Modulation of lipid metabolism by mixtures of protamine and chitooligosaccharide through pancreatic lipase inhibitory activity in a rat model

Abstract

Overweight and obesity are usually related with high fat and calorie intake, and seriously causative of lifestyle-related diseases such as cardiovascular disorders, arteriosclerosis, and colon cancer. In this study, we propose a novel dietary therapy against overweight and obesity using mixtures of protamine and chitooligosaccharide (COS), which are known to interrupt the lipid metabolism in the body. Protamine is a dietary protein originated from salmon reproductive organ, and COS is an oligosaccharide made from chitin or chitosan by chemical or enzymatic hydrolysis. In the enzyme activity analysis in vitro, protamine and COS strongly suppressed the activity of pancreatic lipase, which is the primary enzyme for the digestion and absorption of lipids in the intestine. In in vivo animal test, the mixtures of protamine and COS significantly reduced the serum levels of triglyceride (TG), total cholesterol (T-CHO), and low density lipoprotein-cholesterol (LDLC) and inhibited the accumulation of lipids in liver tissue of Sprague Dawley (SD) rats fed high fat diets. On the other hand, they increased fecal TG and T-CHO contents. From these alterations in lipid metabolism, we verified that protamine and COS mixtures could effectively interrupt the digestion and absorption of dietary lipids in the body by inhibiting pancreatic lipase activity. In addition, protamine and COS mixtures increased the serum level of high density lipoprotein-cholesterol (HDLC), responsible for removing cholesterol from cells and protecting atherosclerosis, and therefore decreased the potential risks of cardiovascular diseases by lowering values of the atherogenic index (AI) and cardiac risk factor (CRF). Taken together, we suggest protamine and COS mixtures as a prominent dietary therapy for the prevention of overweight, obesity, and further cardiovascular diseases related with hyperlipidemia.

Keywords: Protamine; chitooligosaccharide; cholesterol; high fat diet; triglyceride.

Figure 1

Pancreatic lipase activity. (A) The pancreatic lipase activity was inhibited by protamine. Protamine was used at the concentrations of 0, 2, 4, 8, 16, 32, 64, and 100 µg/mL. (B) The pancreatic lipase activity was inhibited by chitooligosaccharide (COS).The COS was used at the concentrations of 0, 5, 25, 50, 75, 100, 150, and 300 µg/mL. Values represent the mean±SD. ^*P<0.05 (Dunnett's multiple comparison test).

Figure 2

Serum lipid analysis. (A) Triglyceride in serum was measured after the oral administration of various mixtures of protamine and chitooligosaccharide (COS) in corn oil suspension. (B) Total-cholesterol in serum (C) High density lipoprotein-cholesterol (HDLC) (D) Low density lipoprotein-cholesterol (LDLC).The ratios of protamine and COS in the mixtures were 1:3, 1:6, and 1:12. The PO2.1 group was treated with 2.1 mg/kg body weight (b.w.) of protamine and 25 mg/kg b.w. of COS in corn oil suspension. The PO4.2 group was treated with 4.2 mg/kg b.w. of protamine and 25 mg/kg b.w. of COS in corn oil suspension. The PO8.3 group was treated with 8.3 mg/kg b.w of protamine and 25 mg/kg b.w of COS in corn oil suspension. Values represent the mean±SEM. ^*P<0.05 (Dunnett's multiple comparison test).

Figure 3

Fecal lipid analysis. (A) Triglyceride concentration in feces. (B) Total-cholesterol concentration in feces. The PO2.1 group was treated with 2.1 mg/kg body weight (b.w.) of protamine and 25 mg/kg b.w. of COS in corn oil suspension. The PO4.2 group was treated with 4.2 mg/kg b.w. of protamine and 25 mg/kg b.w. of COS in corn oil suspension. The PO8.3 group was treated with 8.3 mg/kg b.w. of protamine and 25 mg/kg b.w. of COS in corn oil suspension. Values represent the mean±SEM. ^*P<0.05 (Dunnett's multiple comparison test).

Figure 4

Histological analysis on liver tissues. (A) The liver tissue of vehicle group. (B) The PO2.1 group. (C) The PO4.2 group. (D) The PO8.3 group. After 24 h of oral ministration of protamine and COS mixtures, we harvested the liver tissues from rats. Frozen liver tissues were cryo-sectioned, cut 6-ìm thick, and stained with the 5% Oil-red-O working solution by standard methods of manufacturer's. Counter staining was conducted with the hematoxylin and then mounted with glycerin. The lipid containing cells are shown the red spots under a light microscope (BX51 U-LH100HGWIG, Olympus; ×40, ×200, and ×400 magnification).