**5. Healthspan and Longevity Enhancing Mechanisms**

It is now evident that RJ contains compounds (MRJPs, royalactin, amino acids, 10-HDA, pantothenic acid, Ach) that can modulate major mechanisms of aging. However, the exact mechanism through which RJ may extend lifespan is not well-understood. The mechanisms through which RJ extend lifespan can differ even within single species (Table 1). Several facets through which RJ can function have been proposed (Figure 3).





pRJ-Fr.5:

protease-treated-fraction

 5; 10-HDA:

10-hydroxy-2-decenoic

 acid; IP:

intraperitoneal;

 HET: genetically

heterogeneous

 head tilt.

**Figure 3.** Probable mechanisms through which royal jelly (RJ) and its components extend lifespan. RJ reduces insulin/insulin-like growth factor-1 signaling (*IIS*), which heightens the activity of *DAF-16*—the *C. elegans* counterpart to the mammalian Forkhead Box O transcription factor (*FOXO*). Activation of *DAF-16*/*FOXO* promotes its translocation from the cytoplasm to the nucleus and fosters interactions with its associated transcriptional co-regulator proteins: host cell factor (*HCF-1*), sirtuin homologue (*SIR-2.1*), and *FTT-2* (a 14-3-3 protein). This results in formation of protein complex inside cells and enhancement of the expression of multiple longevity promoting genes as well as regulation of downstream process of dietary restriction signaling (which lowers food-intake) and the mechanistic target of rapamycin (*mTOR*) pathway by extending the lifespan of the control *unc-24*/+ mutants. The interplay among several genes involved in *IIS*, *mTOR*, and dietary restriction signaling boosts key longevity-related cellular processes: DNA repair, autophagy, antioxidant activity, anti-inflammatory activity, stress resistance, and cell proliferation. On the other hand, RJ activates epidermal growth factor receptor (*EGFR*) signaling mainly by activating its receptor (*LET-23*). *EGFR* functions via downstream phospholipase C-γplc-3 and inositol-3-phosphate receptor itr-1 to upregulate elongation factors and chaperonins, which increase protein translation and proteasome activity—a mechanism that entails rebuilding cellular components; enhancement of cellular detoxification, ribosomal function, and muscle maintenance; and reduction of lipofuscin levels (age-pigments that accumulate during senescence).

#### *5.1. Insulin-Signaling*/*insulin like Growth Factor-1 Signaling*

The role of lowered insulin signaling/insulin-like growth factor-1 pathway (*IIS*) in longevity is well documented [101,102]. Findings from studies of *C. elegans* show that *IIS* does not only contribute to prolonged of lifespan, but it also promotes healthspan by resisting different types of stress [102]. The underlying mechanisms include improvement of insulin sensitivity and decrease of insulin levels, suppression of inflammation, reduction of adipose tissue, and increase of adiponectin levels [103]. The lifespan-expanding effect of RJ is associated with an interplay of several genes—expressed in some studies mainly by extending the lifespan of the insulin-like receptor *daf-2* mutants. *daf-2* demonstrates a life-expanding effect by regulating the downstream process of dietary restriction signaling (which lowers food-intake) as well as *TOR* signaling by extending the lifespan of the control *unc-24*/+ mutants [66]. Given that *daf-2* is a key upstream component of *IIS*, downregulation of *daf-2* stimulates a signaling cascade that leads to phosphorylation and fine tuning of the main downstream transcription factors of *IIS* known to promote lifespan: *DAF-16*—the *C. elegans* counterpart to the mammalian Forkhead Box O transcription factor (*FOXO*), heat shock transcription factor 1 (*HSF-1*), and *SKN-1*/*NRF2* [66,67,104]. Among all these pathways, RJ exerted its effect by targeting the activity of *DAF-16*/*FOXO*, which is a key longevity factor in various species ranging from worms to humans [52,67]. The interactions between *DAF-16* and its associated transcriptional coregulator proteins—host cell factor (*HCF-1*), sirtuin homologue (*SIR-2.1*), and *FTT-2* (a 14-3-3 protein)—lead to formation of a protein complex inside cells [67]. Moreover, activation of *DAF-16* promotes its translocation from the cytoplasm to the nucleus, where it stimulates the expression of multiple genes, which regulate processes that promote longevity: DNA repair, autophagy, antioxidant activity, anti-inflammatory activity, stress resistance, and cell proliferation [10,104]. Aging is associated with increased aggregation and insolubility of various RNA granule proteins (e.g., stress granule proteins). Reduction of *daf-2* receptor signaling heightens *DAF-16* activity, which can efficiently extend lifespan in *C. elegans* by preventing the buildup of misfolded proteins that seed the aggregation of insoluble stress granule proteins [105].
