Title:
Fish skin gelatin hydrolysates as dipeptidyl peptidase IV inhibitors and glucagon-like peptide-1 stimulators improve glycaemic control in diabetic rats: a comparison between warm- and cold-water fish
Names of authors:
Tzu-Yuan Wanga; Cheng-Hong Hsiehb; Chuan-Chuan Hungc,d; Chia-Ling Jaoe; Meng-Chun Chend and Kuo-Chiang Hsub,c,d*
Affiliation and address of authors:
a Division of Endocrine and Metabolism, China Medical University Hospital, 2 YuDe Road, Taichung 40447, Taiwan.
b Department of Health and Nutrition Biotechnology, Asia University, 500 Lioufeng Road, Taichung 41354, Taiwan.
c Food Safety and Inspection Center, Asia University, 500 Lioufeng Road, Taichung 41354, Taiwan.
d Department of Nutrition, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan.
e Department of Food and Beverage Management, Tung Fang Design University, 110 Tung-Fang Road, Kaohsiung 82941, Taiwan.
Short title: Peptides improve glycemic control in diabetic rats
*Corresponding author
Address: Department of Nutrition, China Medical University, 91 Hsueh-Shih 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Road, Taichung 40402, Taiwan. Tel.: +886-4-22053366 ext. 7522 Fax: +886-4-22062891
E-mail address：[email protected] (K. C. Hsu) 26
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Abstract
Various warm- and cold-water fish skins were used to prepare gelatin hydrolysates and compare their in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The DPP-IV inhibitory activity of gelatin hydrolysates from warm-water fish was greater than that from cold-water fish. The <1.5 kDa ultrafiltration fractions obtained from halibut skin hydrolysate (HSGH) and tilapia skin gelatin hydrolysate (TSGH) displayed in vitro DPP-IV inhibitory activity of 38.2 and 51.9% at sample concentration of 1 mg solid/mL, respectively; and they were used for in vivo
antihyperglycaemic experiment. The daily administration of TSGH for 30 days was more potent to improve the glucose tolerance in streptozotocin-induced diabetic rats than that of HSGH due to the inhibition of plasma DPP-IV activity, enhancement of glucagon-like peptide-1 (GLP-1) and insulin secretion. Therefore, the warm-water fish skin gelatin rich in imino acid content has the potential to be the precursor of DPP-IV inhibitor for improvement of diabetes.
Keywords: Warm-water fish; cold-water fish; gelatin; proline; DPP-IV; GLP-1 Chemical compounds studied in this article
Streptozotocin (PubChem CID: 29327); Nicotinamide (PubChem CID: 936); Aprotinin (PubChem CID: 22833874)
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1. Introduction
Type 2 diabetes mellitus (T2DM) is the most prevalent form of diabetes and is one of the fastest growing health concerns worldwide (Sebokova, Christ, Boehringer, & Mizrahi, 2007). By 2030, it is estimated that the number of people affected with this chronic metabolic disorder will reach 366 million (Power, Nongonierma, Jakeman, & FitzGerald, 2014; WHO, 2012). T2DM is a complex disease and there are many co-morbidities and complications associated with it including obesity, hypertension, high cholesterol, cardiovascular disease and renal failure (Ben-Avraham, Harman-Boehm, Schwarzfuchs, & Shai, 2009). Over the past decade, a number of new therapies focusing on the modulation of incretin hormones, especially glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have been developed for the management of T2DM (Power et al., 2014). GIP and GLP-1 are natural substrates for dipeptidyl peptidase-IV (DPP-IV), which rapidly cleaves the N-terminal dipeptides of the hormones resulting in loss of their insulinotropic activity (Deacon, Nauck, Toft-Nielsen, Pridal, Willms, & Holst, 1995; Kieffer, McIntosh, & Pederson, 1995). GLP-1 has some physiological actions, such as stimulation of insulin biosynthesis, inhibition of glucagon secretion, decreases of gastric emptying and food intake, enhancement of satiety (Ahren, 2004; De León, Crutchlow, Ham, & Stoffers, 2006; Hansotia & Drucker, 2005). It has been reported that over 95% of the degradation of endogenous GLP-1 is attributed to the action of DPP-IV (McIntosh, Demuth, Pospisilik, & Pederson, 2005). Therefore, DPP-IV inhibitors are proposed to protect incretins, especially GLP-1, from enzyme cleavage; therefore, the utilization of DPP-IV inhibitors is thought to be a new therapeutic approach for management of type 2 diabetes (Deacon & Holst, 2006).
DPP-IV (CD26; E.C. 3.4.14.5) is a plasma membrane glycoprotein ectopeptidase 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
that belongs to the prolyl oligopeptidase family (De Meester, Lambeir, Proost, & Scharpé, 2003). It acts as a cleaving enzyme with the specificity for removing X-Pro or X-Ala dipeptides from the N terminus of polypeptides and proteins. It has a strong preference for Pro > Ala > Ser as the penultimate amino acid residue (De Meester et al., 2003; Lambeir, Durinx, Scharpé, & De Meester, 2003; Mentlein, Gallwitz, & Schmidt, 1993). Some previous studies have shown that specific DPP-IV inhibition increased the half-life of total circulating GLP-1, decreased plasma glucose, and improved impaired glucose tolerance in animal and human experiments (Deacon, Nauck, Meier, Hücking, & Holst, 2000; Mitani, Takimoto, Hughes, & Kimura, 2002).
Several synthetic DPP-IV inhibitors (commonly known as gliptins) have emerged in recent years as potent antidiabetic drugs, such as vildagliptin, saxagliptin, linagliptin, sitagliptin and alogliptin which have been approved for use in the European Union, USA and Japan. Most synthetic DPP-IV inhibitors are generally well tolerated; however, some adverse effects of gliptin-based compounds have been reported, including headaches, urinary and upper respiratory tract infections (Gooben & Gräber, 2012; Krushner & Gorrell, 2010; Scheen, 2013). Therefore, to develop natural DPP-IV inhibitors without side effects as therapeutic agents of type 2 diabetes is necessary.
Previous studies have reported that bioactive peptides possess DPP-IV inhibitory activity. Diprotins A and B are bioactive peptides observed to exhibit DPP-IV inhibitory activity with IC50 values of 1.1 and 5.5 μg/mL; they were identified to be Ile-Pro-Ile and Val-Pro-Leu, respectively (Umezawa, Aoyagi, Ogawa, Naganawa, Hamada, & Takeuchi, 1984). Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, both prepared from β-lactoglobulin showed IC50 values of 49 and 174 μM against DPP-IV, respectively (Tulipano, Sibilia, Caroli, & Cocchi, 2011; Uchida, Ohshiba, & Mogami, 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
2011). In a previous study, we successfully isolated several DPP-IV inhibitory peptides from Atlantic salmon skin gelatin, tuna cooking juice and porcine skin gelatin, and these peptides comprised Pro, Ala, or Gly as the second N-terminal residue, showing the IC50 values between 41.1 and 116.1 μM against DPP-IV (Hsu, Tung, Huang, & Jao, 2013; Huang, Jao, Ho, & Hsu, 2012; Li-Chan, Huang, Jao, Ho, & Hsu, 2012). The Atlantic salmon skin gelatin hydrolysate and the <1 kDa fraction of the porcine skin gelatin hydrolysates have been demonstrated to improve glycaemic control of streptozotocin (STZ)-induced diabetic rats by daily administration for 35 and 42 days, respectively (Hsieh, Wang, Hung, Chen, & Hsu, 2015; Huang, Hung, Jao, Tung, & Hsu, 2014).
Based upon our previous studies, peptides in some protein hydrolysates act as DPP-IV inhibitors to preserve the secreted GLP-1, resulting in insulin secretion and improvement of glycaemic control in diabetic rats (Hsieh et al., 2015; Huang et al., 2014). Furthermore, previous studies have shown that protein hydrolysates, peptones, peptides, amino acids or even intact proteins also had the action to stimulate GLP-1 secretion in vitro and in vivo (Cordier-Bussat et al., 1998; Hall, Millward, Long, & Morgan, 2003; Hira, Mochida, Miyashita, & Hara, 2009; Reimer, 2006). Therefore, the hypoglycaemic effect of DPP-IV inhibitory peptides attributed to the inhibition of DPP-IV or the dual actions with GLP-1 secretion needs to be investigated.
It is well known that the imino acids (proline and hydroxyproline) are rich in gelatin for about 15-23% of amino acid composition (Karim & Bhat, 2009). The total amount of imino acids in warm-water fish gelatin is higher (16-20%) than that in cold-water fish (14-17%). Therefore, fish skin gelatin can be expected as good precursors for DPP-IV inhibitors, especially warm-water fish. Then, we compared the
in vitro DPP-IV inhibitory activity of gelatin hydrolysates of warm- and cold-water
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fish, and the peptide sequences were also identified. We also demonstrated that the hydrolysates had both actions of DPP-IV inhibition and GLP-1 secretion to improve glycaemic control in vivo in STZ-induced diabetic rat model.
2. Materials and methods
2.1. Materials and reagents
The Pacific hake (Merluccius productus) and halibut (Hippoglossus stenolepis) fish skins, the processing byproducts recovered from fresh skin-off fillets, were supplied by Albion Fisheries Ltd. (Vancouver, BC, Canada), Tilapia (Oreochromis
niloticus) skins were supplied by Fortune Life Enterprise Co. Ltd. (Kaohsiung,
Taiwan), the milkfish (Chanos chanos) skins were donated by Simmy Seafood Co. Ltd. (Long An Province, Vietnam). The fish skins were transferred on ice to our laboratory, vacuum packed and stored at -20℃ until use. Flavourzyme® 1000 L (from
Aspergillus oryzae, 1000 LAPU/g) was purchased from Nova Technologies Inc.
(Bagsvaerd, Denmark). One LAPU (leucine aminopeptidase unit) is the amount of
enzyme which hydrolyzes 1 μmole of leucine-p-nitroanilide per min. Dipeptidyl peptidase IV (D7052, from porcine kidney), Gly-Pro-p-nitroanilide hydrochloride, streptozotocin (STZ), nicotinamide (NA), aprotinin (A6103, from bovine) and heparin (H5515, from porcine intestinal mucosa) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and reagents used were of analytical grade and commercially available.
2.2. Extraction of gelatin
The thawed fish skins were gently washed with running tap water, drained, and cut into pieces (about 5 × 10 cm). The fish skins were soaked in 0.2 M NaOH (1:10; 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149
w/v) and stirred in a cold room at 4℃ for 30 min. This procedure was repeated three times to remove non-collagenous proteins and pigments. The skins were washed with running tap water until the pH was neutral. Afterward, the skins were soaked in 0.05 M acetic acid (1:10; w/v), stirred at room temperature for 3 h, and then washed with
running tap water until the pH was neutral. Almost all of the scales could be removed. The gelatin of the swollen skins was extracted in double-distilled water (ddH2O; 1:2; w/v) at 70℃ for 3 h (Cheow, Norizah, Kyaw, & Howell, 2007). The oily and aqueous layers of the extract were separated by using a separatory funnel, and the extract was filtered through two layers of cheesecloth, lyophilized, and stored in a desiccator until use.
2.3. Amino acid analysis
The amino acid compositions of gelatin samples were analyzed using a Waters Pico-Tag system (Waters, Watford, UK) with the technical support from the Advanced Protein Technology Centre, Hospital for Sick Children (Toronto, ON, Canada). The gelatin solutions were hydrolyzed under vacuum in 6 M HCl (1:1; v/v) at 110℃ for 24 h in the presence of 1% phenol (v/v). All amino acids in hydrolyzed samples were derivatized with phenylisothiocyanate (PITC), and both primary and secondary amino acids produced phenylthiocarbamyl derivatives that are amenable to Waters Acquity UPLC with Acquity UPLC BEH C18 column (2.1 mm x 10 cm) and UV detection at 254 nm. The amount of the amino acids was calculated based on Pierce amino acid standard solution (Kaspar, Dettmer, Gronwald, & Oefner, 2009).
2.4. Enzymatic hydrolysis
The weighted gelatin added with 25-fold ddH2O (w/w) was incubated at 50℃ 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174
for 20 min prior to enzymatic hydrolysis. The hydrolysis reaction was started with the addition of enzymes at various enzyme/substrate ratios (E/S of 1, 3 and 5%; w/w). The reactions with Flavourzyme (FLA) were conducted at pH 7.0, and 50℃ for up to 8 h. After hydrolysis, the hydrolysate solutions were heated in boiling water for 15 min to inactivate the enzymes and then cooled in ice water for 20 min. The pH of hydrolysates was adjusted to 7.0 with 2 M NaOH and centrifuged (Centrifuge 05P-21, Hitachi Ltd., Katsuda, Japan) at 10,000 g and 4℃ for 15 min. The supernatant was lyophilized and stored at -20℃. The control was gelatin without any treatment.
2.5. Determination of DPP-IV inhibitory activity (in vitro)
DPP-IV activity determination in this study was performed in 96-well microplates in order to measure the increase in absorbance at 385 nm using
Gly-Pro-p-nitroanilide as DPP-IV substrate according to the method of Kojima, Ham, and
Kato (1980) with some modifications. The lyophilized hydrolysates were dissolved in 100 mM Tris buffer (pH 8.0), and an aliquot of 40 μL was added to 40 μL of 1.59 mM Gly-Pro-p-nitroanilide (in 100 mM Tris buffer, pH 8.0). The mixture was incubated at 37℃ for 10 min, followed by the addition of 80 μL of DPP-IV (diluted with the same Tris buffer to 0.01 Unit/mL). The reaction mixture was incubated at 37℃ for up to 60 min, and the reaction was stopped every 5 min by adding 150 μL of 1 M sodium acetate buffer (pH 4.0). The absorbance of the resulting solution was read at 385 nm with an ELISA reader (Bio Tek μ QUANT; Bio Tek Instruments, Inc., Winooski, VT, USA). Recorded data were plotted versus time, and the DPP-IV activity was quantified from the linear part of the curve. The % DPP-IV inhibition was defined as the percentage of DPP-IV activity inhibited by a given concentration of the hydrolysate. The IC50 value corresponds to the concentration of the sample 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
needed to inhibit DPP-IV by 50%.
2.6. Ultrafiltration (UF)
The DPP-IV inhibitory peptides of the hydrolysates were fractionated by ultrafiltration (Model ABL085, Lian Sheng Tech. Co., Taichung, Taiwan) with spiral wound membranes having molecular mass cutoffs of 2.5 and 1.5 kDa. The fractions were collected as follows: >2.5 kDa, peptides retained without passing through 2.5 kDa membrane; 1.5-2.5 kDa, peptides permeating through the 2.5 kDa membrane but not the 1.5 kDa membrane; <1.5 kDa, peptides permeating through the 1.5 kDa membrane. All fractions collected were lyophilized and stored in a desiccator until use. The fraction with the highest DPP-IV inhibitory activity was used as the sample for identification of amino acid sequence and the animal experiment.
2.7. Identification of amino acid sequence by MALDI-TOF/TOF MS/MS
The peptides in the ultrafiltration fraction which showed the greatest DPP-IV inhibitory activity were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a delayed extraction source and a 335 nm pulsed nitrogen laser. This analysis was carried out using a MALDI-TOF/TOF (UltraFlexIII, Bruker Daltonics Inc., Billerica, MA, USA). Peptides solution (0.6 μL) was mixed with 0.6 μL of saturated α-cyano-4-hydroxycinnamic acid, and a droplet of the resulting solution was placed on the sample target mass spectrometer. The droplet was dried by evaporation at room temperature and then loaded into the mass spectrometer for analysis. The instrument was operated in positive ion reflection mode with the source voltage set at 20 kV. All spectra were the results of signal averaging of 200 shots. Measurements were determined in the mass 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224
range of m/z 0-1,500 Da, while the peptide sequencing was determined by MS/MS spectra processing, using BioTools (Version 3.2; Bruker Daltonics Inc., Billerica, MA, USA).
2.8. Peptide synthesis
Peptides were prepared by the conventional Fmoc solid-phase synthesis method with an automatic peptide synthesizer (Model CS 136, CS Bio Co. San Carlos, CA, USA), and their purity was verified by analytical RP-HPLC-MS/MS.
2.9. Animals (STZ-NA induced diabetic rats)
Male Sprague-Dawley rats (LASCO, Taipei, Taiwan), aged 7 weeks and weighing between 230 and 250 g were used. Animals were fed a 5012-Rat Diet (LabDiet®, St. Louis, MO, USA) consisting (as a percentage of total kcal) of 13% fat, 60% carbohydrate, and 27% protein. Diabetes was induced in overnight fasted rats by a single intraperitoneal injection of a buffer (0.01 M citrate, pH 4.5) solution of STZ at a dosage of 65 mg/kg body weight, 15 min after the i.p. administration of 180 mg/kg body weight of nicotinamide (NA) in normal saline. At 1 week after the injection of STZ, the rats were considered to be diabetic if their plasma glucose levels over 200 mg/dL (Murugan & Pari, 2006). All rats care and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University.
2.10. Experimental design
Animals were divided into 6 groups of 11 rats each, and the rats in experimental groups were administrated samples by oral gavage. The experimental period was 30 days. Group A: normal control rats administered drinking water daily; Group B: 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
normal rats administered TSGH (750 mg/kg/day); Group C: diabetic control rats administered drinking water daily; Group D: diabetic rats administered HSGH (750 mg/kg/day); Group E: diabetic rats administered TSGH (750 mg/kg/day); and Group F: diabetic rats administrated sitagliptin (120 mg/kg/day; positive control).
2.11. Oral glucose tolerance test (OGTT)
The OGTT was performed on day 14 and 28, and GLP-1 secretion test was done on day 28 after induction of diabetes. Both tests were performed in overnight fasted rats from all groups. At -30 min on the day of the experiment, drinking water (normal and diabetic control), HSGH, TSGH or sitagliptin was orally administered to groups of rats. At 0 min, glucose (2 g/kg) was fed to each rat. Blood was withdrawn from tail vein at 0, 30, 60, 90, 120 and 180 min for the assay of glucose levels immediately using a blood glucose meter (TD-4207, Taidoc, New Taipei, Taiwan).
2.12. Biochemical determinations
On the morning after final administration on day 30, the animals were sacrificed by over dose of CO2. Blood samples were collected in chilled blood vases containing ethylenediaminetetraacetic acid (EDTA). Samples were centrifuged (3,000 g, 4℃ 15, min) and stored at -80℃. Plasma DPP-IV activity was measured using a DPPIV/CD26 assay kit (Enzo Inc., Farmingdale, NY, USA). Plasma total and active GLP-1 concentrations were measured using a glucagon like peptide-1 (total) RIA kit and a glucagon like peptide-1 (active) ELISA kit (Millipore Corp., Billerica, MA, USA). Plasma insulin concentration was measured using a Mercodia rat insulin kit (Mercodia Inc., Uppsala, Sweden).
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2.13. Statistical analysis
Each data point representing the mean of three replicates was subjected to analysis of variance (ANOVA) using SAS software version 9.1 (SAS Institute Inc., Cary, NC, USA). A test of comparison of two means was analyzed by Duncan’s test and the significance level of P<0.05 was employed.
3. Results and discussion
3.1. Amino acid composition of fish skin gelatin
The amino acid composition of various fish skin gelatin is presented in Table 1. The contents of glycine, the most dominant amino acid, ranged from 2.59 to 2.90 µmole/mg sample in fish skin gelatin. The warm-water fish, tilapia and milkfish, contained higher amounts of the imino acids (1.95-2.03 µmole/mg sample) than the cold-water fish, halibut and hake (1.77-1.79 µmole/mg sample). Tilapia and milkfish had 1.26 and 1.19 µmole/mg sample of proline, while halibut and hake comprised proline content of 1.16 and 1.15 µmole/mg sample, respectively.
3.2. DPP-IV inhibitory activity of hydrolysates
In our preliminary study, various commercial enzymes, including Alcalase, bromelain, Flavourzyme, pepsin, trypsin and pancreatin, were used to hydrolyze fish skin gelatins and determine their DPP-IV inhibitory activities. The result showed that all the hydrolysates obtained by FLA had greater DPP-IV inhibition rates than those by the other enzymes (data not shown); therefore, FLA was used in this study. The DPP-IV inhibitory activity of fish skin gelatin hydrolyzed with FLA at various E/S ratios for 4 h is shown in Fig. 1A. The DPP-IV inhibition rates increased with E/S ratio, and those showed the highest values of 43.2 and 42.5% (P<0.05) at 3 mg 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299
solid/mL, respectively, in tilapia and milkfish skin gelatin hydrolysates with 5% E/S ratio. Therefore, 5% E/S was adopted to investigate the DPP-IV inhibitory activity of hydrolysates at various hydrolysis time. Fig. 1B shows the DPP-IV inhibition rates of the fish skin gelatin hydrolysates with 5% FLA during hydrolysis up to 8 h. All the fish skin gelatin (control, without hydrolysis) showed limited DPP-IV inhibitory activities lower than 10%, whereas after hydrolysis, DPP-IV inhibition rates increased with time. However, the halibut and hake skin gelatin hydrolysates showed 34.3-39.6% inhibition rates with insignificant differences in all conditions of various hydrolysis time (P>0.05). The tilapia and milkfish skin gelatin hydrolysates after 6 and 8-h hydrolysis showed the greatest DPP-IV inhibitory activities of 45.6-48.1% in all samples; while no significant differences of the inhibition rates were observed between the 4 hydrolysates (P>0.05). In consideration of time and cost saving, tilapia and milkfish skin gelatin hydrolysates with FLA at E/S ratio of 5% and 6-h hydrolysis, halibut and hake skin gelatin hydrolysates with FLA at E/S ratio of 5% and 4-h hydrolysis were used for further fractionation by UF. In our previous study, Atlantic salmon skin gelatin hydrolysate at 5 mg solid/mL showed 45.2% DPP-IV inhibition rate (Li-Chan et al., 2012); therefore, the gelatin hydrolysates of warm-water fish skin had more potential as the precursor of DPP-IV inhibitors than those of cold-water fish.
3.3. DPP-IV inhibitory activity of UF fractions of hydrolysates
The DPP-IV inhibitory activities of the hydrolysates and their UF fractions (>2.5, 1.5-2.5 and <1.5 kDa) of fish skin gelatin are shown in Fig. 2. The result showed that the DPP-IV inhibition rates of all the skin gelatin hydrolysates at 1 mg solid/mL were 14-20%, which were significantly higher than those (8.7-16.2%) of the 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324
fraction >2.5 kDa (P<0.05). As expected, the fraction <1.5 kDa of fish skin gelatin hydrolysates had greater DPP-IV inhibitory activity as compared to the hydrolysates and the other two fractions (P<0.05); while the highest DPP-IV inhibition rate was 51.9% and observed in the fraction <1.5 kDa of tilapia hydrolysate (Fig. 2). The DPP-IV inhibition rate of the fraction <1.5 kDa of halibut hydrolysate was 38.2%, which was slightly but insignificantly (P>0.05) higher than that of hake hydrolysate. Previous studies has reported the preferable DPP-IV inhibitory peptides derived from food protein consisted of 2-8 amino acid residues (Pieter, 2006; Aart et al., 2009), and their molecular weights were supposed between 200 and 1,000 Da. Therefore, in consideration of the high in vitro DPP-IV inhibitory activity and in vivo bioavailability of peptides, the <1.5 kDa UF fractions of halibut and tilapia skin gelatin hydrolysates (HSGH and TSGH) were used to identify the peptide sequences and to compare their antihyperglycemic effect by the animal experiment.
3.4. Amino acid sequences of peptides in UF fractions of TSGH
The amino acid sequences of peptides in HSGH and TSGH were identified by MALDI TOF/TOF MS/MS. Fourteen and thirteen peaks for HSGH and TSGH, respectively, were obtained; the peaks with m/z higher than 1500 were eliminated based upon the peptides with unexpected molecular mass. Three major peaks with strong intensity for both HSGH and TSGH were selected for MS/MS analysis (Fig. 3). After the analysis by MS/MS spectra processing with BioTools database, the amino acid sequences of the 6 peptides were SPGSSGPQGFTG (862.32 Da), GPVGPAGNPGANGLN (1021.42 Da), PPGPTGPRGQPGNIGF (1261.44 Da), IPGDPGPPGPPGP (919.53 Da), LPGERGRPGAPGP (1026.58 Da) and GPKGDRGLPGPPGRDGM (1358.76 Da) (Table 2), which had Pro as the second N-325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349
terminal residue. The second N-terminal residues of the oligo-peptides reported in the literature to present DPP-IV inhibitory activity includes Pro, Trp, Ala, Val, Lys, and Asp (Lacroix & Li-Chan, 2012), and Pro is most preferable. The origins of these 6 peptides were collagen type I α-2 and α-3 of halibut (Hippoglossus olivaceus) and collagen type I α-1 and α-3 of tilapia (Oreochromis niloticus) as verified by comparing the UniProt Knowledgebase of ExPASy Proteomics Server available at http://expasy.org/ (Table 2). The IC50 values against DPP-IV of these peptides ranged from 65.4 to 146.7 µM, which showed similar DPP-IV inhibitory activity as compared to the peptides from various proteins with the IC50 values between 41.9 to 174 µM reported in previous studies (Huang et al., 2012; Lacroix & Li-Chan, 2014; Silveira, Martínez-Maqueda, Recio, & Hernández-Ledesma, 2013). The two N-terminal amino acid residues of these 6 peptides obtained in this study were Ser-Pro, Gly-Pro, Pro-Pro, Ile-Pro and Leu-Pro, which in di-, tri- and oligopeptides have been reported to exhibit DPP-IV inhibitory activity (Lacroix & Li-Chan, 2012). However, these 6 peptides comprised 12-17 amino acid residues which were much longer in length than the preferable DPP-IV inhibitory peptides with 2-8 amino acids (Pieter, 2006; Aart et al., 2009). A challenge of the bioavailability of these peptides is to resist the degradation of digestive enzymes followed by passing through gastrointestinal epithelium (Renukuntla, Vadlapudi, Patel, Boddu, & Mitra, 2013); and therefore, the hypoglyacemic effect of these peptides after digestion was examined by an animal experiment.
3.5. Effect of HSGH and TSGH on blood glucose levels during OGTT in diabetic rats
The OGTT was performed on day 14 and 28 after induction of diabetes. After receiving glucose at 2 g/kg, the blood glucose concentration of the rats was 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374
monitored for 180 min; and the area under curve (AUC) after calculation was also displayed (Fig. 4). On day 14 and 28, the blood glucose levels of the rats in Group C and D still reached over 200 mg/dL, while those of the rats in the other groups were lower than 200 mg/dL, which is the limit we define the rats diabetic. The results of OGTT showed that significant higher plasma glucose levels and AUC were observed in diabetic control rats of group C than normal and diabetic rats of other groups (P<0.05). From the results shown in Fig. 4, both HSGH and TSGH had the ability to lower the blood glucose levels of diabetic rats after 28-day administration; and the blood glucose levels of TSGH-treated diabetic rats in group E were similar to those of sitagliptin-treated diabetic rats in group F (P>0.05). Furthermore, TSGH did not induce hypoglycemia in normal rats (Group B) as compared to normal control rats (Group A) (P>0.05). It has been proposed that blood glucose levels of SFH (silk fibroin hydrolysate)-treated db/db mice were significantly lower than untreated db/db mice, showing that SFH can improve glucose tolerance of diabetic mice (Do et al., 2012). Another study indicated that the chronic administration of whey protein hydrolysates to ob/ob mice improves glucose clearance following a glucose load (Gaudel et al., 2013). This result was similar to our previous study reported that porcine skin gelatin hydrolysates had the effect to improve glycaemic control of STZ-induced diabetic mice (Huang et al., 2014). Although the administration of HSGH for 28 days significantly lowered the blood glucose levels of diabetic rats (P<0.05), the blood glucose levels during OGTT were still over 200 mg/dL. Therefore, we demonstrated that TSGH has the antihyperglycemic effect on diabetic rats as sitagliptin but no hypoglycemic side effect on normal rats.
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3.6. Plasma DPP-IV activity and GLP-1 levels
The effects of administration of HSGH and TSGH after 30 days on the plasma DPP-IV activity and GLP-1 levels in diabetic rats are shown in Fig. 5. The plasma DPP-IV activity of the diabetic control rats in Group C (115.5%) was significantly higher than that of any other groups (P<0.05). The normal rats in group A and B, and the diabetic rats treated with HSGH in group D showed similar plasma DPP-IV activity between 86.6 and 94.6% (P>0.05), while the diabetic rats treated with TSGH (group E) had significantly lower DPP-IV activity of 71.6% (P<0.05) (Fig. 5). Sitagliptin showed the greatest inhibitory effect on plasma DPP-IV (36.6% left) of diabetic rats in this study. A previous study has indicated that the long term (up to 5 weeks) administration of DPP-IV inhibitor sitagliptin greatly inhibited the plasma DPP-IV activity by about 50% in STZ-induced diabetic mouse (Kim, Nian, Doudet, & McIntosh, 2008). In a previous study, porcine skin gelatin hydrolysate and sitagliptin significantly reduced plasma DPP-IV activity for 36.7 and 69.0%, respectively, in STZ-induced diabetic rats (Huang et al., 2014). The result in this study has proven that TSGH had greater inhibitory effect on DPP-IV than HSGH in order to improve the glycemic control in diabetic rats.
The measurement of total GLP-1 levels may reflect the effects of HSGH and TSGH on GLP-1 secretion of diabetic rats after 30-day treatment. Fig. 5 shows that TSGH had no effect on GLP-1 secretion in normal rats (Group A and B; P>0.05); however, HSGH, TSGH and sitagliptin were observed to stimulated GLP-1 secretion in diabetic rats (P<0.05). As expected, GLP-1 secretion in diabetic rats (12.73 pM total GLP-1) was reduced as compared to the normal rats (20.12 pM) (Vilsboll & Holst, 2004). After 30-day treatment, TSGH (Group E) had the greatest increment in total GLP-1 level (27.81 pM); HSGH (Group D) and sitagliptin (Group F) showed 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424
similar effect on GLP-1 secretion (23.46-23.81 pM) in diabetic rats. Sitagliptin was reported to increase total GLP-1 secretion in both mGLUTag and hNCI-H716 cells by activating cAMP and ERK1/2 signaling (Sangle, Lauffer, Grieco, Trivedi, Iakoubov, & Brubaker, 2012). Moreover, GLP-1 secretion enhancement was also observed by dairy proteins, meat and zein hydrolysates, and essential amino acids in both in vitro and in vivo experiments (Chen & Reimer, 2009; Hira et al., 2006; Reimer, 2006). Interestingly, Atlantic salmon skin gelatin hydrolysate was reported to have no action on stimulation of GLP-1 secretion in our previous study (Hsieh et al., 2015). The mechanism of the protein or peptide structures that stimulates GLP-1 secretion is still unclear although several studies have confirmed that leucine, glutamic acid and the mixture of essential amino acids can induce GLP-1 secretion (Chen & Reimer, 2009; Reimer, 2006). On the other hand, the plasma active GLP-1 levels of the diabetic control rats in group C (5.14 pM) was similar to normal control rats in group A (5.61 pM) and normal rats treated with TSGH in Group B (6.14 pM) (P>0.05) (Fig. 5). The diabetic rats treated with HSGH (Group D), TSGH (Group E) and sitagliptin (Group F) had significantly higher active GLP-1 levels than the diabetic control rats (P<0.05); meanwhile, the active GLP-1 level of the rats in Group E (13.32 pM) was higher than that in Group D (7.37 pM). In our previous study, daily administration of porcine skin gelatin hydrolysate for 42 days in STZ-induced diabetic rats increased approximately 10% of plasma active GLP-1 level as well as that of sitagliptin (Huang et al., 2014). Previous studies have indicated that the short-term (9 days) and long-term (up to 1 month) daily administration of DPP-IV inhibitors, e.g. ASP8497, vildagliptin, sitagliptin, resulted in significant increases in plasma active GLP-1 levels in STZ-induced mice (Kim et al., 2008; Matsuyama-Yokono et al., 2009). In the present study, TSGH showed more effective DPP-IV inhibition and GLP-1 secretion than 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449
HSGH in diabetic rats and therefore retained higher active GLP-1 level. This is the first study demonstrated that the peptides had dual actions of DPP-IV inhibition and GLP-1 secretion to improve glycemic control in STZ-induced diabetic rats.
3.7. Plasma insulin levels
The effect of administration of HSGH and TSGH after 30 days on the plasma insulin levels in diabetic rats is shown in Fig. 6. The plasma insulin levels of all diabetic rat groups were significantly lower than normal rats in group A and B (2.25-2.35 µg/L) (P<0.05). The administration of TSGH and sitagliptin showed the similar insulin levels of 1.56-1.64 µg/L, which was higher than that of HSGH (1.14 µg/L) (P<0.05). The diabetic control rats had the lowest insulin level of 0.43 µg/L in this study due to the damage of pancreatic ß-cells by STZ.
Streptozotocin can induce the loss of pancreatic ß-cells and their functional defects which cause postprandial insulin secretion and lead to hyperglycaemia (Matsuyama-Yokono et al., 2009). In the STZ-induced diabetic animal model, chronic daily dosing of DPP-IV inhibitors, e.g. ASP8497, vildagliptin, sitagliptin and isoleucine thiazolodide, for 4 to 7 weeks could increase glucose-dependent insulin secretion and reduce blood glucose levels (Kim et al., 2008; Matsuyama-Yokono et al., 2009; Pospisilik et al., 2003). In our previous study, the diabetic rats administered porcine skin gelatin hydrolysate for 42 days and sitagliptin displayed plasma insulin levels were 4 to 5-fold higher than the diabetic control rats (Huang et al., 2014). The results have confirmed that administration of TSGH was more potent to improve the insulin secretion than HSGH in diabetic rats.
This is the first study to compare in vitro DPP-IV inhibitory activities and in vivo antihyperglycaemic effect of protein hydrolysates based upon the various imino acid
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contents. The results have confirmed the mechanism of glycaemic control in diabetic rats by protein hydrolysates as DPP-IV inhibitors reported in previous studies (Hsieh et al., 2015; Huang et al., 2014). Moreover, TSGH and HSGH also act as GLP-1 stimulator rather than Atlantic salmon skin gelatin hydrolysate (Hsieh et al., 2015). The future study is necessary to characterize the peptides showing dual effects on stimulation of GLP-1 secretion and inhibition of DPP-IV. Gelatin hydrolysates of warm-water fish skins (TSGH) with higher imino acid contents possessed greater in
vitro and in vivo DPP-IV inhibitory activity than those of cold-water fish skins
(HSGH), showing the potency to improve glycaemic control in STZ-induced diabetic rats.
4. Conclusions
This study has clearly demonstrated that fish skin gelatin hydrolysates having dual actions of DPP-IV inhibition and GLP-1 secretion enhancement, and therefore improve glycaemic control in diabetic rats after 30-day treatment. Tilapia skin gelatin hydrolysate (TSGH) is more potent as an antihyperglycaemic agent than halibut (HSGH), owing to the higher content of imino acids. This study provides an outlook on the protein source with high imino acid content having the potential to improve diabetes.
Acknowledgements
The authors thank Dr. Eunice C.Y. Li-Chan (The University of British Columbia) for her technical support as well as Dr. Musleh Uddin (Albion Fisheries Limited) for the kind supply of fish skins. This study was financially supported by China Medical University (Project No. CMU102-ASIA-13 and NSC 102-2313-B-475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499
039-005). 500
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Figure Legends
Fig. 1. DPP-IV inhibition rate of various fish skin gelatin hydrolyzed with FLA. (A) Fish skin gelatin was hydrolyzed at various E/S ratios for 4 h; (B) fish skin gelatin was hydrolyzed at E/S ratio of 5% during hydrolysis up to 8 h. The DPP-IV inhibition rate was determined with the hydrolysates at 3 mg solid/mL. Bars represent standard deviations from triplicate determination. Different letters indicate the significant differences (P<0.05). Control: fish skin gelatin without hydrolysis.
Fig. 2. DPP-IV inhibition rate of various fish skin gelatin hydrolysates fractionated by ultrafiltration at the concentration of 1 mg solid/mL. Bars represent standard deviations from triplicate determination. Different letters indicate the significant differences (P<0.05).
Fig. 3. Mass spectrum of the selected peptides in HSGH and TSGH.
Fig. 4. Effect of HSGH and TSGH on blood glucose levels during OGTT and plasma glucose AUC in diabetic rats at day 14 and 28. Bars represent standard deviations from triplicate determination. Different letters indicate the significant differences (P<0.05). Group A: normal control rats, Group B: normal rats + TSGH, Group C: diabetic control rats, Group D: diabetic rats + HSGH, Group E: diabetic rats + TSGH, Group F: diabetic rats + sitagliptin (n = 11/group).
Fig. 5. Effect of daily administration of HSGH and TSGH for 30 days on plasma DPP-IV activity and GLP-1 levels in diabetic rats. Bars represent standard deviations from triplicate determination. Different letters indicate the significant differences 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689
(P<0.05). Group description is the same as Fig. 4 (n = 11/group).
Fig. 6. Effect of daily administration of HSGH and TSGH for 30 days on plasma insulin levels in diabetic rats. Bars represent standard deviations from triplicate determination. Different letters indicate the significant differences (P<0.05). Group description is the same as Fig. 4 (n = 11/group).
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