Skeletal muscle growth and hypertrophy are largely regulated by insulin-like growth factor 1 (IGF-1) signaling. Previous research demonstrated that muscle disuse upregulates Casitas B-lineage lymphoma b (CBLB), a ubiquitin ligase. CBLB promotes the ubiquitination and degradation of insulin receptor substrate 1 (IRS1), a key component of IGF-1 signaling. This leads to increased expression of muscle atrophy-related ubiquitin ligases such as MAFbx and MuRF1 through FOXO3 activation. Studies using *Cblb*-deficient mice confirmed CBLB's role in muscle atrophy. A CBLB inhibitory peptide, Cblin (Asp-Gly-pTyr-Met-Pro), was developed and shown to prevent muscle atrophy via intramuscular injection, but this method is invasive. Many food-derived di- and tripeptides exhibit bioactivities and are absorbed via PEPT1. However, it is unknown if larger peptides, like Cblin, can be orally absorbed. Prior research indicated that soy glycinin might inhibit muscle atrophy due to the release of Cblin-like peptides. This study aimed to determine if Cblin could be absorbed orally and if Cblin-rich rice (CbR) could prevent muscle atrophy.

The existing literature extensively details the IGF-1 signaling pathway in skeletal muscle growth and atrophy. Previous work by the authors and others highlighted the role of CBLB in mediating muscle atrophy through the degradation of IRS1. The effectiveness of Cblin peptide in preventing muscle atrophy through intramuscular injection had been established. The study reviewed the absorption mechanisms of peptides from food sources, specifically the PEPT1 transporter for di- and tripeptides. Studies demonstrating the absorption of some oligopeptides despite their size were also considered. Prior work by the authors showed that soy glycinin inhibited muscle atrophy, potentially through the release of Cblin-like peptides. The development of a transgenic rice strain enriched in gamma-aminobutyric acid and the potential for plant-based delivery of peptides provided the framework for this study.

The study used Caco-2 cell monolayers to assess Cblin peptide transport. Transepithelial electrical resistance (TEER) was monitored to ensure monolayer integrity. LC-TOF/MS was employed to measure peptide concentrations across the monolayer. The apparent permeability coefficient (Papp) was calculated. The influence of Gly-Sar (a PEPT1 substrate) and cytochalasin D (a tight junction opener) on Cblin transport was investigated. In vivo absorption of Asp-Gly-Tyr-Met-Pro was assessed in Sprague-Dawley rats following oral administration (100 mg/kg BW), with plasma samples analyzed by LC-TOF/MS using a stable isotope-labeled internal standard. CbR was characterized by analyzing glutelin extracts after in vitro digestion with chymotrypsin, using HPLC and LC-TOF/MS. In vivo anti-muscle atrophy effects of CbR were evaluated in mice with sciatic denervation. Mice were fed either CbR or non-transgenic rice for 7 days before denervation. Body weight, food consumption, muscle wet weight, IRS1 protein levels (immunoblotting), and mRNA expression levels of *Mafbx* and *Murf1* (real-time RT-PCR) were measured. Statistical analyses (Student's t-tests, one-way/two-way ANOVAs with post hoc tests) were performed.

Both phosphorylated (Asp-Gly-pTyr-Met-Pro) and non-phosphorylated (Asp-Gly-Tyr-Met-Pro) Cblin peptides crossed Caco-2 cell monolayers, with the non-phosphorylated form exhibiting higher permeability (Papp values: 3.5 ± 1.2 × 10⁻⁷ cm/s and 7.0 ± 0.8 × 10⁻⁷ cm/s, respectively). Gly-Sar did not affect transport, suggesting PEPT1 was not involved. Cytochalasin D significantly enhanced transport, indicating paracellular transport via tight junctions. Oral Asp-Gly-Tyr-Met-Pro was detected in rat plasma, peaking at 15 min (2.78 ± 0.17 pmol/mL). Metabolic fragments (Gly-Tyr-Met-Pro and Met-Pro) were also detected. CbR digestion released a Cblin-like peptide (Gln-Asp-Gly-Tyr-Met-Pro-Trp), confirmed by HPLC and LC-TOF/MS. Mice fed CbR showed no significant difference in body weight or food consumption compared to the control group. However, CbR significantly reduced denervation-induced muscle weight loss in both gastrocnemius (13% reduction) and soleus muscles. CbR also preserved IRS1 protein levels and suppressed the expression of *Mafbx* and *Murf1* genes, indicating the inhibition of muscle atrophy pathways. Analysis of serum and muscle tissue from mice fed CbR showed various metabolic fragments, but detection of the intact heptapeptide was inconsistent.

The findings demonstrate that the Cblin pentapeptide, despite its size, can be absorbed orally. The transport across Caco-2 cells likely occurs primarily via paracellular pathways. Oral administration of CbR effectively prevented muscle atrophy in mice, suggesting the potential of CbR as an alternative treatment for muscle wasting conditions. The mechanism appears to involve the inhibition of CBLB activity, leading to the preservation of IRS1 and the suppression of atrophy-related genes. The identification of various metabolic fragments in serum suggests that multiple shorter peptides derived from the Cblin-like heptapeptide might contribute to the observed effects. Further research is needed to fully elucidate the contributions of individual fragments and to optimize the delivery and bioavailability of the active peptide(s).

This study successfully demonstrated the oral bioavailability of Cblin and the efficacy of CbR in preventing muscle atrophy in a mouse model. CbR provides a novel, non-invasive approach for addressing muscle atrophy. Further studies are needed to fully characterize the metabolism and bioactivity of the various CbR-derived peptides and to explore the potential therapeutic applications of CbR in humans.

The study primarily focused on the effects of CbR on denervation-induced muscle atrophy in mice, which may not fully translate to other types of muscle wasting or human conditions. The detection of specific peptide fragments in mouse serum and muscle was inconsistent, limiting a complete understanding of the metabolic pathways and the contributions of individual fragments to the observed effects. The study did not investigate the long-term effects of CbR consumption or potential side effects with prolonged use.