Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2017 14(4): 1167-1174. OPEN ACCESS

Metabolic fuel level and enzyme activity changes in starved Broadhead ( macrocephalus Gunther, 1864)

Rattanasuda Chaiyachate1 , Bundit Yuangsoi1 Thongchai Champasri1,Chamaiporn Champasri2and Siripavee Charoenwattanasak1

1Department of , Faculty of Agriculture, Khon Kaen University, 40002, Khonkaen, 2Department of Biochemistry, Faculty of Science, Khon Kaen University, 40002, Khonkaen, Thailand

*Correspondence: [email protected] Accepted: 18 nov2017 Published online: 30 Dec. 2017 A study on metabolic fuel level and enzyme activity changes in starved (Clarias macrocephalus) was conducted using 24 aquarium tanks with 3 individuals of Broadhead catfish (average weight 40 grams per fish) per tank. They were divided into 2 groups i.e. fed group (feed for 1-4 weeks) and starved group (starve for 1-4 weeks). The results showed that muscle water content and alanine aminotransferase (ALT) have no significant differences (P>0.05), while hepatosomatic index (HSI), serum glucose, muscle glycogen, liver glycogen, muscle protein, muscle lipid and Hexokinase (HK) activity were decreased due to duration of starvation (P<0.05), and lower in the starved group compared to the fed group in each week (P<0.05). Aspartate aminotransferase (AST) and glutamate dehydrogenase (GDH) activities were higher in the starved group compared to the fed group in the 3rd week and in the 2nd -3rd week, respectively (P<0.05). In conclusion, starvation had significant effects on metabolic fuel levels and enzyme activities in Broadhead catfish. Keywords: starvation, metabolic fuels, enzyme activities, Clarias macrocephalus

INTRODUCTION allocate their time to activities such as Wetlands in the tropics have many species of predator avoidance, thermoregulation, ecdysis, or fishes such as Cyprinid, catfish, Snakehead fish, various behaviors related to reproduction (McCue, climbing perch and . Most were completely 2010). aquatic ecosystems in the rainy season, but in the Starvation is experienced in most species of fish aridity season numerous fish species endure of during certain periods of every year largely due to starvation due to the seasonal changes in food environmental conditions and it affects different availability or seasonal changes in water organs in different ways. Starvation also affects temperature. the physiology and other constituents of fish, Starvation refers to the biological condition where reduced energy requirements due to low are unable to eat so as a result of some metabolic rates and utilization of stored energy extrinsic limitation on food resources. The caused decrease metabolic fuels such as frequency and duration of natural starvation glycogen, protein and lipid, and body mass loss events can vary widely. In contrast, the term (Beamish, 1964; Murray, 1971; Wieser et al, fasting refers to an animals unable to eat so as a 1992; Yang and Somero, 1993). The previous result of some intrinsic mechanism, although food studies on effects of starvation in vary fishes may be available to them. Fasting, occurs when found glucose level was decrease in starved fish Chaiyachate et al. Effects of starvation in Broadhead catfish. i.e. Yellow perch (Perca flavescens), Common Experimental design carp (Cyprinus carpio), White sturgeon (Acipenser The experimental was designed in completely transmontanus), Rainbow trout (Oncorhynchus randomized design (CRD) with 3 replications. It mykiss), Goldfish (Carassius auratus), Snakehead was conducted using 24 aquarium tanks with 3 fish (Ophiocephalus maculatus), Red sea beam individuals of Broadhead catfish (average weight (Chrysophrys major) and Sturgeon (A. fulvescens) 40 grams per fish) per tank. They were divided (Foster and Moon, 1991; Navarro and Gutierrez, into 2 groups i.e. fed group (feed for 1-4 weeks) 1995; Gillis and Ballantyne, 1996; Hung et al, and starved group (starve for 1-4 weeks). 1997; Russell and Gahr, 2000; Friedrich and The Broadhead catfish average weight 35 grams Stepanowska, 2001; Congleton and Wagner, were collected from Fisheries department farm, 2006). In addition, decreasing of protein and faculty of Agriculture, Khon Kaen University. They glycogen in Channa punctatus have also been were acclimatized to experimental condition in reported (Namrata et al, 2011). concrete tank at least for 2 weeks before to Starvation affecting the activities of enzyme were experimentation. During this time, they were fed 3 found in vary fish such as were decrease in citrate times per day on a pellet food for catfish. After synthase, glucose-6-phosphate dehydrogenase acclimation for 2 weeks and fish have average and lactate dehydrogenase activities in Walking weight 40 grams, 9 individuals were sampling for catfish (Clarias batrachus) (Tripathi and Verma, initial group, 36 individuals were randomly 2003). The decreasing in hexokinase activity and assigned into 12 aquarium tanks in fed group, and increasing in aspartate aminotransferase and 36 individuals were randomly assigned into 12 glutamate dehydrogenase activities in African aquarium tanks in starved groups. Each aquarium lungfish (Protopterus dolloi) have also been tank contained 3 individuals with 20 liters of water reported (Frick et al, 2008). quality parameters such as pH 7.5, dissolved The Broadhead catfish (Clarias macrocephalus oxygen 5 mg l-1 and temperature 24 oC. Water Gunther, 1864) is an economically important fish was replaced about 50% every 3 days to avoid that is a native of , for use in accumulation of waste water. Water qualities were human consumption in countries and are common monitored regularly 2 times a week during inexpensive food prepared in a variety of ways. experiment period. Each group was collected They usually are living in canals, marshes, rice weekly, there were contained 9 individuals fed fields, stagnant pools and rivers. Should the water group and 9 individuals starved group. evaporate during the aridity seasons, the fish is able to alive for a long time due to they have Serum and tissues collection accessory air breathing called arborescent organ Fish were anesthetized by lowering the ambient or dendrite (Munshi, 1961). They stand for temperature with ice or cold water. Blood samples starvation condition during aridity season every were taken from anesthetized fish by caudal years and affecting that are determined by puncture using syringes with a 24 gauge needle. o physiological changes, such as body mass loss or Blood samples were centrifuged at 5,000 g at 4 C nitrogen excretion, or by the primary metabolic for 10 min. using a Hettich Rotina 35R centrifuge fuels i.e. glucose, glycogen, protein or lipid used (Germany), and serum was quickly frozen and o during starvation. The problem concerns about stored at -20 C for glucose levels assay. Tissues the great metabolic fuels viz. glycogen, protein or (liver and muscle) were rapidly excised and o lipid, are metabolized transferred to storage immediately frozen in -20 C for water content, resources and converted to energy. Starvation glycogen, protein and lipid levels analysis and also affects the physiology and other constituents hexokinase (HK), aspartate aminotransferase of fish (Deng et al, 1993). In addition, the activities (AST), alanine aminotransferase (ALT) and of metabolic pathways enzymes were considered glutamate dehydrogenase (GDH) activities during starvation. analysis.

MATERIALS AND METHODS Tissue preparations These experiments comply with Ethical Principles For assays of muscle protein levels, and HK activity analysis, muscle was homogenized in 3 and Guidelines for the Use of Animals. The o researchers have been licensed No. U1-01889- volume of ice cold 0.1 M imidazole pH 7.4 at 4 C 2558 of the Institute of Animals for Scientific using a KAI Ultra-Turrax T18 homogenizer Purpose Development (IAD), National Research (Japan). Homogenate were divided 100 l for Council of Thailand (NRCT). protein levels assay. The remaining homogenates

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Chaiyachate et al. Effects of starvation in Broadhead catfish. were centrifuged at 15,000 g 4oC for 10 min. The standard curve is constructed by using BSA resultant supernatant (cytosolic fraction) was used standard (Bradford, 1976). directly in HK activity assay (Singer et al, 1990). Muscle and liver glycogen level were measured For assay of muscle and liver glycogen levels using the anthrone method. Preparing 5 ml analysis, tissues were homogenized in 5% anthrone reagent (0.05% anthrone + 1% thiourea trichloroacetic acid (TCA). Homogenate were + 72% H2SO4) and 1 ml carbohydrate solution. centrifuged at 5,000 g for 5 min, pellets were They were immersed in a boiling water bath to transfer to the blender with an appropriate volume depth a little above the level of the liquid in the of 5% TCA and homogenize again. The resultant tube for 11 min and then removed to cold water supernatant was added 5 volume of 95% ethanol bath and to cool down to room temperature and with careful blowing to effect thorough mixing and measured absorbance at 630 nm. Glycogen level allowed to stand overnight at room temperature. in the samples can be calculated by against a After precipitation was completed, they were reagent blank and determined concentration of centrifuged at 5,000 g for 10 min. The pellets were unknown samples from a standard curve dissolved by addition of 2 ml distilled water. constructed with use of glucose standard (Carroll Carbohydrate solution was used directly in et al, 1955). glycogen level assay (Carroll et al, 1955). For assays of AST, ALT and GDH activity, liver Determination of enzyme activities were homogenized in 3 volume of ice cold 0.1 M Maximal enzyme activities were determined using imidazole pH 7.4 at 4 oC. Homogenate were a Biochrom Libra UV-Visible spectrophotometer centrifuged at 15,000 g 4 oC for 10 min. The (UK). Reaction rates of all enzymes were resultant supernatant (cytosolic fraction) was used determined by changing in absorbance of NADH directly in AST, ALT and GDH activity assay at 340 nm every 30 sec for 5 min. Conditions of (Singer et al, 1990). the enzyme assays were optimized with respect to substrate concentration in 1 ml and are as follows: Determination of hepatosomatic index, muscle HK: 72.6 mM imidazole pH 7.4, 5 mM MgCl2, 0.2 water content and metabolic fuels mM NAD, 1 mM ATP, 1 mM glucose, 1 unit Hepatosomatic index was measured using the G6PDH and 200 l muscle cytosolic. Brown method (Brown, 1957). Muscle water AST: 54.3 mM imidazole pH 7.4, 7 mM α- content was measured by the oven dry method ketoglutarate, 7 mM L-aspartic acid, 0.2 mM and muscle lipid was measured by ether extract NADH, 0.025 mM pyridoxal phosphate, 1 unit method (AOAC, 1980). malate dehydrogenase (MDH) and 100 l liver Serum glucose level was measured by cytosolic. hexokinase method. The reagent volume 1 ml that ALT: 59.1 mM imidazole pH 7.4, 10.5 mM α- contained 83.5 mM imidazole pH 7.4, 5 mM ketoglutarate, 200 mM L-alanine, 0.2 mM NADH, MgCl2, 0.2 mM NAD, 1 mM ATP, 1 unit HK, 1 unit 0.025 mM pyridoxal phosphate, 1 unit lactate glucose-6-phosphate dehydrogenase (G6PDH) dehydrogenate (LDH) and 100 l liver cytosolic. and 100 l serum. The reagent was assayed by GDH: 62.2 mM imidazole pH 7.4, 14 mM α- incubating for 5 min and then measured an ketoglutarate, 250 mM ammonium acetate, 0.1 absorbance at 340 nm using a Biochrom Libra mM EDTA, 0.2 mM NADH, 1 mM ADP and 100 l UV-Visible spectrophotometer (UK). Glucose liver cytosolic. levels in the samples can be calculated by against Enzyme activities in the samples can be a reagent blank and determined concentration of calculated by against a reagent blank and unknown samples from a standard curve determined concentration of unknown samples constructed with use of glucose standard (Singer from a standard curve constructed with use of et al., 1990). NADH standard (Singer et al., 1990). Muscle protein level was measured by Bradford method, the reagent volume 1 ml was contained Statistical analysis 990 l Bradford reagent and 10 l sample Results are expressed as mean±sd. Statistical solution. The reagent was assayed by incubating analyses were performed using the SPSS for 7 min, measured absorbance at 595 nm. software version 19.0. The duration of feeding and Protein levels in the samples can be calculated by starvation period were analyzed by the One-way against a reagent blank and determined ANOVA, mean were compared with Dancan’s concentration of unknown samples from a new multiple rang test. Statistical comparison between fed (control) and starved groups in each

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Chaiyachate et al. Effects of starvation in Broadhead catfish. period were made by the dependent paired hepatosomatic index (HSI), muscle glycogen, liver sample t-test. glycogen, muscle protein and muscle lipid were decreased according starvation duration in the RESULTS starved group (P<0.05) but no significant differences in fed group (P>0.05). The comparison Hepatosomatic index, muscle water content between fed and starved groups in each period, and metabolic fuels the results showed that HSI, serum glucose, The duration of feeding and starvation period, muscle glycogen, liver glycogen, muscle protein the results showed that muscle water content that and muscle lipid were significantly lower in the expressed in percent of wet weight, no significant starved group compared to the fed group (P<0.05) differences were observed in all groups (P>0.05). (figure 1). Serum glucose were decreased according experimental duration in all groups (P<0.05), while

Figure 1. Hepatosomatic index (HSI), muscle water content, serum glucose, muscle glycogen, liver glycogen, muscle protein, and muscle lipid in the Broadhead catfish (Clarias macrocephalus) under fed and starved. (mean±sd)

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Figure 2. Hexokinase (HK), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and glutamate dehydrogenase (GDH) activities in the Broadhead catfish (Clarias macrocephalus) under fed and starved. (mean±sd decrease in HSI of the starved group due to using Enzyme activities the stored fuels in the liver i.e. glycogen, cause to Enzyme activities were expressed in nmol liver mass loss that is the most obvious impact to -1 -1 min mg protein . The duration of feeding and phase 1-2 of starvation (Le Maho et al, 1981; starvation period, the results showed that enzyme Mendez and Wieser, 1993; Bines, 1999; McCue, activities were no significant differences in all 2010). Serum glucose level expressed during groups (P>0.05), except HK activity in the starved starvation due to food deprivation, previous group was decrease according starvation duration studies have also found glucose level was (P<0.05). When comparison between fed and decreased in starved fish i.e. Yellow perch (Perca starved groups in each period, the results showed flavescens), Common carp (Cyprinus carpio), that HK activity was significantly lower in the White sturgeon (Acipenser transmontanus), starved group compared to the fed group in week Rainbow trout (Oncorhynchus mykiss), Goldfish 2-4 (P<0.05), but AST was significantly higher in (Carassius auratus), Snakehead fish the starved group compared to the fed group in (Ophiocephalus maculatus), Red sea beam rd the 3 week and GDH was significantly higher in (Chrysophrys major) and Sturgeon (A. fulvescens) the starved group compared to the fed group in (Foster and Moon, 1991; Navarro and Gutierrez, nd rd the 2 -3 week (P<0.05), while ALT activity was 1995; Gillis and Ballantyne, 1996; Hung et al, no significant different (P>0.05) (figure 2). 1997; Russell and Gahr, 2000; Friedrich and Stepanowska, 2001; Congleton and Wagner, DISCUSSION 2006). Changes in glucose levels may depend on the species and duration of the starvation of Hepatosomatic index and metabolic fuels fishes.When the blood glucose level decrease, the Evidence from starvation studies suggest that body required to use stored metabolic fuels. The the starvation or poor food quality affecting an results showed that muscle glycogen, liver animal may reduce the body weight and fuels glycogen, muscle protein and muscle lipid were metabolism (McCue, 2010). The significant decreased according starvation duration, due to

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Chaiyachate et al. Effects of starvation in Broadhead catfish. glycogen was breakdown to glucose by from stored fuels such as glycogen and derived glycogenolysis and amino acid change to glucose from oxidation of triglycerides (Hoelzer et al, by gluconeogenesis (Chavin and Young, 1970; 1986;McCue, 2010). Sakamoto and Yone, 1978; Woo and Cheung, 1980; McCue, 2010). In addition, lipid was CONCLUSION breakdown to acetyl-CoA by β-oxidation and This study showed that the starvation for 4 weeks excess acetyl-CoA are synthesized as a ketone of the Broadhead catfish as results to insufficient body to be the main metabolic fuel (Masoro, 1968; of nutrients due to serum glucose was decreased. Owen et al, 1969; Robinson and Williamson, It is the cause of metabolic fuels such as muscle 1980; Lindstrom, 1991; Castellini and Rea, 1992; glycogen, liver glycogen, muscle protein and Cherel et al, 1992; Owen et al, 1998; Bairlein, muscle lipid were decreased, because they are 2002; McCue, 2007). Muscle protein is used to used during starvation. In addition, the decreasing provide energy, when glucose, glycogen, and lipid in HK activity, the maintenance of steady levels of are insufficient due to body mass loss (Stickney glucose in the body, called glucoregulation which and Lovell, 1977). is a homeostatic mechanism. The increasing in AST and GDH activities suggests greater amino acid catabolizing capacity during starvation, Enzyme activities leading to the generation of TCA cycle HK is an enzyme that breakdown glucose to intermediate convert to glucose in glucose-6-phosphate in glycolysis, which gluconeogenesis. produces pyruvate. Pyruvate was converted to acetyl-CoA that is energy substance to tri- CONFLICT OF INTEREST carboxylic acid cycle (TCA cycle) to produces The authors declared that present study was chemical energy in the form adenosine performed and published in absence of any triphosphate (ATP). The significant decrease in conflict of interest HK activity during starvation because the serum glucose decreased, that is the maintenance of ACKNOWLEGEMENT steady levels of glucose in the body. This is called The researchers wish to thank the Fisheries glucoregulation which is a homeostatic Department, Faculty of Agriculture, Khon Kaen mechanism (Hoelzer et al, 1986). A decrease in University (KKU) and KKU scholarship for the activity of this enzyme also has been observed in facilities and financial support provide which made African lungfish (Protopterus dolloi) (Frick et al, this research possible. Mr. Kampon Thaiso and 2008). Ms. Sirorat Sribancham are greatly appreciated AST and ALT are enzymes that catalyze the for their field and laboratory assistance. reversible transfer of amino group, such as AST catalyzes the inter conversion of aspartate and α- AUTHOR CONTRIBUTIONS ketoglutarate to oxaloacetate and glutamate, while RC designed and performed the experiments. BY, ALT catalyzes the interconversion of alanine and TC, CC and SC carried out the experiments, RC α-ketoglutarate to pyruvate and glutamate. GDH is and SC also wrote the manuscript. All authors an enzyme that converts glutamate to α- read and approved the final version ketoglutarate and vice versa, that products are Copyrights: © 2017 @ author (s). intermediate substances in TCA cycle. AST and This is an open access article distributed under the GDH activities significant higher in the starved terms of the Creative Commons Attribution License group compared to the fed group in the 3rd week (CC BY 4.0), which permits unrestricted use, and in the 2nd-3rd week respectively, maybe distribution, and reproduction in any medium, because using the sparing protein i.e. aspartate and glutamate were converted to oxaloacetate provided the original author(s) and source are and α-ketoglutarate into TCA cycle during food credited and that the original publication in this deprivation (McCue, 2010). An increase in journal is cited, in accordance with accepted activities of these enzymes also has been academic practice. No use, distribution or observed in African lungfish (Protopterus dolloi) reproduction is permitted which does not comply (Frick et al, 2008). While, ALT activity no with these terms. significantly different may suggest due to the amount of pyruvate and acetyl-CoA are sufficient REFERENCES for TCA cycle requirements, that were converted AOAC (Association of Official Analytical

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