Blood glycemia-modulating effects of melanian snail protein hydrolysates in mice with type II diabetes

Abstract

Freshwater animal proteins have long been used as nutrient supplements. In this study, melanian snail (Semisulcospira libertina) protein hydrolysates (MPh) were found to exert anti-diabetic and protective effects against liver and kidney damage in mice with type II diabetes adapted to a 45% kcal high-fat diet (HFD). The hypoglycemic, hepatoprotective and nephroprotective effects of MPh were analyzed after 12 weeks of the continuous oral administration of MPh at 125, 250 and 500 mg/kg. Diabetic control mice exhibited an increase in body weight, and blood glucose and insulin levels, with a decrease in serum high-density lipoprotein (HDL) levels. In addition, an increase in the regions of steatohepatitis, hepatocyte hypertrophy, and lipid droplet deposit-related renal tubular vacuolation degenerative lesions were detected, with noticeable expansion and hyperplasia of the pancreatic islets, and an increase in glucagon- and insulin-producing cells, insulin/glucagon cell ratios in the endocrine pancreas and hepatic lipid peroxidation, as well as decreased zymogen contents. Furthermore, a deterioration of the endogenous antioxidant defense system was observed, with reduced glucose utilization related hepatic glucokinase (GK) activity and an increase in hepatic gluconeogenesis-related phosphoenolpyruvate carboxykinase (PEPCK) and glucose‑6-phosphatase (G6pase) activity. However, all of these diabetic complications were significantly inhibited by oral treatment with MPh in a dose-dependent manner. In addition, the marked dose-dependent inhibition of hepatic lipid peroxidation, the depletion of the liver endogenous antioxidant defense system, and changes in hepatic glucose-regulating enzyme activities were also observed. The results of this study suggest that MPh exerts potent anti-diabetic effects, along with the amelioration of related complications in mice with type II diabetes. The overall effects of MPh at a dose of 125 mg/kg on HFD-induced diabetes and related complications were similar or more potent than those of metformin (250 mg/kg).

Figure 1

Changes in (A) blood glucose, (B) insulin, and (C) HbA1c levels in mice with type II diabetes. Values are expressed as the means ± SD of 8 mice. HFD, 45% kcal high-fat diet; MPh, melanian snail (Semisulcospira libertina) protein hydrolysates, test material. Metformin was administered at a dose of 250 mg/kg. ^ap<0.01 and ^bp<0.05 as compared with vthe intact control by Mann-Whitney U (MW) test; ^cp<0.01 as compared with the HFD control by MW test.

Figure 2

Histological images of the pancreas. Note that the noticeable decrease in the exocrine pancreas zymogen granule content (the percentages of exocrine pancreas occupied by zymogen granules) may be due to the release of zymogen granules, and an increase in pancreatic islet numbers and mean diameters results from marked hyperplasia of the pancreatic islet itself or component endocrine cells detected in the HFD control as compared with the intact control. However, exocrine pancreas zymogen granule contents were markedly increased in all test material-treated mice as compared with the HFD control, in which the percentages of exocrine pancreas occupied by zymogen granules were not significantly altered as compared to those of HFD control mice. In addition, expansions of pancreatic islets were also meaningfully inhibited by treatment with all test materials in the present study. (A) Intact control: mice supplied normal pellet diet (vehicle control mice); mice administered 10 ml/kg of distilled water orally. (B) HFD control: mice administered 10 ml/kg of distilled water orally with HFD supply. (C) Metformin, mice administered 250 mg/kg of metformin orally with HFD supply. (D) MPh 500, mice administered 500 mg/kg of MPh orally with HFD supply. (E) MPh 250, mice administered 250 mg/kg of MPh orally with HFD supply. (F) MPh 125, mice administered 125 mg/kg of MPh orally with HFD supply. HFD, 45% kcal high-fat diet; MPh, melanian snail (Semisulcospira libertina) protein hydrolysates, test material; IS, pancreatic islet. PD, pancreatic secretory duct. All images show hematoxylin and eosin staining. Scale bars, 80 µm.

Figure 3

Histological images of the insulin- and glucagon-immunoreactive cells in the pancreas. Significant increases of insulin and glucagon-immunoreactive cells, and also insulin/glucagon cells were detected in the HFD control mice as compared with the intact control. However, these abnormal increases in insulin and glucagon-immunostained cells and their ratio (insulin/glucagon cells) were significantly normalized by treatment with all test materials, including MPh 250 mg/kg as compared with the HFD control.(A) Intact control: mice supplied normal pellet diet (vehicle control mice); mice administered 10 ml/kg of distilled water orally. (B) HFD control: mice administered 10 ml/kg of distilled water orally with HFD supply. (C) Metformin, mice administered 250 mg/kg of metformin orally with HFD supply. (D) MPh 500, mice administered 500 mg/kg of MPh orally with HFD supply. (E) MPh 250, mice administered 250 mg/kg of MPh orally with HFD supply. (F) MPh 125, mice administered 125 mg/kg of MPh orally with HFD supply. HFD, 45% kcal high-fat diet. MPh, melanian snail (Semisulcospira libertina) protein hydrolysates, test material. All images show immunostaining with avidin-biotin-peroxidase complex. Scale bars, 80 µm.

Figure 4

Histological images of the liver. Note that marked increases in steatohepatitis and in the percentages of fatty changed regions in liver parenchyma, were detected in the HFD control as compared with the intact control, resulting from severe hypertrophy of hepatocyte related to intracellular lipid depositions. However, steatohepatitis was normalized by treatment with all test materials, including MPh 250 mg/kg treated mice. In particular, the HFD-fed mice treated with MPh at 125, 250 and 500 m/kg also exhibited noticeable decreases in steatohepatitis regions and related hepatocyte hypertrophies as compared with HFD-fed mice, in this experiment. (A) Intact control: mice supplied normal pellet diet (vehicle control mice); mice administered 10 ml/kg of distilled water orally. (B) HFD control: mice administered 10 ml/kg of distilled water orally with HFD supply. (C) Metformin, mice administered 250 mg/kg of metformin orally with HFD supply. (D) MPh 500, mice administered 500 mg/kg of MPh orally with HFD supply. (E) MPh 250, mice administered 250 mg/kg of MPh orally with HFD supply. (F) MPh 125, mice administered 125 mg/kg of MPh orally with HFD supply.. HFD, 45% kcal high-fat diet; MPh, melanian snail (Semisulcospira libertina) protein hydrolysates, test material; CV, central vein; PT, portal triad. Scale bars, 80 µm.

Figure 5

Histological images of the kidney revealed that significant increases in degenerative vacuolated renal tubules were detected in the HFD control as compared with the intact control, resulting from lipid droplet deposited diabetic nephropathies; however, these diabetic nephropathies were significantly normalized by treatment with all test materials as compared with the HFD control, in our experiment. (A) Intact control: mice supplied normal pellet diet (vehicle control mice); mice administered 10 ml/kg of distilled water orally. (B) HFD control: mice administered 10 ml/kg of distilled water orally with HFD supply. (C) Metformin, mice administered 250 mg/kg of metformin orally with HFD supply. (D) MPh 500, mice administered 500 mg/kg of MPh orally with HFD supply. (E) MPh 250, mice administered 250 mg/kg of MPh orally with HFD supply. (F) MPh 125, mice administered 125 mg/kg of MPh orally with HFD supply. HFD, 45% kcal high-fat diet; MPh, melanian snail (Semisulcospira libertina) protein hydrolysates, test material. All images show hematoxylin and eosin staining. Scale bars, 80 µm.