Caffeine-Induced Upregulation of pas-1 and pas-3 Enhances Intestinal Integrity by Reducing Vitellogenin in Aged Caenorhabditis elegans Model

<b>Background:</b> Intestinal aging is characterized by declining protein homeostasis via reduced proteasome activity, which are hallmarks of age-related diseases. Our previous study showed that caffeine intake improved intestinal integrity with age by reducing vitellogenin (VIT, yolk protein) in <i>C. elegans</i>. In this study, we investigated the regulatory mechanisms by which caffeine intake improves intestinal integrity and reduces vitellogenin (VIT) production in aged <i>Caenorhabditis elegans</i>. <b>Methods:</b> We performed RNA-seq analysis, and qRT-PCR to validate and confirm the RNA-seq results. Transgenic worms with VIT-2::GFP and VIT-6::GFP were used for measuring VIT production. dsRNAi was conducted to elucidate the roles of <i>pas-1</i> and <i>pas-3</i> genes. <b>Results:</b><i>pas-1</i> and <i>pas-3</i>, a <i>C. elegans</i> ortholog of human <i>PASM4</i>, was upregulated by caffeine intake. They reduced VIT production by repressing <i>unc-62</i>, a transcriptional activator of <i>vit</i> expression. Interestingly, <i>vit-2</i> was required for <i>pas-1</i> and <i>pas-3</i> expression, and RNAi of <i>pas-1</i> and <i>pas-3</i> promoted intestinal atrophy and colonization, suggesting a balancing mechanism for VIT levels in intestinal health. Additionally, lifespan was extended by caffeine intake (2 ± 0.05 days), however, this effect was not observed by <i>pas-1</i> but not <i>pas-3</i> RNAi, suggesting that the mode of action for an anti-aging effect of caffeine through <i>pas-1</i> and <i>pas-3</i> is distinctive. The lifespan extended by <i>pas-1</i> was mediated by SKN-1 activation. <b>Conclusions:</b> Caffeine intake enhances intestinal health through proteasome activity and extends lifespan in aged <i>C. elegans</i> by upregulating <i>pas-1</i> and <i>pas-3</i>. These findings suggest that caffeine consumption mitigates age-related proteasome impairment and maintains intestinal integrity during aging.