*5.2. The Mechanistic Target of Rapamycin Signaling*

The mechanistic target of rapamycin (*mTOR*) pathway is another signaling cascade that contributes to RJ enhancement of lifespan extension [17,39,66]. The underlying mechanism involves suppression of *mTOR* gene expression by MRJPs and freeze-dried RJ [17,39]. The nutrient sensor *mTOR* is a serine/threonine protein kinase that comes in two structurally and functionally distinct forms: *TOR* Complex 1 (*TORC1*) and *TORC2*. Mammalian *TORC1* (*mTORC1*)—not *TORC2*—plays a major role in aging. *mTORC1* comprises three components, the catalytic subunit mammalian *TOR* (*mTOR*), regulatory-associated protein of target of rapamycin (*RAPTOR*), and mammalian lethal SEC13 protein 8 (*mLST8*) [106,107]. Evidence denotes that inhibition of *mTORC1* with rapamycin extends lifespan up to the double in model organisms [4]. *mTORC1* signaling is regulated by endocrine signaling, especially growth factors and *IGF-1*, as well as nutrients e.g., amino acids, lipids, and glucose, in addition to cellular energy and oxygen levels [107,108]. In this respect, 10-HDA supplementation to *C. elegans* extended the lifespan of the control *unc-24*/+ mutants but did not extend the lifespan of the long-lived heterozygous mutants in *daf-15*, which encode *RAPTOR* [66]. The anti-aging activity of the protein kinase *mTOR* originates from its ability to function both in a cell autonomous manner and a non-cell autonomous manner to regulate growth, protein translation, ribosomal biogenesis, autophagy, and cellular metabolism in response to both environmental and hormonal signals [106,108]. It is thought that *mTORC1* inhibition restores cellular physiological integrity, and hence delays age-related pathologies. In details, *mTORC1* promotes translation initiation of mRNAs of metabolism-related genes and ribosomal-related proteins via phosphorylation of two main ribosomal proteins: *S6K1* and *S6K2*. Thus, *mTORC1* inhibition is associated with enhanced endogenous protein degradation as well as less aggregation of proteotoxic and oxidative stress wastes, which in turn preserve homeostasis in the face of oxidative damage. Autophagy genes play a major role in these processes [4,109]. For a detailed description of mechanisms through which *mTOR* functions, we refer readers to these reviews [4,107,108].

#### *5.3. Dietary Restriction Signaling*

Dietary restriction is another mechanism that promotes longevity in various species. It involves prolonged reduction of intake of most dietary elements except vitamins and minerals (without getting into malnutrition)—it is equivalent to voluntary intermittent fasting in humans, which is reported to prevent numerous debilitating disorders such as abdominal obesity, diabetes, hypertension, and cardiovascular diseases [10]. Recent reviews point out that dietary restriction in humans exhibits similar effects to model organisms in terms of body composition, circulating lipoprotein, inflammatory and metabolite profiles, energy expenditure, and oxidative stress [110,111]. *C. elegans eat-2* mutant is considered a model of dietary restriction; its acetylcholine receptor mutation hinders pharyngeal pumping and limits intake of nutrients [109]. The main lipid of RJ, 10-HDA, which exhibited a lifespan-extending effect did not extend the lifespan of the *eat-2* mutants in *C. elegans*, which denotes that feeding impairment-related dietary restriction signaling was involved in the lifespan extending mechanism. However, progeny production was not delayed in 10-HDA-treated

worms—unlike dietary-restricted worms—which indicated that the lifespan-extending effect of 10-HDA was related to the downstream process of the dietary restriction signaling [66]. Indeed, the longevity effect of *eat-2* mutant is mediated by compensatory changes of several energy sensing effectors: dietary restriction downregulates *IIS*, which activates *FOXO*, whereas the energy sensor for cellular AMP/ATP ratio known as *AMPK* gets activated to stimulate catabolic reactions for energy gain by phosphorylating *DAF-16*/*FOXO*. It also downregulates *AKT*/*mTOR* through *PHA-4*/*FOXA* transcription factor and *S6K* (in a *DAF-16*/*FOXO*-independent manner), and stimulates the expression of autophagy genes—*unc-51*/*ULK1*, *bec-1*/*Beclin1*, *vps-34*, *atg-18*, and *atg-7*—which inhibit general protein translation and simultaneously stimulate the translation of specific mRNAs involved in cellular homeostasis [5,10,109]. In addition, dietary restriction causes activation of *NRF2* transcription factor, which suppresses inflammation and counteracts oxidative damage [112].

## *5.4. Epidermal Growth Factor Signaling*

RJ, royalactin in particular, extended lifespan of various species by activating the epidermal growth factor receptor (*EGFR*) signaling. As shown in Table 1, RJ activation of *EGFR* signaling involves upregulation of *S6K* and *MAPK*, which results in enhanced locomotor activity and antioxidant capacity; increased *20E* titre, which stimulates growth (increased body size); and increased juvenile titre, which increased fertility—all are effects that indicate healthspan. Royalactin might interact with *LIN-3* to promote the binding of the ligand to the extracellular domain of the *EGFR*, which stimulate *EGF* signaling [68]. Still, the exact mechanism through which RJ affects *EGFR* to promote longevity is not clear. However, it has been recently reported that, royalactin-related *EGFR* signaling induces longevity in *C. elegans* via upregulation of elongation factors and chaperonins, which increase protein translation and proteasome activity—a mechanism that entails rebuilding cellular components and enhancement of cellular detoxification, ribosomal function, and muscle maintenance—rather than stabilizing the existing proteome [113]. Furthermore, activation of *MAPK*—which is stimulated by *EGF*—increases the lifespan of *daf-2* mutants and thus controls the expression of pathogen response genes (*C-type lectins*, *ShK toxins*, and *CUB-like* genes); such increased resistance to pathogens increases lifespan in *C. elegans* [114]. *EGF* pathway functions in a manner that is independent of the *insulin*/*IGF-like* pathway i.e., activation of its receptor (*LET-23*) is necessary for its action [68]. In addition, it is not affected by deficiency of the *DAF-16*/*FOXO* transcription factor and it exerts more effects when *daf-2*/*InR* activity is inhibited [115]. Moreover, royalactin is thought to affect regulators of *EGF* signaling—such as high performance in advanced age genes (*HPA-1* and *HPA-2*)—to stimulate the release of *LIN-3* [68]. In fact, *HPA-1* and *HPA-2* genes negatively regulate *EGF* signaling by binding and sequestering *EGF*. While *HPA-1* is thought to contribute to longevity, *HPA-2* is reported to induce healthspan benefits in *C. elegans* by encoding secreted proteins similar in sequence to extracellular domains of insulin receptor. *EGF* signaling functions via downstream phospholipase C-γplc-3 and inositol-3-phosphate receptor itr-1 to promote healthy aging associated with low lipofuscin levels (age pigments that accumulate during senescence), enhance physical performance, and extend lifespan [115,116]. *EGFR*-mediated antiaging effects seem to be evolutionally conserved from worms to humans. In humans, a recent study tested single-nucleotide polymorphisms (*SNPs*) in *EGFR* for association with longevity. Comparison of genotype frequencies of 41 *EGFR SNPs* between 440 American males of Japanese ancestry aged ≥95 years and 374 men of average lifespan (whites and Koreans) revealed a significant association with longevity for seven *SNPs* in *EGFR*—evidence that genetic variation in *EGFR* contributes to lifespan extension in Japanese people [101].
