Influence of Dietary Energy Level on Hepatic70-kDa Heat Shock Protein Expression in Broiler Chickens Submitted to Acute Heat Stress

Influência do Nível de Energia da Dieta sobre a Expressão Hepática de Hsp70-kDa em Frangos Submetidos ao Estresse Calórico Agudo

Abstracts

This experiment was carried out to study the effect of dietary energy on the colonic temperature and hepatic Hsp70 content in broiler chicken at room temperature and after heat stress conditions. Broiler chickens were reared up to 51 days of life, and fed diets containing high (HE -13,186 kJ ME/kg) or low (LE -12,139 kJ ME/kg) energy. At 21 and 51 days of age, the colonic temperature was measured at room temperature and liver samples were obtained for Hsp70 quantification by Western blotting analysis. It was also investigated at these ages the time course response of colonic temperature and hepatic Hsp70 level during heat stress (35º C/5 h). The data showed that at early age, at room temperature, colonic temperature or hepatic Hsp70 levels were not affected by dietary energy, but at 51 days of life low energy fed broilers had lower Hsp70 concentration in the liver. During heat stress, the increase in both colonic temperature and hepatic Hsp70 concentration were significantly less in high energy fed birds. The findings of this study suggest that hepatic Hsp70 synthesis is affected by dietary energy, and that broiler chicken fed high-energy diet can change the thermoresistance during acute heat stress.

broiler chicken; dietary energy level; heat shock protein; heat stress

Este trabalho foi desenvolvido com o objetivo de pesquisar o efeito da energia da dieta sobre a temperatura do cólon e concentração de proteína de choque térmico (Hsp70) de frangos à temperatura ambiente, bem como durante o estresse calórico agudo. Os frangos foram criados até 51 dias de idade e alimentados com dietas contendo nível de energia alto (13.186 kJ EM/kg) ou baixo (12.139 kJ EM/kg). No 21º e 51º dias de idade, a temperatura do cólon foi medida e amostras de fígado foram obtidas para quantificação da Hsp70 através da análise por Western Blotting.. Nessas mesmas idades, a resposta das aves ao estresse calórico agudo (37º C/5 h) foi avaliada (temperatura colón e Hsp70 no fígado). Os resultados mostraram que aos 21 dias de idade, à temperatura ambiente, a temperatura do cólon e a concentração de Hsp70 hepática não foram afetadas pela energia da dieta, mas, aos 51 dias de idade, os frangos alimentados com baixos teores de energia apresentaram menores concentrações de Hsp70 no fígado. As respostas ao estresse calórico agudo mostraram que as aves alimentadas com dietas de alta energia tiveram menor incremento na temperatura do cólon, bem como no conteúdo de Hsp70 hepático. Os resultados desse estudo sugerem que a síntese de Hsp70 no fígado pode ser afetada pela energia da dieta e que frangos alimentados com altos níveis de energia podem ter a termotolerância alterada em condições de estresse agudo pelo calor.

energia da dieta; estresse pelo calor; frangos; proteína de choque térmico

Influência do Nível de Energia da Dieta sobre a Expressão Hepática de Hsp70-kDa em Frangos Submetidos ao Estresse Calórico Agudo

Influence of Dietary Energy Level on Hepatic70-kDa Heat Shock Protein Expression in Broiler Chickens Submitted to Acute Heat Stress

Autor(es) / Author(s)

Gabriel JE¹

Ferro MIT¹

Ferro JA¹

Silva MM¹

Givisiez PEN²

Macari M²

1-Depto. de Tecnologia - UNESP

2-Depto.de Morfologia-Fisiologia Animal - UNESP

Corresponência / Mail Address

Jesus Aparecido Ferro

Depto. de Tecnologia, FCAV- UNESP

Via de Acesso Prof. Paulo Donato Castellane s/n

14870-000 - Jaboticabal - SP - Brasil

E-mail: [e-mail]

Unitermos / Keywords

energia da dieta, estresse pelo calor, frangos, proteína de choque térmico

broiler chicken, dietary energy level,

heat shock protein, heat stress

Observações / Notes

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support. J.E. Gabriel was a fellowship recipient from CNPq.

RESUMO

Este trabalho foi desenvolvido com o objetivo de pesquisar o efeito da energia da dieta sobre a temperatura do cólon e concentração de proteína de choque térmico (Hsp70) de frangos à temperatura ambiente, bem como durante o estresse calórico agudo. Os frangos foram criados até 51 dias de idade e alimentados com dietas contendo nível de energia alto (13.186 kJ EM/kg) ou baixo (12.139 kJ EM/kg). No 21^o e 51^o dias de idade, a temperatura do cólon foi medida e amostras de fígado foram obtidas para quantificação da Hsp70 através da análise por Western Blotting.. Nessas mesmas idades, a resposta das aves ao estresse calórico agudo (37^o C/5 h) foi avaliada (temperatura colón e Hsp70 no fígado). Os resultados mostraram que aos 21 dias de idade, à temperatura ambiente, a temperatura do cólon e a concentração de Hsp70 hepática não foram afetadas pela energia da dieta, mas, aos 51 dias de idade, os frangos alimentados com baixos teores de energia apresentaram menores concentrações de Hsp70 no fígado. As respostas ao estresse calórico agudo mostraram que as aves alimentadas com dietas de alta energia tiveram menor incremento na temperatura do cólon, bem como no conteúdo de Hsp70 hepático.

Os resultados desse estudo sugerem que a síntese de Hsp70 no fígado pode ser afetada pela energia da dieta e que frangos alimentados com altos níveis de energia podem ter a termotolerância alterada em condições de estresse agudo pelo calor.

ABSTRACT

This experiment was carried out to study the effect of dietary energy on the colonic temperature and hepatic Hsp70 content in broiler chicken at room temperature and after heat stress conditions. Broiler chickens were reared up to 51 days of life, and fed diets containing high (HE -13,186 kJ ME/kg) or low (LE –12,139 kJ ME/kg) energy. At 21 and 51 days of age, the colonic temperature was measured at room temperature and liver samples were obtained for Hsp70 quantification by Western blotting analysis. It was also investigated at these ages the time course response of colonic temperature and hepatic Hsp70 level during heat stress (35^o C/5 h). The data showed that at early age, at room temperature, colonic temperature or hepatic Hsp70 levels were not affected by dietary energy, but at 51 days of life low energy fed broilers had lower Hsp70 concentration in the liver. During heat stress, the increase in both colonic temperature and hepatic Hsp70 concentration were significantly less in high energy fed birds.

The findings of this study suggest that hepatic Hsp70 synthesis is affected by dietary energy, and that broiler chicken fed high-energy diet can change the thermoresistance during acute heat stress.

INTRODUCTION

All the organisms have developed a great number of different strategies to deal with adverse changes in their environment characterising what has been called as stress response. This response is a universal mechanism that results in the activation of specific genes, providing the synthesis of a highly conserved set of proteins known as the heat shock proteins (hsps) (Craig, 1985; Lindquist, 1986). Among the different heat shock proteins, the Hsp70 has been the subject of intensive scrutiny, since this protein is the most abundant translational product in stressed cells from eukaryotes.

Several investigations have elucidated the functions of heat shock proteins in stressed cells and organisms, and it has been suggested that Hsp70 serves as a molecular chaperone, preventing the aggregation of denaturated proteins and promoting their proper refolding (Beckmann et al., 1990; Georgopoulos & Welch, 1993; Hendrick & Hartl, 1993; Hightower et al., 1994). In addition, some studies have correlated an increased synthesis of this protein with the acquisition of the stress tolerance (Lee & Dewey, 1987; Lazlo, 1988; Parsell & Lindquist, 1994).

Considering the possible participation of Hsp70 in the cellular mechanisms of temperature regulation in stressed organisms, some studies have been carried out to investigate the expression pattern of this protein in broilers chickens, since the exposure to high ambient temperature directly affects the growth and development of these animals. Wang & Edens (1994) showed an enhancement in the levels of this protein and its mRNA in different tissues of broiler chickens exposed to high environmental temperatures. Gabriel et al. (1996) also demonstrated a time-dependent increase in the levels of Hsp70 and its mRNA in the liver of broilers chickens submitted to acute heat stress.

Dietary energy interferes on basal metabolic rate and on plasma levels of different metabolic hormones (Macari et al., 1983; LeBlanc et al., 1986). Other researches have demonstrated a relationship between environmental temperature and dietary energy levels that affect broiler chicken performance (Reece & McNaughton, 1982; Lott et al., 1992). Thus, the purpose of this study was to investigate, in vivo, the effect of dietary energy on the increment in the colonic temperature and hepatic Hsp70 expression of broiler chickens submitted to acute heat stress.

MATERIAL AND METHODS

Feeding and heat stress

Male broiler chickens from Hubbard-Peterson strain, reared up to 51 days of age and fed high (13,186 kJ ME/kg, n = 120 birds) or low (12,139 kJ/ME/kg, n = 120 birds) energy diets containing 220 and 200 g crude protein/kg in the initial (1-28 days) and final (29-49 days) growing phases, respectively, were used in this experiment. The diets were based on corn and soybean meal to which vitamin and mineral mix were added. The birds were fed ad libitum and the ambient temperature varied between 31 to 18^º C according to bird’s age. Broiler chicken body weight, feed intake and feed conversion were determined for the initial and final phases. The birds at 21 and 51 days of age were submitted to acute heat stress by exposure to a climatic chamber at ambient temperature of 37^º C for 5 hours using 30 birds for each age. Before (n = 5) and after the beginning of heat stress, the colonic temperature was measured every each hour (n = 5) by inserting a thermistor probe connected to a telethermometer into the bird’s colon. During acute heat stress, food and water were not available. After measuring the colonic temperature the broilers were sacrificed by cervical dislocation and liver sample obtained, frozen in liquid nitrogen and maintained at –80^º C for further Hsp70 analysis.

Total protein extraction and electrophoresis

Liver samples (1 g) were homogenised in 10 mL lysis buffer (20 mM Tris-HCl, pH 7.5; 9 g/L NaCl; 2 mM b-mercaptoethanol) using an Ultra-turrax homogenizer, at 20,000 rpm for 3 times (30 seconds each), with ice-bath intervals of 30 seconds. Cell lysates were centrifuged at 31,000Xg for 30 minutes at 4^º C and, the supernantant was manually homogenised for 10 times using Potter-Elvehjem homogenizer. Two 300 mL aliquots were separated for total protein determination and electrophoresis analysis. Electrophoresis samples were obtained by the addition of 300 mL of two times concentrated sample buffer (125 mM Tris-HCl, pH 6.8; 20% glycerol; 4% SDS; 0.002% bromophenol blue) and 40 mL b-mercaptoethanol to one of the 300 mL aliquot. These samples were boiled for 2 minutes and stored at –20^º C until electrophoresis analysis. Total protein concentration of supernatant aliquots was determined in quintuplicate, according to the method described by Hartree (1972). The standard curve was produced with bovine serum albumin (BSA, Sigma) in triplicate samples containing 0, 20, 40, 60, 80 and 100 mg of this protein. Thirty micrograms total protein were loaded and separated on 9% polyacrylamide gels under denaturing conditions (Laemmli, 1970), using the Mini-Protean II apparatus (Bio-Rad) at a constant voltage (200 V). Before loading, the samples stored at –20^º C were reboiled for 2 minutes and a sample (pool of five birds) of supernatant from control (not stressed) birds was loaded on all gels, as a reference standard. A pre-stained molecular weight standard (Gibco-BRL) was used in all gels.

Western blotting analysis and Hsp70 quantification

After fractionation through SDS-polyacrylamide gels, proteins were electrophoretically transferred to polyvinylene di-fluoride (PVDF) membranes using the procedure describe by Towbin et al. (1979). Transference was performed for 12 hour at 4^º C at constant voltage (90 V), using a mini-trans-blot cell (Bio-Rad). The membranes were stained with 0.5 g/L Ponceau S in 10 g/L acetic acid, for 3 min, to evaluate the transference efficiency. After several washings with deionised water, non-specific interaction sites were blocked using 10 mL of cold TBS buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl) containing 50 g/L non-fat dried milk and 0.2 g/L Tween-20, for 1 hour at room temperature, in a shaker (100 rpm, approximately). The membranes were then incubated with 10 mL of monoclonal anti-Hsp70 antibody (H-5157, Sigma) in 10 mL of cold TBS-milk solution (1:1000 dilution) containing 0.2 g/L Tween-20, for 1 hour at room temperature in a shaker. Four washings of 5 minutes each using 10 mL TBST (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5 g/L Tween-20) and one washing of 10 minutes using 10 mL of cold TBS buffer were performed. The membranes were incubated with 2 mL of secondary anti-mouse antibody conjugated to alkaline phosphatase (A-5153, Sigma) diluted in 10 mL of cold TBS-milk solution (1:5000 dilution), for 1 hour at room temperature with constant shaking. After rinsing with cold TBST and TBS as described above, the colour reaction was developed for 2 minutes by addition of 33 m L of nitro-blue tetrazolium chloride solution (50 g/L dissolved in dimethylformamide) and 66 mL of 5-bromo-4-chloro-3-indolylphosphate p-toluidine (50 g/L dissolved in dimethylformamide 700 g/L) in 10 mL AP buffer (100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl₂). The colour development reaction was blocked by addition of a solution of trichloroacetic acid (30 g/L). The membranes were washed with deionised water and dried at room temperature, protected from light. The colour signal of the bands corresponding to Hsp70 was analysed by a densitometer at 525 nm (Shimadzu CS-9301) using reflection model and zigzag scanning. Hsp70 levels were quantified following the procedure described by Givisiez et al. (1999) and the concentrations of hepatic Hsp70 were expressed as ng Hsp70. m g total protein^-1.

Statistical analysis

The data of performance (body weight, feed intake and feed conversion) were submitted to analysis of variance and difference between means verified by Tukey's test (p < 0.05). Colonic temperature and hepatic Hsp70 concentrations are shown as means ± SE according bird’s age and time of heat exposure. The difference between means, for each time, during heat-exposure was verified by Student’s t-test at 5% probability (p £ 0.05)

RESULTS

Broiler chicken performance is shown in Table 1. Broiler chicken fed ration containing high energy level had better performance (p < 0.05) than birds fed low energy ration for all phases.

The data obtained in this experiment also show the effect of dietary energy level on colonic temperature and on hepatic Hsp70 content of broiler chickens before and after acute heat stress (Table 2 and, Figures 1 and 2 A-B). Dietary energy did not affect (p > 0.05) colonic temperature at thermoneutral temperature, but hepatic Hsp70 content was different (p < 0.05) according bird’s age; at early age (21 days) liver Hsp70 content was not affected (p > 0.05) by dietary energy, but at old age (51 days) the liver content of this heat shock protein was higher (p < 0.05) for high energy fed animals (Table 2).

Figure 1 (A) shows that dietary energy level did affect (p < 0.05) colonic temperature rise during acute heat stress at 21 days of ages, with low energy fed birds showing high increment in the colonic temperature when compared with high energy fed ones. At this age, the increase in hepatic Hsp70 content was different (p < 0.05) between high and low energy fed birds (Fig. 1 B). The time course response showed that low energy fed birds had an abrupt increase in the liver Hsp70 level until three hour beginning heat stress (+ 1.735 ng. m g total protein^-1 from basal value), and the same response was observed for high energy fed animal, but with lower magnitude. At 51 days of age, an abrupt increase (± 1,0^º C) was seen in colonic temperature for both low and high energy fed birds during the first hour of heat stress (Fig. 2A). After that time, the rise in colonic temperature was still present in low energy fed broilers (maximum increase of 1.6º C at 4 h), but for high energy fed ones the colonic temperature did not change until 4 hours of heat stress (maximum of +0.8^º C), decreasing afterwards. The increase in colonic temperature of low energy fed birds was accompanied by an enhancement in hepatic Hsp70 level (Fig. 2B: +3.218 and +3.521 ng. m g total protein^-1 from basal value, at the third and fourth hour beginning of heat stress, respectively) but, for high energy fed broilers the liver content of Hsp70 did not change significantly (p > 0.05) during the heat stress episode (maximum increase of +1.146 ng. m g total protein^-1). A significant difference (p < 0.05) was seen between the third and fourth hour of heat stress for hepatic Hsp70 level between high and low energy fed birds.

DISCUSSION

The findings of this study revealed that dietary energy affects broiler chicken performance by increasing body weight and improving feed conversion, which is related to the availability of energy for growth. The data also show that dietary energy did not affect basal colonic temperature when the birds are kept at thermoneutral ambient temperature, giving evidence that the animals are able to maintain the balance between heat loss and heat production fairly constant. On the other hand, the hepatic Hsp70 content was influenced by dietary energy level at 51 days of age, but not at early age (21 days). Since the liver is the most important metabolic organ in the body, generating a great amount of heat, the increase in the synthesis of Hsp70 might be related to this activity especially during the growing phase of the broiler chickens (from 29 to 51 days of life). However, further investigation is necessary to establish cell metabolic activity and Hsp70 synthesis.

The findings also show that the increment in colonic temperature interferes with Hsp70 expression when broiler chickens are exposed to acute heat stress. The increase in colonic temperature was followed by an increase in hepatic Hsp70 content suggesting that the synthesis of this protein is dependent on the increment in internal temperature regardless broiler chicken age, as previously reported by Givisiez et al (1999). At early age (21 days), the effect of dietary energy on both colonic temperature and hepatic Hsp70 rise was less pronounced than at later age (51 days of life). These findings suggest that the magnitude of heat stress at early age is less severe than at old ages, since young birds have less external insulation, higher area/volume ratio and can lose more heat than old birds.

The effect of dietary energy on both colonic temperature and hepatic Hsp70 level during the time course of heat stress was also seen in this investigation. The influence of dietary energy on gene expression is poorly investigated. Heydari et al. (1993) reported that there was an increase in the Hsp70 mRNA levels in adult rats submitted to dietary restriction; however, nothing was shown regarding the translational mechanism of this protein. Our investigation also showed that dietary energy was able to alter the hepatic Hsp70 synthesis, especially at late age (51 days of life), since the highest hepatic Hsp70 level was found in high energy fed broilers. Thus, high-energy diet seems to increase the transciptional and translational cellular mechanisms in broiler chickens with accumulation of the heat shock protein. However, further investigations are necessary to identify the triggering stimuli involved in this process.

The lack of increase in both colonic temperature and hepatic Hsp70 in high energy fed broiler during heat stress could be related to the low magnitude of stress, or to the increased thermoresistance developed by these birds when fed high energy diet.

In conclusion, the findings of this study show that dietary energy is able to change the synthesis of Hsp70 in the liver of broiler chickens which is age dependent. The lack of increase in colonic temperature and liver Hsp70 in birds fed high energy diet during heat stress seen in this study might be related to the change in thermoresistance in these birds.

References

• Beckman RP, Mizzen LA, Welch WJ. Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 1990; 248: 850-854.
• Craig EA. The heat shock response. Critical Review in Biochemistry 1985; 18: 239-280.
• Gabriel JE, Ferro JA, Stefani RMP, Ferro MIT, Gomes SL, Macari M. Effects of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers. British Poultry Science 1996; 37: 443-449.
• Givisiez PEN, Ferro JA, Ferro MIT, Kronka SN, Decuypere E, Macari M. Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments. British Poultry Science 1999; 40: 292-296.
• Georgopoulos C, Welch WJ. Role of major heat shock proteins as molecular chaperones. Annual Review of Cell Biology 1993; 9: 601-635.
• Hartree EF. Determinations of protein: a modification of the Lowry method that gives a linear photometric response. Analytical Biochemistry 1972; 48: 422-427.
• Hendrick JP, Hartl FU. Molecular chaperone functions of heat-shock proteins. Annual Review of Biochemistry 1993; 62: 349-384.
• Heydari AR, Wu B, Takahashi R, Strong R, Richardson A. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Molecular and Cellular Biology 1993; 13: 2909-2918.
• Hightower LE, Sadis SE, Takenaka IM. Interactions of vertebrate hsc70 and hsp70 with unfolded proteins and peptides. In: The Biology of Heat Shock Proteins and Molecular Chaperones, ed. R. Morimoto, A. Tissières and C. Georgopoulos. Cold Spring Harbor Laboratory Press; 1994; p.179-207.
• Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 226: 112-115.
• Lazlo A. The relationship of heat shock proteins, thermotolerance and protein synthesis. Experimental Cell Research 1988; 178: 401-414.
• LeBlanc J, Lupien D, Diamond P, Macari M. Thermogenesis in response to various intake of palatable food. Canadian Journal of Physiology and Pharmacology 1986; 64: 976-982.
• Lee YJ, Dewey W. Effect of cycloheximide or puromycin on induction of thermotolerance by heat in chinese hamster ovary cells: dose fractionation at 45.5^o C. Cancer Research 1987; 47: 5960-5966.
• Lindquist SL. The heat-shock responses. Annual Review Biochemistry 1986; 55: 1151-1191.
• Lott BD, Day EJ, Deaton JW, May JD. The effect of temperature, dietary energy level, and corn particle size on broiler performance. Poultry Science 1992; 71: 618-624.
• Macari M, Ingram DL, Dauncey MJ. Influence of thermal and nutritional acclimatization on body temperature and metabolic rate. Comparative Biochemistry Physiology 1983; 74A: 549-553.
• Parsell DA, Lindquist S. Heat shock proteins and stress tolerance. In: The Biology of Heat Shock Proteins and Molecular Chaperones, ed. R. Morimoto, A. Tissières and C. Georgopoulos. Cold Spring Harbor Laboratory Press; 1994; p.457-494.
• Reece FN, McNaughton JL. Effects of dietary nutrient density on broiler performance at low and moderate environmental temperatures. Poultry Science 1982; 61: 2208-2211.
• Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 1979; 76: 4350-4354.
• Wang S, Edens FW. Hsp70 mRNA expression in heat-stressed chickens. Comparative Biochemistry Physiology 1994; 107(B): 33-37.

Publication Dates

• Publication in this collection
  31 Oct 2002
• Date of issue
  Sept 2000