Identification and anti-oxidative potential of milk fat globule membrane (MFGM)-derived bioactive peptides released through in vitro gastrointestinal digestion.

This study investigated the stability of milk fat globule membrane (MFGM) protein under simulated gastrointestinal conditions using an in vitro enzymatic digestion method. The optimal hydrolysis conditions were determined by monitoring the changes in particle size and zeta-potential of MFGM protein hydrolysates over time. Furthermore, the distribution of small molecular weight peptides with antioxidant activity was explored through DEAE-52 combined with in vitro cell experiments. Two novel antioxidant peptides (TGIIT and IITQ) were identified based on molecular docking technology and evaluated their potential scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+) radicals. TGIIT and IITQ also demonstrated remarkable abilities in promoting mitochondrial biogenesis and activating Keap1/Nrf2 signaling pathway, which can effectively counteract skeletal muscle dysfunction induced by oxidative stress. Thus, MFGM-derived antioxidant peptides have the potential to be employed in food to regulate muscle protein metabolism and alleviate sarcopenia.

Synergistic effects of MFG-E8 and whey protein on mitigating d-galactose-induced sarcopenia through PI3K/AKT/PGC-1α and MAPK/ERK signaling pathways.

Milk fat globule epidermal growth factor 8 (MFG-E8) and whey protein have emerged as promising bionutrient supplements for enhancing skeletal muscle mass and function. In the present study, aging-related sarcopenia rat model was employed to elucidate the effects of the combined administration of MFG-E8 and whey protein on the catabolism and anabolism of gastrocnemius protein. Combined intervention led to notable enhancements in the antioxidative stress status and mitochondrial biogenesis capacity of gastrocnemius muscle fibers in the aging rats, concomitant with a significant inhibition of lipid accumulation. Moreover, the synergistic effect of MFG-E8 and whey protein was found to exert modulatory effects on key signaling pathways, including PI3K/Akt/PGC-1α pathway and MAPK/ERK signaling pathways in the gastrocnemius muscle of the aging rats. Specifically, this combined intervention was observed to promote mitochondrial biogenesis and regulate the expression of protein anabolism and catabolism-related regulators, thereby facilitating the alleviation of mitochondrial oxidative stress and enhancing biogenesis in gastrocnemius tissues. The findings of our study provide compelling evidence for the potential of MFG-E8 as a promising dietary supplement with antisarcopenic properties to ameliorate muscle protein metabolism disorders and mitigate mitochondrial-mediated myoblast apoptosis induced by oxidative stress.

Identification and antioxidative mechanism of novel mitochondria-targeted MFG-E8 polypeptides in virtual screening and in vitro study.

Milk fat globule-EGF factor VIII (MFG-E8) has been identified as an important source of bioactive peptides, which may exert a pivotal role in regulating biologic redox equilibrium. However, the composition of MFG-E8 polypeptides and their mechanisms on mitigating sarcopenia remain unknown. The aim of this study was to identify the composition of MFG-E8 polypeptides and its effects against oxidative stress in dexamethasone-induced L6 cell injury. Simulated digestion in vitro and liquid chromatography-tandem mass spectrometry were used in this investigation. A total of 95 peptides were identified during complete simulated digestion; among them, the contents of 21 peptides were analyzed, having been determined to exceed 1%. Molecular docking assay found that IDLG, KDPG, YYR, and YYK exhibited high binding affinity with keap1. MTT, dichlorodihydrofluorescein diacetate, mito- and lyso-tracker, and transmission electron microscope assay demonstrated that IDLG and KDPG can alleviate oxidative stress-injured L6 cell vitality, mitochondria activity, vacuolation, and function decrease, and increased autophagy, thereby improving mitochondrial homeostasis. From a molecular perspective, IDLG and KDPG can decrease the expression of keap1 and increase the expression of Nrf2, HO-1, and PGC-1α. Therefore, MFG-E8-derived IDLG and KDPG could be potential polypeptides countering oxidative stress in the treatment of sarcopenia, via the keap1/Nrf2/HO-1 signaling pathway.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells.

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid ß-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

Whey protein peptides PEW and LLW synergistically ameliorate hyperuricemia and modulate gut microbiota in potassium oxonate and hypoxanthine-induced hyperuricemic rats.

Pro-Glu-Trp (PEW) and Leu-Leu-Trp (LLW) are peptides derived from whey protein digestive products; both peptides exhibit xanthine oxidase inhibitory activity in vitro. However, it remains unclear whether these peptides can alleviate hyperuricemia (HUA) in vivo. In this study, we investigated the roles of PEW and LLW, both individually and in combination, in alleviating HUA induced by potassium oxonate and hypoxanthine. Together, PEW and LLW exhibited synergistic effects in reducing the serum levels of uric acid (UA), creatinine, and blood urea nitrogen, as well as increasing the fractional excretion of UA. The combined treatment with PEW and LLW inhibited UA synthesis, promoted UA excretion, and restored renal oxidative stress and mitochondrial damage. Moreover, the combined treatment alleviated dysbiosis of the gut microbiota, characterized by increased helpful microbial abundance, decreased harmful bacterial abundance, and increased production of short-chain fatty acids. Taken together, these results indicate that the combination of PEW and LLW mitigate HUA and kidney injury by rebalancing UA synthesis and excretion, modulating gut microbiota composition, and improving oxidative stress.

Novel xanthine oxidase inhibitory peptides derived from whey protein: identification, in vitro inhibition mechanism and in vivo activity validation.

As the development of hyperuricemia (HUA) and gout continues to accelerate worldwide, there is increasing interest in the use of xanthine oxidase (XO) inhibitors as therapeutic agents for the management of HUA and gout. In the present study, XO inhibitory peptides were identified from whey protein isolate (WPI) hydrolysates, and the underlying inhibitory mechanism and in vivo activities was investigated. WPI hydrolysates were isolated and purified, and two peptides (ALPM and LWM) with lower binding energy were screened by molecular docking. The result showed that these two peptides interacted with residues around the active site of XO through hydrogen bond and hydrophobic interaction. The IC50 values of ALPM and LWM were 7.23 ± 0.22 and 5.01 ± 0.31 mM, respectively. According to the Lineweaver-Burk curve, the inhibition types of ALPM and LWM were non-competitive inhibition. Circular dichroism (CD) spectra indicated ALPM and LWM could change the secondary structure of XO. Molecular dynamics simulations revealed that XO-peptide complexes were more stable and compact than XO. Moreover, animal studies have shown that ALPM and LWM have anti-hyperuricemia effects in vivo. This study suggested that ALPM and LWM can be considered as natural XO inhibitors for the treatment of HUA.

Anti-hyperuricemic and nephroprotective effects of whey protein hydrolysate in potassium oxonate induced hyperuricemic rats.

BACKGROUND: Hyperuricemia (HUA) is a serious public health concern globally that needs to be solved. It is closely related to gout and other metabolic diseases. To develop a safe and effective dietary supplementation for alleviating HUA, we investigated the effects of whey protein hydrolysate (WPH) on HUA and associated renal dysfunction and explored their underlying mechanism. RESULTS: Potassium oxonate was used to induce HUA in model rats, who were then administered WPH for 21 days. The results showed that WPH significantly inhibited xanthine oxidase and adenosine deaminase activity in serum and liver, decreased uric acid (UA), creatinine, and blood urea nitrogen levels in serum, and increased the UA excretion in urine. In addition, WPH downregulated the expression of urate transporter 1 and upregulated the expression of organic anion transporter 1, adenosine triphosphate binding cassette subfamily G member 2, organic cation/carnitine transporters 1 and 2, and organic cation transporter 1 in kidneys. CONCLUSION: These findings demonstrated for the first time that WPH could alleviate HUA by inhibiting UA production and promoting UA excretion, and improve the renal dysfunction caused by HUA. Thus, WPH may be a potential functional ingredient for the prevention and treatment of HUA and associated renal dysfunction. © 2021 Society of Chemical Industry.

Whey Protein Peptide Pro-Glu-Trp Ameliorates Hyperuricemia by Enhancing Intestinal Uric Acid Excretion, Modulating the Gut Microbiota, and Protecting the Intestinal Barrier in Rats.

Hyperuricemia (HUA) is a metabolic disorder characterized by an increase in the concentrations of uric acid (UA) in the bloodstream, intricately linked to the onset and progression of numerous chronic diseases. The tripeptide Pro-Glu-Trp (PEW) was identified as a xanthine oxidase (XOD) inhibitory peptide derived from whey protein, which was previously shown to mitigate HUA by suppressing UA synthesis and enhancing renal UA excretion. However, the effects of PEW on the intestinal UA excretion pathway remain unclear. This study investigated the impact of PEW on alleviating HUA in rats from the perspective of intestinal UA transport, gut microbiota, and intestinal barrier. The results indicated that PEW inhibited the XOD activity in the serum, jejunum, and ileum, ameliorated intestinal morphology changes and oxidative stress, and upregulated the expression of ABCG2 and GLUT9 in the small intestine. PEW reversed gut microbiota dysbiosis by decreasing the abundance of harmful bacteria (e.g., Bacteroides, Alloprevotella, and Desulfovibrio) and increasing the abundance of beneficial microbes (e.g., Muribaculaceae, Lactobacillus, and Ruminococcus) and elevated the concentration of short-chain fatty acids. PEW upregulated the expression of occludin and ZO-1 and decreased serum IL-1ß, IL-6, and TNF-α levels. Our findings suggested that PEW supplementation ameliorated HUA by enhancing intestinal UA excretion, modulating the gut microbiota, and restoring the intestinal barrier function.

Novel antioxidant peptides from MFGM protein Hydrolysates: Separation, identification and effects on Dexamethasone-induced mitochondrial dysfunction.

Milk fat globule membrane (MFGM) protein is a complex milk protein system with antioxidant property, which can alleviate skeletal muscle dysfunction caused by oxidative stress. In this study, peptide products of MFGM protein obtained through in vitro digestion were isolated and purified, and the composition and antioxidant activities of MFGM peptides (MFGMP) were identified and assessed using LC-MS/MS combined with molecular docking and in vitro approach. Three novel antioxidant peptides TGIIT, YAR and YYK were identified from MFGMPF1, among which TGIIT and YAR exhibited excellent antioxidant effects and protected dexamethasone (Dex)-induced L6 cells by enhancing mitochondrial function and biogenesis involving modulating Sirt-1/PGC-1α signaling pathway. Furthermore, YAR and TGIIT also significantly decreased expression of pro-apoptotic factors such as cyt-c, cleaved caspase-3 and caspase-9. Therefore, YAR and TGIIT, two novel antioxidant peptides, are expected to be utilized in functional food or medicine, providing an emerging role of MFGMP in maintaining anti-oxidant/oxidant status.

Identification of mitochondria-related hub genes in sarcopenia and functional regulation of MFG-E8 on ROS-mediated mitochondrial dysfunction and cell cycle arrest.

Sarcopenia has high prevalence in the elderly population, but the genes and pathways related to aging in elderly patients with sarcopenia are poorly understood. Milk fat globule epidermal growth factor 8 (MFG-E8) is a peripheral membrane glycoprotein isolated from the milk fat globule membrane (MFGM). It has been found to exhibit various nutritional effects, including antibacterial, anti-cancer, anti-oxidant, anti-sarcopenia, and improving brain development and cognitive effects. This study aimed to investigate key differentially expressed genes (DEGs) and pathways associated with the progression of sarcopenia using bioinformatics analysis and in vitro myoblast experiment. The gene expression profiles of GSE8479 and GSE9676, which includes 40 young normal samples and 55 elderly samples, were downloaded from the Gene Expression Omnibus Database (GEO). Over 3253 DEGs were identified in the young and elderly samples (adjusted p value <0.05). A total of 213 co-expressed significantly DEGs were identified with Venn diagrams, including 82 up-regulated DEGs and 131 down-regulated DEGs. Based on the analysis of Gene Ontology (GO), protein-protein interaction (PPI) networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, 10 hub genes screened by our study have been proved to play a role in regulating the occurrence and development of aging-related sarcopenia mainly via metabolic pathways, Huntington's disease, Parkinson's disease, oxidative phosphorylation and non-alcoholic fatty liver disease pathways. To further verify the protective effect of MFG-E8 on oxidative stress injured myoblasts, the cell cycle distribution, cell viability and reactive oxygen species (ROS) production were measured. The protein and mRNA levels of Akt, extracellular regulated protein kinases (ERK), p21Cip1, p27Kip1, cyclin D1, cyclin E1, cyclin-dependent kinase (CDK) 2 and 4 were quantified using qRT-PCR and western blot analysis. The results indicated that MFG-E8 has potential anti-sarcopenia effects by promoting ERK and Akt activation-mediated cell proliferation and cell cycle progression in myoblasts.

Potential of food-derived bioactive peptides in alleviation and prevention of Alzheimer's disease.

Memory and cognitive impairment are the principal clinical symptoms of Alzheimer's disease (AD). Cholinergic deficiency, amyloid-beta (Aß) toxicity, tau protein hyperphosphorylation, synaptic dysfunction, oxidative stress, and neuroinflammation can all exacerbate the development of AD. With the increased number of AD patients and the frequency of AD complications, people are more inclined to select hydrolyzed proteins or bioactive peptides derived from natural foods as intervention agents to combat this type of neurological disease. Currently, our lack of understanding of the complex pathological mechanisms of the disease has led to a high failure rate in the generation of anti-AD food-derived peptides. Accordingly, this review describes the specific regulatory mechanisms of food-derived bioactive peptides on AD-related therapeutic targets over the past decade and highlights the pathogenesis of AD, potential food sources of anti-AD bioactive peptides, methods for evaluating memory efficacy, and regulatory pathways of food-derived bioactive peptides against AD disease.

Identification, inhibitory mechanism and transepithelial transport of xanthine oxidase inhibitory peptides from whey protein after simulated gastrointestinal digestion and intestinal absorption.

In the present study, xanthine oxidase (XO) inhibitory peptides were identified from peptides that survived in whey protein isolate (WPI) simulated gastrointestinal digestion and passed through the Caco-2 cell monolayer, and their inhibitory mechanism and transepithelial transport were investigated. After in silico screening and activity validation, PEW and LLW showed the highest XO inhibitory activity with 50 % inhibitory concentrations (IC50) of 3.46 ± 0.22 and 3.02 ± 0.17 mM, respectively. Molecular docking, molecular dynamics simulation, and circular dichroism (CD) results revealed that these two peptides could interact with the residues in the XO active cavity via hydrogen bonds and hydrophobic forces to form a more stable protein-ligand complex, thus affecting the binding of the substrate to XO. Furthermore, PEW and LLW were transported across Caco-2 cell monolayers intact through the paracellular route and peptide transporter 1 (PepT1), and tight junction proteins zonula occludens 1 (ZO-1) and occludin were not disrupted by PEW and LLW. This study suggests that PEW and LLW potentially regulate XO activity in vivo to exert antihyperuricemia effects.

TMT-based quantitative proteomics analysis reveals the effect of bovine derived MFG-E8 against oxidative stress on rat L6 cells.

Sarcopenia is an aging-associated oxidative stress-induced mitochondrial dysfunction characterized by a decline in skeletal muscle mass, strength and function. Milk fat globule-EGF factor 8 (MFG-E8) is a secreted matrix glycoprotein that plays a crucial role in regulating tissue homeostasis and protecting against skeletal muscle injury. To explore the molecular mechanism of MFG-E8 in ameliorating the rotenone (Rot)-induced L6 skeletal muscle cell oxidative stress injury, differential proteomics of inner L6 cells was conducted. Tandem mass tag (TMT) labeling combined with mass spectrometry (MS) was performed to find associations among control, Rot and Rot + MFG-E8 groups. Over 3248 proteins were identified in the L6 cells. A total of 639 significantly differential proteins were identified, including 294 up-regulated proteins (>1.2 fold) and 345 down-regulated proteins (<0.83 fold) after the exogenous intervention of MFG-E8. Based on the analysis of Gene Ontology (GO), STRING and KEGG databases, MFG-E8 relieves oxidative stress induced-L6 cell damage by regulating the expression of these differential proteins mainly via carbon metabolism, glutathione metabolism and mitochondria-mediated metabolic pathways, e.g. carbohydrate, lipid and amino acid metabolism. Furthermore, to verify the protective effect of MFG-E8 on oxidative stress injured L6 cells, the levels of intracellular reactive oxygen species (ROS), nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH) contents and the protein expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were detected.

MFG-E8 induced differences in proteomic profiles in mouse C2C12 cells and its effect on PI3K/Akt and ERK signal pathways.

Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C2C12 myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative approach was used to analyse the myogenic C2C12 proliferation program. Over 1987 proteins were identified in C2C12 cells. The MFG-E8 (200 µg/mL) and MFG-E8 (500 µg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C2C12 cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C2C12 cell proliferation process. Based on the analysis of KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.