Bioactive Peptides from Meretrix lusoria Enzymatic Hydrolysate as a Potential Treatment for Obesity in db/db Mice

Abstract

Obesity is acknowledged as a significant risk factor for cardiovascular disease, often accompanied by increased inflammation and diabetes. Bioactive peptides derived from marine animal proteins show promise as safe and effective anti-obesity agents by regulating adipocyte differentiation through the AMPK signaling pathway. Therefore, this study aims to investigate the anti-obesity and anti-diabetic effects of bioactive compounds derived from a Meretrix lusoria Protamex enzymatic hydrolysate (MLP) fraction (≤1 kDa) through a 6-week treatment (150 mg/kg or 300 mg/kg, administered once daily) in leptin receptor-deficient db/db mice. The MLP treatment significantly decreased the body weight, serum total cholesterol, triglycerides, and LDL-cholesterol levels while also exhibiting a beneficial effect on hepatic and serum marker parameters in db/db mice. A histological analysis revealed a reduction in hepatic steatosis and epididymal fat following MLP treatment. Furthermore, poor glucose tolerance was improved, and hepatic antioxidant enzyme activities were elevated in MLP-treated mice compared to db/db control mice. Western blot analysis showed an increased expression of the AMPK protein after MLP treatment. In addition, the expression of lipogenic genes decreased in db/db mice. These findings indicate that bioactive peptides, which are known to regulate blood glucose levels, lipid metabolism, and adipogenesis, could be beneficial functional food additives and pharmaceuticals.

Keywords: AMPK pathway; bioactive peptides; db/db mice; enzymatic hydrolysate; obesity.

Figure 1

MLP hydrolysate fraction scavenging abilities of (a) DPPH radicals, (b) ABTS radicals, and (c) OH radicals (%); (d) IC₅₀ values (mg/mL) of all MLP fractions, (e) reducing power, and (f) cell viability assay. Each value represents the mean ± SEM (n = 3). Tukey’s multiple comparison test indicates that values not sharing a common letter are significantly different at p < 0.05.

Figure 2

Meretrix lusoria hydrolysate fractionation. (a) Chromatogram of MLP sub-fractions (1–4) evaluated at 254 and 280 nm using RP-HPLC analysis; (b) DPPH radical scavenging activity of four sub-fractions, and highlighted sub-fraction 3 was exhibited high protein yield (4452.7 mg/mL) with effective DPPH radical scavenging ability; (c,d) Sub-fraction 3 base peak and extracted ion chromatogram from LC-MS analysis, including identified bioactive peptides (1) LDDLKRN, (2) VDDLTRQ, (3) LGVOGPQ, (4) KDLEL and (5) LELELRE; (e,f) LC-MS chromatogram and spectrum of Sequence 1 (m/z 437.24, peak at t_R = 11.6 min); (g,h) LC-MS chromatogram and spectrum of Sequence 2 (m/z 423.72, peak at t_R = 12.8 min); (i,j) LC-MS chromatogram and spectrum of Sequence 3 (m/z 683.37, peak at t_R = 15.6 min); (k,l) LC-MS chromatogram and spectrum of Sequence 4 (m/z 617.35, peak at t_R = 16.9 min); (m,n) LC-MS chromatogram and spectrum of Sequence 5 (m/z 451.25, peak at t_R = 19.7 min).

Figure 3

Effect of MLP treatment on obese db/db mice. (a–d) Representative liver and adipose tissues from week 6 of WT, db/db CON, and MLP-treated db/db mice (e–h) Representative images of H&E staining at 20× magnification depicting db/db CON mouse hepatic steatosis, and MLP therapy in a dose-dependent manner. (i–l) MLP alleviates hepatic TC and TG contents, including serum AST and ALT levels. (m–p) Representative images of H&E staining at 10× magnification depicting db/db CON mice adipocyte area was increased with fat deposition, MLP therapy reduces the adipocyte area in a dose-dependent manner, and (q) adipocyte area. (r–t) MLP alleviates serum TC, TG, and LDL-cholesterol levels. db/db MLP-L (150 mg/kg); db/db MLP-H (300 mg/kg). Each value represents the mean ± SEM (n = 6). Tukey’s multiple comparison test shows a significant difference (p < 0.05) between values that do not share a common letter.

Figure 4

Effect of MLP therapy on antioxidants: (a) GSH contents, (b) GPx, (c) GST, (d) GR, (e) SOD, and (f) CAT enzyme activities in obese db/db mice. Each value represents the mean ± SEM (n = 6). Tukey’s multiple comparison test shows a significant difference (p < 0.05) between values that do not share a common letter.

Figure 5

MLP regulated protein expression within the AMPK signaling pathway. (a) Proteins AMPK, SREBP-1, FAS, and ACC were identified using a Western blot, and the (b) AMPK levels and (c) levels of SREBP-1, ACC, and FAS in each group were determined. Each value represents the mean ± SEM (n = 4). Tukey’s multiple comparison test shows a significant difference (p < 0.05) between values that do not share a common letter.

Figure 6

MLP treatment regulated the expressions of genes involved in gluconeogenesis-related metabolic pathways, specifically (a) PEPCK, (b) G6Pase, (c) LGP, (d) GS and lipogenic enzymes (e) FAS, (f) ACC, (g) SCD, (h) PDK and (i) CPT in liver tissues. Each value represents the mean ± SEM (n = 4). Tukey’s multiple comparison test shows a significant difference (p < 0.05) between values that do not share a common letter.

Figure 7

MLP ameliorated glucose homeostasis in db/db mice. IPGTT and area under the curve (AUC) of IPGTT. db/db MLP-L (150 mg/kg); db/db MLP-H (300 mg/kg). Each value represents the mean ± SEM (n = 6). Tukey’s multiple comparison test shows a significant difference (p < 0.05) between values that do not share a common letter.

Figure 8

Proposed mechanism for the role of AMPK on lipid and glucose metabolisms in liver tissue. The MLP treatment activates AMPK activity, which leads to downregulated lipogenic enzymes such as ACC and FAS. The results indicate that the AMPK pathway may increase glucose transport as well as fatty acid oxidation.