Oral intake of rice overexpressing ubiquitin ligase inhibitory pentapeptide prevents atrophy in denervated skeletal muscle

Skeletal muscle growth is largely governed by IGF-1 signaling. Under unloading/disuse conditions (e.g., space flight, tail suspension, sciatic denervation), expression of the ubiquitin ligase CBLB increases, which promotes ubiquitination and degradation of IRS1, attenuating IGF-1 signaling and driving upregulation of atrophy-related ubiquitin ligases (MAFbx/atrogin-1, MuRF1) via FOXO3 activation. Prior work showed Cblb-deficient mice resist unloading-induced atrophy and that a designed inhibitory pentapeptide (Cblin; Asp-Gly-pTyr-Met-Pro), which mimics pTyr608 of IRS1 and binds the CBLB TKB domain, prevents IRS1 ubiquitination and muscle loss when injected intramuscularly. However, injections are invasive. While di- and tripeptides are well-known to be absorbed via PEPT1, absorption of longer oligopeptides (≥4 residues), including Cblin, is less clear though some can cross epithelia via paracellular routes. The authors also hypothesized that dietary proteins releasing Cblin-like sequences could protect muscle. This study asks whether Cblin pentapeptides can be absorbed intact from the intestine and whether a transgenic rice enriched in a Cblin-like peptide (CbR) can prevent denervation-induced skeletal muscle atrophy in rodents.

Background literature establishes: (1) the central role of IGF-1/IRS1 signaling in muscle hypertrophy/atrophy; (2) unloading increases CBLB, promoting IRS1 degradation and induction of MAFbx and MuRF1 via FOXO3; (3) Cblb-deficiency confers resistance to disuse atrophy; (4) a CBLB inhibitory peptide (Cblin) prevents atrophy when injected into muscle; (5) food-derived di-/tripeptides are absorbed via PEPT1 and can be bioactive in vivo, while some longer oligopeptides may traverse intestinal monolayers via paracellular routes; (6) prior evidence that methylated model pentapeptides can be absorbed, and that soy glycinin may release Cblin-like fragments; (7) plant biotechnology can embed repeats of functional peptides into rice storage proteins, enabling their release upon digestion. These inform the hypothesis that orally delivered Cblin or Cblin-like sequences from engineered rice may be bioavailable and functional against muscle atrophy.

In vitro intestinal transport: Caco-2 monolayers (TEER > 300 Ω·cm^2) were exposed apically to 1 mM Asp-Gly-pTyr-Met-Pro or Asp-Gly-Tyr-Met-Pro. Basolateral samples were collected at 15, 30, 45, and 60 min and analyzed by LC-TOF/MS monitoring [M+H]+ m/z 662.1625 (pTyr form) and 582.2228 (Tyr form). Apparent permeability coefficients (Papp) were calculated. Transport pathway interrogation included addition of 10 mM Gly-Sar (PEPT1 substrate/inhibitor) and cytochalasin D (0.5 µg/mL, 30 min preincubation) to modulate tight junctions. Fragment ions were searched by targeted m/z extraction to assess peptide integrity and dephosphorylation. Rat oral absorption: Male Sprague–Dawley rats were fasted 16 h and orally dosed with Asp-Gly-Tyr-Met-Pro at 100 mg/kg BW (additional doses 50 and 200 mg/kg referenced). Blood was collected at 0, 15, 30, 45, and 60 min. Absolute plasma concentrations were quantified by LC-TOF/MS using an internal standard (50 pmol/mL Asp-Gly-(13C2;15N)-Tyr-Met-Pro). Pharmacokinetic parameters (Cmax, Tmax, AUC0–60, t1/2) were derived. Potential metabolites were screened by extracted ion chromatograms (e.g., Gly-Tyr-Met-Pro m/z 467.1959; Met-Pro m/z 247.1111). Transgenic rice characterization: Cblin peptide-enriched rice (CbR) was engineered with 15 repeats of Gln-Asp-Gly-Tyr-Met-Pro-Trp (Cblin-like) inserted into the glutelin gene to enable release by chymotrypsin. Glutelin was extracted from CbR and non-transgenic rice (non-Tg) and digested in vitro with chymotrypsin. Digests were profiled by HPLC (220 nm). The RT 27.0 min fraction (matching synthetic Gln-Asp-Gly-Tyr-Met-Pro-Trp) was analyzed by LC-TOF/MS monitoring [M+2H]2+ m/z 448.6840 to confirm peptide identity. Mouse denervation model: Six-week-old male ICR mice were fed a standard diet containing 50% (w/w) powdered CbR or non-Tg rice for 7 days prior to unilateral sciatic nerve transection under anesthesia; contralateral muscles served as sham-operated controls. Mice continued on the assigned diet for 7 additional days. Body weight and food intake were recorded. Gastrocnemius (Ga) and soleus (Sol) muscles were dissected and weighed. Ga muscles were analyzed for IRS1 protein by immunoblotting (anti-IRS1; ACTB loading control). mRNA expression of Mafbx and Murf1 was quantified by real-time RT-PCR (SYBR-Green), normalized to Actb. Attempts were made to detect CbR-derived peptides and metabolites in serum and skeletal muscle by LC-TOF/MS via targeted m/z searches. Animal experiments were approved by institutional ethics committees (Permissions T27-113 and A30-015-4). Statistics: Data are means ± SEM. Caco-2 Papp comparisons used one-way ANOVA with Tukey post hoc. Effects of Gly-Sar or cytochalasin D were tested with appropriate comparisons; significance indicated at P < 0.05 or P < 0.01. Mouse diet effects analyzed by two-way ANOVA with Tukey–Kramer post hoc. Rat PK summarized from n=5.

- Caco-2 transport: Both Asp-Gly-pTyr-Met-Pro and Asp-Gly-Tyr-Met-Pro appeared intact in basolateral samples with time-dependent increases (RT ~19.8 and 19.4 min, respectively). The pTyr peptide underwent dephosphorylation during transport, yielding Asp-Gly-Tyr-Met-Pro at levels comparable to intact peptide after 60 min. No other fragments were detected. Papp values: 3.5 ± 1.2 × 10−7 cm/s (Asp-Gly-pTyr-Met-Pro) and 7.0 ± 0.8 × 10−7 cm/s (Asp-Gly-Tyr-Met-Pro), similar order to model pentapeptide Gly-Sar-Sar-Sar-Sar (8.6 ± 0.6 × 10−7 cm/s). Transport was unaffected by 10 mM Gly-Sar (PEPT1 substrate), indicating PEPT1 independence, but was significantly increased by cytochalasin D, implicating enhanced paracellular permeability. - Rat oral absorption: Orally administered Asp-Gly-Tyr-Met-Pro was detectable in plasma, peaking at 2.78 ± 0.17 pmol/mL at 15 min (Cmax), with t1/2 ~28 min and AUC0–60 of 100.35 ± 23.40 pmol·min/mL (n=5). Metabolites Gly-Tyr-Met-Pro and Met-Pro were detected, suggesting partial enzymatic processing during absorption. - Transgenic rice digestion: Chymotrypsin digestion of CbR glutelin produced an HPLC peak at RT 26–27 min matching synthetic Gln-Asp-Gly-Tyr-Met-Pro-Trp; LC-TOF/MS confirmed [M+2H]2+ m/z 448.6840 in CbR digests but not in non-Tg, indicating successful incorporation and releasability of the Cblin-like peptide. Compositional analyses from prior work indicate CbR and non-Tg have equivalent macro- and micronutrient profiles aside from the inserted peptide. - Mouse denervation study: Denervation reduced Ga and Sol muscle wet weights by 24% and 45%, respectively, in non-Tg-fed mice. CbR feeding significantly attenuated Ga atrophy, with denervated Ga showing only ~13% loss versus 24% in controls. CbR also reduced Sol loss, though denervated vs sham remained significantly different. In Ga, denervation decreased IRS1 protein in non-Tg-fed mice; CbR preserved IRS1 levels post-denervation. Mafbx and Murf1 mRNAs were each ~2.7-fold higher in denervated Ga with non-Tg diet; this upregulation was suppressed in CbR-fed mice (Mafbx not significantly different from sham; Murf1 significantly reduced). No adverse effects on body weight gain or food intake were observed.

The study demonstrates that Cblin pentapeptides can traverse intestinal epithelial barriers intact, predominantly via a paracellular mechanism rather than PEPT1-mediated transport. Oral dosing in rats yields measurable, albeit low, plasma levels and identifiable metabolites, supporting in vivo absorption. Engineering rice to overexpress a releasable Cblin-like peptide results in a functional food (CbR) that, when included in the diet, mitigates denervation-induced muscle atrophy in mice. Mechanistically, CbR intake preserved IRS1 protein and prevented the denervation-induced transcriptional activation of atrophy-associated ubiquitin ligases (Mafbx, Murf1), consistent with inhibition of CBLB-mediated IRS1 ubiquitination and maintenance of IGF-1 signaling. The effects are expected to be muscle-state selective, given low basal Cblb expression in normal muscle, implying minimal off-target effects in healthy tissue. Challenges in detecting intact heptapeptide and all metabolites in serum/muscle suggest low bioavailability and rapid metabolism; nonetheless, peptides retaining Tyr residues may contribute to CBLB inhibition even without phosphorylation. Matrix effects, digestive enzyme specificities, and peptide deposition in glutelin (PB-II) likely facilitate release and activity. Overall, CbR offers a non-invasive, dietary strategy to prevent disuse atrophy, with translational potential pending further pharmacokinetic and mechanistic clarification.

This work establishes that a CBLB-inhibitory pentapeptide (Cblin) can be absorbed from the gut and that a transgenic rice enriched with a Cblin-like sequence (CbR) effectively attenuates denervation-induced muscle atrophy in mice by preserving IRS1 and suppressing Mafbx/Murf1 expression. The findings provide proof-of-concept for a functional food approach to prevent muscle atrophy. Future research should (1) quantify the bioavailability and tissue distribution of intact peptides and key metabolites; (2) identify the most bioactive fragments and define their individual contributions; (3) elucidate transport pathways beyond paracellular diffusion; (4) optimize peptide expression, release, and stability in food matrices; and (5) evaluate efficacy and safety in longer-term and clinically relevant models.

- Plasma concentrations of the pentapeptide after oral dosing were low, and overall bioavailability remains unclear. - Intact CbR-derived heptapeptide and all anticipated metabolites were difficult to detect reproducibly in serum and especially in skeletal muscle. - The precise intestinal transport pathways beyond paracellular diffusion were not fully delineated. - Mouse denervation is an acute atrophy model; longer-term and diverse atrophy contexts (e.g., aging, disease, immobilization) were not assessed. - Sample sizes were modest; detailed dose–response and pharmacodynamics of CbR were not fully characterized. - Human relevance requires further study, including variability in digestion, absorption, and matrix effects.