Potato protease inhibitors inhibit food intake and increase circulating cholecystokinin levels by a trypsin-dependent mechanism

Abstract

Objective: To investigate the mechanisms underlying the satiety-promoting effects of a novel protease inhibitor concentrate derived from potato (PPIC).

Methods: The acute and prolonged effects of oral PPIC administration (100 mg kg(-1) per day) on food intake, body weight and gastric emptying were evaluated in healthy rats. Parameters of body weight, food intake, plasma glucose, insulin and cholecystokinin (CCK) were measured. Duodenal proteolytic activity and CCK expression were determined in tissue extracts. Intestinal STC-1 cell culture model was used to investigate the direct effect of PPIC on CCK transcript level and secretion.

Results: Acute oral administration of PPIC reduced immediate food intake during the first 2 h following the treatment, delayed gastric emptying and decreased proteolytic activity in the duodenum. Repeated oral ingestion of PPIC reduced weight gain in male rats and significantly elevated the plasma CCK levels. Although duodenal mucosal CCK mRNA levels increased in response to PPIC administration, the concentrate failed to elevate CCK expression or release in STC-1 cells. The 14-day ascending dose range study (33-266 mg kg(-1) PPIC per day) showed no adverse side effects associated with PPIC administration.

Conclusion: These findings provided evidence that PPIC is effective in reducing food intake and body weight gain in healthy rats when administered orally by increasing circulating CCK levels through a trypsin-dependent mechanism.

Figure 1

Total protein content (a) and trypsin inhibitory activity (b) of PPIC (lane 2) and purified potato protease inhibitor II (lane 3). Twenty µg of total protein was loaded per well. Samples were separated on 10% PAGE and stained with Coomassie Blue for total protein; or treated with trypsin and developed in the presence of N-Acetyl-phenylalanine-naphthylester and o-dianisidine tetrazotized to visualize trypsin inhibitor activity (light bands). Lane 1, broad range protein molecular weight marker (kDa).

Figure 2

Changes in food intake 1, 2, and 24 h after oral administration of a single dose of 100 mg/kg PPIC or purified potato protease inhibitor II to healthy rats (n=6–8). Results are expressed as % of food intake reduction relative to baseline food intake in the absence of treatment (means ± SEM). Asterisks indicate a significant difference (*p<0.05, **p<0.01) from control animals receiving 100 mg/kg casein.

Figure 3

Effect of a single dose of PPIC (100 mg/kg) on gastric emptying (a) and trypsin proteolytic activity in the duodenum (b) of healthy rats (n=6–8). All values are means ± SEM. Asterisks indicate a significant difference (*p<0.05) from control animals receiving 100 mg/kg casein.

Figure 4

Oral administration of 100 mg/kg PPIC for 10 days decreases body weight gain (a) and cumulative food intake (b) in healthy rats. All values are means ± SEM (n=6–8). Asterisks indicate a significant difference (*p<0.05) from control animals receiving 100 mg/kg casein.

Figure 5

Increased plasma CCK (a) and CCK mRNA levels (b) in the duodenal mucosa associated with PPIC administration. On the last day of the 10-day experiment, plasma CCK levels were measured in submundibular vein blood samples immediately before and 15 min after the animals were dosed with 100 mg/kg PPIC. Two control animals (lanes 1–2) and two PPIC-treated animals (lanes 3–4) were randomly selected to generate representative Northern blot results for CCK and β-actin expression levels. All values are means ± SEM (n=6–8). Asterisks indicate a significant difference (*p<0.05) from control animals receiving 100 mg/kg casein.

Figure 6

Dose-response curve of body weight changes to PPIC administration in male rats during the 14-day ascending dose range study aimed at evaluation of safety and toxicity of oral PPIC (33–266 mg/kg per day). All values are means ± SEM (n=5). Asterisks indicate a significant difference (*p<0.05) from control animals receiving 100 mg/kg casein.