3.2.6. Metabolic Syndrome
Due to multiple physiological mechanisms, aging is becoming one of the most critical risk factors for the occurrence and progression of metabolic syndrome, which is an age-related disease consisting of obesity, glucose intolerance, insulin resistance, dyslipidemia, and hypertension 
. Endoplasmic reticulum (ER) stress is disturbed homeostasis leading to an impaired protein synthesis process such as the accumulation of unfolded and misfolded proteins, which is especially associated with the onset of metabolic disorders such as diabetes mellitus and disorderly hepatic lipid metabolism 
. Nonalcoholic fatty liver disease has been regarded as a novel component of metabolic syndrome, with attenuated hepatic ketogenesis, insulin sensitivity, and abnormal fat accumulation 
. NK has been found to improve metabolic and inflammatory markers, as well as lower insulin levels and promote β-HB production particularly during KD 
. Of note, β-HB administration has already been reported to inhibit inflammasome formation, lipid accumulation, and oxidative stress by binding to specific HCARs, and inhibiting HDACs, FFARs, and NLRP3, to suppress ER stress, suggesting the beneficial effects of β-HB supplementation on liver steatosis and restoration of liver functions in aging progression 
4. Therapeutic Application of β-HB in Aging
4.1. Endogenous Ketosis
Endogenous ketosis can be achieved by using various durations of fasting, CR, or CHO restriction and particularly by a KD (Figure 5).
4.1.1. Intermittent Fasting and Caloric Restriction
Energy-restricted metabolic states such as intermittent fasting (IF) or CR, have obvious characteristics of increased ketosis and could extend lifespan in animals . Ketogenesis is believed to increase with prolonged starvation. IF is relatively easy to practice for a long time, with alternative periods of feeding or fasting which can last 24 h from one to four days per week, such as alternate-day fasting, whole-day fasting (periodic fasting), and time-restricted feeding [120,121]. IF has been proven to be safe in monitored patients and the hormonal level of this state is typically marked by a low-insulin, high-glucagon, and increased plasma fatty acids and cortisol environment which heavily promote lipolysis [122,123]. It is currently gaining more popularity and is being considered as a potential non-pharmacological way to promote healthy aging . CR is achieved by reducing energy intake of about 25–30% without lacking essential nutrients, which has also been observed to improve age-related mortality and morbidity, delay aging progression, and result in healthspan in invertebrate and vertebrate species [125,126]. Circulating β-HB level is elevated as a beneficial metabolite and mediator during these two states, which are widely accepted as anti-aging interventions . However, before these regimens, it is hard to maintain long-term ketosis. The mechanism of β-HB as a potential CR mimic to slow aging has yet to be explored further.
4.1.2. Ketogenic Diets
A large amount of data on the effects of β-HB metabolism comes from studies on KD, especially in rodents. KD is not an energy-limiting state, yet the related phenotype replicates some of the biochemical properties of IF and CR which are strongly associated with longevity. KD is composed of high-fat, adequate-protein, and a very low level of CHO (typically about 88%, 10%, and 2%). The KD promotes endogenous ketogenesis without fasting . In terms of lifespan extension, β-HB has been proposed to promote longevity in worms via two different anti-aging pathways, which are inhibiting HDACs, leading to increased DAF-16/FOXO activity, as well as involving the mitochondrial metabolism of β-HB, and activating the SKN-1/Nrf2 antioxidant response pathway . KD has also
been demonstrated to improve the longevity and survival of mice, together with increased protein acetylation and decreased activation of tissue-specific mTOR complex 1 . KD has been mechanistically investigated to improve neuroprotection and mitochondrial metabolism, activate autophagy, enhance antioxidative and anti-inflammatory capability, and inhibit insulin/insulin-like growth factor signaling, which contributes to the anti-aging process . Although KD has already been clinically used as a therapy, which is easier to sustain than CR, it is in some ways difficult to rigorously follow and requires specific medical guidance and strong motivation .
4.2. Exogenous Ketosis and Supplementation of β-HB
It has been demonstrated that not only KD, but also exogenous ketone supplements (EKSs) can increase and maintain blood KB level, especially β-HB, so as to promote anti-aging effects . β-HB supplements are now being commercially marketed as an alternative to KD. The supplements are commonly present in either a powder form of ketone salts (KS) or a liquid form of ketone ester (KE). In addition, medium-chain triglycerides (MCTs) or their combination with MCTs oil are also usually used to induce and sustain NK to improve ketotic response [132,133]. The production of β-HB from these supplements would not be affected by CHO, thus administration of EKSs may be practical and alternative when maintaining a normal diet to achieve therapeutic ketogenesis (Figure 5) .
4.2.1. Ketone Salts
Oral administration of isolated β-HB would be the most direct method of exogenously inducing NK. However, KB in its free acid form can be expensive, unstable, and ineffective at producing sustained ketosis. Thus, ketone acids buffering with sodium, potassium, calcium, or other electrolytes have been explored to enhance efficacy, inhibit overload of any single mineral, and these compounds are commercially available. It is reported that co-ingestion with MCTs may improve the efficiency of increasing β-HB relatively, at least in rats . However, a few undesirable adverse effects exist while consuming large doses of KS, which usually results in gastrointestinal distress, and inappropriate cation overload or acidosis .
4.2.2. Ketone Esters
Several existing synthetic KEs prove to be the most effective agents to induce immediate, sustained, and dose-dependent elevation in serum ketones concentration, which provides an alternative way to increase β-HB and is well-tolerated in rodents and humans [137,138]. Ester bonds hold KEs together and are cleaved by gastric esterases to release KB in their free acid form from the backbone molecule, which is often a ketogenic precursor molecule R,S-1,3-butanediol (BD). Two prominent KEs in the recent research studies are (R)-3-hydroxybutyl (R)-3-hydroxybutyrate ketone monoester (KME) and R,S-1,3-butanediol acetoacetate ketone diester (KDE), the former appears safer and superior at appropriate doses in healthy adults, whatever acutely or daily sustained up to 28 days [139–142]. NK produced by KEs is therefore achieved without prolonged fasting or KD yet it is currently the most potent method of EKSs.
4.2.3. Medium Chain Triglycerides
MCTs have a much greater ketogenic potential than long-chain fatty acids since they are rapidly absorbed, energy-dense, water-miscible, and tasteless. 6 to 12 carbons of fatty acids are contained in MCTs in length. MCTs can be hydrolyzed to medium-chain fatty acids by lipases in the gastrointestinal tract, and then rapidly metabolized to Ac-CoA, finally to KB in the liver . MCTs are consequently regarded as ketogenic fats due to their ability of ketogenesis without the restriction of dietary CHO intake . Unfortunately, high MCTs consumption are often not well adopted because of their gastrointestinal side effects, including diarrhea, dyspepsia, and flatulence, which could be alleviated through a progressive 1 or 2-week period . In addition, the generation of β-HB by supplementation of MCTs is at a low level in the blood .
4.2.4. R, S-1,3-Butanediol
BD is an organic butyl alcohol approved by the Food and Drug Administration, which is metabolized to produce two isoforms of β-HB, D- and L-β-HB or R- and S-β-HB via hepatic conversion, even though it is not a fatty acid or MCT . Oral administration of BD could achieve ketosis and approach a KD state in dogs . It was demonstrated that a dose-dependent elevation of KB in a ratio of 6:1 of R-β-HB to AcAc could be produced by BD in rodents . BD is often utilized as a backbone in the synthesis of KE. Gut or tissue esterases could easily break the ester bond and release KB and BD without the involvement of salts or acid . A variety of preclinical toxicology studies have found that BD is safe and tolerable .
4.2.5. β-HB Enantiomers
β-HB is a chiral molecule, with two enantiomers, R/D and S/L, which is an important characteristic in terms of its signaling activities as well as possible therapeutic applications . Currently, a racemic mixture enantiomer of β-HB is the most commercially available ingestible EKSs apart from KS and KME, as its synthesis is more affordable than pure enantiomers. The chiral specificity is introduced by BDH1, determining that only R-βHB is the normal product of human metabolism and could be readily catabolized into ATP and Ac-CoA . IF, CR, KD, exercise, or any other situation which leads to endogenous β-HB would produce only R-β-HB. It is reported that ingestion of the same amount of racemic EKSs may produce higher and more sustained S-β-HB in blood circulation due to its slower metabolization compared to R-β-HB [154,155]. Despite divergent metabolic effects, these two enantiomers have similar molecular interactions as well as intracellular signal transduction cascades, which remains a hotly debated topic.
4.3. Comparisons between Endogenous and Exogenous Ketosis Induced by NK
In essence, the metabolic conditions of chronic endogenous dietary ketosis are in obvious contrast to the rapid exogenous ketosis delivered by ketone bodies. The following are the differentiations. Firstly, KD elevates blood β-HB level to a range of 0.5 mM to 3.0 mM, whereas EKSs approximately elevate to 0.3 mM to 1.0 mM . Secondly, KD requires a couple of days to achieve sustained NK state, while EKSs elevate β-HB concentration acutely. Thirdly, KD need to follow strict CHO intake while EKSs require no direct CHO restriction, which determines EKSs have higher compliance, especially in short term supplementation . Fourthly, circulating glucose concentrations are therefore divergent because of the diverse CHO requirement. Fifthly, their similar anabolic and
anticatabolic effects have as well been demonstrated [157,158]. Finally, KD and EKSs have been reported to reduce the substrate utilization of CHO during exercise, yet under distinct metabolic states [159,160]. NK is defined as a metabolic state, exerting physiological changes at both systemic and cellular level wherein β-HB concentration is over 0.5 mM regardless if induced by endogenous or exogenous ketones. These effects can be similar or different and can be universal or tissue-specific. NK is believed to be a potential state for performance-enhancing or therapeutic benefits under endogenous or exogenous ketosis, which needs further evaluation.
5. Future Perspectives
Future studies are necessary to further elucidate the following practical issues: activating endogenous NK in a normal dietary context to sustain a steady state of metabolism; improving the targeted delivery of β-HB prodrugs or precursors to avoid excessive salt load or acidosis; bringing β-HB to the sites of action by using existing endogenous transporters and metabolite gradients to explore specific downstream signaling events; confirming whether the synthetic KE compounds need been strictly pure due to its enormous financial burden for the majority of patients and health systems; investigating if S-β-HB has a better pharmacokinetic than R-β-HB, which might help reduce cost and discover another signaling function; establishing methodologies to quantify β-HB flux rates and differentiate these two enantiomers in terms of concentrations and impacts. Besides, the recommended dose and timing of β-HB supplements, the short half-life and bitter taste of KME as well as the interaction with other substrates in various nutritional surroundings are also needed to be specified. β-HB is emerging as vitally important regulators of metabolic health and longevity, alleviating aging phenotypes via multiple and yet unknown molecular mechanisms. By modulating lipolysis, energy expenditure, metabolic rate, insulin resistance, autophagy, feeding behavior, as well as exercise performance, β-HB might serve as a signaling biomolecule to affect cellular function and human healthspan. The evaluation of β-HB may be a crucial approach for the treatment of the aging population.
In conclusion, β-HB is the most abundant KB and plays a vital role as an energetic metabolite, a signaling molecule, as well as an epigenetic regulator, which could be used as a therapeutic agent in a range of cancers, neurodegeneration, traumatic brain disorders, cardiac diseases, muscle dysfunction, metabolic syndrome, and inflammation. Endogenous ketosis and an exogenous supplement may be promising strategies for numerous diseases. Further research is needed to investigate whether ketotherapeutics can promote healthy aging, and to figure out the specific relationship and underlying mechanisms between β-HB and the aging process, which may offer a novel way in delaying the onset and development of age-associated dysfunctions.