Mitochondrial and metabolic dysfunction in ageing and age-related diseases

Abstract

Organismal ageing is accompanied by progressive loss of cellular function and systemic deterioration of multiple tissues, leading to impaired function and increased vulnerability to death. Mitochondria have become recognized not merely as being energy suppliers but also as having an essential role in the development of diseases associated with ageing, such as neurodegenerative and cardiovascular diseases. A growing body of evidence suggests that ageing and age-related diseases are tightly related to an energy supply and demand imbalance, which might be alleviated by a variety of interventions, including physical activity and calorie restriction, as well as naturally occurring molecules targeting conserved longevity pathways. Here, we review key historical advances and progress from the past few years in our understanding of the role of mitochondria in ageing and age-related metabolic diseases. We also highlight emerging scientific innovations using mitochondria-targeted therapeutic approaches.

Ageing is characterized by a gradual loss of normal physiological function, culminating in frailty, a lack of resilience and increased susceptibility to several diseases, including cancer, as well as neurodegenerative, cardiovascular and metabolic diseases¹, culminating in death^2,3.

The increase in life expectancy observed since the beginning of the 20th century has been so rapid that the possibility that ageing is simply caused by genetic factors can be ruled out⁴. Moreover, individuals who survive to particularly old ages (>90 years of age), who often do not develop health problems, are predominantly in the so-called ‘blue zones’ of the world (for example, Ikaria in Greece and Okinawa in Japan)⁵. Remarkably, these populations are not genetically different from their neighbours, but their environment and lifestyles, including social networks, might have a role in their ability to stay healthy for longer than people in other areas⁵. Additional factors, such as diet, education, consistent physical activity⁶, conditions during early life and parental health⁷, have considerable effects on mortality. Although people from most nations are living longer than previously, their health span is not increasing⁸. Thus, preventing late-life morbidities should be an urgent goal in the field of gerontology.

Nine hallmarks of mammalian ageing have been described and are now generally accepted² (BOX 1). Notably, cellular metabolism interconnects these hallmarks of ageing². Here, we review the role of mitochondria in metabolism and how mitochondria contribute to age-related metabolic diseases, which might guide us in designing therapies targeting metabolic disorders to extend human health span and lifespan.

Box 1 |. The hallmarks of ageing.

In a seminal review in 2013, López-Otín and colleagues took on the task of providing some order to the multitude of biological pathways that are involved in the ageing process by proposing that all factors implicated in ageing are organized into nine categories, or hallmarks. Criteria for being hallmarks are that the process has to be present during normal ageing and affects the pace of ageing, if experimentally manipulated. The list includes primary hallmarks, whose effects are undoubtedly negative, such as: genomic instability, telomere loss, epigenetic alteration and loss of proteostasis. Antagonistic hallmarks, whose effects can be beneficial or deleterious depending on their intensity, are deregulated nutrient sensing, mitochondrial dysfunction and cellular senescence. Last are the integrative hallmarks that affect the homeostasis and functionality of the tissue and include stem cell exhaustion and altered intercellular communication².

Notably, even though several interventions that target one of the hallmarks have dramatic benefits on the ageing process in different organisms, such as improving nutrient sensing pathways or proteostasis^299,300, not all hallmarks completely fulfil the last criterion, which consists of slowing down the ageing process by ameliorating the hallmark. This latter requirement remains the most difficult to achieve, mainly due to the pervasive interconnectedness among the ageing hallmarks. The challenge is to improve our understanding of the exact causal network and the interdependence among the hallmarks to develop targeted therapies to improve their function and slow down the ageing process.

Mitochondria

Mitochondria are essential for life, as they have a pivotal role in most fundamental energy transformation processes within a cell. Mitochondrial dysfunction can lead to severe impairment of cellular energy conversion, especially in tissues that rely heavily on chemical energy generated by mitochondria throughout life⁹. Hence, regulation of energy metabolism by environmental variations and excessive levels of the by-product reactive oxygen species (ROS) might be associated with age-related metabolic diseases through constitutive changes in mitochondrial oxidative phosphorylation (OXPHOS), that ultimately lead to mitochondrial dysfunction.

Oxidative phosphorylation.

(OXPHOS). A metabolic pathway in which nutrients are oxidized, releasing energy in the form of ATP resulting from electron transfer from NADH and FADH₂ to O₂.

Crosstalk with longevity pathways

In the past few years, advances in ageing research have indicated that interventions to treat diseases affecting multiple organs could be developed, thus increasing health span and delaying the onset of later-life morbidities. The effects of these novel interventions can be, to some extent, interpreted within the realm of mitochondrial physiology. Ageing and age-related diseases are tightly related to an imbalance in energy supply and demand. This imbalance might be alleviated by various interventions, including physical activity, calorie restriction and agents such as metformin, resveratrol and rapamycin¹⁰.

Although the mechanisms behind calorie restriction-driven longevity extension are still poorly understood, mitochondrial physiology is involved in the beneficial effects of calorie restriction in budding yeast¹¹ and Caenorhabditis elegans¹², as well as in mammals¹³. When carbohydrates and, consequently, calories are in excess, OXPHOS and antioxidant defences are downregulated, and the electron transport chain (ETC) remains in a chronically reduced state. This environment favours ROS production, mitochondrial DNA (mtDNA) mutagenesis and, consequently, premature cell death. By reducing the excess of ingested calories, calorie restriction interventions move the ETC away from a reduced state, lower ROS production, induce OXPHOS and antioxidant defences, prevent age-related diseases and increase lifespan¹⁴.

Electron transport chain.

(ETC). A series of electron transporters (complexes I–IV) embedded in the inner mitochondrial membrane that transport electrons from NADH and FADH₂ to O₂, in which O₂ is reduced to water. In parallel, a proton efflux is driven from the mitochondrial matrix towards the intermembrane space via proton pumps in complexes I, III and IV. The movement of protons back into the matrix occurs through ATP synthase (also known as complex V or FoF1 ATPase).

Evidence for reciprocal and multilevel interactions between longevity pathways, together with the intricate regulation of mitochondrial physiology under calorie restriction interventions¹⁵, has opened the door to the hypothesis that mitochondria might be involved in the lifespan modulation of these pathways. Altogether, this system is multifactorial and highly dynamic, and controls the ageing process (FIG. 1).

Fig. 1 |. Longevity signalling pathways control mitochondrial health.

Caloric restriction reduces the concentrations of glucose, amino acids and lipids, while raising the concentrations of key metabolites, such as NAD⁺ and AMP. These metabolites modulate the activity of metabolic sensors, such as the sirtuins (SIRTs), AMP kinase (AMPK), the target of rapamycin (TOR) and insulin–IGF1 signalling (IIS). Downstream to metabolic sensors, transcription factors, such as forkhead box O (FOXO) and the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), orchestrate mitochondrial physiology and homeostasis. Dysregulation of this multilevel regulatory system reduces mitochondrial homeostasis, resulting in frailty and disease. Ac, acetyl group; ACC, acetyl-CoA carboxylase; LKB1, liver kinase B1; ULK1, UNC51-like kinase 1.

IIS

Suppression of the insulin–insulin-like growth factor 1 (IGF1) signalling (IIS) pathway dramatically extends lifespan in multiple organisms, which suggests that it is an evolutionarily conserved mechanism¹⁶.

Extensive overlap in gene expression has been found between the C. elegans insulin receptor DAF-2 mutant and long-lived mitochondrial mutant strains¹⁷, with the DAF-2 mutant showing an overall decrease in mitochondrial protein turnover¹⁸. Moreover, DAF-2 mutants have increased abundance of proteins involved in OXPHOS, increased respiratory capacity and increased membrane potential¹⁹. The detected increase in respiratory capacity in DAF-2 mutants, contrary to what was observed in adult wild-type N2 worms, is in agreement with findings showing increased mitochondrial ROS production¹⁹. Importantly, this increase in ROS production was observed without any detectable adverse effect in mtDNA integrity and oxidative protein damage¹⁹. In addition, increased ROS levels induced expression of antioxidant enzymes, followed by a decrease in ROS levels²⁰. Thus, these results suggest that inhibition of IIS improves mitochondrial bioenergetics together with a ROS-mediated adaptive response, contributing to the lifespan extension effects observed in C. elegans.

Mitochondrial morphology and dynamics, which undergo several changes in the neurons of C. elegans during ageing²¹, are also affected by IIS. In DAF-2 mutants, age-related changes in mitochondrial morphology and density are mitigated, and mitochondrial trafficking is maintained at under normal levels in adulthood²¹. Moreover, mitophagy is enhanced in DAF-2 mutants²², and exposure to mitophagy modulators increases lifespan in worms^23,24. Manipulation of a gene involved in mitophagy, dct-1, a nematode orthologue of the mammalian receptors NIX and BNIP3L²², also shortens lifespan in DAF-2 mutants²².

Mitophagy.

A process that selectively degrades damaged mitochondria following damage or stress.

Work in mammals has also shown an important connection between IIS and mitochondrial physiology. In the long-lived Ames dwarf mice and growth hormone receptor knockout (GHR-KO) mice, which develop defects in the GH–IGF1 signalling pathway, mitochondrial bioenergetics is markedly improved. This improvement was demonstrated by the metabolic shift to β-oxidation, increased oxygen consumption rate and decreased respiratory quotient²⁵, accompanied by increased expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) as well as proteins involved in OXPHOS²⁶. Likewise, fat-specific insulin receptor knockout (FIRKO) mice have increased lifespan compared with wild-type mice despite having a leaner phenotype from a young age (3 months of age) and normal food intake throughout life²⁷. Expression of genes with protein products involved in key metabolic processes, such as glycolysis, the TCA cycle, β-oxidation and OXPHOS were increased in 2.5–3.0-year-old FIRKO mice compared with age-matched control mice²⁸. Interestingly, the same phenotype was not observed when the insulin receptor was disrupted in liver, adipose and muscle tissues in adult mice²⁹, denoting the complex role of the mammalian IIS pathway.

β-Oxidation.

A metabolic process involving multiple steps of fatty acid molecule breakdown to produce energy.

Respiratory quotient.

The ratio used to measure basal metabolic rate by estimating the volume of carbon dioxide released in relation to the volume of oxygen produced during respiration.

TCA cycle.

A series of chemical reactions taking place in the mitochondrial matrix that uses acetyl-CoA derived from carbohydrates, fat and proteins to provide electrons to the electron transport chain.

mTOR

Mammalian TOR (mTOR) is a ubiquitously expressed serine–threonine protein kinase, belonging to the PI3K family, that has a vital role in regulating cytoplasmic nutritional and growth factor status, as well as cellular energy levels and overall cellular function³⁰. mTOR is present in two complexes, mTORC1 (associated with ‘raptor’) and mTORC2 (associated with ‘rictor’), accounting for the diversity, specificity and selectivity of rapamycin-mediated inhibition of TOR signalling, which was discovered after it was shown that rapamycin is able to inhibit mTORC1 via binding to the protein FKBP12 (REFS^31,32).

Despite the fact that a definite mechanism has not yet been elucidated, the inhibition of mTORC1 and consequent increase in autophagy³³ has gained some attention from the mitochondrial field. Decreased clearance of accumulating damaged organelles, such as mitochondria, has been highly associated with ageing and age-associated diseases³⁴ and might explain some of the beneficial effects that mTOR inhibition has on lifespan extension. Nevertheless, the mTORC1 complex is required for PGC1α binding to the transcription factor Ying Yang 1, which induces expression of nuclear-encoded mitochondrial genes³⁵. Moreover, modulation of mTOR activity results in decreased mitochondrial bioenergetic capacity. Deletion of the mTORC1 component raptor in mouse skeletal muscle affects PGC1α expression, reducing oxidative capacity³⁶, and consequently inducing premature ageing.

In the context of mitochondrial diseases, inhibition of mTOR using rapamycin improves mitochondrial function and alleviates symptoms of Leigh syndrome³⁷, a disease caused by mtDNA deletions, in a mouse model of the syndrome. Additionally, a low dose of rapamycin extended lifespan in a mouse model of mtDNA depletion syndrome³⁸. Therefore, mitochondria might be the critical mediator for the effects of mTOR inhibition on longevity, and might represent a notable therapeutic target in ageing and age-related diseases. However, additional research is necessary to understand the implications of mTOR inhibition in the context of both health and disease.

AMPK

AMPK is considered the primary sensor of cellular AMP to ATP and ADP to ATP ratios and a key regulator of cellular energy homeostasis and metabolism³⁹.

Due to the central role of AMPK in the regulation of cellular metabolism, AMPK activity is closely linked to mitochondrial physiology. Experiments in mice with a reduced ATP to AMP ratio in skeletal muscle showed an increase of mitochondrial biogenesis via AMPK induction of PGC1α expression⁴⁰. Subsequent studies also elucidated this signalling pathway and indicated that activated AMPK phosphorylates PGC1α, which consequently induces mitochondrial gene expression⁴¹. Additionally, mitochondrial quality control (BOX 2) is also under the control of AMPK. AMPK is believed to regulate the autophagic turnover of dysfunctional mitochondria through initiation of mitophagy by phosphorylation and activation of the pro-mitophagic factor UNC51-like kinase 1 (ULK1)⁴². Under mitochondrial stress, fission is activated by AMPK-mediated phosphorylation of mitochondrial fission factor, facilitating the removal of damaged mitochondria⁴³. Moreover, work on C. elegans showed that deficient mitochondrial dynamics are sufficient to block AMPK-mediated and calorie restriction-mediated increases in longevity⁴⁴, demonstrating the close relationship between mitochondrial dynamics and the role of AMPK in regulating longevity. Therefore, decreased AMPK activity might be a contributor to the reduced mitochondrial function and dynamics observed during ageing⁴⁵.

Box 2 |. Mitochondrial quality control.

To ensure a healthy mitochondrial network, cells have quality control mechanisms that preserve mitochondrial integrity (see the figure) by refolding misfolded proteins, removing those that are not recoverable, and eliminating dysfunctional complexes³⁰¹.

The ubiquitin proteasome system (UPS) exerts a first level of surveillance on nuclear-encoded mitochondrial proteins immediately after their translation, ensuring that misfolded or mis-localized proteins are removed before they become a threat to proteostasis. Outer membrane proteins facing the cytoplasm can also be ubiquitinated and removed by the UPS in coordination with an ATpase complex, valosin-containing protein (VCP) in mammals or CDC48 in yeast. in addition to the removal of damaged proteins, UPS also regulates mitochondrial dynamics by degrading mediators of the fission and fusion processes through MITOL-mediated and MUL1-mediated ubiquitination^302,303.

Misfolded proteins within the mitochondrial compartments are processed by a set of chaperones and proteases that are found exclusively in mitochondria for either refolding or degradation³⁰⁴. When chaperones fail in refolding and restoring the function of compromised proteins, a set of proteases are present within the mitochondrial matrix, as well as on both sides of the inner membrane, to eliminate dysfunctional proteins before they threaten the functionality of the organelle³⁰⁵. Soluble chambered proteases that form ring-shaped structures have their active domains inside the barrel-shaped oligomer; thus, proteins need to be unfolded and inserted inside the chamber for degradation through an ATP-dependent reaction. This chaperone-like activity can be an intrinsic property of the complex, as it is for the two mitochondrial AAA proteases, i-AAA and m-AAA, and the Lon proteases³⁰⁶, or provided by a second enzyme, such as in the CLPX–CLPP complex³⁰⁷. The key role of the CLPX–CLPP complex in maintaining mitochondrial homeostasis emerged from studying the CLPP-knockout mouse^308,309.

When the mitochondrial quality control mechanisms fail to resolve protein aggregation, and dysfunctional proteins accumulate at levels that are incompatible with the functionality of the organelle, terminally damaged mitochondria can be removed and degraded by a selective form of macroautophagy, termed mitophagy.

Mitochondrial biogenesis.

A process in which the mitochondrial mass of cells is increased.

Sirtuins

Another indicator of nutrient deficiency is a rise in the NAD⁺ to NADH ratio. Sirtuins, a conserved family of deacetylase and deacylase proteins, require NAD⁺ for their function. In addition, increased AMPK activity also correlates with conditions of nutrient depletion (that is, an increased NAD⁺ to NADH ratio).

In response to calorie restriction, the yeast NAD biosynthetic gene that catalyses the first and rate-limiting step in NAD⁺ biosynthesis, PNC1, is induced⁴⁶. The resulting availability of NAD⁺ increases SIR2 activity, which boosts mitochondrial respiration and suppresses recombinant DNA recombination^11,46, a known cause of ageing in yeast⁴⁷. Interestingly, increased expression of mitochondrial biogenesis transcription driver HAP4 resulted in lifespan extension under normal caloric conditions in yeast (Saccharomyces cerevisiae), with calorie restriction failing to further extend this longevity benefit¹¹.

In rodents, the age-dependent decline in NAD⁺ levels decreases SIRT1 activity, ultimately affecting mitochondrial homeostasis⁴⁸. Moreover, SIRT1 regulates mitochondrial physiology by deacetylating and activating PGC1α and FOXO^49,50, allowing control over a broad range of mitochondrial physiology.

Mitochondrial sirtuins also control mitochondrial physiology⁵¹. Notably, SIRT3 regulates ATP levels in multiple tissues⁵², as well as AMPK activation⁵³. SIRT4 interacts with adenine nucleotide translocator, possibly regulating the ADP to ATP ratio and mitochondrial physiology⁵⁴. Interestingly, SIRT3 has been suggested to be the mediator of some of the longevity benefits of calorie restriction through its targeting and deacetylation of mitochondrial proteins⁵⁵.

In summary, the regulation of mitochondrial physiology by pathways associated with longevity has relevelled the complex network of interactions between those pathways and mitochondrial function during ageing (FIG. 1). Failure to regulate energy homeostasis by these pathways is highly linked with mitochondrial dysfunction, which ultimately leads to age-related metabolic diseases.

Mitochondrial dysfunction in ageing

Different theories have been postulated to explain the evolutionary basis of ageing. The ‘antagonistic pleiotropy’ hypothesis provides a meaningful context for understanding the role of mitochondria in ageing.

Antagonistic pleiotropy.

A theory that describes opposing effects, both harmful and beneficial, on an organism, in which a given gene or process controls more than one trait, which are maintained in the population due to a decline in the force of natural selection. At least one of these traits is beneficial to the organism’s fitness early on in life, and at least one is detrimental to the organism’s fitness later in life.

The antagonistic effects of mitochondria have long been appreciated in organismal ageing and has led many researchers to view the organelle as more than just an energy supplier, but also seeing mitochondria as having an essential role in ageing and age-related diseases. The accumulation of mitochondrial oxidative damage, which is the basis for Harman’s ‘free radical theory of ageing’⁵⁶, has been suggested as a driver of mitochondrial dysfunction and the observed age-related decline in organ function during ageing. However, while several studies have supported the free radical theory of ageing^57,58, others have shown that increased ROS production does not always shorten lifespan⁵⁹. Research findings in multiple species question the role of oxidative stress on mtDNA mutagenesis⁶⁰. These findings have helped guide the field to hypothesize that ROS production might not be the leading cause of the phenotypes correlated with ageing and that other factors might contribute.

mtDNA mutations

In 1972, Harman expanded the free radical theory of ageing with the ‘mitochondrial theory of ageing’, which included the possibility that mtDNA mutations might in fact be the drivers behind the mitochondrial dysfunction observed during ageing⁶¹. Inherited mtDNA and nuclear DNA determine the energetic capacity of an individual during the individual’s lifetime⁶². The accumulation of somatic mtDNA mutations that occurs with ageing leads to a loss of mitochondrial function. The resultant decline in energy capacity, increase in oxidative damage and ultimately increase in apoptosis lead to cellular loss, which results in organ failure. Closely linked to mitochondrial ROS production, the number of mtDNA mutations is known to increase with age⁶³. However, while some reports favour the idea that mitochondrial mutations contribute to ageing and age-related diseases⁶⁴, others have questioned this hypothesis due to the level of mutations observed⁶⁵.

Evidence for a contribution of mtDNA mutations to the mammalian ageing phenotypes has come from the mtDNA mutator mouse, which carries a mutated, proofreading-deficient form of the nuclear-encoded mtDNA polymerase (POLGγ). The mtDNA mutator mouse was shown to accumulate considerable levels of mtDNA mutations and deletions and to recapitulate several signs of premature ageing, such as reduced lifespan, reduced fertility, anaemia, sarcopenia, osteoporosis, hair greying, hair loss⁶⁶, hearing loss⁶⁷ and elevated lactate levels⁶⁸. Moreover, it has been demonstrated that ageing phenotypes (such as spine curvature, reduced body weight and hair loss) and lifespan can be influenced by mtDNA mutation levels in mtDNA mutator mice⁶⁹ and surprisingly also in mice with maternally-inherited mtDNA mutations but an otherwise wild-type nuclear genome⁷⁰. However, the role of mtDNA mutations as a primary driver of the ageing process is still debated, in part because the number of mutations observed in normal aged tissues is seemingly below the threshold required to affect mitochondrial function⁷¹. That said, patients with HIV, who also present with phenotypes of premature ageing⁷² and premature age-associated cardiovascular disease⁷³, have mitochondrial mutations and OXPHOS dysfunction⁷⁴, possibly caused by anti-retroviral drugs that inhibit POLGγ⁷⁵.

Genomic instability

Genomic instability in the nucleus can impact mitochondrial physiology during ageing via what is known as anterograde signalling⁷⁶. Supporting this hypothesis, DNA repair-deficient premature ageing diseases, such as ataxia–telangiectasia (ATM), Cockayne syndrome group A and xeroderma pigmentosum group A, have been linked to changes in mitochondrial physiology, which are indicative of physiological responses to anterograde stimuli⁷⁷. Moreover, most of the mitochondrial proteins are encoded by nuclear DNA, and mutations on those genes are typically associated with mitochondrial diseases⁷⁸.

Genomic instability induced by single-strand breaks or double-strand breaks triggering the DNA damage response (DDR) is a hallmark of ageing², and can arise from both exogenous and endogenous sources⁷⁹. Fortunately, organisms have evolved a variety of nuclear DNA repair mechanisms to ensure that the alterations caused by agents in the nuclear genome are properly repaired⁸⁰. Nuclear DNA repair is energy-demanding⁸⁰, and persistent DNA damage can lead to mitochondrial dysfunction and metabolic deficits⁷⁶.

In addition to its critical role in organismal health and mitochondrial physiology, NAD⁺ is a rate-limiting metabolite for the poly(ADP-ribose) polymerases (PARPs)⁸¹, which are key DDR proteins. When DNA breaks occur, PARP1 senses the breaks and initiates DNA repair signalling. Activated PARP1 will consume NAD⁺, generating PAR through a process called PARylation⁸².

An interplay between PARP1 and mitochondria might explain aspects of age-related functional decline. Indeed, deletion of the Parp1 gene in mice increases NAD⁺ levels, SIRT1 activity and mitochondrial biogenesis⁸³. In nematodes, pharmacological inhibition of PARP1 increases mitochondrial biogenesis and extends lifespan⁸⁴. Additionally, studies conducted in animal models and cells derived from people affected by xeroderma pigmentosum and Cockayne syndrome found decreased SIRT1 activity and persistent PARP activation due to unrepaired DNA damage. Pharmacological NAD⁺ supplementation or inhibition of PARP1 was able to re-establish SIRT1 activity and recover mitochondrial homeostasis^85,86.

Additional evidence for the link between PARP1 and mitochondrial physiology has come from work done in ATM, which is caused by a defect in the ATM gene, which encodes one of the DNA repair proteins recruited by PARP1 to the DNA break site. Analysis of an ATM mouse model showed dysfunctional mitochondria with increased ROS levels and the accumulation of damaged mitochondria⁸⁷. Similarly, Atm^−/− mice also have mitochondrial defects and interventions based on NAD⁺ supplementation⁸⁸ attenuate their health decline.

Cellular senescence

Cellular senescence is characterized by an irreversible cell cycle arrest, changes in gene expression, tumour suppression activation, altered chromatin, resistance to apoptosis and increased protein synthesis⁸⁹. The build-up of senescent cells is considered a driver of the ageing process⁹⁰. No universal marker has been discovered for senescent cells but cell cycle arrest, DDR, β-galactosidase activity and the pro-inflammatory senescence-associated secretory phenotype are common features of cells undergoing senescence⁹⁰.

Senescent cells exhibit numerous changes to mitochondria, including their structure, dynamics and function. For example, defects in mitochondrial OXPHOS are observed in the early stages of cellular senescence^91–93. Cellular senescence leads to decreased mitochondrial membrane potential, increased proton leakage and increased ROS production⁹⁴, thus enhancing DNA damage and DDR⁹⁵. Moreover, cells undergoing senescence display altered mitochondrial metabolite homeostasis⁹⁶, as well as decreased fatty acid oxidation, leading to accumulation of lipids⁹⁷. In addition to functional alterations, the mitochondria in senescent cells show dramatic changes in morphology and dynamics. During senescence, mitochondrial biogenesis is upregulated, leading to an unhealthy mitochondrial network. This network is crucial to cells meeting their metabolic demands through mitochondrial fission and fusion events^95,98. Furthermore, pathogenic mtDNA has also been reported to be involved in cellular senescence⁹⁹. Nevertheless, the contribution of mitochondria to the development of the senescent phenotype, as well as the underlying mechanism driving mitochondrial dysfunction in ageing and age-related diseases, require further elucidation, as this knowledge might contribute to the discovery of new targets for possible anti-ageing interventions.

Epigenetic alterations

Whether epigenetic changes in mtDNA are relevant to health has been debated for years, especially questions about the extent of mtDNA methylation and the relative percentages of the methylated (5-mC) cytosine content in mtDNA and nuclear DNA. Additionally, the absence of CpG islands and the organization of mtDNA into so-called nucleoids has contributed to the controversy. However, in 2011, Shock and colleagues showed that both 5-mC and hydroxymethylated (5-hmC) cytosine residues, along with DNA methyltransferase 1 (DNMT1), are present in mtDNA from human and mouse cell lines¹⁰⁰. Both 5-mC and 5-hmC have been found in mtDNA, as well as DNMT3A and B DNA methyltransferases^101–103. Interestingly, it has been hypothesized that transcription from the heavy-strand promoter, which is regulated by the mitochondrial transcription factor A (TFAM), could be modulated by epigenetic markers on mtDNA; methylation levels could influence where TFAM binds and affects the transcriptional response¹⁰⁴.

CpG islands.

Regions with a high frequency of CpG sites, which correspond to a cytosine nucleotide followed by a guanine nucleotide, in the 5′ to 3′ direction.

Furthermore, mtDNA methylation is disturbed by environmental and other factors, such as traffic-derived elemental carbon¹⁰⁵, maternal smoking¹⁰⁶, endogenous metabolites¹⁰⁷, cell senescence¹⁰⁸ and ageing¹⁰⁹. Human diseases, including cardiovascular diseases¹¹⁰, colorectal cancer¹¹¹, non-alcoholic steatohepatitis¹¹², obesity-induced insulin resistance¹¹³ and Down syndrome¹¹⁴, have also been associated with changes in mtDNA methylation patterns.

In age-related diseases, such as neurodegenerative diseases, perturbations to the mtDNA epigenome have been linked to human diseases. For example, there is a decrease in the methylation of the mtDNA D-loop region in patients carrying the amyotrophic lateral sclerosis-linked superoxide dismutase 1 (SOD1) mutations¹¹⁵. Patients with Parkinson disease have decreased methylation of the D-loop¹¹⁶ and patients with Alzheimer disease-related pathology have reduced levels of mt-ND1 methylation¹¹⁶. Additional research is necessary to elucidate the functional role of epigenetic alterations in mtDNA in the context of ageing and age-related diseases.

D-loop region.

A non-coding region of 1,124 base pairs, containing essential transcription and replication elements, that acts as a promoter for both heavy and light strands in mtDNA.

Age-related metabolic disorders

Maintenance of healthy mitochondria and metabolic regulation is key to long-term health during ageing. Due to the high responsiveness of mitochondria to environmental variations, it is now acknowledged that several factors might contribute significantly to mitochondrial and metabolic dysfunction during ageing, ultimately leading to age-related metabolic disease (FIG. 2).

Fig. 2 |. Mitochondrial dysfunction in age-related metabolic diseases.

Mitochondrial dysfunction during ageing predisposes the elderly to age-related metabolic diseases. Diseases associated with metabolic deregulation can occur in various tissues, especially organs that have high energy demands. NAFLD, non-alcoholic fatty liver disease.

Metabolic syndrome

The metabolic syndrome, which occurs increasingly with age, is associated with obesity, hyperglycaemia, dyslipidaemia, insulin resistance and cardiovascular diseases^117,118. An excess in total body mass, especially visceral adipose tissue, is associated with increased morbidity¹¹⁹ as well as metabolic inflexibility¹²⁰, a phenomenon caused by the inability to transition between fatty acid and glucose metabolism. Consequently, individuals with obesity tend to oxidize lipids instead of metabolizing glucose under insulin-stimulated conditions¹²¹. Indeed, an association between dyslipidaemia and oxidized LDL has been observed in individuals with prediabetes¹²².

Prediabetes.

A condition characterized by moderately elevated fasting or postprandial blood levels of glucose.

In one study, insulin resistance in older patients (mean age 70 ± 2 years) was associated with decreased mitochondrial ATP production and modifications in mitochondrial OXPHOS¹²³. Changes in energy metabolism, especially in tissues dependent on insulin-stimulated glucose disposal¹²⁴, lead to mitochondrial deregulation and increased ROS generation. Consequently, an observed decrease in fatty acid oxidation and an increase in cytosolic levels of free fatty acids ultimately lead to pathologies such as type 2 diabetes mellitus (T2DM), muscle dysfunction and insulin resistance¹²⁵.

Interestingly, mtDNA polymorphisms have been associated with an increased risk of metabolic diseases. Analysis of both European¹²⁶ and Asian populations¹²⁷ showed that the 16189T>C substitution in the D-loop region of mtDNA is associated with obesity and T2DM. Additionally, mtDNA analysis showed that the haplogroup N9A is likely to be protective against the metabolic syndrome in Japanese women¹²⁸, as well as being protective against T2DM in Japanese and Korean populations¹²⁹. Furthermore, in a white family, the T4291C mtDNA mutation has been associated with the metabolic syndrome, and the T16189C mtDNA variant has been reported to be associated with abnormalities in body mass, insulin resistance and increased cardiomyopathy, as well as increased risk of the metabolic syndrome¹³⁰. In addition, the A8890G mtDNA mutation was found in a juvenile patient with the metabolic syndrome phenotype¹³¹. Apart from variations in mtDNA, patients with the metabolic syndrome show a reduced number of mtDNA copies in peripheral blood and adipose tissue¹³², a physiological change typically observed before the development of T2DM¹³³.

Haplogroup.

A genetic population comprising a group of people who share a common ancestor on the patriline or the matriline.

Type 2 diabetes mellitus

T2DM is a chronic disease characterized by dysregulated blood levels of glucose, ultimately impairing insulin action and insulin secretion. Mitochondrial dysfunction and the associated impairment in fuel oxidation are considered to be notable contributors to the development of insulin resistance, non-alcoholic fatty liver disease (NAFLD) and T2DM. Mitochondrial dynamics are crucial to T2DM and related vascular complications, given the central role of mitochondria in ATP production, insulin secretion and ROS formation¹³⁴. Chronic elevation of glucose levels stimulates ROS production and induces oxidative stress, which causes tissue damage. Individuals with prediabetes and T2DM have an increased risk of presenting with hyperglycaemia associated with a progressive rise in oxidative stress and altered mitochondrial morphology¹³⁵. Decreased mitochondrial oxidative capacity and mitochondrial content, reduced oxidative capacity, defective lipid metabolism and increased lipid peroxidation are also seen in skeletal muscle from patients with obesity and insulin resistance compared with healthy and lean individuals^136,137.

Data published in 2017 regarding ³¹P-magnetic resonance spectroscopy from individuals without diabetes mellitus in the Baltimore Longitudinal Study of Aging (BLSA) showed that a higher impairment of mitochondrial capacity is associated with greater insulin resistance and a higher likelihood of prediabetes than in participants classified as being ‘normal’ according to the American Diabetes Association criteria using fasting plasma glucose levels and/or glucose levels 2 h after oral glucose tolerance testing¹³⁸. Furthermore, decreased mitochondrial respiration was found in first-degree relatives of individuals with T2DM¹³⁹. The association between mitochondrial dysfunction and T2DM is also supported by the downregulation of genes involved in oxidative metabolism and under the control of PGC1α in the skeletal muscle of patients with T2DM¹⁴⁰.

Insulin resistance and β-cells

β-cells respond to increased metabolic demands by increasing their mass and insulin synthesizing and secretory activity. Long-term and chronic exposure to hyperglycaemia can cause desensitization and even depletion of β-cells, making them unable to secrete insulin due to the depletion of intracellular reserves¹⁴¹. Both desensitization and depletion of β-cells are reversible states; however, if the hyperglycaemic situation persists, it can lead to the irreversible loss of insulin production and, ultimately, the death of β-cells¹⁴².

Mitochondrial dysfunction is increasingly thought to be a central contributor to β-cell failure in the evolution of T2DM, with ROS production induced by metabolic stress as a key event leading to β-cell failure. In islets from patients with T2DM, the ATP content, and the increase in the ATP to ADP ratio stimulated by glucose, was lower than in islets from control individuals¹⁴³. Impairment in β-cell metabolism observed in islets from donors with T2DM and in β-cells from a mouse model of human neonatal diabetes upon exposure to hyperglycaemia support the notion that inhibition of mitochondrial metabolism is central to β-cell dysfunction^143,144. While there is no evidence for mitochondrial depletion in β-cells from patients with T2DM, the mitochondrial density volume is considerably higher in islets from patients with T2DM than in islets from those without T2DM, with both morphological and functional abnormalities that have not been observed in β-cells from control individuals¹⁴³. In agreement with reduced insulin secretion, PGC1α expression was found to be reduced by 90% in islets from patients with T2DM. In addition, PGC1α silencing in healthy human islets resulted in reduced insulin secretion¹⁴⁵, a phenomenon also found in patients with a common mtDNA variant in mitochondrial transcription factor B1 (TFB1M)¹⁴⁶.

Additionally, analysis of insulin-secreting pancreatic β-cells and islets from patients with T2DM showed alterations in mitochondrial physiology, such as hyperpolarization of the mitochondrial membrane and decreased activities of the enzymes glycerol phosphate dehydrogenase, pyruvate carboxylase and succinyl-CoA–3-ketoacid-CoA transferase^147,148. Moreover, increased expression of uncoupling protein 2 (UCP2), complex I and ATP synthase have been observed in islets from patients with T2DM¹⁴³. Increased ROS levels due to hyperglycaemia activate UCP2, resulting in proton leak across the mitochondrial inner membrane, which leads to reduced ATP synthesis and oxidative damage to the membrane, cytochrome c release and the induction of apoptosis¹⁴⁹.

Proton leak.

The dissipation of proton motive force (Δp), where protons migrate into the mitochondrial matrix without producing ATP.

Cytochrome c.

A haem protein localized in the mitochondrial intermembrane space that functions as an electron transporter between complex III and complex IV of the electron transport chain.

Non-alcoholic fatty liver disease

NAFLD is the most common form of chronic liver disease¹⁵⁰. Epidemiological studies have suggested that the presence of NAFLD might be a precursor to the development of other conditions, such as insulin resistance and T2DM¹⁵¹.

Age-associated liver diseases, such as NAFLD, have been described as hepatic examples of the metabolic syndrome¹⁵², where insulin resistance is also a key pathological feature. Patients with NAFLD present with decreased ETC complex activity^153,154 and a fatty liver-associated decrease in SIRT3 activity, which leads to hyperacetylation of mitochondrial proteins and mitochondrial dysfunction¹⁵⁵. In white adipose tissue, the uncoupling of OXPHOS has been shown to decrease fatty acid synthesis¹⁵⁶, leading to increased energy expenditure and reduced lipogenesis, which provides an important energy source to cells through oxidation of fatty acids¹⁵⁷. Lipid accumulation in hepatocytes has been linked to insulin resistance and the development of T2DM¹⁵⁸. It is likely that the lack of oxidative capacity to overcome the excess fatty acids that are transported to the liver during conditions such as obesity might have a vital role in the progression of NAFLD accompanied by insulin resistance¹⁵⁹.

Studies of obesity in rats and a T2DM model known as the OLETF obese hyperphagic rat showed that reduced hepatic mitochondrial content and function precede the development of NAFLD and insulin resistance, supporting a crucial role for mitochondria in the pathophysiology of NAFLD¹⁶⁰. Decreased ATP content, impaired mitochondrial bioenergetics and increased ROS generation have all been found in patients with non-alcoholic steatohepatitis, and in fatty liver models in the rat^161–163. These biochemical changes are associated with ultrastructural abnormalities that indicate an impaired OXPHOS. Ultrastructural changes include the presence of enlarged mitochondria, loss of cristae organization and crystalline inclusion bodies in the matrix in patients with NAFLD¹⁶⁴. Thus, uncoupling of OXPHOS and increased ROS generation, with oxidative damage to ETC complexes and mtDNA, further compromise mitochondrial function and perpetuate ROS formation, thus destabilizing the redox environment of the cell. Interestingly, in work performed in humans with NAFLD, a rapid elevation of liver levels of triglycerides was observed and is associated with stimulation of mitochondrial oxidative function¹⁶⁵, suggesting a compensatory stimulation of mitochondrial β-oxidation. In line with these arguments, both hepatic insulin resistance and simple steatosis were already established in mice fed a high-fat diet for 8 weeks, well before changes in mitochondrial bioenergetics became evident^166,167.

Vascular diseases

Vascular ageing is the major risk factor for vascular diseases and health status in the aged population¹⁶⁸, contributing to the age-dependent increased risk of pathological conditions of vascular dysfunction, such as hypertension and atherosclerosis, and are also characteristic of cardiovascular risk factors, such as obesity, smoking and a sedentary lifestyle¹⁶⁹.

Oxidative stress, a characteristic of T2DM, hypertension and atherosclerosis, is also the dominant feature of vascular ageing¹⁷⁰. Indeed, elevated ROS levels lead to endothelial dysfunction¹⁷¹, an early event in atherosclerosis pathogenesis that occurs mainly in atherosclerotic lesion formation^172,173. Electron microscopy analysis has shown abnormal ultrastructural lesions in mitochondria from human aortic atherosclerotic lesions, compared with mitochondria from normal parts of the aortic intima¹⁷⁴. Moreover, mtDNA mutations have been associated with the development of chronic human diseases, such as atherosclerosis, hypertension, T2DM, cardiovascular disease and obesity^175–178. Finally, a reduction in mtDNA copy number has also been observed in cardiovascular risk factors independent of atherosclerosis, such as hyperlipidaemia¹⁷⁹.

Under conditions of an imbalanced redox state, increased mitochondrial ROS production might lead to modifications of the arterial walls¹⁸⁰, followed by an inflammatory environment¹⁸¹. Manganese-dependent superoxide dismutase (MnSOD) activity exhibits a negative feedback with atherogenesis, by modulating ROS levels and, consequently, increased LDL oxidation and endothelial dysfunction. In one study, 16-month-old MnSOD^+/− mice consistently showed elevated ROS production and mtDNA damage, followed by the development of dramatic endothelial dysfunction¹⁸². Similarly, genetic ablation of mitochondrial GPX1 in mice triggered noticeable vascular dysfunction, coinciding with increased oxidative stress and impaired nitric oxide metabolism¹⁸³, thus linking perturbations in redox status to vascular ageing.

Closely related to ROS production, the mitochondrial Src homology–collagen (Shc) adaptor protein p66^Shc might be the link between ageing and metabolic and cardiovascular diseases. Induction of Ser26 phosphorylation of p66^Shc by protein kinase C-β (PKCβ) triggers the transport of p66^Shc from the cytosol to mitochondria, where it promotes ROS accumulation by oxidizing cytochrome c¹⁸⁴, ultimately leading to apoptosis.

Mutant p66^Shc−/− mice have been reported to be resistant to oxidative stress and to have an increased lifespan¹⁸⁵, as well as reduced age-dependent endothelial dysfunction¹⁸⁶. Additional work has revealed that p66^Shc activation is involved in adipogenesis, insulin resistance and cardiovascular complications associated with diabetes mellitus^187–189. Moreover, vascular levels of p66^Shc were increased in animal models of obesity and had a role in endothelial insulin resistance¹⁸⁹, an effect restored when p66^Shc was silenced in vivo¹⁸⁹. In humans, the first evidence for a role of p66^Shc in diabetes mellitus came with the finding that patients with T2DM have increased expression of p66^Shc in circulating peripheral blood mononuclear cells¹⁹⁰. Consequently, work has shown that p66^Shc−/− mice are protected against streptozotocin-induced diabetic cardiomyopathy¹⁹¹, diabetes mellitus-induced vascular endothelial dysfunction and oxidative stress¹⁸⁷. Nevertheless, contradictory findings regarding the role of p66^Shc in obesity have been reported. In one study using animal models and human samples from patients with obesity undergoing elective surgery, it was demonstrated that reduced p66^Shc levels could protect from obesity but failed to prevent obesity-associated metabolic dysfunction¹⁹², highlighting the need for further studies to dissect the role of p66^Shc in metabolism.

Chronic inflammation

Mitochondrial dysfunction is a hallmark of many chronic inflammatory diseases¹⁹³, including those caused by the innate immune response¹⁹⁴. Innate immunity has a central role in the inflammatory process and coordinates the three stages of inflammation: initiation, propagation and resolution¹⁹⁵. Unlike the adaptive immune response, the innate immune response is rapid (<90 h) and non-specific, being activated by a broad range of molecules that comprise viruses and bacteria. Pathogen-associated molecular patterns (PAMPs) share conserved features among microbes not present in mammalian cells¹⁹⁶. Once these PAMPs are recognized by specific receptors belonging to the pattern recognition receptor (PRR) family, an immune response is activated and escalates to induce inflammation and the release of numerous cytokines for recruitment of the more specific adaptive immune system¹⁹⁷. Different classes of PRRs, including the membrane-bound Toll-like receptors (TLRs), the C-type lectin receptors and the cytosolic NOD (nucleotide-binding oligomerization domain)-like receptors, have been described elsewhere¹⁹⁸.

In addition to PAMPs, other endogenous molecules are released in the presence of cellular damage, known as damage-associated molecular patterns or alarmins. Alarmins bind to and activate PRRs, thus triggering an immune response and propagating the inflammation¹⁹⁹. Several alarmins consist of mitochondrial molecules, and the ability of these molecules to activate PRRs is dependent on their similarity to bacterial components, reaffirming once again the prokaryotic origin of mitochondria²⁰⁰. One of the strongest alarmins released by stressed mitochondria is mtDNA, which has emerged as a strong activator of innate immunity, particularly of the interferon signalling pathway²⁰¹. Several PRRs bind to mtDNA in the cytoplasm, triggering an innate immune signalling pathway. For example, TLR9 binds to mtDNA and activates the NF-κB signalling pathway, inducing the release of pro-inflammatory cytokines²⁰². Similar to bacteria, mitochondrial protein translation initiates with an N-formylated methionine instead of a regular methionine as in eukaryotic cells. Thus, the leakage of N-formylated peptides outside the mitochondria activates a specific family of PRRs, the formyl peptide receptors, which are potent inducers of the innate immune system²⁰³. Another alarmin of mitochondrial origin is cardiolipin, a unique phospholipid abundant in prokaryotic membranes and a constituent of the mitochondrial inner membrane²⁰⁴.

Other alarmins of mitochondrial origin, including ATP, succinate, TFAM and ROS, all induce and support inflammation by different means²⁰⁵. Extracellular ATP binds to purinergic P2 receptors that are present on several cell types, including macrophages, and promotes chemotaxis and phagocytosis²⁰⁶. Succinate, an intermediate of the TCA cycle, is emerging as an important signalling molecule²⁰⁷. One of succinate’s actions is the stabilization of HIF1α by inhibiting the prolyl hydroxylase domain enzyme activity in macrophages. This action promotes the secretion of pro-inflammatory chemokines, such as IL-1β²⁰⁸. TFAM is a DNA-binding protein belonging to the same family as high mobility group box 1 protein and is a very important alarmin involved in inflammation and autoimmune disorders²⁰⁹. Moreover, mitochondrially-derived ROS are a key component of the immune system. Hydrogen peroxide, a reaction product of superoxide radical (O₂^•−) conversion by MnSOD, crosses the mitochondrial membranes into the cytoplasm, activating the inflammasome and redox-sensitive transcription factors, such as HIF1α and NF-κB^210–212.

Given the central role of mitochondria in regulating the inflammatory response, it is not surprising that altering their function and activity has beneficial and deleterious effects. An example of a beneficial response is the release of mitochondrial alarmins in the context of pathogen invasion or tissue damage to help fight infections. By contrast, an uncontrolled release of mitochondrial components into the cytoplasm or the extracellular space due to, for example, mitochondrial dysfunction, can induce chronic inflammation, a condition associated with several diseases, such as rheumatoid arthritis, atherosclerosis, liver injury, obesity and ageing, an effect known as ‘inflammaging’.

Targeting mitochondria

Lifestyle interventions

Exercise is a well-known lifestyle intervention that prevents metabolic disorders, such as obesity and T2DM, whereas reduced physical activity and a sedentary lifestyle are key contributing factors²¹³. In patients with T2DM, walking and ergometer cycling exercise increased mitochondrial muscle respiration and mitochondrial content and density, as well as the activity of oxidative enzymes^214,215. Daily voluntary exercise also ameliorates progeroid mouse phenotypes, such as locomotor activity, alopecia and kyphosis, and the deregulated proteomic profiles in muscle and brain in mtDNA mutator mice²¹⁶.

Calorie restriction is another widely known lifestyle intervention that can prevent age-related metabolic disorders in multiple species²¹⁷. Under calorie restriction, OXPHOS and antioxidant defences¹⁴, as well as mitochondrial biogenesis, are induced²¹⁸. Endothelial nitric oxide and PGC1α also have key roles. Decreased mitochondrial ROS production and macromolecular oxidative damage due, in part, to mitochondrial uncoupling might also explain the benefits of calorie restriction^219,220. Moreover, calorie restriction stimulates mitochondrial turnover by boosting mitochondrial biogenesis and the clearance of damaged mitochondria via mitophagy^221,222.

Pharmacological interventions

Small molecules.

The ageing research field is driven by the goal of finding better medicines that can target multiple age-related diseases and prevent many others. Among them, several polyphenols that improve mitochondrial physiology have been described²²³. One of the most well-known polyphenols that improves mitochondrial physiology is resveratrol^224,225. Resveratrol enhances mitochondrial biogenesis and oxidative capacity in animal models of diet-induced obesity and ageing^226,227. In humans, resveratrol improves mitochondrial function, reduces inflammation and decreases lipid levels in patients with obesity²²⁸. Moreover, resveratrol-dependent activation of SIRT1 has also been shown to trigger mitochondrial biogenesis both in vitro and in transgenic rats with human genes for renin and angiotensinogen by inducing mRNA expression of mitochondrial biogenesis markers, such as PGC1α²²⁹. Mitophagy can also be induced by resveratrol via FOXO3 activation, which triggers the PINK1–Parkin pathway²³⁰. Additionally, the non-naturally occurring sirtuin-activating compound, SRT1720, has enhanced insulin sensitivity and oxidative metabolism in animal models of obesity, in part, by improving mitochondrial oxidative capacity²³¹. Notably, SRT2104, another non-naturally occurring sirtuin-activating compound, has been tested in the clinic but did not improve glucose homeostasis and insulin sensitivity in patients with T2DM²³². Punicalagin, the most abundant ellagitannin in the pomegranate fruit, stimulates mitochondrial biogenesis in cardiomyocytes from obese rats²³³. Also, the administration of epicatechin-rich cocoa stimulated mitochondrial biogenesis in patients with T2DM²³⁴. Finally, oleuropein, a polyphenol present in the leaves of fruits and olives, promotes phosphorylation and consequent activation of mitophagy-related proteins by activation of the AMPK–ULK1 pathway in a diet-induced mouse model of obesity²³⁵.

Activation of AMPK increases mitochondrial chemical energy generation by increasing the AMP to ATP ratio²³⁶. The AMPK agonist 5-aminoimidazole-4-carboxamide rescues mitochondrial dysfunction and improves exercise performance capacity in Cox-deficient mice²³⁷. Additionally, metformin, which is widely used to treat T2DM, activates AMPK, improves mitochondrial function, decreases age-associated inflammation and improves hyperglycaemia in people with obesity and prediabetes, as well as in HFD-fed mice^238,239. Interestingly, a newly reported molecule, imeglimin, a tetrahydrotriazine-containing oral glucose-lowering agent, decreased ROS production and prevented hyperglycaemia-induced cell death via complex l by inhibiting reverse electron transfer-mediated ROS production in preclinical studies²⁴⁰, and its efficacy and favourable safety and tolerability profile have been confirmed in patients with T2DM²⁴¹. Similarly, chronic moderate inhibition of cytochrome c oxidase by TPP–thiazole improves mitochondrial bioenergetics and glucose homeostasis and reduces visceral adipose tissue in 14–18-month-old mice, limiting age-associated obesity and glucose irregularities²⁴².

In addition, inhibition of mTOR by rapamycin, which also extends the lifespan of model organisms (for example, yeast, nematodes, fruit flies and mice) and prevents age-related diseases, is also currently being tested in multiple clinical trials²⁴³. The health benefits provided by rapamycin, however, seem to be tissue-specific. Knocking out the Raptor complex in adipose tissue enhances mitochondrial respiration²⁴⁴, but muscle-specific knockout causes muscular dystrophy in mice³⁶. Adipose-specific Raptor knockout mice (Raptor^ad−/−) have a lean phenotype and are protected against diet-induced obesity, due to enhanced energy expenditure facilitated by UCP1-mediated mitochondrial uncoupling²⁴⁴. Interestingly, the phenotype of mice with knockout of a downstream target of mTOR, S6K1, mimics the lean phenotype of Raptor^ad−/− mice, and the mice are protected against diet-induced obesity²⁴⁵. In db/db mice, rapamycin exhibits anti-obesity properties by improving whole-body insulin sensitivity and increasing markers of fatty acid oxidation and mitochondrial biogenesis in white adipose tissue²⁴⁶. Finally, leucine, which is sensed by mTOR²⁴⁷, increases NAD⁺ levels, mitochondrial biogenesis, insulin sensitivity and lipid disposal when given to obese mice²⁴⁸. However, leucine supplementation has not improved muscle physiology, glycaemic control or lipidaemia in older patients (aged 71 ± 1 years) with T2DM²⁴⁹, highlighting the need for further studies to dissect amino acid supplementation as a therapy for age-associated metabolic diseases. Interestingly, a human clinical trial testing leucine–resveratrol combinations showed improved glucose homeostasis and insulin sensitivity in patients with prediabetes, suggesting that synergistic approaches might be a good alternative to treat age-associated metabolic diseases²⁵⁰.

NAD⁺-boosting molecules.

Systematic NAD⁺ decline and decreased sirtuin activity are considered among the molecular events that occur during ageing, possibly underlying the increase in age-related diseases that limit organismal lifespan²⁵¹. A large body of evidence from animal studies supports NAD⁺-boosting therapies as a way to ameliorate a wide variety of age-related metabolic disease.

Supplementation of the precursor nicotinamide mononucleotide (NMN) re-established β-cell glucose-stimulated insulin secretion as well as hepatic and skeletal muscle insulin sensitivity in mouse models with impaired glucose homeostasis^252,253. In 22-month-old mice, NMN restored NAD⁺ levels and markers of mitochondrial decline during ageing⁴⁸, while reducing age-associated body weight gain and increasing energy metabolism and physical activity²⁵⁴. Moreover, NMN²⁵² and nicotinamide riboside²⁵⁵, another NAD⁺ precursor, increased mitochondrial function and improved glucose homeostasis in obese mice. Currently, several ongoing clinical trials are investigating the safety and tolerability of NMN and similar molecules²⁵⁶. In hyperglycaemic mice, nicotinamide riboside administration improved liver steatosis and glucose tolerance, and reduced weight gain²⁵⁷. To some extent, this effect can be explained by the elicited mitochondrial unfolded protein response mediated by nicotinamide riboside²⁵⁸. However, results from human studies testing nicotinamide riboside supplementation at 2,000 mg per day showed that although safe, nicotinamide riboside does not improve insulin sensitivity and whole-body glucose metabolism in patients with obesity and insulin resistance²⁵⁹.

The upregulation of the NAD salvage pathway, which recycles NAD⁺ from nicotinamide, was first shown to extend lifespan and mimic caloric restriction in yeast cells⁴⁶. Interestingly, treatment of obese rats with nicotinamide induces mitochondrial biogenesis and improves insulin sensitivity in a SIRT1-dependent manner²⁶⁰. Additionally, inhibition of NAD⁺-consuming enzymes has been shown to be a promising approach to treat mitochondrial dysfunction in age-related metabolic dysfunction²⁶¹. Indeed, PARP inhibition in mice with NAFLD restores NAD⁺ levels, thereby improving mitochondrial function and insulin sensitivity, and decreasing hepatic lipid accumulation²⁶¹.

Mitochondrial uncouplers.

Mitochondrial uncoupling is defined as a process that uncouples nutrient oxidation from ATP production²⁶². One of the first molecules to be described as having potential therapeutic benefits in obesity and metabolic diseases is the mitochondrial uncoupler 2,4-dinitrophenol (DNP)²⁶³. Mice fed a high-fat diet have improved metabolic parameters, such as reduced body weight, improved glucose homeostasis, decreased serum levels of insulin and decreased liver levels of triglycerides^264,265. However, the potential adverse effects of DNP led to the development of derivatives of DNP, for example a liver-targeted methyl ether derivative of DNP (DNP methyl ether; DNPME) and a controlled-release mitochondrial uncoupler (controlled release mitochondrial protonophore; CRMP). Both DNPME and CRMP decreased triglyceride accumulation in liver and improved insulin sensitivity in rat models of T2DM and NAFLD^266,267. Importantly, in two different nonhuman primate models of the metabolic syndrome, high-fat, fructose-fed cynomolgus macaques and spontaneously obese dysmetabolic rhesus macaques, CRMP was shown to reduce hepatic steatosis and insulin resistance and increase the rates of β-oxidation²⁶⁸.

FDA-approved drugs that have uncoupling activity have also been tested in animal models. Niclosamide ethanolamine reversed hyperglycaemia, hyperinsulinaemia and hepatic steatosis in mice fed a high-fat diet and delayed hyperglycaemia in db/db mice²⁶⁹. Another promising candidate is 1,3-bis(dichlorophenyl)urea, which reduces body weight gain and body adipose tissue mass, improving insulin sensitivity and glucose homeostasis, and decreasing hepatic lipid content²⁷⁰. CZ5, an orally bioavailable mitochondrial uncoupler²⁷¹, also reduces body weight, total adipose tissue mass, fasting glucose levels and insulin levels²⁷¹.

Two studies published in the past few years have investigated additional potential candidates for the treatment of age-related metabolic diseases. Kanemoto and colleagues²⁷² described a liver-localized mitochondrial uncoupler, OPC-163493, which was shown to have various benefits in multiple rodent models of the metabolic syndrome, such as decreasing hepatic steatosis and lowering fasting levels of glucose and HbA_1c, as well as increasing nitric oxide bioavailability and reducing oxidative stress in blood vessels. These findings demonstrate the cardiometabolic benefits of OPC-163493. Alexopoulus and colleagues²⁷³ found that the orally available BAM15, a pH-dependent self-limiting uncoupler, improved insulin sensitivity and decreased oxidative stress, body adipose tissue mass, hepatic triglyceride accumulation and inflammatory lipids in diet-induced obese mice. In an attempt to optimize BAM15 limitations, such as tissue distribution and uncoupling effects, 10b and 12i (SHS4121705) derivatives are being tested and are being shown to be efficacious in the STAM mouse model of non-alcoholic steatohepatitis, by decreasing hepatic levels of triglycerides, improving serum levels of alanine transaminase and improving NAFLD activity score^274,275. Another compound that has been synthesized to oppose the possible adverse effects of uncontrolled mitochondrial uncoupling (for example, when mitochondrial uncoupling exceeds the ability of mitochondria to produce ATP), is the dual-action compound 6j, which has both mitochondrial uncoupling activity and pyruvate dehydrogenase activation effects²⁷⁶. Studies performed in diabetic mice have shown that compound 6j is able to improve insulin sensitivity, glucose homeostasis and liver steatosis²⁷⁶.

Collectively, these observations suggest that this class of drugs might hold the potential to treat obesity and metabolic diseases. However, because the use of mitochondrial uncouplers in humans has led to severe adverse effects in the past, safety studies will be particularly important. In 2021, positive results from a phase I clinical trial testing mitochondrial uncoupler HU6 were presented. HU6 was well tolerated without serious adverse effects, and provided additional improvements in resting energy expenditure and dose-dependent weight loss²⁷⁷, supporting the potential for the development of mitochondrial uncouplers for the treatment of metabolic dysfunction in humans.

Mitochondrially-targeted antioxidants.

Due to the central role of mitochondrial ROS and mitochondrial oxidative damage in disease, mitochondrial-targeted antioxidant therapies have been explored as potential treatments²⁷⁸. Traditional exogenous antioxidants (for example, vitamins E, C and D), as well as chemical compounds with described antioxidants properties (for example, coenzyme Q, α-lipoic acid and N-acetylcysteine), have been used to reduce oxidative stress^279,280, but their cellular uptake is limited and they do not accumulate where needed²⁸¹. Mitochondrially-targeted coenzyme Q (MitoQ) or mitochondrially-targeted vitamin E (MitoVitE) have been shown to target mitochondrial dysfunction^282,283. MitoQ, one of the best-characterized mitochondrially-targeted antioxidants, has been shown to protect against oxidative stress²⁸⁴ and offers neuroprotection²⁸⁵ in preclinical studies. In humans, MitoQ improves vascular ageing in middle-aged and older adults (aged 60–79 years)²⁸⁶. MitoVitE decreases hepatic oxidative stress and inhibits hepatic lipid deposition in mice²⁸². Interestingly, MitoQ also alleviated Alzheimer disease-like pathology in the 3×Tg-AD mouse model²⁸⁷ by improving cognition, decreasing oxidative stress and increasing survival. These findings support the idea that targeting mitochondria to relieve symptomology in patients with Alzheimer disease is worthwhile. Another promising mitochondrial target antioxidant is elamipretide (SS-31), a cell-permeable tetrapeptide that localizes to the inner mitochondrial membrane, where it associates with and stabilizes cardiolipin. Clinical studies using elamipretide are more advanced for hereditary mitochondrial diseases than for metabolic diseases. Nevertheless, concerning T2DM, studies using animal models and human islet and leukocyte samples support the beneficial effects of elamipretide in improving mitochondrial function^288,289, restoring glucose levels²⁹⁰, decreasing oxidative stress²⁹¹ and preventing atherosclerosis²⁹². Interestingly, one clinical study published in 2021 showed that elamipretide reverses mitochondrial dysfunction in ageing human muscle, paving the way to test this molecule against age-associated metabolic diseases²⁹³.

Conclusions

One of the most important discoveries in the field of ageing is that the rate of ageing is coordinated, at least in part, by conserved genetic and biochemical pathways, many of which control mitochondrial physiology. The modulation of these pathways could be the key to improving human health and preventing major age-related diseases, especially those associated with metabolic dysfunction. That said, how important mitochondria are to the ageing process is still under debate. Nevertheless, the maintenance of healthy mitochondria and metabolic regulation are key to long-term health.

A summary of different approaches targeting mitochondria in ageing and age-associated metabolic disease, as well as their clinical stage, can be found in TABLE 1. Despite continuing progress over the past two decades in the field of ageing, combatting mitochondrial dysfunction in age-associated metabolic diseases has proven challenging. To some extent, this can be explained by the ubiquitous nature of mitochondria and the heterogeneous clinical presentations associated with multi-organ involvement in age-associated metabolic diseases. In the preclinical context, one of the main challenges in the field of ageing is adequate disease modelling when considering biological age. Moreover, it has been challenging to focus on specific pathologies in the clinical context because of the functional decay of multiple organs during ageing. Therefore, new digital health technologies, such as artificial intelligence and big data, could pave the way to understanding the complex nature of these diseases, providing associations between specific biological or clinical phenotypes and corresponding biomarkers, which could help future preclinical research as well as clinical trial design and change the health span panorama in the aged population.

Table 1 |.

Current clinical status of therapies targeting mitochondria in age-associated metabolic dysfunction

Drug type	Drug name	Most advanced stage	Refs
Small molecule	Resveratrol	Clinical	294
	SRT1720	Preclinical	231
	SRT2104	Clinical	232
	Punicalagin	Preclinical	233
	Epicatechin	Clinical	295
	Oleuropein	Preclinical	296
	Metformin	Clinical	297
	Imeglimin	Clinical	241
	TPP–thiazole	Preclinical	242
	Rapamycin	Clinical	243
	Leucine	Clinical	249
NAD+-boosting molecules	Nicotinamide mononucleotide	Clinical	256
	Nicotinamide riboside	Clinical	298
	Nicotinamide	Preclinical	260
Mitochondrial uncouplers	DNP methyl ether	Preclinical	267
	Controlled release mitochondrial protonophore	Preclinical	268
	Niclosamide ethanolamine	Preclinical	269
	1,3-bis(dichlorophenyl)urea	Preclinical	270
	CZ5	Preclinical	271
	OPC-163493	Preclinical	272
	BAM15 and derivates	Preclinical	273–275
	Compound 6j	Preclinical	276
	HU6	Clinical	277
Mitochondrially-targeted antioxidants	Mitochondrially-targeted coenzyme Q	Clinical	286
	Mitochondrially-targeted vitamin E	Preclinical	282
	Elamipretide	Clinical	293

In conclusion, of all the processes that affect ageing, those controlled by mitochondria are the most responsive to environmental variations, making the mitochondria an excellent model for elucidating the effects of environment on longevity and disease. In the long term, a fundamental understanding of the role of mitochondria in human health and ageing will be needed if there are to be breakthroughs in combatting frailty and age-related diseases.

Key points.

• The rate of ageing is coordinated, at least in part, by conserved genetic and biochemical pathways.

• A complex network of interactions between longevity pathways reveals an intricate regulation of mitochondrial physiology during ageing.

• Cellular metabolism interconnects the nine hallmarks of ageing, and deregulation of energy metabolism by environmental variations is an essential process leading to mitochondrial dysfunction during ageing.

• A better understanding of mitochondrial dysfunction during ageing and age-related metabolic diseases will provide fundamental knowledge to develop therapies to combat late-life morbidities.

• Human longitudinal studies will be essential to understand individuals’ risk of diseases much earlier in life, and will inform health choices and medical care options.