these findings, as emphasized recently in some reviews (López-Otín et al., 2016;Mottis et al., 2019;Quirós et al., 2016).In addition, MOTS-c protein levels were reported to reduce in mouse skeletal muscle and blood with age, with a concomitant increase in the age-dependent development of insulin resistance (Lee et al., 2015).Cross-sectional studies have also shown that circulating MOTS-c protein levels are lower in male children and adolescents with obesity and in adults with T2D (Du et al., 2018;Ramanjaneya, Bettahi, et al., 2019).Additionally, MOTS-c was reported to be negatively correlated with markers of insulin resistance and T2D and to be positively associated with body mass index, total cholesterol and LDL in humans (Du et al., 2018;Ramanjaneya, Bettahi, et al., 2019).In contrast, some recent studies reported that the plasma MOTS-c concentration was unaltered in human obesity but was positively associated with insulin resistance in lean individuals, and with android and liver fat in people without diabetes (Cataldo et al., 2018;Sequeira et al., 2021).Sequeira et al. (2021) also reported that individuals with metabolic syndrome had elevated plasma MOTS-c protein levels, which were positively associated with markers of metabolic syndrome, such as triglycerides, glucose, diastolic blood pressure and waist circumference.The varied findings in previous studies regarding MOTS-c protein levels could be attributed to divergent reasons.For example, these studies were conducted on different populations at diverse stages of various pathologies.Additionally, elevated MOTS-c levels might have functioned as a compensatory response to pathological conditions, such as mitochondrial stress and dysfunction.However, persistent stress conditions might lead to receptor desensitization, ultimately resulting in reduced MOTS-c protein levels.Nevertheless, these assumptions remain speculative.Hence, further research in this field would be valuable to understand fully the mechanisms underlying the action of MOTS-c.
Mitochondrial open reading frame of the 12S rRNA-c and exercise.Given that MOTS-c activates AMPK, an important 'energy sensor' (Lee et al., 2015), it has been speculated that MOTS-c might contribute to the putative role of mitochondria in transmitting exercise-induced signalling cascades (Friedman & Nunnari, 2014) and thereby stimulating physiological adaptation and increased tolerance to exercise (Merry et al., 2020;Reynolds et al., 2021).However, there have been few studies investigating the effect of exercise on MOTS-c expression in circulating blood and in skeletal muscle (Table 2); hence, it is not fully resolved whether exercise regulates the secretion and/or production of MOTS-c.For example, after a single session of HIIE, consisting of 10 × 60 s at peak aerobic power, with 75 s of recovery between each interval, there was a significant increase in the relative protein abundance of MOTS-c in human skeletal muscle immediately after exercise (11.9-fold) and after 4 h of rest (∼18 fold), and circulating serum MOTS-c concentrations also increased during (1.6-fold) and after (1.5-fold) the HIIE and returned to baseline after 4 h of rest (Reynolds et al., 2021).However, these findings should be interpreted with caution, because the reported changes had a high variability among participants and across technical replicates.Considering the potential influence of biological variability in MOTS-c protein changes in response to HIIE, further studies are necessary to replicate the findings to ensure conclusive results.In the only other human study, it was reported that there was no change in plasma or skeletal muscle protein levels of MOTS-c immediately and 3 h after 45 min of cycling at 70% of VO 2 max or immediately and 3 h after resistance exercise involving four sets of seven maximum-weight repetitions of leg-press and knee-extension exercise, with 5 min of rest between exercises and 2 min of rest between sets (von Walden et al., 2021).
In addition to exercise-induced changes in MOTS-c, a few studies have also investigated training-induced changes in MOTS-c in humans and mice.For example, training-induced increases in the skeletal muscle and plasma concentrations of MOTS-c in mice were reported after 4-8 weeks of treadmill running (Guo et al., 2020;Hyatt, 2022;Yang et al., 2021).In breast cancer survivors, Dieli-Conwright et al. (2021) reported that 16 weeks of resistance training combined with aerobic training significantly increased (1.6-fold) resting plasma MOTS-c protein levels.However, it is currently unknown whether the reported increase in plasma MOTS-c levels can be attributed to changes occurring within muscle tissue.Further investigation is needed to shed light on this potential connection.In contrast, Ramanjaneya, Jerobin, et al. (2019) observed that 8 weeks (three times per week) of aerobic training at 60% of VO 2 max for 60 min did not change resting plasma MOTS-c concentrations in healthy women or in women with PCOS.However, the study by Ramanjaneya, Jerobin, et al. (2019) might have been underpowered to determine the effects of the applied training protocol on MOTS-c owing to a relatively small sample size (n = 12) and groups that were not well matched for body mass index and age.Furthermore, another MDP, HN, has also been reported to be more responsive to resistance training compared with walking-based aerobic training in individuals with prediabetes (Gidlund et al., 2016).Given that their signalling pathways are similar, both HN and MOTS-c might have a greater response to resistance exercise, yet this assumption awaits to be tested.

Summary.
To summarize, MOTS-c is a recently discovered MDP that has attracted considerable attention owing to its effects on metabolism, such as improving glucose metabolism, fat oxidation, insulin sensitivity, body composition and mitochondrial homeostasis.However, our understanding of how MOTS-c treatment and/or restoring circulating and muscle levels of MOTS-c improves human health is still limited.
Small humanin-like peptides.Researchers have recently identified another group of MDPs, called SHLPs (SHLP1, SHLP2, SHLP3, SHLP4, SHLP5 and SHLP6), which are located within the same 16S rRNA region of mtDNA as HN (Cobb et al., 2016).Each SHLP consists of 20-38 amino acids, and the expression of SHLPs varies in different tissues of mice.Small humanin-like peptides are expressed in the kidney, liver and spleen, with SHLP1 also being detected in the heart, SHLP2 in skeletal muscle, SHLP3 in the brain, SHLP4 in the prostate and SHLP6 in the heart, liver and kidney (Cobb et al., 2016).Different SHLP transcripts were also detected in the human genome, as shown by the HUGO gene nomenclature committee (HGNC) (2023).

Mechanisms of action.
To date, there is limited research available on the mechanisms of action of SHLPs.For instance, one study presented evidence that the biological functions of SHLP2 might be mediated, in part, through the activation of ERK and STAT3 signalling pathways (Cobb et al., 2016).Additional investigations are needed to expand on these initial findings.

Role in metabolism.
Only a few in vitro and in vivo studies have investigated the cellular actions of SHLPs, and the exact metabolic actions of SHLPs are currently unknown.For example, overexpression of both SHLP2 and SHLP3 was reported to increase mitochondrial oxygen consumption and cellular ATP levels in 22Rv1 cells and to enhance insulin sensitivity in adipose tissue in vitro and in vivo (Cobb et al., 2016).Moreover, infusion of SHLP2 in rats improved glucose infusion rate, suppressed hepatic glucose production and increased glucose uptake in peripheral tissues (Cobb et al., 2016).A recent study investigating the mitochondrial action of SHLP2 has shown that SHLP2 treatment restored the levels of mitochondrial oxidative phosphorylation proteins, increased mtDNA copy number, prevented loss of mitochondria and increased PGC-1α mRNA levels in a human transmitochondrial age-related macular degeneration cell model (Nashine et al., 2018).Additional research to elucidate the cellular and systemic impacts of SHLPs moght reveal new targets for preventing and treating metabolic diseases, particularly those linked to disrupted energy regulation and mitochondrial dynamics.

Role in disease.
Of the limited evidence based on animal research, Cobb et al. (2016) reported that circulating SHLP2 protein levels were lower in old mice (18 months old) compared with young mice (3 months old).In contrast, plasma SHLP2 levels were elevated in patients diagnosed with metabolic syndrome, with an average age of 44 years, and in individuals with non-alcoholic fatty liver disease and liver damage (Sequeira et al., 2021).The elevation of SHLP2 levels in humans could potentially serve as a compensatory mechanism to counteract the metabolic dysfunction linked to these conditions.Similar to human studies, mice fed a high-fat choline-deficient diet had higher protein levels of SHLP2 in plasma than mice fed a chow diet (control) (Sequeira et al., 2021).In summary, the few studies conducted have identified the potential role of SHLPs, particularly SHLP2 and SHLP3, in mitochondrial metabolism and glucose metabolism, suggesting their potential roles in metabolic diseases.However, clinical trials on SHLPs and/or their analogues are needed to investigate whether there are protective effects of these peptides in clinical settings.
Small humanin-like peptides and exercise.Considering that SHLPs and exercise exert similar important metabolic effects, such as improved mitochondrial function and insulin sensitivity (Fig. 2), it is important to determine whether there is an association between exercise and SHLP protein levels.The only study investigating the effects on SHLPs of exercise, performed by young healthy untrained men, reported no change in plasma SHLP2 protein levels in response to a single session of HIIE (10 × 1 min at VO 2 peak oxygen uptake and power output) or after six sessions of HIIE over 2 weeks (three times per week) (Woodhead et al., 2020).This might be attributable to the relatively short duration of the training intervention and/or the nature of the study population, suggesting the need for additional exercise studies involving longer training programmes and in different populations.In contrast, a single session of the same HIIE increased plasma SHLP6 protein levels, followed by a rapid return to baseline, and six sessions of HIIE led to an overall lower plasma concentration of SHLP6 protein compared  (von Walden et al., 2021;Woodhead et al., 2020) or via exogenous injection.B, mitochondrially derived peptides increase mitochondrial biogenesis (Qin et al., 2018), fat oxidation (Kim et al., 2021) and endurance capacity (Dieli-Conwright et al., 2021;Reynolds et al., 2021).C, increased protein levels of mitochondrially derived peptides reduce weight gain (Gong et al., 2015), lipid accumulation (Kwon et al., 2020) and insulin resistance (Cobb et al., 2016;Gong et al., 2015;Kim, Miller, Kumagai, et al., 2019;Kim et al., 2021;Reynolds et al., 2021).
with pretraining levels (Table 2) (Woodhead et al., 2020).This inconsistency between the plasma levels of SHLP2 and SHLP6 in response to exercise might reflect the tissue-specific expression of these two MDPs.
Summary.Small humanin-like peptides might be exercise-induced MDPs that potentially have effects on improving metabolism and treating metabolic diseases.Notably, given that SHLP2 treatment increases the gene expression of PGC-1α, the master regulator of mitochondrial biogenesis, the role of SHLPs in exercise-induced mitochondrial biogenesis awaits to be determined.Furthermore, age-dependent changes in the protein levels of SHLPs might contribute to the development of age-related diseases in humans.However, this area of research awaits further investigation to explore the potential therapeutic role of SHLP treatment in mitigating ageing-induced metabolic dysfunction and to determine whether replenishing SHLPs levels might have therapeutic potential in certain age-related diseases.

Mechanisms of action.
The present evidence suggests that MTLN exhibits its biological activity (e.g.enhancing β-oxidation) through its specific interaction with the mitochondrial trifunctional protein (the enzymatic complex that catalyses the last three steps of long-chain fatty acid oxidation) in the inner mitochondrial membrane (Makarewich et al., 2018).This suggests that MTLN has an important role in metabolism by engaging with a larger protein complex.Further research should be undertaken to determine whether additional protein complexes are involved in the biological functions of MTLN.
Role in metabolism.Mitoregulin has been reported to alter metabolism via effects on substrate utilization and mitochondrial respiratory function (Lin et al., 2019;Makarewich et al., 2018;Stein et al., 2018).For example, heart and skeletal muscle mitochondria isolated from MTLN knockout (KO) mice exhibit a reduced capacity to metabolize fatty acids, whereas MTLN overexpression in transgenic mice results in increased β-oxidation (Makarewich et al., 2018).This supports the role of MTLN in fatty acid oxidation and in maintaining energy homeostasis during increased metabolic demand.In addition to its role in cellular metabolism, studies in cultured human HeLa cells show that MTLN is also engaged in the regulation of Ca 2+ metabolism and the mitochondrial membrane potential (Stein et al., 2018).In mice, silencing of MTLN results in a decrease in basal oxygen consumption and VO 2 max and in lower ATP production by mitochondria, whereas overexpression of MTLN increases basal oxygen consumption, VO 2 max and ATP production (Lin et al., 2019).These actions are potentially mediated by interactions of MTLN with various mitochondrial molecules in striated muscle cells, such as cardiolipin, cytochrome B5 reductase 3 and the mitochondrial trifunctional protein (Chugunova et al., 2019;Makarewich et al., 2018;Stein et al., 2018), which have been proposed to influence mitochondrial protein complex assembly and/or stability (Chicco & Sparagna, 2007;Martin-Montalvo et al., 2016;Rector et al., 2008).Notably, a recent study by Friesen et al. (2020) reported that in human pluripotent stem cell-derived adipocytes, MTLN controls mitochondrial β-oxidation of long-chain fatty acids and triglyceride clearance by regulating lipolysis.The regulation of lipolysis by MTLN is supported by a greater accumulation of triglycerides in adipocytes of MTLN KO compared with MTLN WT mice fed a high-fat diet (Averina et al., 2023;Friesen et al., 2020).

Role in disease.
Although there has been some encouraging progress in unravelling the effects of MTLN on β-oxidation, we are still far from a complete understanding of the potential role of MTLN in disease.Based on these findings, it is likely that clearance of excess lipids in adipose tissue by MTLN treatment could help to combat the obesity pandemic, and this promising action of MTLN leaves this area of research fertile for drug discovery and development.However, gaps in the literature remain, and further studies should determine whether this small molecule could be a target peptide in the prevention of obesity and insulin resistance by improving β-oxidation.
Mitoregulin and exercise.The important role of MTLN in regulating metabolism supports the hypothesis that MTLN might also be an exercise-sensitive peptide that helps to regulate exercise-induced physiological adaptations.Two studies have described the potential role of MTLN on exercise capacity in mice (Makarewich et al., 2018;Wang et al., 2020).One study has reported a significant decrease in exercise capacity and running distance and time in MTLN KO mice compared with WT mice (Makarewich et al., 2018).Another study confirmed the reduced treadmill run time to exhaustion of MTLN KO versus MTLN WT mice (Wang et al., 2020).This improved endurance performance was associated with increased β-oxidation (Makarewich et al., 2018;Wang et al., 2020), which results in more efficient energy utilization during exercise, attributable, in part to less skeletal muscle glycogen use, less muscle lactate production and a lower respiratory exchange ratio.However, no study to date has investigated the response of MTLN in humans to either a single session of exercise or a period of exercise training.Summary.To summarize, MTLN is a recently identified micropeptide that translocates to the inner mitochondrial membrane, where it exerts significant impacts on β-oxidation, energy metabolism and exercise capacity by interacting specifically with the mitochondrial trifunctional protein.Additionally, given that MTLN enhances mitochondrial efficiency by increasing basal oxygen consumption, VO 2 max and ATP production and that there is improved or impaired exercise performance with MTLN overexpression or silencing of MTLN, respectively, it is highly likely that MTLN is involved in mitochondrial regulation.However, these assumptions based on limited studies remain speculative and await further research to draw a firm conclusion on the role of MTLN in exercise-induced mitochondrial adaptations.
Dwarf open reading frames.A recently discovered peptide, dwarf ORF (DWORF), is a nuclear peptide of 34 amino acids in mice and 35 amino acids in humans that is encoded by a muscle-specific long non-coding RNA and has a molecular weight of 3.8 kDa (Nelson et al., 2016).DWORF RNA has been detected in the heart, soleus and diaphragm of mice, with robust expression in slow-twitch muscle tissues (Nelson et al., 2016).DWORF mRNA was reported to be reduced in human ischaemic heart failure tissue (Nelson et al., 2016).

Mechanisms of action.
Dwarf open reading frames is a direct activator of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) pump and is the only micropeptide known to activate muscle-specific SERCA, making DWORF an endogenous enhancer of SERCA activity.DWORF, a novel transmembrane peptide that is localized to the sarcoplasmic reticulum membrane, has been reported to increase Ca 2+ uptake and myocyte muscle contractility mainly by counteracting the effects of phospholamban, sarcolipin and myoregulin, i.e.SERCA inhibitor peptides (Fisher et al., 2021;Li et al., 2021;Nelson et al., 2016).
Role in metabolism.Activity of SERCA contributes ≤20% of daily total energy expenditure and 40-50% of resting metabolic rate in mouse fast-and slow-twitch skeletal muscle (Gamu et al., 2020;Smith et al., 2013).Also playing a pivotal role in Ca 2+ homeostasis, SERCA contributes to muscle contraction by transporting cytosolic Ca 2+ back into the lumen of the sarcoplasmic reticulum (Xu & Van Remmen, 2021).In this context, the appropriate operation of SERCA appears essential for preservation of skeletal muscle health, consequently enhancing the overall well-being of the individual (Morales et al., 2023).Therefore, enhancing SERCA activity through the upregulation of DWORF expression might offer a significant therapeutic strategy for increasing energy expenditure and facilitating enhanced energy use, ultimately leading to a decrease in fat accumulation.However, this assumption requires in-depth research with a focus on mechanistic insights.Also, previous findings demonstrated that impaired SERCA activity and expression resulted in impaired Ca 2+ handling in obese and diabetic skeletal muscle (Eshima, 2021).It was shown that pharmacological activation of SERCA reduces body weight increases in obese mice (Maurya et al., 2015).Although increases in energy expenditure with DWORF therapy might be possible, this area of research has yet to be explored in either rodents or humans, and more comprehensive research is needed to determine the precise physiological role of DWORF in energy metabolism.

Role in disease.
Excess fat storage, caused by a chronic imbalance of energy intake and energy expenditure, contributes to obesity and many metabolic diseases.Given this, there is much interest in therapies or pharmacological interventions that can reduce fat storage and improve health.Although there is evidence that pharmacological activation of SERCA can ameliorate diabetes-associated conditions in a genetic model of insulin resistance and T2D (Gamu et al., 2020;Kang et al., 2016), it is not known whether similar effects can be achieved safely via DWORF therapy.Furthermore, although mitochondrial Ca 2+ overload attributable to high levels of cytosolic Ca 2+ uptake into mitochondria can lead to impaired mitochondrial function, reduced ATP production and increased release of reactive oxygen species (Hyatt & Powers, 2020;Powers et al., 2020;Santulli et al., 2015), it is not known whether exogenous treatment with Dwarf open reading frames and exercise.Acute deterioration in the sarcoplasmic reticulum Ca 2+ release rate is one of the essential factors for the development of muscle fatigue, resulting in reduced Ca 2+ release and Ca 2+ uptake, especially during high-intensity exercise (Li et al., 2002;Place et al., 2015).In contrast, exercise training is a preventive mechanism known to improve the sarcoplasmic reticulum Ca 2+ release rate.In this context, Gejl et al. (2020) recently reported a 10% increase in the sarcoplasmic reticulum Ca 2+ release rate in trained road cyclists and triathletes after 4 weeks (three times per week) of endurance training in combination with high-intensity exercise cycling.Furthermore, DWORF overexpression is associated with enhanced SERCA activity, peak Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load, with a concomitant reduction in the time constant of cytosolic Ca 2+ decay during the contraction-relaxation cycle (Nelson et al., 2016).Therefore, the extent to which DWORF mediates the exercise-induced increase in Ca 2+ release and Ca 2+ uptake remains to be determined.
Several attempts have been made at understanding the effects of exercise on SERCA and Ca 2+ activation in human and mouse skeletal muscle (Duhamel et al., 2007;Kinnunen & Mänttäri, 2012).For instance, both a single exercise session and exercise training have been reported to elevate SERCA levels in both humans and rats (Bupha-Intr et al., 2009;Kubo et al., 2003;Morales-Alamo et al., 2020;Morissette et al., 2014).In addition, although no study has investigated exercise-induced changes in DWORF directly, it was reported that seven sessions of sprint exercise (four to six 30 s all-out sprints, separated by 4 min of recovery) led to a 40% reduction in the protein expression of sarcolipin, a direct target of DWORF (Fisher et al., 2021), in the vastus lateralis muscle in young men with a body mass index <25 kg m −2 .However, sarcolipin expression did not change with 4 days of low-intensity exercise involving 45 min of one-arm cranking followed by an 8 h walk in overweight individuals (Morales-Alamo et al., 2020).Moreover, Morales et al. (2023) revealed that DWORF gene therapy improved treadmill performance in mice.However, it is not known whether different types of exercise or training interventions would induce DWORF expression and increase the amount of Ca 2+ pumped back into the sarcoplasmic reticulum in humans or rodent models.Although the direct physical interaction between DWORF and SERCA raises the possibility that exercise might exert similar effects on DWORF, this hypothesis has not been tested.
Summary.The micropeptide DWORF is a positive regulator of the SERCA pump, exhibiting the capacity to amplify SERCA activity.Despite certain attempts to probe into the role of DWORF in metabolic diseases and metabolism, further comprehensive investigations are required in this area of research.
BRAWNIN.In the last few years, sORF-encoded peptides have received scientific interest from researchers studying the human proteome.This has led to the identification of a new member of sORF-encoded peptides called BRAWNIN (BR) (Zhang et al., 2020).BRAWNIN is a mitochondrial localized sORF peptide of 71 amino acids encoded in the nucleus by C12orf73.BRAWNIN is present in mouse skeletal muscle and in human skeletal and cardiac muscle, where it displays similar staining patterns to mitochondria using immunofluorescence imaging techniques (Zhang et al., 2020).
Mechanisms of action.Zhang et al. (2020) reported that activation of AMPK using 5-aminoimidizole-4-carboxamide-1-β-d-riboside (AICAR) in HEK293T cells and PGC-1α in mouse myotubes resulted in a robust increase in protein levels of BR, with depletion of BR causing impaired mitochondrial ATP production.BRAWNIN has been shown to be essential for mitochondrial respiratory complex III (Liang et al., 2022;Zhang et al., 2020).Based on these findings, it seems tempting to speculate that BR is highly regulated by the AMPK-PGC-1α energy homeostasis axis, and it is likely that a loss of BR would lead to a certain degree of deterioration in cellular bioenergetics and mitochondrial ATP production.Further studies should investigate in detail the potential molecular mechanisms through which BR achieves its full function at the cellular level.
Role in metabolism.BRAWNIN is highly responsive to energy status, as reflected by an increase in BR levels in glucose, serum and fatty acid with starvation, in parallel with AMPK activation (Zhang et al., 2020).It is also well documented that the energy-sensing AMPK regulates energy homeostasis, stimulates ATP production when required, improves glucose and lipid metabolism, and alleviates pathological conditions, such as obesity, insulin resistance and T2D (Herzig & Shaw, 2018;Jeon, 2016;O'Neill, 2013).Similar to AMPK, PGC-1α increases fatty acid oxidation, oxidative phosphorylation and mitochondrial energetics and improves diabetes and obesity-associated conditions (Gureev et al., 2019;Handschin & Spiegelman, 2008;Huang et al., 2017).In this context, considering that regulation of BR is mediated by the AMPK-PGC-1α axis (Liang et al., 2022), it is likely that BR is a promising peptide that could play a pivotal role in metabolic processes.However, although overexpression of AMPK and PGC-1α resulted in a significant increase in BR levels, the extent to which BR mediates the metabolic roles of AMPK and PGC-1α is largely unknown.In addition to the new findings presented by Liang et al. (2022), overexpression or silencing of BR in cell culture experiments and BR transgenic rodent models might be valuable to discover the potential crosstalk of BR with other signalling pathways and molecules to gain a better understanding of its role in metabolism.

Role in disease.
The limited number of in vitro and in vivo studies available in the literature suggests that the absence of BR is closely linked to an impairment of mitochondrial function, which has been proposed to be a major factor leading to a number of metabolic diseases, such as insulin resistance, obesity and diabetes (Kim et al., 2008).Additionally, BR protein levels have been reported to be correlated with the levels of mitochondrial respiratory chain proteins in brown adipose tissue, cardiac tissue and skeletal muscle (Zhang et al., 2020).However, no study has yet explored the role of BR in tackling the pathological conditions that underlie metabolic diseases through the enhancement of mitochondrial functions, pointing to this important area for future research.
BRAWNIN and exercise.Although the findings of Zhang et al. (2020) suggest that exercise might increase BR induction by activation of the AMPK-PGC-1α axis, it is not unknown whether exercise can induce BR expression or to what extent exercise-induced physiological adaptations are mediated by BR.Nonetheless, a recent study reported that compared with WT mice, BR KO mice had reduced exercise capacity, as reflected by a decrease in time to exhaustion and maximal running speed (Liang et al., 2022).
Summary.BRAWNIN is a mitochondrial localized sORF peptide encoded in the nucleus that has a significant potential impact on mitochondrial ATP production, which is likely to be mediated by the AMPK-PGC-1α energy homeostasis axis.Further research is required to advance our understanding of the role and regulation of BR in pathological conditions and exercise-mediated physiological adaptations.

Mitochondrially derived peptides and nuclear-encoded small peptides in inter-organ/inter-tissue communication
Accumulating research suggests the impact of exercise on the release of extracellular vesicles into the circulation (Vechetti et al., 2021).These systemic messengers can carry a variety of exerkines, which are signalling molecules released in response to either acute or chronic exercise that exert their effects through endocrine, paracrine and/or autocrine pathways (Chow et al., 2022) and provide a means for tissue communication during and after exercise (Safdar & Tarnopolsky, 2018;Vechetti et al., 2021).Additionally, although skeletal muscle contraction is widely considered to be a potent stimulus for mitochondrial stress during exercise, leading to the release into the circulation of MDPs with potential endocrine, paracrine and autocrine functions, there is currently no solid evidence regarding the intercommunication between MDPs and tissue-tissue interactions.However, data from various animal models have shown that the administration of exogenous MDPs and analogues improved metabolic function in adipose tissue, liver, brain, pancreas, heart and skeletal muscle (Cobb et al., 2016;Hoang et al., 2010;Xu et al., 2008;Zhong et al., 2022).Furthermore, a few studies have reported increased protein levels of MDPs, both in the circulation and in skeletal muscle, after exercise and exercise training (Hyatt, 2022;von Walden et al., 2021;Woodhead & Merry, 2021;Woodhead et al., 2020).These studies emphasize the potential significance of the release of MDPs from the skeletal muscle, facilitating communication with various tissues and organs.From this perspective, investigating non-skeletal organs as potential sources of release of MDPs and nuclear-encoded small peptides and the intercommunication between these peptides and tissue-tissue interactions remain crucial avenues for future research.Moreover, additional research is required to enhance our understanding of the relationship between these exercise-induced peptides and tissue-tissue crosstalk and to ascertain whether extracellular vesicles serve as carriers for the transport of MDPs and nuclear-encoded small peptides.

Future perspectives
Recognizing their pivotal role in cellular energy regulation and metabolism, mitochondria hold potential as a promising therapeutic target for the prevention of metabolic diseases.However, despite the encouraging findings thus far, existing research on the metabolic role of MDPs and nuclear-encoded small peptides in humans remains limited.It is likely that additional research will elucidate the potential of these peptides in preventing metabolic diseases in humans, in addition to their roles in exercise-induced physiological adaptations.In this section, we highlight numerous research questions and propose new avenues and approaches for future studies to address knowledge gaps regarding MDPs and nuclear-encoded small peptides.
Directions for future studies on MDPs and nuclear-encoded small peptides.To gain a better understanding of the promising therapeutic effects of these peptides and their analogues, further investigation is warranted.For example, further in-depth mechanistic studies are needed to uncover receptors and downstream targets of these peptides and the signalling pathways with which they interact, contributing to an understanding of the intracellular action of MDPs and nuclear-encoded small peptides.These investigations might contribute to J Physiol 602.4 the development of new pharmacological agents, remedies and more potent agonists that can be implemented in the treatment of metabolic diseases.
The origin and the tissue-specific expression of MDPs and small peptides in response to physiological (i.e.exercise) and pathophysiological (i.e.hyperglycaemia, hyperinsulinemia) states remain largely unknown owing to technical difficulties, such as the lack of validated antibodies, enzyme-linked immunosorbent assay kits and primers designed for gene-expression analysis, owing to poor gene annotation.To address these gaps, traditional genome editing tools, such as mitochondria-targeted zinc-finger nucleases or mitochondria-targeted transcription activator-like effector nucleases, might be useful (Yin et al., 2022).Likewise, CRISPR/Cas9 genome editing technology might contribute to a better understanding of the role of these peptides in metabolism (Uyhazi & Bennett, 2021).These tools can be used to identify whole-body or tissue-specific effects of these peptides through diverse techniques, such as gene KO experiments, analysis of proteomic changes to the cell using mass spectrometry, conducting mass spectrometry-linked protein pulldown assays that look for proteins bound to MDPs and small peptides, and identifying proteins that are up-or downregulated as part of the response induced by MDPs and small peptides.Nevertheless, these techniques have some limitations, including their high cost, the labour-intensive nature of their implementation and the often minimal area of DNA available for designing primers for micropeptides (Yin et al., 2022).Functional screening and genetic manipulation in human pluripotent stem cells might enable the determination of gene function, with further research required to understand the mechanistic action of these peptides (Zhu & Huangfu, 2013).Likewise, investigating the metabolic effects of the gain or loss of function of MDPs and small peptides is an additional crucial area that awaits more investigation.
Given that multiple animal models for different types of metabolic diseases are available, further investigations should explore the role of MDPs and nuclear-encoded small peptides as therapeutic treatments.Notably, considering that mtDNA is susceptible to oxidative stress and exhibits higher mutation rates than nuclear DNA (Yakes & Van Houten, 1997;Zempo et al., 2021), it is important to understand sequence variants of MDPs coded within mtDNA.This would probably enable the determination of potential protective or deleterious mutations and polymorphisms.Furthermore, the cellular action of MDPs and nuclear-encoded small peptides in metabolically active cell lines derived from skeletal muscle, adipose tissue and the liver, especially in humans, remains a fruitful area for further research.
An understanding of how MDP treatment and/or restoration of the circulating and muscle protein levels of MDPs improves human health is also lacking.Given their cytoprotective functions (Gong et al., 2014;Hazafa et al., 2021;Kuliawat et al., 2013), further research should explore whether the notable effects exerted by these promising peptides are present in humans, particularly among individuals with metabolic diseases.Also, elucidating the mRNA and protein levels of MDPs and small peptides in different tissues or even in extracellular vesicles of clinical populations might reveal new interactions and roles of these peptides in health and disease, thereby providing different possibilities to enable disease prevention or management.Such investigations would contribute to development of a deeper understanding of the role of these promising peptides in the human health span.Additionally, they could help to determine whether metabolic disorders involving mitochondrial dysfunction are linked to MDPs and could be prevented by administration of MDPs.Moreover, an increase in MDPs in response to certain disease states could be linked to the potential involvement of these peptides in compensating for metabolic stress.However, the present body of research is limited, making it challenging to draw solid conclusions.Cross-sectional research should be conducted to determine circulating levels of MDPs and nuclear-encoded small peptides in individuals with various metabolic diseases (i.e.insulin resistance, T2D and obesity) and the association of MDPs with the metabolic markers linked to these diseases and impaired energy metabolism.
Directions for future exercise studies.Despite some attempts made to identify effects of exercise on MDP expression, additional studies are warranted to understand the role of MDPs in mediating the therapeutic benefits of exercise.As mentioned above, there are several varying findings in previous studies, some of which might be attributed to different participant characteristics (prediabetic vs. healthy) and the study design used (acute vs. chronic interventions).From this point of view, randomized controlled and well-planned human studies that compare the effects of different exercise interventions on MDPs will contribute significantly to clarifying these inconsistent results.
It is important to elucidate what changes occur in the mRNA and protein expression of these peptides in both serum/plasma and skeletal muscle after a single session of exercise and exercise training.This is an important area of research that will contribute to determining the extent to which MDPs and nuclear-encoded small peptides mediate exercise-induced metabolic benefits, such as an increase in β-oxidation and endurance capacity.In addition, research is needed to shed light on whether alterations in expression of MDPs and nuclear-encoded small peptides after exercise are correlated with athletic performance.Further studies involving different types of exercise and training interventions with/without infusions of MDPs, micropeptides and their analogues are warranted.Studies involving individuals who are overweight or obese and have mitochondrial dysfunction are particularly needed to explore the extent to which these peptides mediate the health benefits of exercise.
Owing to the complex nature of skeletal muscle, which is composed of multiple cell types, single-cell RNA sequencing and proteomics could offer new insights into the cellular response of MDPs and small peptides to exercise and training.Owing to the diverse range of pathways in which micropeptides and MDPs have been implicated, there might be fibre-specific patterns of expression that contribute to the glycolytic and oxidative phenotypes present in type I and type II fibre types, respectively.We are not aware of any studies in humans that have investigated the response of MDPs or nuclear-encoded small peptides in different skeletal muscle fibre types to either a single session of exercise or a period of exercise training

Conclusion
In the last 10 years, the beneficial effects of exercise on mitochondria and energy metabolism have been outlined by several exemplary reviews (Bishop et al., 2019;Gabriel & Zierath, 2017;Granata et al., 2018;Hawley et al., 2014;Pedersen & Febbraio, 2012).Recent research has focused increasingly on the identification of new genes and proteins in the nuclear or mitochondrial genome with roles in exercise-induced adaptation (Miller et al., 2020;Pillon et al., 2020;Williams et al., 2021).From the perspective of the interaction between mitochondria and exercise, the relationship between MDPs and exercise has received considerable attention (Fig. 3).Despite being a new area of research, the number of studies investigating the role of MDPs and nuclear-encoded small peptides has increased rapidly in recent years.Given the limited information highlighting their significant roles in metabolism, specifically involving the mitochondria, there is likely to be ongoing scientific inquiry into the role of these peptides in human health.Notably, investigation of the possible pathways through which MDPs enhance metabolic well-being remains an important area of research, with significant implications for the management of diseases characterized by mitochondrial dysfunction.Moreover, in-depth investigations should explore the possibility that additional transcripts currently designated as non-coding might contain sORFs that encode functional proteins.Micropeptides of this type could potentially serve as pivotal regulators within essential biological processes, in addition to offering exciting opportunities for future targeted therapeutics for the management or treatment of numerous diseases.Also, recently discovered nuclear-encoded small peptides (MTLN, DWORF and BR) seem to have a potential impact on mitochondrial function and metabolism similar to exercise interventions (Fig. 3).To our knowledge, no data have conclusively shown interactions of these small peptides with exercise in humans (Fig. 3).By exploring the interaction of MDPs and nuclear-encoded small peptides with exercise and how these peptides respond to various exercises and training interventions, it is likely that some of the unexplored molecular mechanisms responsible for the many beneficial adaptations to exercise would be elucidated.

Figure 1 .
Figure 1.Schematic representation of the location of mitochondrially derived and nuclear-encoded small peptides in the human mitochondrial genome Mitochondrial open reading frames of the 12S rRNA-c (MOTS-c) is encoded from the 12S region.Humanin and small humanin-like peptides (SHLPs) are encoded from different 16S regions of mitochondrial DNA.Abbreviations: BR, BRAWNIN; DWORF, dwarf open reading frames; MTLN, mitoregulin; sORFs, small open reading frames.

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Physiol 602.4    DWORF can alleviate such effects of mitochondrial Ca 2+ overload.

Table 1 . Gene names, protein names, genomic regions, synonyms, localization and universal protein resource knowledgebase identifiers of mitochondrially derived peptides and small peptides
* Gene name, protein name, genomic region and Genbank Accession are according to the Human Genome Organisation (HUGO) nomenclature committee.†Peptidesused in this work are listed with their universal protein resource knowledgebase (UniProtKB) identifiers.Abbreviation: CDS, coding sequence.

Table 2 . Characteristics and main findings of studies investigating responses of humanin, MOTS-c, SHLP2 and SHLP6 to a single session of exercise and exercise training in humans Peptide
Abbreviations: F, female; HIIE, high-intensity interval exercise; HN, humanin; HR max , maximal heart rate; M, male; MOTS-c, the mitochondrial open reading frame of the 12S rRNA-c; NR, not reported; RM, maximum repetition; SHLP, small humanin-like peptide; VO 2 max , maximal oxygen uptake; VO 2 peak , peak oxygen uptake.