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. 2023 Jun 24;15(13):2863.
doi: 10.3390/nu15132863.

Whey Protein Hydrolysate Ameliorated High-Fat-Diet Induced Bone Loss via Suppressing Oxidative Stress and Regulating GSK-3β/Nrf2 Signaling Pathway

Tingting Bu  1 , Ju Huang  1 , Yue Yu  1 , Peilong Sun  1 , Kai Yang  1
Affiliations

Whey Protein Hydrolysate Ameliorated High-Fat-Diet Induced Bone Loss via Suppressing Oxidative Stress and Regulating GSK-3β/Nrf2 Signaling Pathway

Tingting Bu et al. Nutrients. .

Abstract

Long-term hypercaloric intake such as a high-fat diet (HFD) could act as negative regulators on bone remodeling, thereby inducing bone loss and bone microarchitecture destruction. Currently, food-derived natural compounds represent a promising strategy to attenuate HFD-induced bone loss. We previously prepared a whey protein hydrolysate (WPH) with osteogenic capacity. In this study, we continuously isolated and identified an osteogenic and antioxidant octapeptide TPEVDDA from WPH, which significantly promoted the alkaline phosphatase activities on MC3T3-E1 cells and exerted DPPH radical scavenging capacity. We then established an HFD-fed obese mice model with significantly imbalanced redox status and reduced bone mass and further evaluated the effects of different doses of WPH on ameliorating the HFD-induced bone loss and oxidative damages. Results showed that the administration of 2% and 4% WPH for 12 weeks significantly restored perirenal fat mass, improved serum lipid levels, reduced oxidative stress, and promoted the activity of antioxidant enzymes; meanwhile, WPH significantly preserved bone mass and bone mechanical properties, attenuated the degradation of trabecular microstructure, and regulated serum bone metabolism biomarkers. The protein levels of Runx2, Nrf2, and HO-1, as well as the phosphorylation level of GSK-3β in tibias, were notably activated by WPH. Overall, we found that the potential mechanism of WPH on ameliorating the HFD-induced bone loss mainly through its antioxidant and osteogenic capacity by activating Runx2 and GSK-3β/Nrf2 signaling pathway, demonstrating the potential of WPH to be used as a nutritional strategy for obesity and osteoporosis.

Keywords: glycogen synthase kinase-3β; high-fat diet; nuclear factor-erythroid 2-related factor 2; obesity; osteoporosis; oxidative stress; runt-related transcription factor 2; whey protein hydrolysate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Separation and identification of the osteogenic and antioxidant peptides from WPH. (a) Separation of WPH on the RP-HPLC equipped with a C18 column. (b) Evaluation of WPH fractions on the ALP activity in MC3T3-E1 osteoblasts and the DPPH radical scavenging capacity. (c) Total ion chromatograms of WPH-F9 and the MS/MS spectra of the octapeptide TPEVDDEA. (d) Evaluation of TPEVDDEA (5–80 μM) on the ALP activity in MC3T3-E1 osteoblasts and the DPPH radical scavenging capacity. All data were shown as mean ± SD. The # (*), ## (**), and ### (***) indicated p < 0.05, p < 0.01, and p < 0.001, as compared to the control group, respectively.
Figure 2
Figure 2
Effect of HFD feeding on the body weights, bone microstructures, and redox status of mice. (a) Scheme of experiment design. (b) Changes of body weight between ND- and HFD-fed mice. (c) Serum SOD, OCN, and RANKL levels. (d) Transverse images of distal metaphyseal femurs. (e) H&E-stained images of proximal tibia (5×). (f) Quantitation of vBMD and trabecular area ratio obtained from μCT and H&E analysis. All data were shown as mean ± SD. The * and *** indicated p < 0.05 and p < 0.001 between ND and HFD groups.
Figure 3
Figure 3
Effects of WPH on bone microstructure in HFD-fed mice. (a) Transverse images of distal metaphyseal femurs. (b) H&E staining of trabecular microstructure of proximal tibia (5×). (c–h) Quantification of bone morphometric parameters in μCT and H&E analysis. All data were shown as mean ± SD (n = 3). The #, and ### indicated p < 0.05 and p < 0.001, as compared to the ND group. The *, **, and *** indicated p < 0.05, p < 0.01, and p < 0.001, as compared to the HFD control group. The ns indicated p > 0.05 among groups.
Figure 4
Figure 4
Effects of WPH on biomarkers of bone remodeling and antioxidant capacity in HFD-fed mice. (a) Bone remodeling biomarkers of OCN, TRACP, OPG, and RANKL in serum. (b) Antioxidant capacity biomarkers of SOD, GSH-PX, CAT, and MDA in serum. All data were shown as mean ± SD (n = 8). The #, and ### indicated p < 0.05 and p < 0.001, as compared to the ND group. The *, **, and *** indicated p < 0.05, p < 0.01, and p < 0.001, as compared to the HFD control group. The ns indicated p > 0.05 among groups.
Figure 5
Figure 5
Effects of WPH on protein expressions in HFD-fed mice. (a–f) Protein expression levels of β–catenin, Nrf2, HO-1, and GSK-3β and the phosphorylated forms GSK-3β. β–Actin was used as the loading control. All data were shown as mean ± SD (n = 3). The ### indicated p < 0.001, as compared to the ND group. The *, **, and *** indicated p < 0.05, p < 0.01, and p < 0.001, as compared to the HFD control group.
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