Analisis zimografico de metaloproteasas y catepsina en higado graso de pato criollo (Cairina moschata).
Fatty liver or steatosis is the accumulation of triglycerides in the hepatic cells, normally as the result of high-carbohydrate force-feeding . Under biological stress, such a lipid excess exists as a response of the endoplasmatic reticulum, which includes the activation of proteins that causes resistance to insulin, caspase-4 mediated-apoptosis, inflamation and mithochondrial disfunction . However, in palmipeds, the liver is where most fat is formed, being very sensible to fat degeneration, consisting in an abnormal fat reserve in the cytoplasm of parenchymatous cells. This is an anatomic consequence, given that the bird lacks a lymphatic system, fats from the feed goes to and store directly in the liver. Still, they possess an adipose tissue less able to synthesize fats, compared to mammals, thus being the liver the ultimate organ for fat metabolism [1,9]. The increase in liver weight is not due solely to the triglyceride accumulation (normally by a factor of 180), but also due to the increase of other components (proteins, phospholipids, cholesterol, DNA, RNA). Hence, in the particular case of palmipeds [i.e. species from the family of Anatidae, like ducks (e.g. Cairina moschata), or geese (e.g. Anser spp.)], a steatotic liver is not a pathological organ, but one that presents an enhanced metabolic activity; in contrast with the classical fatty liver pathological steatosis diagnosed in humans (fatty liver syndrome and fatty liver hemorrhagic syndrome).
Proteolytic activity has an important role in the regulation of a wide range of biological processes, given the high specificity associated to the hydrolysis of the peptide bond. The proteolytic mechanism regulates the action of a variety of proteins, and thus, also diverse cellular processes. This hydrolytic activity is given by a family of enzymes and zymogens present in the cell, called widely 'proteases', with the purpose to hydrolyze the peptide bond [7,21].
Several techniques have been used to determine the presence of proteases in biological samples. The use of electrophoresis for this purpose is a simple alternative widely use in the study of many types of proteases and its inhibitors [23,29]. Zymography is a valuable tool for the visualization of proteolytic activity on an electrophoretic gel matrix, by using a given protein substrate . In order to characterize the proteolytic profile present in hepatic cells under steatosis, one-dimensional zymography (1DZ) and two-dimensional zymography (2DZ) approaches were introduced, with the goal to demonstrate the feasibility of this technique to determine proteolytic activity in fatty liver in a semi-quantitative way. For this, an extraction procedure was performed, commercial inhibitors were used to identify the type of protease, and the total proteolytic activity was quantified by photometry.
MATERIAL AND METHODS
All experiments described fully comply with legislation on research involving animal subjects according to the European Communities Council Directive of November, 24 1986 (86/609/ EC) , the Venezuelan Law of Science & Technology  and the Venezuelan Code of Bioethics and Biosecurity .
2-mercaptoethanol, acetic acid, hydrochloric acid, phosphoric acid, casein, gelatin, tyrosine, 2-propanol, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA); Coomassie brilliant blue (CBB)-R250 and CBB-G250 were from Pierce (Rockford, IL, USA); sodium acetate, trichloroacetic acid (TCA), sodium chloride, calcium chloride, ethanol, sodium hydroxide, ammonium persulfate and tetramethylethylenediamine (TEMED) were purchased from Riedel de Haen (Seelze, Germany); glycerol, glycine, bisacrylamide, dithiothreitol (DTT) and Triton X-100 were from Promega (Madison, WI, USA). The electrophoresis miniprotean III model used was from Bio-Rad (Hercules, CA, USA). All chemicals used were of analytical grade, and deionized water was used.
Three liver samples were collected from 3 Muscovy ducks (Cairina moschata) from different origins. The first sample was treated as reported by Awde et al. , and was designated as a positive control sample for fatty liver. Two other liver samples from Muscovy ducks were grown similarly until week 10. Then, one continued with regular feeding (negative control) and the other was force-fed until week 14. At this point, both birds were sacrificed. Livers were removed from carcasses, weighted, and samples were harvested, immediately frozen in liquid nitrogen and stored at -20[degrees]C (Whirlpool, Model ED2CHOXKO07, Whirlpool Corporation, USA) until analysis.
Three types of extracts were obtained from the three liver samples. First, an extract in buffer without inhibitors was designated as plain homogenate, second, an extract in buffer selective for metalloproteases [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Ca[Cl.sub.2], 10 [micro]g/mL (21 [micro]M; MW 475.6) leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF)], and Anally a cathepsin buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Ca[Cl.sub.2], 20 mM ethylenediaminetetraacetic acid (EDTA)] . For each extract, 400 mg were homogenized at 4[degrees]C in a potter (Thomas Corning Potter-Elvehjem Tissue Grinder, 7725T-17, Thomas Scientific, USA) with the specified buffer. Afterwards, suspensions were centrifuged (Thermo Electron IEC Centra CL5R Refrigerated Centrifuge, ThermoFisher Scientific, USA) at 10,000 x g for 20 minutes (min) at 4[degrees]C. Resulting supernatants were collected and stored at -20[degrees]C (Whirlpool, Model ED2CHOXKO07, Whirlpool Corporation, USA) until assayed.
The total amount of protein was determined according to Bradford . A calibration curve (triplicate) was plotted employing 1 g/L BSA as standard protein. Samples were assayed by duplicate; absorbance was measured at 595 nm (Genesys 10-S spectrophotometer, Thermo Scientific, Thermo Electron Corporation, USA).
A modification of the Kunitz  method was employed, with casein as substrate. Five hundred pL casein [1% (m/v) dissolved in 50 mM Tris-HCl pH 8.0] were preincubated at 37[degrees]C for 15 min, then 200 [micro]L of the supernatant containing the enzymatic activity was added and the mixture was incubated at the same temperature with gentle and constant agitation for 20 min. Reaction was stopped by addition of 500 [micro]L TCA (5% m/v), an placed over ice for 30 min. Samples were centrifuged for 20 min at 2,500 x g. Finally, absorbance at 280 nm was measured to the resulting supernatant. Assays were done by triplicate. A negative control was performed by submitting TCA (5% m/v) directly to enzyme fraction and then the substrate was added. Enzymatic units were defined as the amount of [micro]mol of tyrosine released as hydrolysis product from the casein per min. For this purpose, a calibration curve was plotted employing a 1 g/L tyrosine stock solution .
Polyacrylamide gel electrophoresis
Electrophoresis was performed by a modified procedure described by Laemmli , under non-reducing conditions (i.e. absence of 2-mercaptoethanol, no sample heating). Unless elsewhere stated, 7.5 or 12% (m/v) resolving gels were used and 5% (m/v) as stacking gel. The Mini-Protean III (Bio-Rad) chamber was used. About 30 [micro]g protein dissolved in 4x sample loading buffer [Tris-HCl 200 mM pH 6.8, 4% (m/v) SDS, 40% (v/v) glycerol, 0.02% (m/v) bromophenol blue] were applied per well. Five [micro]L of molecular weight standards (16.5-210 kDa range, ThermoScientific, USA) were used. Running buffer [Tris 25 mM, glycine 192 mM, 0.1% (m/v) SDS] was added to the chamber and electrophoresis was performed at 90 V the first 15 min, and then raised to 120 V, always at 4[degrees]C, until the blue front reached the bottom of the gel. After the run, gels were stained with CBB-R250 [0.125% (m/v), 40% (v/v) 2-propanol, 10% (v/v) acetic acid] and destained [40% (v/v) 2-propanol, 10% (v/v) acetic acid] until optimal band patterns were observed. Digitalization of the gels was done with a transiluminator (Bio-Rad, Chemidoc, 1708280, USA) and images were saved as jpeg Ales. For molecular weight (MW) determination, a calibration curve with the standard proteins was performed, relating the log MW vs. relative mobility.
For two-dimensional electrophoresis (2DE) purposes, a rehydration buffer [8 M urea, 1% (m/v) CHAPS, 40 mM DTT, 0.25% (v/v) Biolytes 3-10] was added to the liver extract (100-400 [micro]g), avoiding to exceed 150 [micro]L of deposited sample. The immobilized pH gradient (IPG) strip was placed over the sample and left 30 min for absorption, then mineral oil was added. Strips were rehydrated under active conditions: 50 V, 19[degrees]C, 11 h, without pause after rehydration, and with 50 [micro]A/strip. The voltage was programmed as follows: S1 200 V, linear, 1 h; S2 500 V, linear, 1 h, S3 1000 V, linear 1 h; S4 4000 V, linear 30 min, and S5 4000 V, ramping 5 h. After focusing, strips were equilibrated for 20 min in equilibration buffer I [8 M urea, 0.375 M Tris pH 8.8, 2% (m/v) SDS, 20% (v/v) glycerol, 130 mM DTT] and then in equilibration buffer II [8 M urea, 0.375 M Tris pH 8.8, 2% (m/v) SDS, 20% (v/v) glycerol, 270 mM iodoacetamide (IAA)], and Anally rinsed with water. The strips were then placed over the 10% (v/v) polyacrylamide gels, containing 0.15% (m/v) gelatin as substrate, and overlaid with heated agarose solution [1% (m/v) agarose, 0.025% (m/v) bromophenol blue] . Gels were run as described before.
Metalloproteases and cathepsin activity was visualized by 1DZ and 2DZ. For metalloproteases, a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) copolymerized with 0.2% gelatin was pre-run in running buffer at 4[degrees]C for 20 min at 125 V. About 50 [micro]g total proteins were applied and the system was run at 4[degrees]C for 120 min at 125 V. After run, gels were incubated twice in renaturing buffer [100 mM glycine pH 8.0, 2.5% Triton X-100] for 30 min each. Then, solution was discarded and gel was placed in activating buffer [100 mM glycine pH 8.0] at 37[degrees]C for 18 h. Finally, gels were stained with CBB-R250 0.5% (m/v) and destained until pale bands under a deep blue background were observed [10,27].
On the other hand, the zymography method proposed by Platt, Randall and Hanjoong  for cathepsin detection was used. PAGE (12.5% m/v) copolymerized with 0.2% (m/v) gelatin was run at 100 V, 4[degrees]C, for 2 h. Sample buffer and running buffer were the same as previously described. Renaturing buffer [Tris 50 mM pH 7.4, 20% (v/v) glycerol] was used with mild agitation and then incubated with activation buffer [0.1 M sodium acetate pH 5.5, 1 mM EDTA, 2 mM DTT] at 37[degrees]C for 18 hours (h). Afterwards, gels were briefly soaked in water (dd) and submitted to the same staining procedure previously described. For the 2DZ protocol, after the 2DE run, gels were treated with 1% (v/v) Triton-X 100, and then incubated in a suitable activation buffer [22,24]. Here, gels were washed and incubated with the same solutions used for the 1DZ.
Proteolytic activity inhibition
To test the effect of specific inhibitors over the proteolytic activity, 200 [micro]L of enzyme samples were pre-incubated with inhibitor [10 mM EDTA, 20 [micro]M leupeptin or 1 mM PMSF] for 30 min at 37[degrees]C, and then the photometric assay for activity determination was performed. The inhibition percentage was calculated against a control without inhibitor. Additionally, the effect of EDTA was visualized by zymography. Inhibitors were prepared as following: 30 mM EDTA (MW 372.24) stock solution. 0.0448 g EDTA disodium salt was dissolved in water, the pH adjusted to 7.4, final volume 4 mL. One mg/mL leupeptin (MW 475.59) stock solution was prepared in distilled water. 100 mM PMSF stock solution (MW 174.19) was prepared by weighing 0.0358 g and dissolving in 2-propanol in a final volume of 2 mL.
Densitometry of zymograms
The proteolytic activity detected by zymography was quantified according to band intensity, employing the Scion Image software, version BETA 4.0.2 (Scion Corporation) following the supplier's instructions. After desaturation of the image, colors were inverted, so that now black bands with a white background were obtained. Images were converted to .tiff files and imported to the software. Bands to be analyzed were defined and intensity was measured using the integral density modus. The data from the areas of the densitograms were plotted to compare the activity quantified in each band.
RESULTS AND DISCUSSION
Extraction and enzyme activity
The different extraction methods employed were efficient for the separation of the different proteolytic activities present in the hepatic cells. Fatty liver samples were determined to be richer in total protein amounts, confirming previous reports indicating protein content increased by a factor of 1.6 in fatty liver compared with lean liver (TABLE I) . All samples had protease activity, but those from fatty liver gave higher activity values. Metalloproteases and cathepsin extracts gave higher specific activities (TABLE I).
SDS-PAGE and zymography
The results of the analysis of the liver extracts on SDS-PAGE 7.5% (m/v) are shown in FIG. 1A. A ~90 kDa band was observed by zymography corresponding to metalloprotease activity (FIG. 1B). SDS is commonly used for electrophoresis, but is known to produce alterations in the protein tertiary structure, interrupting the interaction with the catalytic site of the protein. Thus, to maintain enzymatic activity, non-reducing conditions were employed. As result, the proteolytic profile of the sample was observed (FIG. IB), and bands of the optimized color-inverted image (FIG. IC) were analyzed by densitometry. Zymograms showed an active protease with molecular mass of ~90 kDa, present in the metalloprotease extract. The band intensity is ~100% higher in the sample from fatty liver, compared with the lean liver (calculations not shown). According to these results, it is proved that hepatic cells under steatosis hold a higher metalloprotease activity. This result varies from the one previously reported , where a 72 kDa metalloprotease is detected. There may be a number of factors involved in this observation, as the method proposed here has several different features, including differences in activation buffer components, gel %, running conditions, and different incubation times. Although biological samples were treated similarly, geographic alterations may be present as well, as ducks raised in Venezuela may be metabolically distinct as those raised in France due to climate changes, diet variations, and duck farming techniques. For these reasons, comparisons between both works are limited. Beyond this, it has been reported strong variability in the processing yield of fatty liver from mule ducks, even if overfeeding and processing conditions are controlled .
Cathepsins belong to the cysteine protease family, and its presence was demonstrated in the samples analyzed. Former studies in animal models suggest that an increase in cathepsin activity have a potential implication in promoting adipogenesis and growth of adipose tissue . FIG 2B shows the results obtained when lean and steatotic liver samples were loaded in 12.5% (m/v) zymograms. A ~37 kDa band containing cathepsin activity was detected, and was further corroborated by SDS-PAGE. Here is also detected an increase in proteolytic activity in samples from fatty liver, compared with lean liver. Differences in band intensity analyzed by densitometry gave ~100% increase in cathepsin activity in fatty livers (calculations not shown). This result is similar with that obtained by Awde et al. [2, 3], although they were able to see two extra bands of lower molecular mass, that were not detected with the method developed here. However, again, similarities are restricted for the same reasons explained formerly when discussing metalloproteases. zymography (FIG. 3C) gave as result a pl ~5.5 and a smear with a [M.sub.r] between 25 and 50 kDa. This new value strongly contrasts with the originally obtained by 1DZ, namely 90 kDa (FIG. 1B). Probably the harsh conditions employed during isoelectric focusing (IEF) might have driven to a protein denaturation, originating smaller polypeptide fragments. Similar values have been published , where several spots corresponding to cysteine protease had pl values in the range 4.8-6.3. Further assays were performed with the metalloproteins and cathepsin extracts from the lean and steatotic liver. However, reproducibility was an issue when comparing spots (data not shown). Though qualitative results may be given, difficulties where seen by enzyme proper activation (no spots on the gel), or deviations on the pl and/or MW were registered (harsh denaturing conditions proper of IEF, effect of urea, prolonged exposure to high voltage, etc.). It is advised to take care when submitting samples to IEF, as this type of variations had been reported before .
To further characterize the proteolytic activities, 2DZ was performed. As a first step, only the control liver was assayed. The electropherogram corresponding to 2DE analysis of the control liver is shown in FIG. 3A. Not many spots are present, but well defined protein spots are present in the region between pi 5.1 and 7.0, and [M.sub.r] 50-100 kDa. A ladder pattern is found at pl ~8.5, and some weak bands are seen in the acidic region. For cathepsin activity (FIG. 3B) a middle intensity spot was obtained, indicating a pl ~4.4 and a [M.sub.r] ~37 kDa, which agrees with the value obtained by 1DZ (FIG. 2B). Other weaker spots at the same pi are seen as well. On the other hand, metalloprotease activity analyzed by 2D
EDTA, leupeptin and PMSF were tested to analyze its inhibitory effect over the proteolytic activities. The residual activity present in each sample was assayed by duplicates. Results are shown in TABLE II. It can be stated that no true inhibition was observed by any of the inhibitors when the cathepsin extract was used, about 37% of inhibition was calculated when treated with PMSF. True inhibition was detected when testing EDTA with the metalloprotease extracts of both liver samples (~90%). Bax et al.  report an evolutive profile of proteins present in steatotic liver from duck. By coupling 2DE with Matrix-assisted laser desorption/ ionization--time of flight--mass spectrometry (MALDI-TOF-MS), they identified 14 proteins, including proteases like calpains. This study may give rise to further analysis by submitting the resulting spots to mass spectrometry. A recent report describes the importance of employing MS technology to explain fat loss during cooking of foie gras and the fact that it is most likely associated with different proteolytic patterns .
The main producer of 'foie gras' is France, being the fatty liver of ducks the most valuable product in these production systems. The quality determination of fatty liver is important for legal and economical reasons. Though variabilities in the analysis of fatty liver from ducks are expected to arise, even when overfeeding and processing conditions are controlled, comparisons among samples can be established. This investigation showed that all liver samples tested contained protease activity, albeit those from fatty liver gave a mean value of 2.5 times higher activity values when compared to lean liver samples. Metalloprotease activity showed a ~90 kDa band observed by 1DZ, but this value shifts when analyzed by 2DZ, obtaining a smear pattern with [M.sub.r] 25-50 kDa at pl ~5.5. On the other hand, cathepsin showed a ~37 kDa band during 1DZ, a value that differs from others published. Yet 2DZ analysis of cathepsin gave a spot corresponding to pi ~4.4 and a Mr ~37 kDa. Metalloprotease activity was inhibited in the presence of EDTA, however leupeptin failed to inhibit cathepsin activity. The protease analysis between the steatotic and non-steatotic liver was appreciated in this research, however further work must be performed in order to better characterize these activities, e.g. by MS. Recent publications further support the importance of this kind of research, where protease activities are screened to And paths responsible for quality losses during the cooking of ducks' fatty livers. Also semi-quantifications of proteins by electrophoresis and proteolytic activities by mono-dimensional zymography for metalloproteases and cathepsins have been reported in order to study the effect of chilling rates on the quality features of fatty livers.
The authors thank the staff from GenPhySE lab for technical assistance, and the staff from BIOMOLP-UC for equipment allowance. For laboratory supplies we thank the staff from Lab. de Biotecnologia-Facyt-Biologia-UC. This work was partially funded by Consejo de Desarrollo Cientifico y Humanistico, Universidad de Carabobo CDCH-AM-030-11 and CDCH-187-2015.
Note: Partial results shown in this article were presented at the 13th International Congress on Amino Acids, Peptides and Proteins (ICAPP), Galveston, Texas, USA. The authors declare that they have no competing interests.
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Recibido: 12/05/2016 Aceptado: 08/07/2016
Jeff Wilkesman (1) *, Maria Fernanda Padron (1), Liliana Kurz (1), Sahar Awde (2) and Herve Remignon (2)
(1) Universidad de Carabobo, Facultad de Ciencias y Tecnologia, AMBIOQUIM, Valencia, 2005 Venezuela. (2) GenPhySE, Universite de Toulouse, INPT, iNp-ENVT, INRA, 31320 Auzeville-Tolosan, France. * Corresponding Author: Dr. Jeff Wilkesman, Telf. +58.241.6005000 ext.375.062. E-mail: firstname.lastname@example.org.
Caption: FIGURE 1. ELECTROPHEROGRAMS OF THE EXTRACTS AND ZYMOGRAPHY OF METALLOPROTEASES. A) SDSPAGE (7.5%). LANES 1-3, EXTRACTS FROM LEAN LIVER; LANES 4-6 EXTRACTS FROM FATTY LIVER. LANES 1 AND 4, PLANE HOMOGENATE, 2 AND 5: METALLOPROTEASE, 3 AND 6: CATHEPSIN. B) ZYMOGRAM (7.5% AND 0.2% GELATIN) OF ONLY THE METALLOPROTEASE EXTRACT (100 MG). LANE 2, LEAN LIVER, 5: FATTY LIVER. C) COLOR-INVERTED IMAGE OF THE ZYMOGRAM SHOWN IN B). M, MOLECULAR WEIGHT MARKER (KDA).
Caption: FIGURE 2. ELECTROPHEROGRAMS FOR CATHEPSIN ANALYSIS. A) SDS-PAGE (12.5%), 30 MG OF TOTAL PROTEIN FROM CATHEPSIN EXTRACT WAS APPLIED. LANE 1: LEAN LIVER, 2: FATTY LIVER. B) ZYMOGRAPHY OF 80 MG CATHEPSIN EXTRACTS. LANE 1: LEAN LIVER; 2: FATTY LIVER. M, MOLECULAR WEIGHT MARKER (KDA).
Caption: FIGURE 3. ELECTROPHEROGRAMS FROM 2DE AND 2DZ OF THE CONTROL LIVER. THE FIRST DIMENSION WAS RUN WITH IEF STRIPS PH 3-10. SECOND DIMENSION WAS RUN WITH SDS-PAGE 10%. A) 2DE OF THE PROTEIN EXTRACT FROM LIVER CONTROL. B) 2DZ OF THE CATHEPSIN EXTRACT. C) 2DZ OF THE METALLOPROTEASE EXTRACT. IN EACH GEL ~250 MG TOTAL PROTEINS WERE LOADED.
TABLE 1 PROTEIN CONCENTRATION AND SPECIFIC ENZYMATIC ACTIVITY OF THE LIVER EXTRACTS Protein concentration (mg/mL) Liver sample Extract Control Lean Fatty Plain homogenate 31 [+ o -] 2 42 [+ o -] 2 68 [+ o -] 2 Metalloprotease 44 [+ o -] 1 41 [+ o -] 1 61 [+ o -] 1 Cathepsin 59 [+ o -] 2 60 [+ o -] 2 79 [+ o -] 1 Specific activity (a,b) (U/mg) Liver sample Extract Control Lean Fatty Plain homogenate 1.7 [+ o -] 0.1 1.4 [+ o -] 0.1 3.0 [+ o -] 0.1 Metalloprotease 12.4 [+ o -] 0.3 8.5 [+ o -] 0.3 20.5 [+ o -] 0.4 Cathepsin 10.2 [+ o -] 0.1 7.2 [+ o -] 0.3 21.5 [+ o -] 0.5 (a) Mean values from triplicates [+ o -] s.d. (b) U= pmoles tyrosine at 37 [grados] C, 20 min TABLE II INHIBITOR EFFECT OVER THE PROTEOLYTIC ACTIVITY Inhibition (a) (%) Extracts Lean liver Inhibitors EDTA Leupeptin Plain homogenate 28.2 [+ o -] 0.4 13.4 [+ o -] 0.1 Metalloprotease 90.1 [+ o -] 0.2 6.9 [+ o -] 0.4 Cathepsin 2.8 [+ o -] 0.3 5.2 [+ o -] 0.1 Inhibition (a) (%) Extracts Lean Liver Fatty liver Inhibitors PMSF EDTA Plain homogenate 17.4 [+ o -] 0.1 21.4 [+ o -] 0.1 Metalloprotease 5.6 [+ o -] 0.5 89.4 [+ o -] 0.2 Cathepsin 37.2 [+ o -] 0.4 4.0 [+ o -] 0.2 Inhibition (a) (%) Extracts Fatty liver Inhibitors Leupeptin PMSF Plain homogenate 16.1 [+ o -] 0.4 15.0 [+ o -] 0.6 Metalloprotease 4.5 [+ o -] 0.3 3.0 [+ o -] 0.6 Cathepsin 6.4 [+ o -] 0.3 24.9 [+ o -] 0.3 (a) Mean values from duplicates [+ o -] s.d.