Effects of Tripeptide IRW (Ile-Arg-Trp) on metabolism, mitochondrial biogenesis, and lifespan extension

Abstract

Bioactive tripeptide IRW (Ile-Arg-Trp) was initially identified as an ACE (angiotensin-converting enzyme) inhibitory peptide from egg protein ovotransferrin. The pharmacological spectrum of IRW extends towards metabolic boosting and anti-diabetic properties as well. The overall objectives of this thesis were to understand the anti-aging effects of IRW via the study of its impact on the NAD axis, mitochondrial biogenesis, and lifespan extension. Firstly, treatment of muscle (L6) cells with IRW increased intracellular Nicotinamide phosphoribosyltransferase (NAMPT) protein levels and boosted Nicotinamide adenine dinucleotide (NAD+). Both immunoprecipitation and recombinant NAMPT assays indicated the possible NAMPT activating ability of IRW. Similarly, IRW increased NAMPT mRNA and protein levels in liver and muscle tissues of C57BL/6J mice fed a high-fat diet (HFD). A significant increased level of circulating NAD+ was also observed following IRW treatment. Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT) and NAD+ in vivo. However, IRW treatment did not boost NAMPT levels in Sirtuin 1 (SIRT1) KO cells, indicating a possible SIRT1-dependency for the pharmacological effect. Overall, these data indicate that IRW is a novel small peptide booster of the NAMPT pool. Next, IRW was also investigated for its impact on mitochondrial biogenesis. Results showed that IRW activates mitochondrial biogenesis resulting in increased mitochondrial DNA, ATP surge, improved metabolic and microbiome function. IRW activated the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC1α), the master regulator of mitochondrial biogenesis, in multiple cell lines and tissues of C57BL/6J HFD mice. It also increased mitochondrial DNA in muscles of aged Drosophila fed with IRW for a week. The CRISPR-Cas9 experiments elucidated the underlying mechanism as Family with Sequence Similarity 120B (FAM120B) dependent, a constitutive activator of peroxisome proliferator activated receptor gamma (PPARγ). To the best of our knowledge, IRW is the first bioactive peptide to induce mitochondrial biogenesis in vitro and in vivo. Finally, the ability of IRW to extend lifespan was evaluated using the Drosophila model in vivo. Different fly lines, such as w, wy, and Wdah were fed regular fly medium supplemented with IRW. IRW treatment at 50 and 100 μM concentrations prolonged the median life span of white mutant (w) by 5.1 and 12.08% (respectively) and of yellow mutant (wy) by 12.1 and 22.9% (respectively). Likewise, midlife IRW feeding in Wdah flies improved lifespan significantly as well. Also, IRW treatment at these concentrations significantly improved the histone markers in flies and activated the expression of multiple gene pathways involved in sirtuins (SIRT1), antioxidant defense (SOD2), autophagy (ATG7), and insulin signaling (dInR). Together, our study identifies the first bioactive peptide with the ability to extend lifespan in vivo and suggests an important prospective role of IRW intake for healthy aging in humans. Overall, this research demonstrated the potential of IRW as an anti-aging functional food due to its role in promoting NAD+ levels and mitochondrial biogenesis leading to extension in lifespan.