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

Preparation of potato proteinase inhibitors concentrate (PPIC)

PPIC was prepared using an improved purification method based on previously reported extraction procedures (9, 12) and relevant information on abundance and distribution of protease inhibitors in potato juice (11). One kg of potato tubers was homogenized with 400 ml of the extractant buffer (sodium chloride: acetic acid: water 1:1:9, w:v:v) using a handheld commercial blender for 5 min at room temperature. The resulting mixture was centrifuged at 13,000g for 10 min and filtered through several layers of Whatman filter paper. The resulting liquid (450 ml) was transferred to an Erlenmeyer flask and heated to 70 °C on a hot plate while stirring. The solution was rapidly cooled to 30 °C and centrifuged at 13,000g for 10 min to pellet the visible precipitate of denatured proteins. Thermostable proteins from the clarified potato extract (400 ml) were precipitated with addition of 100 g of sodium chloride and centrifugation at 13,000g for 10 minutes to isolate the visible pellet, designated as the wet PPIC. At the final step, the wet PPIC was dissolved in 50 ml of water and loaded onto a gel filtration column (a total bed volume of 150 ml) packed with Bio-Gel P-6 DG matrix (Bio-Rad, Hercules, CA). The first three 20 ml fractions were discarded, while the next six fractions were pooled, lyophilized, and designated as PPIC used in animal and cell culture studies. Potato protease inhibitor II (PI2) was further isolated from PPIC concentrate according to the method of Pouvreau et al (11).

PPIC analysis

PPIC samples were analyzed on 12% polyacrylamide gel/ Coumassie Brilliant Blue G-250 staining for protein content and protease inhibitory activity was visualized using a slightly modified method (20). Briefly, nondenaturing PAGE gels were treated with trypsin or chymotrypsin and subsequently developed in the presence of N-Acetyl-phenylalanine-naphthylester and o-dianisidine tetrazotized for 10 min at 37 °C (21). After visualization, major protein bands were excised from the SDS-polyacrylamide gel, and MALDI-TOF protein spectra were recorded on Bruker Reflex III mass spectrometer following the service protocols of Mass Spectrometry Core Facility (University of Guelph, Canada).

Animals

Male Wistar rats (180–200 g) were obtained from Charles River Laboratories (Wilmington, MA). Rats were housed individually under controlled temperature (24 ± 2 °C) and light (12 h light-dark cycle, lights on at 0200 h) with access to AIN-93 standard diet (Research Diets, New Brunwick, NJ) and tap water ad libitum. Following an adaptation period of 1 week, animals were introduced to daily 6 h food deprivation (beginning at 0800 h) and a daily gavage at 1400 h until it was determined that the process of gavaging did not significantly affect the daily food uptake (1 week). The 6 h fasting time prior to PPIC administration was introduced to eliminate variation in feeding patters among different animals by ensuring that all animals did not consume food immediately before the treatment. For all studies, animals were randomized into groups (n=6–8) with approximately equal mean body weight (within 2 g). Body weight and food consumed by each animal were determined daily by differential weighing after correcting for spillage on balances accurate to 0.01 g. All animal experimental procedures were carried out at the AAALAC-accredited Cook Animal Facility in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and approved by Rutgers IACUC.

Acute administration of PPIC and PI2 to rats

Three groups of rats fasted for 6 h received a single per os dose of control solution (100 mg/kg casein), PPIC concentrate (100 mg/kg), or purified PI2 (100 mg/kg) in a total volume of 2 ml distilled water at the beginning of the feeding cycle (lights off, 1400 h). Food intake was measured at 1, 2, and 24 h postgavage and corrected for spillage. We included casein in the control treatment to account for possible effect of protein supplementation on food intake and satiety (22).

Effect of PPIC administration on gastric emptying and proteolytic activity

Gastric emptying was measured essentially as described by Shi el al (23). In short, the pre-warmed 40% peptone meal suspended in 1 ml of distilled water and supplemented with 1 mg/ml of phenol red (non absorbable dye marker) was given orally through a stainless steel tube immediately following the gavage of 100 mg/kg of PPIC (treatment group) or 100 mg/kg casein (control group) in 2 ml of distilled water. One hour after the treatment, all animals were sacrificed. Their stomachs were immediately ligated, opened, and gastric content was collected in graduated tubes. The phenol red concentration in each stomach was determined spectrophotometrically at 520 nm (Molecular Devices, Sunnyvale, CA). Phenol red concentration found in stomachs of animals sacrificed immediately after administration of the peptone meal (baseline group) served as a 100% reference point.

In the same set of experiments, the remaining proteolytic activity in the duodenum washes was recorded 1 hour following the treatment. Duodenum was ligated, opened, and duodenal content was collected in Eppendorf tubes with 0.5 ml of PBS buffer. Total serine protease activity in the samples was estimated as described elsewhere (20), using standard curve of known trypsin concentrations. Proteolytic activity found in duodenum of animals treated with casein (control group) served as a 100% reference point.

Repeated PPIC administration

Two groups of rats fasted for 6 h were provided with a single per os dose of control solution (100 mg/kg per day casein) or PPIC concentrate (100 mg/kg per day) in a total volume of 2 ml distilled water for 10 days. Food intake and body weight gain were recorded daily prior to the treatment. At the end of experiment, plasma CCK levels were measured immediately before and 15 min after the animals were dosed with PPIC in submundibular vein blood samples using indirect quantification of CCK-induced amylase release from isolated rat pancreatic acini (13). Blood was collected by cardiac puncture at the time of sacrifice into EDTA-coated tubes. Plasma glucose (colorimetric assay kit, Sigma, St. Louis, MO) and insulin (ELISA kit, Linco Research, St. Charles, MO) levels were measured.

PPIC toxicity studies

PPIC toxicity was investigated following daily oral gavage administration to healthy rats for 14 consecutive days by ITR Laboratories, Quebec, Canada (study no 7392). Twenty five male and 25 female rats were randomized among five groups of rats (n=10) and treated with 0, 33, 66, 133, and 266 mg/kg body weight of PPIC. In-life observations included mortality, cage-side clinical signs (ill health, behavioral changes), body weights and daily food consumption. Clinical pathology testing was performed on all animals at termination and included hematology (red blood cell count, white blood cell count, hematocrit, hemoglobin, mean corpuscular hemoglobin, cell morphology, platelet count), coagulation (activated partial thromboplastin time, prothrombin time), blood chemistry (total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, total bilirubin, creatinine, triglycerides, total cholesterol, urea, inorganic phosphorus, potassium, sodium, calcium, chloride) and urianalysis (volume, color and appearance, specific gravity, pH, bilirubin, protein, blood, sediment microscopy, glucose, urobilinogen, ketone). Gross pathology consisted of an external examination, including identification of all clinically recorded lesions, as well as a detailed internal examination including organ weights (adrenals, heart, kidney, liver, lungs, ovaries, pituitary, spleen, testes, thymus, and thyroid).

The mutagenic potential of PPIC concentrate was evaluated in Ames test using five histidine-dependent strains of Salmonella up to the maximum dose of 5 mg per plate either with or without metabolic activation by MDS Pharma Services, Lyon, France (study no AA41707).

Cell culture

The enteroendocrine STC-1 cell line that expresses CCK mRNA and secretes the biologically active form of the peptide, CCK-8 (24) was a kind gift from Dr. Douglas Hanahan, University of California, San Francisco). Cells were maintained in DMEM supplemented with 4.5% glucose and 10% fetal bovine serum at 37 °C in 5% CO₂, and passaged every 3–4 days. Routinely, 5×10⁵ cells were plated on 24-well culture dishes and were used for studies 24 h after plating. Cell viability was estimated by the MTT assay (25). After dimethyl sulfoxide was added to solubilize formazan crystals, absorbance was read at 550 nm on a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

Gene expression analysis

Total RNA was isolated from the scraped mucosa layer of duodenum samples rinsed with ice-cold PBS buffer or cell culture using Trizol reagent (Invitrogen, Carlsbad, CA) and quantified by absorption measurements at 260 and 280 nm using the NanoDrop system (NanoDrop Technologies Inc., Wilmington, DE). Total RNA (20 µg) were loaded and subjected to electrophoresis on a 1% denaturing agarose gel containing formaldehyde before capillary blotting onto a Hybond N+ nylon membrane (Amersham, Piscataway, NJ). Hybridizations with ³²P-labeled cDNA CCK and β-actin probes, respectively, were performed according to published procedures (26). Results were expressed as ratio of CCK to β-actin mRNA to normalize any differences in RNA loading and transfer.

For cDNA synthesis and quantitative PCR analysis, RNA was treated with DNAse I, reverse-transcribed to cDNA, and amplified essentially as described (27) using the following primers: rat CCK precursor (Genbank X01032), forward 5’-CAC GAC CCC TCG CCT CTA A-3’ and reverse 5’-GGC TGC ATT GCA CAC TCT GA-3’; and β-actin (Genbank NM_031144), forward 5’-GGG AAA TCG TGC GTG ACA TT-3’ and reverse 5’-GCG GCA GTG GCC ATC TC-3’. Samples were subjected to a melting curve analysis to confirm the amplification specificity. The relative change in the target gene with respect to the endogenous control gene was determined using 2ΔΔCT method (28).

Statistics

Data are represented as mean ± s.e.m. Statistical analyses were performed using GraphPad Prism 4.0 (San Diego, CA) using Student’s t-test or one-way ANOVA (as appropriate). Body weight was analyzed with one-way repeated-measures ANOVA with post hoc testing to localize significant differences at each time point. P-values of less than 0.05 were considered significant.

PPIC treatment reduces food intake in rats

Potato proteinase inhibitors concentrate (PPIC) prepared from acidified potato tuber juice yielded 0.15–0.25 mg total protein per gram of potato tuber fresh weight (data not shown). PPIC contained essentially thermostable proteins in the molecular weight range of 5–160 kDa (Figure 1a) and showed a prominent inhibitory activity against trypsin in vitro (Figure 1b). Potato proteinase inhibitor II, a dimeric protein with two 10 kDa subunits (9), was identified in PPIC based on its molecular weight and anti-trypsin activity and isolated to 90% purity (Figure 1) to serve as reference treatment in subsequent studies. MALDI-TOF mass spectrometry identified three major proteins in the PPIC extract as potato protease inhibitor II (PI2), potato protease inhibitor I, and potato kunitz-type protease inhibitor.

A significant reduction in food intake was seen in the first (41.2%) and second (42.6%) hour following oral administration of 100 mg/kg PPIC to 6 h fasted rats in the early dark phase (p<0.01, Figure 2). A residual non-significant 6.9% decrease in food intake was also observed at the 24 h time point. To account for a possible effect of protein intake on satiety (22), 100 mg/kg casein solution served as a negative control that resulted in weak food intake reduction of 16.9% and 12.1% in the first and second hour only. Purified potato protease inhibitor II (PI2) was used as a positive control (6) to ensure that the observation system used was sensitive enough to detect significant changes in food intake. Oral administration of 100 mg/kg PI2 significantly reduced food intake in rats (25.2% and 17.4% in the first and second hour, respectively), albeit to a lesser extent than PPIC at the same concentration (p<0.05, Figure 2).

Effect on gastric emptying and proteolytic activity

The acute effect of oral PPIC administration on gastric emptying and proteolytic activity in the duodenum was also investigated. Approximately 53.8% of the peptone test meal supplemented with the inert dye left the stomach of control animals treated with 100 mg/kg casein within 1 hour of treatment. Under identical conditions, PPIC-treated animals exhibited a significant delay in gastric emptying by 20.1% over the control group (p<0.05, Figure 3a). Since one week of feeding with diets of different protein content was not sufficient to achieve a statistically significant deceleration of gastric emptying (23), the observed delay in gastric emptying can be attributed to the PPIC treatment alone.

An isolated duodenum system was used to assess the effect of PPIC on proteolytic activity in the gastrointestinal tract. A single dose of 100 mg/kg decreased trypsin-like activity in duodenum by 47.3% one hour after PPIC administration (p<0.05, Figure 3b).

Repeated PPIC administration

Following confirmation of the anorexigenic effect of oral administration of PPIC, the effects of repeated administration of PPIC were investigated. After 10 days of PPIC treatment (100 mg/kg per day), initial body weight of rats increased by 36.9%, while that of control rats receiving 100 mg/kg casein increased by 48.2%, suggesting that PPI administration was associated with 11.3% decrease in body weight gain (p<0.05, Figure 4a). At the same time, PPIC-treated animals significantly decreased cumulative food intake by 6.9% (p<0.05, Figure 4b).

Basal plasma CCK levels in control animals measured on the last day of the treatment were 0.96 pmol/l and were not significantly different from those of test animals prior to PPIC administration. Fifteen minutes after PPIC application, plasma CCK levels increased strongly and significantly higher as compared to control animals (p<0.05; Figure 5a). The postprandial mRNA levels (expressed as CCK mRNA to β-actin mRNA ratio) in the duodenal mucosa of PPIC-treated animals were elevated 2.4-fold compared to casein-treated controls (Figure 5b). Plasma glucose levels in control rats (6.4 ± 0.2 mM) were similar to those of rats treated with PPIC (6.6 ± 0.4 mM). Animals treated with PPIC exhibited a lower plasma insulin concentration (112 ± 26 pM) as compared to the control group (167 ± 19 pM; p<0.05). As a result, insulin to glucose ratio was lower in animals treated with PPIC.

PPIC toxicity

An ascending dose range study was performed to determine toxicity of PPIC following a daily oral gavage administration to the rat for 14 consecutive days, and to exclude the possibility that PPIC inhibits food intake and causes weight loss due to its toxicity to the animals of both sexes. Parameters evaluated in this study included monitoring of mortality, behavioral changes, clinical signs, body weight, food consumption, clinical pathology investigation and post-mortem examinations. There were no deaths during the course of the study. Reduced food intake up to 11.4% was observed in both male and female rats treated with PPIC at 133 and 266 mg/kg throughout the treatment period, but only the males showed a significantly reduced weight gain at the highest dose levels (Figure 6). There were no treatment-related effects on hematology parameters, clinical chemistry, or urinalysis. Apparent marginal elevations in mean activated partial thromboplastin times were noted in PPIC-treated groups, however these elevations were minor, did not achieve statistical significance, were not associated with any other changes, and did not exhibit a correlation to dose (not shown). No organ weight differences, macroscopic, or microscopic changes were noted at necropsy.

When tested up to the maximum dose level of 5 mg per plate using both the plate incorporation and pre-incubation methods, PPIC did not induce any biologically significant increases in the number of revertants in 5 Salmonella strains used, either with or without metabolic activation (data not shown).

Effect of PPIC on CCK expression in vitro

To answer the question whether PPIC increases CCK production directly or through a trypsin-dependent mechanism by preserving trypsin-sensitive CCK-releasing intestinal factor (18, 19), we examined changes in CCK expression and release in murine enteroendocrine STC-1 cell line. This cell line is derived from an intestinal tumor arising in double transgenic mice carrying the rat insulin II promoter cloned upstream to the coding region of the SV 40 T antigen and the polyoma small t antigen genes (26) and secrets the biologically active form of the peptide, CCK-8 (24). No significant difference in both CCK mRNA levels and CCK release was observed in PPIC-treated cells in the dose range 0.1 to 100 µg/ml (data not shown), suggesting that PPIC did not directly stimulate CCK expression and release in vitro. No cytotoxic effect of PPIC on STC-1 cells was noted at these concentrations.