1 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL AJUSTE METABÓLICO E RESPOSTAS IMUNES DE PACUS JUVENIS ALIMENTADOS COM DIFERENTES NÍVEIS DE CARBOIDRATOS E SUBMETIDOS A JEJUM PROLONGADO Rodrigo Yukihiro Gimbo Zootecnista 2015 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL AJUSTE METABÓLICO E RESPOSTAS IMUNES DE PACUS JUVENIS ALIMENTADOS COM DIFERENTES NÍVEIS DE CARBOIDRATOS E SUBMETIDOS A JEJUM PROLONGADO Rodrigo Yukihiro Gimbo Orientadora: Porfa. Dra. Elisabeth Criscuolo Urbinati Tese apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Doutor em Zootecnia. 2015 3 DADOS CURRICULARES RODRIGO YUKIHIRO GIMBO – nascido na cidade de Taubaté-SP, no dia 29/07/1985. Em março de 2004 ingressou na UNESP, campus de Dracena, concluindo o curso de Zootecnia em janeiro de 2009. Durante a graduação, participou de diversos congressos, realizou estágios na área de produção animal e iniciação científica sob orientação do Prof. Dr. Leonardo Susumu Takahashi. Em março de 2009, iniciou o curso de mestrado em Zootecnia pela UNESP, Faculdade de Ciências Agrárias e Veterinária em Jaboticabal, sob orientação da Profa. Dra. Elisabeth Criscuolo Urbinati. Após conclusão do mestrado em 2011, ingressou no curso de doutorado em Zootecnia, na mesma instituição, e sob mesma orientação. No dia 13 de fevereiro de 2015, submeteu o presente estudo para avaliação pela banca examinadora com parte desta tese já publicada ou em processo de publicação. i Aos meus pais, Francisco e Nelza E a todos amigos de pós graduação, Dedico ii AGRADECIMENTOS Agradeço aos meus pais por sempre incentivar a busca por conhecimento. À minha orientadora Profa. Dra. Elisabeth Criscuolo Urbinati, por todo o ensinamento e confiança que depositou em mim enquanto estive presente em seu laboratório. Aos Prof. Dr. Dalton José Carneiro por emprestar as estruturas do Laboratório de Nutrição de Organismos Aquáticos para a realização de parte deste projeto e sugestões como membro da banca do Exame Geral de Qualificação. Aos Profs. Drs. João Martins Pizzauro, Monica Serra e Luis Roberto Furlan por suas sugestões durante o Exame Geral de Qualificação. Foi um momento de grande aprendizado. Aos membros da banca de defesa, Profs. Drs. Ana Paula Baldan, Maria Célia Portella, Marco Antônio Belo pelos conselhos e análise crítica da tese. Ao Prof. Dr. Leonardo Susumu Takahashi, pelos ensinamentos desde a graduação. À técnica de laboratório Damares, pelo apoio nas pesquisas e pela amizade. Aos amigos de laboratório, Luis Benavides, Gisele Fávero, Rafael Sabioni, Talisia Martins, Leonardo Sabbag, Fábio Zanuzzo, Isabela Olmos, Mayara Nicolau, Isabela Leirão, Natalia Franco, Mariana Maluli e Soliris Castilho por fazerem do trabalho no laboratório serem divertidos. Obrigado pelo aprendizado e pela confiança que depositaram em mim. Já sinto saudades de todos vocês!!! Aos funcionários do Centro de Aquicultura da UNESP, Valdecir, Márcio e Seu Mauro pela disponibilidade e empenho em todas os momentos que precisei da ajuda de vocês. Aos amigos de pós graduação, Gustavo Squassoni, Thiago Freitas, Amanda Halum, Thyssia Bonfim, Ivã Guidini, Caroline Nebo, Natália Leitão, Lidiane Sandre, Hellen Buzzolo, Ligia Neira, Thiago Torres, Rudney Weiber e Juliano Coutinho iii Aos amigos do Judô, Wesllen, Thiago, Renan, Fabrício, Nathan e Anderson. O Judô vai muito além de uma luta! E esta filosofia ajudou bastante durante meu doutorado. À minha amiga, companheira e namorada, Juliana Tomomi Kojima, principalmente pelo apoio, e compreensão nos momentos de cortisol elevado. Às oficinas da Publicase financiadas pelo Programa de Pós Graduação em Zootecnia. Ao CNPq, pelo apoio financeiro. iv SUMÁRIO CAPÍTULO 1 – Considerações gerais ......................................................................... 5 1. Introdução e justificativa ....................................................................................... 5 2. Revisão de literatura ............................................................................................. 6 2.1. Homeostase da glicose em peixes ................................................................. 6 2.2. Jejum e realimentação ................................................................................... 8 2.3. Sistema imune em peixes ............................................................................ 10 2.4. Modulação do sistema imune pelo estresse .................................................... 11 2.5. Estresse nutricional ......................................................................................... 12 2.6. Pacu (Piaractus mesopotamicus) .................................................................... 13 3. Objetivos gerais .................................................................................................. 14 3.1. Objetivos específicos ................................................................................... 14 4. Referências bibliográficas ................................................................................... 14 CAPÍTULO 2 – Juvenile pacu fish efficiently utilize high levels of dietary carbohydrates for growth under different feeding strategies ..................................... 22 Abstract ..................................................................................................................... 22 Introduction................................................................................................................ 22 Material and methods ................................................................................................ 24 Results ...................................................................................................................... 27 Discussion ................................................................................................................. 30 CAPÍTULO 3. Serum ammonia as indicator of unbalanced diet in pacu (Piaractus mesopotamicus) ........................................................................................................ 42 Short communication ................................................................................................. 42 Introduction................................................................................................................ 42 Material and methods ................................................................................................ 42 Results ...................................................................................................................... 45 Discussion ................................................................................................................. 45 References ................................................................................................................ 46 CAPÍTULO 4. Energy deficit does not affect immune responses of experimentally infected pacu (Piaractus mesopotamicus)* ............................................................... 48 Abstract ..................................................................................................................... 48 Introduction................................................................................................................ 49 v Material and methods ................................................................................................ 50 Results ...................................................................................................................... 53 Discussion ................................................................................................................. 56 References ................................................................................................................ 60 CAPÍTULO 5 – Considerações finais ........................................................................ 63 vi Índice de figuras Capítulo 2 Figure 1. Effects of carbohydrates (CHO) and feeding strategy on weight gain (WG), specific growth rate (SGR) and protein efficiency rate (PER). 39 Figure 2. Effects of carbohydrates (CHO) and feeding strategy on blood glucose, triglycerides, cholesterol and non-esterified fatty acids (NEFA) levels. 40 Figure 3. Effects of carbohydrates (CHO) and feeding strategy on liver glycogen, liver lipids, hepatossomatic index (HSI) and mesenteric fat index (MFI). 41 Figure 4. Effects of carbohydrates (CHO) and feeding strategy on the activities of hexokinase (HK), glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PDH) and aspartate amino transferase (AST). 42 Capítulo 3 Figure 1. AST (aspartate aminotransferase) activity and serum ammonia of pacu fed with 25 and 45% CHO during 30 days. Different letters indicate statistical difference 46 Capítulo 4 Figure 1. Innate immune indicators of pacu starved for 30 days or fed continuously 56 Figure 2. Metabolic parameters of pacu starved for 30 days or fed continuously 57 Figure 3. Serum cortisol levels of pacu starved for 30 days or fed continuously 58 vii Tabela de abreviaturas* CHO Carboidrato CRH Hormônio liberador de corticotropina HPI Hipotálamo-pituitária-interrenal WG Weight gain SGR Specific growth rate PER Protein eficiency rate HK Hexoquinase GK Glicoquinase G6PDH Glicose 6-fosfato desidrogenase AST Aspartato aminotransferase *As abreviações podem ser utilizadas em sua respectiva tradução em ingês 1 AJUSTE METABÓLICO E RESPOSTAS IMUNES DE PACUS JUVENIS ALIMENTADOS COM DIFERENTES NÍVEIS DE CARBOIDRATOS E SUBMETIDOS A JEJUM PROLONGADO Resumo Este trabalho está dividido em três artigos no formato para a submissão e publicação. No primeiro, testamos níveis de carboidratos (CHO) associados ao jejum e realimentação como estratégia de induzir as vias catabólicas e anabólicas dos diferentes nutrientes e auxiliar no entendimento da intolerância dos peixes aos CHOs; no segundo, avaliamos o uso da amônia sérica como indicador de dietas desbalanceadas; e, ainda, no terceiro artigo avaliamos como o jejum afeta a imunidade e a estratégia metabólica adotada pelos peixes para sustentar a resposta imune em condições de déficit energético. Os resultados indicam que o pacu pode tolerar altos níveis de CHO, uma vez que os valores de glicemia e glicogênio hepático foram semelhantes entre os peixes que ingeriram 25 e 45% CHO, além de suportar longos períodos de jejum (30 dias), sem comprometer a capacidade de resposta de ganho em peso. Entretanto, 25% CHO resultou em menor acúmulo de lipídeo visceral, associado com maior lipólise (mais ácidos graxos livres circulantes) e menores níveis de triglicerídeo e colesterol circulantes após 30 dias de alimentação. Após 30 dias de jejum, os peixes consumiram as reservas energéticas avaliadas, mas após um dia de re-alimentação, apenas as reservas de glicogênio e os níveis circulantes de triglicerídeo e colesterol normalizaram, enquanto as reservas lipídicas teciduais foram restauradas ao final dos 30 dias de realimentação. As atividades da hexoquinase (HK), glicoquinase (GK), glicose 6-fosfato desidrogenase (G6PDH) e aspartato aminotransferase (AST) acompanharam o perfil das variáveis metabólicas, reforçando que o pacu é capaz de usar altos níveis de CHO e ajustar seu metabolismo para suportar longos períodos de jejum. Como foram utilizadas duas dietas, uma balanceada em proteína e energia (45% CHO), e outra desbalanceada (25% CHO), testamos a determinação da concentração da amônia plasmática para validar o uso deste parâmetro como indicador de dietas desbalanceadas na alimentação de pacu. A atividade da AST foi usada para comprovar a ocorrência do catabolismo de proteínas após 30 dias ingerindo ambas as dietas. Ao final deste período, observamos o aumento da amônia plasmática e da atividade da AST nos peixes que ingeriram 25% CHO, indicando que a determinação da amônia plasmática é um eficaz indicador da utilização de dietas desbalanceadas. Por fim, para avaliar o custo metabólico da resposta imune em condição de déficit energético, dois grupos de peixes foram alimentado durante 30 dias ou submetido ao jejum pelo mesmo período. Após 30 dias, os peixes foram amostrados e inoculados com A. hydrophila e amostrados novamente após 3 e 24 horas. A atividade respiratória dos leucócitos foi menor nos peixes 2 submetidos ao jejum, entretanto, após a inoculação da bactéria ambos grupos foram capazes de elevar a atividade respiratória dos leucócitos, atingindo o mesmo nível. A atividade do sistema complemento, reduzida pelo jejum, aumentou em resposta à inoculação da bactéria. A concentração de lisozima foi mais elevada nos peixes em jejum antes e após 3 horas da inoculação da bactéria, e o grupo alimentado alcançou os mesmos níveis do grupo em jejum apenas após 24 horas da inoculação. Os peixes alimentados sustentaram a resposta imune num primeiro momento graças às reservas de glicogênio e enquanto os peixes em jejum dependeram principalmente das reservas lipídicas e num segundo momento, ambos os grupos dependeram de lipídeos para fornecer energia para os processos imunes. Assim, mostramos que construir uma resposta imune é um processo caro, entretanto, o pacu, mesmo em condição de déficit energético é capaz de mobilizar suas reservas de energéticas para sobreviver após uma infecção bacteriana. Palavras chave: Imunidade de peixe, metabolismo de carboidrato, metabolitos sanguíneos, peixe tropical 3 METABOLIC ADJUST AND IMMUNE RESPONSE OF PACU JUVENILES FED WITH DIFFERENT CARBOHYDRATES LEVELS AND SUBMITTED TO LONG- TERM FASTING Abstract This study is divided in three papers formatted to be submitted and publishing. In the first one, we tested carbohydrate (CHO) levels associated to fasting and refeeding as strategy to induce catabolic and anabolic pathways of different nutrients and assist in understanding of CHO intolerance in fish; in the second, we evaluated the serum ammonia as indicator of unbalanced diets utilization; and also, in a third paper, we evaluated how fasting affect the fish immunity and the metabolic strategies adopted by fish to sustain the immune response under energy deficit conditions. The results indicate pacu use efficiently high CHO levels, once values of blood glucose and liver glycogen were similar between fish fed with 25 and 45% CHO, besides this, pacu tolerates long-term fasting (30 days), without compromise the ability of weight gain response. However, 25% CHO resulted in lower mesenteric fat accumulation and higher lipolysis (more current non-esterified fatty acids) and lower levels of triglycerides and cholesterol after 30 days feeding. After 30 days fasting, fish consumed the evaluated energy reserves, but after one day re-feeding, only reserves of liver glycogen, triglycerides and cholesterol normalized, while the tissue lipid reserves were reestablished at the end of 30 days re-feeding. The hexokinase (HK), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH) and aspartate aminotransferase (AST) follow the metabolic variables profile, reinforcing that pacu is able to use high CHO levels and adjust their metabolism to tolerate long-term fasting. As we used two diets with different protein/energy ratio, we tested the serum ammonia determination to validate the use of this parameter as indicator of unbalanced diets utilization in pacu. The AST activity was used to prove the occurrence of protein catabolism after feeding fish during 30 days with both diets. At the end of period, we observed increasing in serum ammonia and AST activity in fish fed with 25% CHO diet, indicating the serum ammonia determination is a effective indicator of unbalanced diet utilization. Lastly, to evaluate the metabolic cost of immune response under energy déficit, two fish groups were fed during 30 days or submitted to fasting by same period. After 30 days, fish were sampled and inoculated with A. hydrophila and than sampled again after 3 and 24 hour. The leukocytes respiratory activity was lower in fish submitted to fasting, however, after bacteria inoculation, both groups increased leukocytes respiratory 4 activity to the same level. The complement system activity was reduced in fish submitted to fasting, but increased in response to bacteria inoculation. Lysozyme concentration was elevated in fasted fish before and 3 hours after inoculation, and fed group reached the same activity of fasted fish only at 24 hours after inoculation. Fed fish sustained the immune response in a first moment due to glycogen reserves while fasted fish depended on lipid reserves and in a second moment, both groups used lipid to provide energy to immune process. Thus, we showed that build an immune response is an expensive process, however, the pacu, even under energy deficit condition is able to mobilize energy reserves to survive after bacterial infection Keywords: Fish immunity, blood metabolites, carbohydrate metabolism, tropical fish 5 CAPÍTULO 1 – Considerações gerais 1. Introdução e justificativa A glicose possui função fundamental como fonte de energia para a maioria dos mamíferos, entretanto, sua importância em peixes aparenta ser limitada (Wilson, 1994; Hemre et al., 2002; Stone, 2003). Em algumas espécies de peixes, principalmente em espécies carnívoras é possível observar uma hiperglicemia pós- prandial prolongada após alimentação com dietas ricas em carboidratos (Cowey e Walton, 1989; Wilson, 1994; Moon, 2001). Já em outras espécies, estudos mostraram a capacidade dos carboidratos dietéticos em reduzir o catabolismo de proteínas (Cho e Kaushik, 1990; Wilson, 1994, Baldan, 2008). Ao contrário dos animais terrestres, os peixes dependem da proteína dietética como fonte primária de energia. A proteína da dieta por sua vez é o item mais oneroso e pode contribuir significativamente para o custo da dieta. Assim, é desejável que a proteína seja utilizada para crescimento e não para suprimento da necessidade energética dos peixes (Kumar et al., 2010). Existem algumas hipóteses para explicar a baixa utilização de glicose dietética pelos peixes. Dentre elas, podemos citar a capacidade dos aminoácidos dietéticos de estimularem mais a secreção de insulina que a glicose (Mommsen e Plisetskaya, 1991), o menor número relativo de receptores de insulina no músculo de peixe em comparação com ratos (Párrizas et al., 1994), a baixa capacidade de fosforilação da glicose (Cowey e Walton, 1989), o baixo número de transportadores de glicose em músculo de peixe (Wright et al., 1998) e uma inadequada regulação da homeostase da glicose em resposta a um desbalanço entre a glicólise e a gliconeogênese (Panserat et al., 2000). Neste estudo, associamos o uso de carboidratos com o jejum e realimentação como estratégia para ativar as vias catabólicas e anabólicas dos diferentes nutrientes e auxiliar no entendimento da questão da tolerância/intolerância aos carboidratos. Entretanto, devido à pouca informação na literatura (Martin et al., 2010) sobre o efeito 6 do jejum na imunidade, procurou-se estudar também a estratégia metabólica adotada pelos peixes para sustentar a resposta imune em condição de déficit energético. 2. Revisão de literatura 2.1. Homeostase da glicose em peixes Carboidratos (CHOs) são bastante usados em dietas de animais domésticos como fonte de energia. Apesar de não existir exigência de carboidratos em dietas para peixes, sua inclusão em níveis adequados pode assegurar melhor eficiência na utilização de outros nutrientes (Wilson, 1994). A utilização de CHOs pode reduzir o catabolismo de proteínas para síntese de glicose (Suarez e Mommsen, 1987), além de melhorar a eficiência de retenção proteica e diminuir as perdas metabólicas de nitrogênio no ambiente (Cowey e Walton, 1989; Wilson, 1994). A melhora no crescimento e o efeito poupador de proteína podem estar relacionados ao fato da glicose ser um importante combustível metabólico para os tecidos glicose-dependentes, tais como células vermelhas e tecido nervoso, entre outros. Desta forma, CHOs presentes na dieta de peixes podem reduzir a atividade gliconeogênica, afastando aminoácidos da via oxidativa (Cowey et al., 1977). Para que a glicose possa ser utilizada como fonte de energia, é necessário que esta molécula seja transportada para o citoplasma da célula. Com exceção das células presentes no epitélio gastrintestinal e nos túbulos renais (transporte ativo secundário, co-transporte de sódio e glicose), a glicose entra nas células por difusão facilitada mediada por proteínas transportadoras de glicose (GLUTs). Em mamíferos, já foram identificadas 14 isoformas de GLUT, cada uma expressa por genes diferentes e presentes em diferentes tecidos (Mueckler e Thorens, 2013). O metabolismo da glicose em peixes é bastante estudado devido a sua importância como fonte de energia (Wilson, 1994) e à baixa capacidade dos tecidos periféricos em utilizarem carboidratos dietéticos, quando comparados com aves e mamíferos (Wilson, 1994; Henre et al., 2002; Enes et al., 2009). Apesar da baixa taxa de absorção de carboidratos, há evidências de que a glicose entra na célula por meio de GLUTs. Wright et al. (1998) observaram proteínas reagindo com anticorpo contra GLUT-1 (de mamífero) no coração e no cérebro de tilápia. Já Krasnov et al. (1999) 7 observaram aumento na absorção metabolismo de carboidratos em embriões transgênicos de truta arco-íris expressando genes de GLUT-1 humano. Em estudo com pacu (Piaractus mesopotamicus), Baldan (2008) identificou uma proteína com alta homologia com a GLUT-4 do músculo esquelético de rato, porém com intensidade de banda reduzida. Em condições aeróbicas, a glicose é catabolizada na via glicolítica, ciclo do ácido cítrico e fosforilação oxidativa para a produção de ATP, ou pode seguir a via das pentoses fosfato para a produção de NADPH e ribose 5-fosfato, necessários para a biossíntese de lipídeos e nucleotídeos, respectivamente. O excesso de glicose pode ser armazenado na forma de glicogênio (glicogênese) ou convertido a lipídeo (Enes et al., 2009). Em condições de jejum, a necessidade de glicose utilizada no metabolismo pode ser obtida pela degradação do glicogênio (glicogenólise) ou pela síntese de novo de glicose através da gliconeogênese (Pilkis e Granner, 1992). No fígado de vertebrados, a glicoquinase ou hexoquinase IV, uma das três enzimas que regulam a glicólise, atua na taxa de utilização de glicose para controle de sua homeostase, pela fosforilação da glicose a glicose-6-fosfato. Esta, por sua vez, atua como inibidor da hexoquinase quando em concentrações elevadas (Berg et al., 2002). Outro ponto de controle da via é a fosforilação da frutose 6-fosfato, catalisada pela fosfo-frutoquinase. Esta enzima pode ser inibida por altas concentrações de ATP, ou ativada pela frutose 1,6-bifosfato. O último ponto de controle da via é a conversão de fosfoenolpiruvato a piruvato, catalizada pela enzima piruvato-quinase, reação que ocorre a favor da formação de ATP, sendo a frutose 1,6-bifosfato um importante ativador da via (“feed-foward regulation”) (Berg et al., 2002). Estudos com peixes teleósteos, na maioria carnívoros, mostram que diferentes espécies, mesmo com o mesmo hábito alimentar, possuem capacidades distintas de aproveitamento de carboidrato, com variações qualitativas e quantitativas da atividade da glicoquinase, sugerindo que esta pode ser uma explicação para a tolerância/intolerância ao carboidrato (Panserat et al., 2000). 8 2.2. Jejum e realimentação Por estarem sujeitos a longos períodos de privação alimentar durante parte do ciclo de vida, os peixes desenvolveram habilidades para suportar períodos de jejum, sem comprometer a capacidade de sobrevivência (Love, 1970). O jejum envolve uma série de adaptações fisiológicas para promover o ajuste biológico do animal nesta condição e suas consequências finais são altamente dependentes da espécie considerada, da idade do peixe e de condições experimentais como temperatura da água, fotoperíodo, dieta pré-jejum, e duração do jejum (Love, 1970; Weatherley e Gill, 1987; Blasco et al., 1991, Souza et al., 2000). A estratégia de jejum seguida de realimentação é uma ferramenta para manipular as alterações bioquímicas e metabólicas (Baldan, 2008). Em algumas espécies, a primeira reserva energética a ser mobilizada é o glicogênio (Hung, et al., 1997; Méton et al., 2003). Esta hidrólise, além de fornecer substrato energético, ajuda a manter a homeostase da glicemia durante os primeiros estágios do jejum. Paralelamente à mobilização de glicogênio, reservas de lipídeos são usadas para obter energia e o uso da proteína muscular como fonte de energia só é utilizada em situações extremas (Navarro e Gutiérrez, 1995). Por outro lado, algumas espécies tentam preservar as reservas de glicogênio, degradando proteína para gliconeogênese e mobilizando lipídeos como substrato energético (Sheridan e Mommsen, 1991; Gillis e Ballantyne, 1996). Os precursores gliconeogênicos são moléculas de não-carboidratos que podem ser usadas para produzir glicose. Estão inclusos nesta lista todos os intermediários da via glicolítica, do ciclo do ácido cítrico, além do glicerol, lactato e α- ceto ácidos, provenientes da deaminação dos aminoácidos não essenciais (Berg et al., 2002). Na via glicolítica, existem sete reações reversíveis, usadas para a síntese de glicose a partir do piruvato ou lactato. Entretanto, três reações são irreversíveis e devem ser contornadas por quatro reações alternativas que são energeticamente a favor da síntese de glicose. Assim, o piruvato é, primeiro, carboxilado pela enzima piruvato carboxilase, formando oxalacetato e posteriormente é convertido a fosfoenol piruvato pela fosfoenol piruvato carboxiquinase. Outra enzima importante é a frutose 1,6-bifosfatase que hidrolisa a frutose 1,6-bifosfato, formando frutose 6-fosfato. Por último, ocorre a hidrólise da glicose 6-fosfato pela glicose 6-fosfatase. Em conjunto, 9 essas enzimas fornecem uma via energeticamente favorável para a formação de glicose na forma livre. Por outro lado, a realimentação dispara uma variedade de respostas que depende de fatores como espécie, idade, condições ambientais, período de jejum e o histórico alimentar anterior ao jejum (Navarro e Gutiérrez, 1995). No geral, os peixes realimentados mostram rápida recuperação do peso, conhecido como ganho compensatório, sustentado pela rápida recuperação do perfil metabólico (Soengas et al., 1996; Méton, et al., 2003; Morales et al., 2004). Com relação à atividade de enzimas que regulam as vias glicolítica e gliconeogênica, o jejum (Caseras et al., 2000; Kirchner et al., 2003a; Metón et al., 2004; Kirchner et al., 2005; Soengas et al., 2006), assim como a restrição no fornecimento de energia na dieta (Caseras et al., 2000), reduzem de forma significativa a atividade e a expressão gênica da glicoquinase (hexoquinase IV) em truta arco-íris e “European seabream” (Caseras et al., 2000). Além disso, é possível observar o aumento da expressão desta enzima algumas horas após a realimentação (Soengas et al., 2006). Comportamento semelhante foi observado por Fideu et al. (1983), Bonamusa et al. (1992), Metón et al. (2003) e Kirchner et al. (2003b), sendo que a atividade da piruvato quinase no fígado de truta arco-íris reduziu após o jejum, e foi restaurada apenas após 20 dias de realimentação. De acordo com Caseras et al. (2000), este atraso na expressão gênica após a realimentação pode contribuir para explicar a hiperglicemia prolongada após a ingestão de alimentos. Curtos períodos de privação alimentar (2 a 9 dias) não foram suficientes para produzir mudanças significativas na atividade e a expressão da glicoquinase (GK) (Pérez-Giménez et al., 2007) e na atividade da piruvato quinase (PK) (Metón et al., 2003). Já as enzimas gliconeogênicas fosfoenol piruvato caboxiquinase (PEPCK) (Kirchner et al., 2003), frutose 1,6-bifosfatase (FBFase) (Morata et al., 1982; Metóm et al., 1999; Metóm et al., 2003; Kirchner et al., 2003) e glicose 6-fosfatase (G6Fase) (Morata et al., 1982; Caseras et al., 2002; Metóm et al., 2003; Kirchner et al., 2003) possuem atividade e expressão gênicas aumentadas após jejum prolongado quando comparados com peixes continuamente alimentados. 10 2.3. Sistema imune em peixes O sistema imune dos peixes, como nos demais vertebrados, apresenta respostas imunes inatas ou não especificas que funcionam como uma primeira barreira contra microrganismos, e respostas imunes específicas, mais lentas, dependentes do reconhecimento de antígenos, produção de anticorpos específicos e formação de memória imunológica (Bernstein et al., 1998). A imunidade inata tem origem em células e moléculas presentes em tecidos e fluidos corporais que desempenham ação protetora. Algumas proteínas foram identificadas em peixes, a exemplo da mucotripsina, transferrina, lisozima, proteínas do sistema complemento e lectinas (Dalmo et al., 1997). A imunidade inata celular é aquela gerada pela ação de monócitos, macrófagos e granulócitos (neutrófilos, eosinófilos e basófilos) e células citotóxicas (“natural killers”) (Secombes, 1996), que atuam no reconhecimento e na eliminação de patógenos, funções essenciais nesta etapa de defesa do organismo (Zaccone et al., 2009). A inflamação em peixe inicia- se como nos mamíferos, com o aumento na liberação de neutrófilos, resultando em um aumento do número das células circulantes, podendo seu perfil ser usado como indicativo de infecção (Kindt et al., 2006). Os fagócitos também desempenham um papel importante em função de sua atividade respiratória. Durante a fagocitose, ocorre a produção de espécies reativas de oxigênio que desempenham função bactericida (Afonso et al., 1998). Muitas moléculas e células que atuam na resposta imune inata podem ser utilizadas como indicadores no monitoramento da saúde dos peixes (Robertsen, 1999). Composto por várias proteínas solúveis, o sistema complemento desempenha um papel importante na imunidade não específica atuando nos processos biológicos de fagocitose, opsonização, quimiotaxia de leucócitos e inativação de toxinas liberadas por bactérias (Secombes, 1996; Claire et al., 2002; Boshra e Sunyer, 2006, Nakao et al., 2011). As proteínas do sistema complemento também estão envolvidas nos mecanismos de recrutamento de células fagocíticas em reações inflamatórias e na exposição de antígenos aos linfócitos, atividades relacionadas com a via clássica do sistema (Claire et al., 2002; Boshra e Sunyer, 2006). O sistema complemento, mais estudado e conhecido em mamíferos, é composto por 30 proteínas plasmáticas e de membrana, que são ativadas em três vias de reações distintas as quais convergem em C3, uma convertase, pivô central do sistema. A ação das três vias, clássica, 11 alternativa e das lectinas forma o complexo de ataque à membrana (CAM), responsável pela atividade lítica em patógenos (Nonaka e Smith, 2000; Nakao et al., 2011). O sistema complemento dos peixes é funcionalmente similar ao de mamíferos, sendo que as principais vias descritas em mamíferos, clássica, alternativa e lítica, foram identificadas em nível funcional em peixes ósseos e cartilaginosos (Nonaka e Smith, 2000). Biller-Takahashi et al. (2012) observaram aumento da atividade lítica da via alternativa das proteínas no sistema complemento em pacus após desafio com Aeromonas hydrophila. A lisozima é uma molécula importante na defesa do organismo contra patógenos. É uma enzima produzida pelos leucócitos e apresenta atividade lítica sobre membranas de diversas espécies de bactérias, tanto em bactérias Gram positivas quanto negativas. Em peixes, a enzima encontra-se amplamente distribuída sobre a pele, muco, brânquias, trato intestinal, soro, tecidos linfóides e outros fluidos corporais. Variações nos níveis séricos de lisozima podem ocorrer devido à sazonalidade, sexo, maturação sexual, alimentação, temperatura da água, estresse e infecções (Hernández e Tort, 2003). A mensuração da concentração sérica de lisozima pode ter valor diagnóstico na determinação da condição imunológica e resistência a doenças (Saurabh e Sahoo, 2008). Embora ambas as respostas, inatas e específicas, tenham papel fundamental na defesa contra patógenos, acredita-se que, para os peixes, as respostas inatas sejam mais importantes quando comparados com mamíferos (Saurabh e Sahoo, 2008; Urbinati et al., 2014). O entendimento da biologia dos peixes, em particular da resposta imune, é importante para um manejo sanitário apropriado. O estudo da resposta inata nestes animais tem gerado interesse crescente nos últimos anos e pode ser considerado um fator chave na defesa primária e na organização da imunidade adquirida (Whyte, 2007). 2.4. Modulação do sistema imune pelo estresse A ativação da resposta de estresse pode provocar tanto ativação como inibição do sistema imune. Em um primeiro momento, ocorre ativação, principalmente provocada por catecolaminas e pelo hormônio liberador de corticotropina (CRH) e, posteriormente, ocorre inibição, provocada pela liberação dos hormônios do eixo hipotálamo-pituitária-interrenal (HPI), particularmente relacionada à ação do cortisol 12 (Tort, 2011). Em situações de estresse, o aumento das catecolaminas atua nos tecidos hematopoiéticos, aumentando a libração de eritrócitos na circulação, e o aumento de cortisol diminui a produção de leucócitos. Da mesma forma, monócitos e linfócitos circulantes podem ser diretamente afetados pelos hormônios (Ellis, 1981). Em caso de estresse agudo, foi observado aumento no número de receptores de glicocorticoides nos leucócitos do rim cefálico (Maule e Schreck, 1991). Apesar de poucas informações em peixes, alguns estudos mostram alterações em número e padrão de distribuição de células brancas decorrentes de estresse, pela necessidade de mobilização de células para locais afetados e aumento da eficiência de defesa (Wojtaszek et al., 2002; Dhabhar, 2002). A fase aguda do estresse favorece a mobilização de células de defesa e a distribuição dos diferentes tipos celulares, de acordo com a necessidade (Dhabhar, 2002). Seguindo o aumento inicial de leucócitos, e com a permanência da ativação do eixo HPI, ocorre uma diminuição geral do número de células circulantes, reflexo da migração e permanência das mesmas nos órgãos afetados (Tort, 2011). A redução da quantidade e atividade dos componentes do sistema imune pode diminuir a resistência dos peixes às doenças e facilitar a infecção por microrganismos patogênicos oportunistas, como a Aeromonas hydrophila é um bastonete ou coco- bastonete Gram negativo aeróbio ou anaeróbio facultativo, agente etiológico da enfermidade conhecida como septicemia hemorrágica. Está presente em praticamente todos os ambientes aquáticos, assim como na pele e no trato intestinal dos peixes de água doce (Holliman, 1993). A ampla distribuição da bactéria e sua adaptação a mudanças ambientais deve-se a ampla variedade de enzimas secretadas por suas cepas (Pemberton et al., 1997). A manifestação da septicemia hemorrágica está normalmente relacionada a situações estressantes como a ocorrência de parasitoses (Martins et al., 2000), condições inapropriadas da água, tais como grande quantidade de matéria orgânica, baixa concentração de oxigênio dissolvido, oscilações térmicas e outras formas de fragilidade dos hospedeiros (Austin e Austin, 2007). 2.5. Estresse nutricional A nutrição e o manejo alimentar dos peixes têm como principal objetivo melhorar a eficiência produtiva, entretanto, se inadequadas estas ferramentas podem 13 se tornar fatores estressores durante a criação. Devido ao grande número de espécies de peixe criadas no Brasil, o mercado de ração oferece aos produtores dietas formuladas de acordo com o hábito alimentar do peixe (carnívoro ou onívoro), mas que podem não suprir as exigências nutricionais de algumas espécies ou até induzir os animais a um quadro de estresse metabólico em função do excesso ou deficiência de algum nutriente na dieta. Pouco se sabe como dietas desbalanceadas podem afetar a resposta de estresse (Serra et al., in press). O manejo alimentar também pode levar os peixes a um quadro de estresse. Tanto o jejum e a restrição alimentar quanto alterações na frequência de alimentação dos peixes são muito utilizados em piscicultura para melhorar o desempenho produtivo (ALI et al., 2003) e a qualidade de água. Entretanto, estes manejos podem alterar vias metabólicas, e a regulação hormonal do metabolismo de peixes ocorre por processos complexos que envolve diversos fatores, incluindo o cortisol (MOMMSEN et al.,1999). Os efeitos da restrição de alimentos na liberação de cortisol são variáveis. Em ”Artic charr” Salvelinus alpinus submetidos a restrição alimentar por 141 dias (JØRGENSEN et al., 1999) e em “longjaw mudsucker” Gillichthys mirabilis após 20 dias de jejum, os níveis de cortisol aumentaram, e houve normalização dos níveis do hormônio com o retorno da alimentação (KELLEY et al., 2001). Já em bagre do canal, houve diminuição dos níveis de cortisol após 21 dias de jejum (SMALL, 2005). Em truta arco íris, o jejum não mostrou ser um estressor nutricional, e a mobilização de energia durante o jejum, no inverno, pode ser alcançada sem o envolvimento do cortisol (POTTINGER et al., 2003). Apesar dos resultados contraditórios, o cortisol possui importância na mobilização de reservas energéticas dos peixes em jejum, estimulando a glicogenólise e a gliconeogênese hepáticas (MOMMSEN et al., 1999), e podem ser dependentes da espécie, fatores ambientais e intensidade da restrição. Outro indicador de estresse que se modificou com a restrição alimentar foi a expressão gênica de proteínas de choque térmico HSP70 e HSP90, em larvas de truta arco íris submetidas a privação alimentar por sete dias (CARA et al., 2005). 2.6. Pacu (Piaractus mesopotamicus) O pacu é encontrado nas bacias dos rios Paraná, Paraguai e Uruguai (Godoy, 1975). Dentre as espécies nativas exploradas comercialmente, ele é um dos peixes 14 mais estudados nas regiões Sul, Sudeste e Centro-oeste do Brasil (Urbinati et al., 2013). Esta espécie é capaz de alimentar-se tanto de pequenos animais como também de folhas, caules, flores, frutos e sementes, mostrando que ao contrário da maioria das espécies de peixes, principalmente espécies carnívoras, é capaz de tolerar altos níveis de carboidrato dietético (Urbinati et al., 2013). Pacus alimentados com altos níveis de carboidratos mostraram boa capacidade de aproveitar este ingrediente, sendo observadas melhores taxas de crescimento (Figueiredo-Garutti, 1996) e melhor conversão alimentar (Baldan, 2008). Por apresentar estas características, o pacu mostra-se um excelente modelo biológico em estudos de tolerância a carboidratos em peixes. 3. Objetivos gerais O objetivo deste estudo foi avaliar a capacidade de aproveitamento de carboidratos dietéticos em juvenis de e verificar a relação do jejum com as respostas imunes inatas. 3.1. Objetivos específicos Avaliar o desempenho de juvenis de pacu alimentados com diferentes níveis de carboidratos e submetidos à restrição alimentar, seguido de realimentação. Estudar as respostas metabólicas sanguíneas (glicemia, ácidos graxos, triglicerídeos, colesterol e proteínas totais) e teciduais (lipídeo e glicogênio) dos peixes. Verificar o uso da amônia plasmática como indicador da utilização de dietas desbalanceadas em pacu. Avaliar as respostas imunes inatas (atividade respiratória de leucócitos, concentração de lisozima e atividade do sistema complemento) do pacu após período prolongado de restrição alimentar. 4. Referências bibliográficas AFONSO, A.; LOUSADA, S.; SILVA, J.; ELLIS, A.E.; SILVA, M.T. Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and electron microscopic cytochemical study. Diseases of Aquatic Organisms, v.34, p.27-37, 1998. 15 ALI, M.; NICIEZA, A.; WOOTTON, R.J. Compensatory growth in fishes: a response to growth depression. Fish and Fisheries, v.4, p.147-190, 2003. AUSTIN, B.; AUSTIN, D.A. Bacterial Fish Pathogens, Diseases of Farmed and Wild Fish. Chichester,UK PraxisPublishing Ltd, 2007. BALDAN, A.P. 2008. Avaliação da tolerância do pacu (Piaractus mesopotamicus) a carboidratos. 119p. Tese (Doutorado em Aqüicultura) – Centro de Aqüicultura da Unesp, Jaboticabal, 2008. BERG, J.M.; TYMOCZKO, J.L.; STRYER, L. Biochemistry. 5th edition. New York: W H Freeman; 2002. BERNSTEIN, R.M.; SCHLUTER, S.F.; MARCHALONIS, J.J. Immunity. In: EVANS, D. H. (Ed.). The physiology of fishes. 2. Boca Raton: CRC Press, 1998. p.215- 242. BILLER-TAKAHASHI, J. D.;TAKAHASHI, L. S.; MARZOCCHI-MACHADO, C. M.; ZANUZZO, F. S.; SABIONI, R. E.; URBINATI, E. C. Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus). Revista Brasileira de Zootecnia, v. 41, n. 2, p. 237-241, 2012. BLASCO, J.; FERNÁNDEZ, J.; GUTIÉRREZ, J. The effects of starvation and refeeding on plasma amimo acid levels, Cyprinus carpio L., 1758. Journal of Fish Biology, v.38, p.587-598, 1991. BONAMUSA, L.; FRUTOS, P.G.; FERNANDEZ, F.; BAANANTE, I.V. Nutritional effects on key glycolytic-gluconeogenic enzyme activities and metabolite levels in the liver of the teleost fish Sparus aurata. Molecular Marine Biology and Biotechnology, v.1, p.113-124, 1992. BOSHRA, H.; LI, J.; SUNYER, J. O. Recent advances on the complement system of teleost fish. Fish & Shellfish Immunology, v.20, n.2, p.239-262, 2006. CARA, J.B.; ALURU, N; MOYANO, F.J.; VIJAYAN, M.M. Food-deprivation induces HSP 70 and HSP 90 protein expression in larval gilthead sea bream and rainbow trout. Comparative Physiology and Biochemistry Part B, v.142, p.426-431, 2005. CASERAS, A.; METÓN, I.; FERNÁNDEZ, F.; BAANANTE, I.V. Glucokinase gene expression is nutritionally regulated in liver of gilthead sea bream (Sparus aurata). Biochemistry and Biophysics Acta, v.1493, p.135–141, 2000. CASERAS, A.; METÓN, I.; VIVES, C.; EGEA, M.; FERNÁNDEZ, F.; BAANANTE, I.V. Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream (Sparus aurata). British Journal of Nutrition, v.88, p.607-614, 2002. CHO, C. Y.; KAUSHIK, S. J. Nutritional energetic in fish. Energy and protein utilization in rainbow trout (Salmo gairdneri). World Review of Nutrition and Dietetics, v.61, p.132-172, 1990. 16 CLAIRE, M.; HOLLAND, H.; LAMBRIS, J. D. The complement system in teleosts. Fish & Shellfish Immunology, v.12, p.399-420, 2002. COWEY, C., DE LA HIGUERA, M., ADRON, J.W. The effect of dietary composition and of insulin on gluconeogenesis in rainbow trout (Salmo gairdneri). British Journal of Nutrition, 38, 385-395, 1977. COWEY, C.B.; Walton, M.J. Intermediary metabolism. In: Halver JE (ed) Fish nutrition. Academic Press, San Diego, p 260–329, 1989. DALMO, R.A.; INGEBRIGTSEN, K.; BOGWALD, J. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). Journal of Fish Diseases, v.20, n.4, p.241-273, 1997. DHABHAR, F.S. Stress-induced augmentation of immune function—The role of stress hormones, leukocyte trafficking, and cytokines. Brain, Behavior, and Immunity, v.16, n.6, p.785-798, 2002. ELLIS, A.E. Stress and the modulation of defence mechanisms in fish. In: PICKERING, A. D. (Ed.). Stress and Fish. London: Academic Press, 1981. p.147- 165. ENES, P.; PANSERAT, S.; KAUSHIK, S.; OLIVA-TELES, A. Nutritional regulation of hepatic glucose metabolism in fish. Fish Physiology and Biochemistry, v. 35, p.519- 539, 2009. FIDEU, M.D.; SOLER, G.; RUIZ-AMIL, M. Nutritional regulation of glycolysis in rainbow trout (Salmo gairdneri R.). Comparative Physiology and Biochemistry Part B, v.74, p.795-799, 1983. FIGUEIREDO-GARUTTI, M.L. Carboidrato como fonte de energia, o efeito do cromo trivalente na dieta e ação da insulina em juvenis de pacu, Piaractus mesopotamicus. 65f. Dissertação (Mestrado em Zootecnia) – Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, Universidade Estadual Paulista, 1996. GILLIS, T.E.; BALLANTYNE, J.S. The effects of starvation on plasma free amino acid and glucose concentrations in lake sturgeon. Journal of Fish Biology, 1996; 49:1306–1316. HEMRE, G-I., MOMMSEN, T.P., KROGDAHL, A. Carbohydrates in fish nutrition: effects on growth, glucose metabolism and hepatic enzymes. Aquaculture Nutrition, v.8, p.175-194, 2002. HERNÁNDEZ, A.; TORT, L. Annual variation of complement, lysozyme and haemagglutinin levels in serum of the gilthead sea bream Sparus aurata. Fish & Shellfish Immunology, v.15 p.479-481, 2003. 17 HIDALGO, M.C., UREA, E., SANZ, A. Comparative study of digestive enzymes in fish with nutritional habits. Proteolytic and amylase activities. Aquaculture, v.170, p.267- 283, 1999. HOLLIMAN, A. The veterinary approach to trout. In: BROWN, L. (Ed.). Aquaculture for veterinarians: fish husbandry and medicine. Oxford: Pergamon Press, 1993. cap.14, p.223-247. HUNG S.S.O., LIU W., LI H., STOREBAKKEN T., CUI Y. Effect of starvation on some morphological and biochemical parameters in white sturgeon, Acipenser transmontanus. Aquaculture, v. 151 (1–4), p. 357–363, 1997. JØRGENSEN, E.H.; BYE, B.E.; JOBLING, M. Influence of nutritional status on biomarker responses to PCB in the Arctic charr (Salvelinus alpinus). Aquatic Toxicology, v.44, p.233-244, 1999. KELLEY, K. M.; HAIGWOOD, J. T.; PEREZ, M.; GALIMA, M. M. Serum insulin-like growth factor binding proteins (IGFBPs) as markers for anabolic and catabolic condition in fishes. Comparative Biochemistry and Physiology Part B, v.129, p.229-236, 2001. KINDT, T.; OSBORNE, B.; GOLDSBY, R. Kuby – Immunology. W. H. Freeman, 2006. KIRCHNER, S.; KAUSHIK, S.; PANSERAT, S. Effect of partial substitution of dietary protein by a single gluconeogenic dispensable amino acid on hepatic glucose metabolism in rainbow trout (Oncorhynchus mykiss). Comparative Physiology and Biochemistry Part A, v.134, p.337-347, 2003. KIRCHNER, S.; KAUSHIK, S.; PANSERAT, S. Low protein intake is associated with reduced hepatic gluconeogenic enzyme expression in rainbow trout (Oncorhynchus mykiss). Journal of Nutrition, v.133, p.2561-2564, 2003. KIRCHNER, S.; SEIXAS, P.; KAUSHIK, S.; PANSERAT, S. Effects of low protein intake on extra-hepatic gluconeogenic enzyme expression and peripheral glucose phosphorylation in rainbow trout (Oncorhynchus mykiss). Comparative Physiology and Biochemistry Part B, v.140, p.333-340, 2005. KRASNOV, A.; PITKÄNEN, T.I.; REINISALO, M.; MÖLSÄ, H. Expression of human glucose transporter type 1 and rat hexokinase type II cDNAs in rainbow trout embryos; effects on glucose metabolism. Marine Biotechnology, v. 1, p. 25-32, 1999. KUMAR, V.; SAHU, N.P.; PAL, A.K.; KUMAR, S.; SINHA, A.K.; RAJAN, J.; BARUAH, K. Modulation of key enzymes of glycolysis, gluconeogenesis, amino acid catabolism, and TCA cycle of the tropical freshwater fish Labeo rohita fed gelatinized and non- gelatinized starch diet. Fish Physiology and Biochemistry, v. 36, p. 491-499, 2010. LOVE, R.M. The chemical biology of fishes. Vol. 2. London: Academic Press, 1980. p.133-229. 18 MARTIN, S.A.M.; DOUGLAS, A.; DOMINIC, H.F.; SECOMBES, C.J. Starvation alters the liver transcriptome of the innate immune response in Atlantic salmon (Salmo salar). BMC Genomics, p.11-418, 2010. MARTINS, M. L.; MORAES, F. R.; FUJIMOTO, R. Y.; ONAKA, E. M.; NOMURA, D. T.; SILVA, C. A. H.; SCHALCH, S. H. C.Parasitic infections in cultivated freshwater fishes. A survey of diagnosticated cases from 1993 to 1998. Revista Brasileira de Parasitologia Veterinária, v.9, n.1, p.23-28, 2000. MAULE, A.G.; SCHRECK, C.B. Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leukocytes and gill of coho salmon. General and Comparative Endocrinology, v. 84, n.1, p.83-93, 1991. METÓN, I.; MEDIAVILLA, D.; CASERAS, A.; CANTÓ, E.; FERNÁNDEZ, F.; BAANANTE, I.V., 1999. Effect of diet composition and ration size on key enzyme activities of glycolysis–gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream (Sparus aurata). British Journal of Nutrition, v. 82, p. 223–232. METÓN, I.; CASERAS, A.; FERNÁNDEZ, F.; BAANANTE, IV. Molecular cloning of hepatic glucose-6-phosphate catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition. Comparative Physiology and Biochemistry Part B, v.138, p.145– 153, 2004 METÓN, I.; FERNÁNDEZ, F.; BAANANTE, I.V. Short- and long-term effects of refeeding on key enzyme activities in glycolysis-gluconeogenesis in the liver of gilthead sea-bream (Sparus aurata). Aquaculture, v.225, p.99-107, 2003. MOMMSEN, T.P.; VIJAYAN, M.M.; MOON, T.W. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Reviews in Fish Biology and Fisheries, v.9, p.211-268, 1999. MOMMSEN, T.P.; PLISETSKAYA, E.M. Insulin in fishes and agnathans: history, structure and metabolic regulation. Reviews in Aquatic Science, v.4, p.225–259, 1991. MORALES. A.E.; PÉREZ-JIMÉNEZ, A.; CARMEN, HIDALGO, M.; ABELLÁN, E.; CARDENETE, G. Oxidative stress and antioxidant defenses after prolonged starvation in Dentex dentex liver. Comparative Biochemistry and Physiology Part C, v. 139, n. 1-3, p. 153–161, 2004.. NAVARRO I.; GUTIÉRREZ J. 1995. Fasting and starvation. Pp.393–434. In: Hochachka P.W., Mommsen T. (eds.) Biochemistry and molecular biology of fishes. Vol. 4. Elsevier, New York, NY, USA. MOON, T.W. Glucose intolerance in teleost fish: fact or fiction? Comparative Physiology and Biochemistry Part B, v.129, p.243-249, 2001. 19 MORATA, P.; VARGAS, A.M.; SÁNCHEZ-MEDINA, F.; GARCIA, M.; CARDENETE, G.; ZAMORA, S. Evolution of gluconeogenic enzyme activities during starvation in liver and kidney of the rainbow trout (Salmo gairdneri). Comparative Physiology and Biochemistry Part B, v.71, p.65-70, 1982. MUECKLER M.; THORENS, B. The SLC2 (GLUT) family of membrane transporters. Molecular Aspects of Medicine, v.34, p.121-138, 2013. NAKAO, M.; TSUJIKURA, M.; ICHIKI, S.; VO, T. K.; SOMAMOTO, T. The complement system in teleost fish: Progress of post-homolog-hunting researches. Developmental and Comparative Immunology, v.35, n.12, p.1296-1308, 2011. NONAKA, M.; SMITH, S. L. Complement system of bony and cartilaginous fish. Fish & Shellfish Immunology, v.10, n.3, p.215-28, 2000. PANSERAT, S.; BLIN, C.; MÉDALE, F.; PLAGNES-JUAN, E.; BRÈQUE, J.; KRISHNAMOORTHY, J. Molecular cloning, tissue distribution and sequence analysis of complete glucokinase cDNAs from gilthead seabream (Sparus aurata), rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio). Biochimica et Biophysica Acta, v.1474, p.61–69, 2000. PÁRRIZAS, M.; PLANAS, J.; PLISETSKAYA, E.M.; GUTIÉRREZ, J. Insulin receptors and its tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish. American Journal of Physiology, v.266, p.1944–1950, 1994. PEMBERTON, J.M.; KIDD, S.P.; SCHMIDT, R. Secreted enzymes of Aeromonas. FEMS Microbiology Letters, v. 152, n. 1, p. 1-10, 1997. PÉREZ-JIMÉNEZ, A.; GUEDES, M.J.; MORALES, A.E.; OLIVA-TELES, A. 2007. Metabolic responses to short starvation and refeeding in Dicentrarchus labrax. Effect of dietary composition. Aquaculture, v.265, p.325-335. PILKIS, S.; GRANNER, D.K. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. The Annual Review of Physiology, v.54, p.885- 909, 1992. POTTINGER, T.G.; RAND-WEAVER, M.; SUMPTER, J.P. Overwinter fasting and re- feeding in rainbow trout: plasma growth hormone and cortisol levels in relation to energy mobilization. Comparative Physiology and Biochemistry Part B, v. 136, p. 403-417, 2003. ROBERTSEN, B. Modulation of the non-specific defence of fish by structurally conserved microbial polymers. Fish & Shellfish Immunology, v.9, n.4, p.269-290, 1999 SAURABH, S.; SAHOO, P. K. Lysozyme: an important defence molecule of fish innate immune system. Aquaculture Research, v.39, n.3, p.223-239, 2008. 20 SECOMBES, C.J. The nonspecific immune system: cellular defenses. In: IWAMA, G. e NAKANISHI, T. (Ed.). The fish immune system. London: Academic Press, 1996. p. 95-103. SHERIDAN, M.A.; MOMMSEN, T.P. Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. General Comparative Endocrinology, v. 81, p. 473-483, 1991. SMALL, B.C. Effect of fasting on nychthemeral concentrations of plasma growth hormone (GH), insulin-like growth factor I (IGF-I), and cortisol in channel catfish (Ictalurus punctatus). Comparative Biochemistry and Physiology Part B, v.142, p.217-223, 2005 SOENGAS, J.L.; POLAKOF, S.; CHEN, X.; SANGIAO-ALVARELLOS, S; MOON, T.W. Glucokinase and hexokinase expression and activities in rainbow trout tissues: changes with food deprivation and refeeding. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, v.291, p.810–821, 2006. SOENGAS, J.L.; STRONG, E.F.; FUENTES, J.; VEIRA, J.A.R.; ANDRÉS, M.D. Food deprivation and refeeding in Atlantic salmon, Salmo salar: effects on brain and liver carbohydrate and ketone bodies metabolism. Fish Physiology and Biochemistry, v. 15, n. 6, p. 491-511, 1996. SOUZA, V.L.; OLIVEIRA, E.G.; URBINATI, E.C. Effects of food restriction and refeeding on energy stores and growth of pacu, Piaractus mesopotamicus (Characidae). Journal of Aquaculture in the Tropics, v.15, p.371-379, 2000. STONE, D.A.J. Dietary carbohydrate utilization by fish. Reviews in Fisheries Science, v.11, p.337-369, 2003. SUAREZ, R.K.; MOMMSEN, T.P. Gluconeogenesis in teleost fishes. Canadian Journal of Zoology, v.65, p.1869-1882, 1987. TORT, L. Stress and immune modulation in fish. Developmental and Comparative Immunology, v. 35, n. 12, p. 1366-1375, 2011. URBINATI, E.C.; GONCALVES, F.D.; TAKAHASHI, L.S. Pacu Piaractus mesopotamicus. In: Bernardo Baldisserotto; Levy de Carvalho Gomes. (Org.). Espécies Nativas para piscicultura no Brasil. 2 edição revista e ampliada. Santa Maria: Editora UFSM, 2013, capitulo 8, p.1-606. URBINATI, E.C.; ZANUZZO, F.S.; BILLER-TAKAHASHI, J.D. Estresse e sistema imune em peixes. In: Bernardo Baldisserotto, José Eurico Possebon Cyrino, Elisabeth Criscuolo Urbinati. (Org.). Biologia e Fisiologia de Peixes Neotropicais de Água Doce. 1ed.Jaboticabal: Fundação de Apoio à Pesquisa, Ensino e Extensão (Funep), 2014, v.1, p.87-105. WEATHERLEY, A.H.; GILL, H.S. The biology of fish growth. London: Academic Press. 1987. 443p. 21 WHYTE, S.K. The innate immune response of finfish – A review of current knowledge. Fish & Shellfish Immunology, v.23, n.6, p.1127-1151, 2007. WILSON, R.P. Utilization of dietary carbohydrate by fish. Aquaculture, v.124, p.67- 80, 1994 WOJTASZEK, J.; DZIEWULSKA-SZWAJKOWSKA, D.; LOZINSKA-GABSKA, M.; ADAMOWICZ, A.; DZUGAJ, A. Hematological effects of high dose of cortisol on the carp (Cyprinus carpio L.): Cortisol effect on the carp blood. General and Comparative Endocrinology, v.125, n.2, p.176-183, 2002. WRIGHT, JR.; O’-HALI, W.; YANG, H.; BONEN, A. GLUT-4 deficiency and severe peripheral resistance to insulin in the teleost fish tilapia. General and Comparative Endocrinology, v.111, p.20-27, 1998 ZACCONE, G.; MESEGUER, J.; GARCÍA-AYALA, A.; KAPOOR, B.G. Fish Defenses. 234 May Streem, Enfield, New Hampshire, United States of America: Science Publishers, 2009. 22 CAPÍTULO 2 – Juvenile pacu fish efficiently utilize high levels of dietary carbohydrates for growth under different feeding strategies Abstract This study evaluated metabolic patterns in pacu fed diets containing 25% and 45% carbohydrates (CHO). Fish were fed for 30 days, fasted for 30 days and then were re- fed for 30 days with the same initial diet. Fish were collected and analyzed at days 30, 60, 62 and 90 of the experimental period to assess weight gain (WG), specific growth rate (SGR) and protein efficiency rate (PER), as well as blood glucose, triglycerides, cholesterol, non-esterified fatty acids (NEFA), total protein, liver glycogen, liver fat, mesenteric fat (MFI) and activity of the metabolic liver enzymes of glycolysis (hexokinase, HK; glucokinase, GK), lipogenesis/pentose phosphate pathway (glucose 6 phosphate desidrogenase, G6PDH) and amino acid metabolism (aspartate aminotransferase, AST). Continuously-fed fish on the 45% CHO diet showed better growth performance during the entire experimental period. Fasted fish lost weight at day 60 but, regardless of diet, recovered growth potential. In the initial sample (day 30), levels of blood glucose and liver glycogen did not vary with dietary CHO level. The lack of hyperglycemia in fish fed 45% CHO can be explained by the observed elevation in HK and GK activities, at day 30. Indeed, after fasting, in the acute response to re-feeding, these fish also reacted with a greater elevation of GK. During fasting, fish previously fed either diet reduced G6PDH activity and MFI, reduced blood triglycerides and increased cholesterol levels. However, only in fish fed 25% CHO did fasting induce protein catabolism for energy maintenance, as evidenced by an elevation in AST activity at day 60. Metabolic responses and enzymatic activity demonstrate that pacu modulates energy metabolism according to diet and feeding strategy to accumulate or mobilize reserves. Keyword: metabolism, enzyme activity, growth performance Introduction Protein sources constitute some of the most expensive components of fish diets. Therefore, excess dietary protein, unbalances in the protein to energy ratio, and alterations to dietary amino acids profiles may have a drastic impact on fish farming costs (Kim et al., 2014). Moreover, improper feeding of fish may increase nitrogen 23 excretion to the environment (Cho and Kaushik, 1990; Einen and Roem, 1997, Mente et al., 2003). Thus, optimizing the conversion of feed protein into fish growth by manipulating dietary carbohydrates (CHO) and lipids improves productivity and reduces environmental impacts of farming (Kaushik and Médale, 1994, Kumar et al., 2010). Dietary CHOs represent the cheapest energy source. However, most teleosts do not tolerate high CHO concentrations, and maximum dietary levels depend on fish species (Wilson, 1994; Hemre et al., 2002; Polakof et al., 2010), Formulated diets usually contain less than 20% CHO for carnivorous fish, and between 30% to 40% for omnivorous fish (Wilson, 1994). Fish adjust to different nutritional conditions by changing their metabolic profile (Walton and Cowey, 1982; Metón et al., 1999; Lundstedt et al., 2004). In general, protein-rich diets stimulate the proteolytic and gluconeogenic pathways. On the other hand, the partial replacement of proteins by CHO stimulates glycolysis, glucogenesis and lipogenesis, reducing protein catabolism and gluconeogenesis (Pérez-Jiménez et al., 2009). Until now, the reasons for glucose intolerance in fish are not fully clear (Enes et al., 2009; Pérez-Jiménez et al., in press). The manipulation of dietary CHO and of feeding strategies (e.g., feeding, fasting, re-feeding) provides a tool for the study of metabolic changes associated with glucose intolerance in fish (Wilson, 1994; Moon, 2001; Hemre et al., 2002; Fu and Xie, 2004; Polakof et al., 2010; Pérez-Jiménez et al., 2012). However, most previous studies focused on carnivorous species that do not use dietary CHO efficiently (Wilson, 1994; Fu and Xie, 2004). Usually, feeding or re-feeding these species results in a prolonged postprandial hyperglycemia. On the other hand, carnivorous fish are better adapted to fasting, because of their low feeding frequency under natural conditions, whereas omnivorous fish ingest food continuously (Bond, 1996). In this sense, the study of dietary CHO manipulation in fish with different natural habits may shed light on this important tolerance mechanism. The pacu (Piaractus mesopotamicus), natural to South America, represents one of the most commonly farmed tropical fish. In its natural habitat, it mostly feeds on roots, seeds and fruit, thus, it is highly adapted to dietary CHO. In this study, we investigated the metabolic adjustments of pacu to feeding, fasting and re-feeding with two CHO levels. We assessed fish growth, and the acute 24 and chronic metabolic responses to re-feeding, including changes to blood metabolic parameters, tissue energy reserves and to the activity of metabolic enzymes. Material and methods Fish and experimental conditions We used 300 fish (15.8± 1.2 g), obtained from a fish farm and initially held in 20 100 L polietilene tanks (15 fish tank-1) for one week acclimatization, being fed with a commercial diet. During this period, we observed the appropriate feeding rate (4%) to be used during experiment. Water temperature (29.1 � 0.3�C) and dissolved oxygen (> 5.0 mg L-1) were monitored. Photoperiod was 12 h light: 12 h dark. Experimental design and sampling Fish were distributed in four groups: (1) Fed with a 25 % CHO diet during the 90-days trial; (2) fed with a 45 % CHO diet during the 90-day trial; (3) fed with a 25 % CHO diet for 30 days, followed by a 30-day fasting period and a 30-day re-feeding period with the same diet; (4) fed with 45 % CHO diet for 30 days, followed by a 30- day fasting period and a 30-day re-feeding period with the same diet. Diets were offered twice a day (9:00 AM and 5:00 PM) at 4% of body weight, calculated at each 15 days. At 30 (initial sampling), 60, 62 and 90 days of trial, 10 fish from each treatment (two fish per tank) were anaesthetized (benzocaine, 0.1 g L-1) for blood collection and body measurements. The blood, drawn from the caudal vessel, was dispensed in microtubes containing anticoagulant (plasma) and microtubes without anticoagulant (serum). Plasma was separated by immediate centrifugation of whole blood (3000 rpm during 10 minutes at 4 ºC), and serum after blood remain at room temperature, for 3 h, to clot. Following blood sampling, fish were euthanized (benzocaine, 0.4 g L-1) and mesenteric fat, liver and white muscle from dorsal portion were removed. Mesenteric fat and liver were weighed to calculate the mesenteric fat and hepatosomatc index (MFI or HSI) [(tissue weight / body weight) x 100]. White muscle was processed to determine lipid concentration, a portion of liver was used to determine lipid and glycogen concentration and other portion of liver was used to enzymes activity assays. 25 Specific procedures Experimental diets Two isonitrogenous and isolipidic experimental diets were formulated varying to contain either 25 or 45 % carbohydrate (Table 1). Dry ingredients were weighed and mixed until to obtain a homogenous mixture. Water (40 %) was added to the mixture and then pelletized. After dried, the diets were stored at -20ºC until needed. Table 1. Formulation and nutrient composition of experimental diets. Ingredients Experimental diets 25 % CHO 45 % CHO Fish meal 18.75 13.00 Soybean meal 9.03 14.00 Viscera meal 13.00 15.98 Corn 11.00 14.00 Rice meal 16.60 3.00 Wheat meal 9.00 9.00 Corn starch 2.00 26.00 Soybean oil 0.40 3.00 Bicalcic phosphate 0.50 0.50 Premix1 0.50 0.50 BHT 0.02 0.02 Caulim 19.20 1.00 Proximate composition (% dry matter) Digestive protein (%) 21.70 21.80 Digestive energy (kcal kg- 1) 2,438.20 3,339.40 Fat (%) 7.50 7.50 Carbohydrate (%) 25.00 44.50 1Premix: vitamin A 860,000 UI; vitamin D3 240,000 UI; vitamin E 10,500 UI; vitamin K3 1,400 mg; vitamin B1 2,100 mg; vitamin B2 2,150 mg; vitamin B6 2,100 mg; vitamin B12 2,200 mcg; Niacin 10,000mg; calcium pantotenate 5,600 mg; folic acid 580 mg; biotin 17mg; vitamin C 18,000 mg; metionin 100,000 mg; colin 60,000 mg; cooper 1,800 mg; manganese 5,000 mg; zinc 8,000 mg; iodine 90 mg; cobalt 55 mg; selenium 30 mg. Growth performance Data from 30, 60 and 90 days of experiment were used to calculate the growth performance, as follow: weight gain (g) (WG) = final body weight – initial body weight; specific growth rate (%) (SGR) = ((ln final body weight – ln initial body weight)/days) x100) and protein efficiency rate (%) (PER) = (weight gain/consumed protein) x 100. 26 Metabolic assays Plasma was used to determine blood glucose and triglycerides concentrations (kit Labtest, Sao Paulo, Brazil, code 84 and 87, respectively) and serum to determine cholesterol and total protein (kit Labtest, Sao Paulo, Brazil, code 76 and 99). Liver glycogen level was measured according to Moon et al. (1989) and liver and muscle lipid levels following Bligh and Dyer (1959). Enzyme activity assays The enzymes activity was determined in liver. Tissue samples were homogenized according proposed by Pérez-Giménez et al. (2009) and enzimes activity were performed as follow: Hexokinase (HK, 2.7.1.1) activity was determined as previously described by Vijayan et al. (1990). The reaction mixture contained 50 mM imidazole–HCl buffer (pH 7.4), 2.5 mM ATP, 5 mM MgCl 2 , 0.4 mM NADP, 2 units mL−1 G6PDH and 1 mM D- glucose. Glucokinase (GK, 2.7.1.2) activity was determined as previously described by Vijayan et al. (1990). The reaction mixture contained 50 mM imidazole–HCl buffer (pH 7.4), 2.5 mM ATP, 5 mM MgCl 2 , 0.4 mM NADP, 2 units mL−1 G6PDH and 100 mM D-glucose. Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity was measured as previously described by Morales et al. (1990), using a reaction mixture containing 50 mM imidazole–HCl buffer (pH 7.4), 5 mM MgCl 2 , 2 mM NADP and 1 mM glucose-6-phosphate. Aspartate aminotransferase (AST, EC 2.6.11) activity was determined as described by Singer et al. (1990). The reaction mixture contained 50 mM imidazole– HCl buffer (pH 7.4), 10 mM α-ketoglutarate, 0.3 mM NADH, 0.05 mM pyridoxal phosphate, 3 units mL−1 MDH and 25 mM L-aspartate. The enzymatic reactions were initiated by the addition of the tissue extract, following adaptations of Moura (unpublished data) to HK, GK and G6PDH in pacu. All enzyme activities are expressed as milliunits per milligram of soluble protein (specific activity). One unit of enzyme activity was defined as the amount of enzyme required to transform 1 μmol of substrate per min under the above assay conditions. Soluble 27 protein concentration was determined according to Bradford (1976), with bovine serum albumin used as the standard. Statistical analysis The experiment was conducted in an entirely randomized design and results were analyzed by a three-way ANOVA. Two feeding strategies (fasted and fed) x two CHO levels (25 and 45 % CHO) x three or four periods of sampling factorial, followed by Tukey’s post-hoc tests, after being tested for normality (Cramer Von Mises) and homoscedasticity tests (Brown-Forsythe). P < 0.05 was used as the level of statistical significance. Values in figures are means ± standard error (S.E.) of the mean for growth performance and means ± standard deviation (S.D.) of the mean for other variables. Bioethical statement The experimental procedures were approved by the Comissão de Ética no Uso de Animais (CEUA – Protocol 002112/12) and performed in accordance with the Guidelines of the Ethical Principles in Animal Experimentation, adopted by the Colégio Brasileiro de Experimentação (COBEA). Results Fish performance To evaluate growth performance of pacu fed with different CHO levels followed by long-term fasting and re-feeding, we collected and analyzed fish at days 30, 60 and 90 of the experimental period. Feeding strategy and CHO only had independent effects on all tested variables. Inclusion of 45% CHO resulted in higher values (P<0.05) of WG, SGR and PER at the initial 30 day sampling (Fig. 1). At day 60 – after 30 days of fasting – no differences were observed between diets within the same feeding strategy. At this sampling, both fasted fish lost weight. At day 90 – after 30 days of re-feeding – fish continuously-fed with 45% CHO had higher WG, whereas no other differences were detected among groups. Metabolic responses To evaluate the metabolic adjustment of pacu fed with different CHO levels followed by long-term fasting and re-feeding, we collected and analyzed fish at days 30, 60, 62 and 90 of the experimental period. At day 62, we aimed to evaluate acute 28 changes to the metabolic profile, soon after reestablishing the feeding protocol, as opposed to the chronic effects of re-feeding observed at day 90. Blood glucose did not differ (P>0.05) between diets after 30 days of feeding (Fig. 2 A). At day 60, blood glucose was reduced in all animals in comparison to the initial sampling. However, this reduction was significantly greater in fasted fish in comparison to continuously-fed fish, independently of dietary CHO. After one day of re-feeding (day 62), both fasted fish groups showed increased blood glucose levels that were similar to those of continuously-fed fish. At the final sampling, blood glucose of all treatment groups returned to the levels observed at day 30. Fish fed 45% CHO showed higher initial sampling levels of triglycerides than fish fed 25% CHO (Fig. 2 B). After 30 days of fasting, both fasted fish groups had lower triglycerides levels than continuously-fed fish, but there were no differences between diets. Continuously-fed fish maintained the increased triglyceride levels unchanged until the end of the experimental period. On the other hand, regarding fasted fish, at day 62, only in fish re-fed with 45% CHO did triglycerides return to the same levels of fed fish. At day 90, triglyceride levels in previously-fasted fish on both diets had returned to the levels observed in continuously-fed fish. At the initial sampling, cholesterol levels were the lowest observed throughout the experiment, and fish fed a 25% CHO diet had lower levels than fish fed a 45% CHO diet (Fig. 2 C). At day 60, we observed a general increase in cholesterol levels for fish in both diets. However, fasted fish had higher cholesterol levels than those observed in fed fish, but levels were the same for all groups at day 62. At day 90, cholesterol levels were lower for all fish, but only returned to the initial levels in previously-fasted animals. NEFA levels only differed between diets at the initial sampling, when fish fed 25% CHO had higher NEFA levels than fish fed 45% CHO (Fig 2 D). Total protein did not differ among treatments or time points (Data not showed). Liver glycogen did not differ (P>0.05) between diets after 30 days of feeding (Fig. 3 A). At day 60, liver glycogen was reduced in all animals in comparison to the initial sampling. However, this reduction was significantly greater in fasted fish in comparison to continuously-fed fish, independently of dietary CHO. After one day of re-feeding (day 62), both fasted fish groups showed increased liver glycogen levels 29 that were similar to those of continuously-fed fish. At the final sampling, liver glycogen levels remained similar to those observed at day 62. Liver lipid levels did not differ among treatments when comparing the initial to the final samples. At days 60 and 62, liver lipids were not statistically analyzed due to the low amount of tissue obtained after fasting, but the fasted group values were obtained from a pool of all samples from each treatment (Fig. 3 B). At day 30, HSI was the highest observed throughout the experiment for all groups, however, HSI of fish fed 45% CHO was higher than seen in fish fed 25% CHO (Fig. 3 C). At day 60, fasted fish on both diets had decreased HSI compared to continuously-fed fish and compared to fish sampled at the first time point. These values remained unaltered one day after re-feeding started. At the end of 90 days, fish re-fed with 45% CHO had higher HIS levels than those observed in continuously-fed fish in the same diet group, and fish re-fed with 25% CHO. At the initial sampling, MFI was higher in fish fed a 45% CHO diet. At days 60, 62 and 90, MFI had the same profile: fasted fish had a lower index than fed fish. Fish re-fed with 25% CHO had reduced MFI at day 62 (Fig 3 D). Comparison among sampling times showed a tendency towards increasing MFI in continuously-fed fish, but not fasted animals. Enzyme activity At the initial sampling, HK activity was higher in fish fed 45% CHO than in those fed 25% CHO (Fig. 4 A). At day 60, both fed fish groups kept the same profile observed at the initial sampling, but fasted fish had significantly reduced HK activity. No alterations to this profile were observed 1 day after re-feeding started, except for a recovery of HK activity in fish re-fed a 45% CHO diet, albeit to a level that was lower than observed in continuously fed fish. At the end of the experiment, we observed increased HK activity in fish re-fed a 45% CHO diet, in comparison to continuously fed fish. In continuously fed fish, HK activity tended to decrease with time, but statistical significance was only observed at the 90-day time point. Fish fed 45% CHO had higher GK at day 30 (Fig 4 B). At day 60, both fasted fish groups reduced GK activity, but only fish previously fed 45% CHO differed significantly from continuously-fed fish on the same diet. At day 62, only fish re-fed 30 45% CHO increased GK activity to values higher than those seen in continuously-fed fish. This pattern was only observed in fish re-fed 25% CHO at day 90. Dietary CHO did not affect G6PDH activity at the initial sampling, and activity remained constant for continuously-fed fish (Fig. 4 C). At day 60, both fasted fish groups had reduced G6PDH activity when compared to continuously-fed fish and with previous sampling time. This pattern remained in at day 62 but disappeared at day 90, when all groups had similar levels of G6PDH activity. AST activity was higher in fish continuously feeding on 25% CHO at the initial sampling and at day 60 compared to fish feeding on 45% CHO. Values for continuously-fed animals were constant throughout the experiment (Fig 4 D). Fish previously fed 25% CHO showed increased AST activity in response to fasting, and both re-fed groups had reduced AST activity at day 62 compared to continuously-fed fish. At the end of the experiment, fish in all treatment groups had the same levels of AST activity. Discussion Dietary manipulation allows for the assessment of glucose intolerance mechanisms in fish (Wilson, 1994; Moon, 2001; Hemre et al., 2002; Fu and Xie, 2004; Polakof et al., 2010; Pérez-Jiménez et al., 2012). To date, however, most studies have focused on carnivorous species that are naturally less adapted to dietary CHO. In this study, we assessed metabolic changes in the fruit-eating fish pacu. We observed that pacu efficiently used high levels of dietary CHO for growth under different feeding strategies. The inclusion of 45% CHO in diets increased the growth performance of pacu juveniles. Higher WG values for fish fed this higher CHO diet compared to the 25% CHO diet were associated with better SGR and PER at day 30. At day 90, differences between fish fed 25% and 45% CHO diets were intensified. Two characids economically important in the Amazon region of South America, the black pacu, Colossoma macropomum, and red pacu, Piaractus brachypomus, both omnivorous species, were fed diets containing Amazonian carbohydrate-rich plant feedstuffs (yucca, Manihot sculenta, plantain, Musa paradisiaca, or pijuayo, Bactris gasipaes) as alternative energy sources for practical diets. The diets contained around 45% CHO. However, differently from our study, the energy in the test feedstuffs was used partly 31 for liver glycogen synthesis rather than for growth because no significant differences in body weight was observed among fish fed the test diets (Lochmann et al., 2009). The 25% CHO diet was intentionally formulated to have a lower amount of energy than that provided by the 45% CHO diet, because dietary CHO but not lipids were altered, resulting in diets with different protein to energy ratios. Some benefits of diets with high-energy content have been reported, such as better growth, food utilization and protein retention besides reduced nutrient excretion (Einen and Roem, 1997). The increased AST activity in fish fed 25% CHO, observed in this study, suggests that the imbalance between energy and protein may have resulted in protein catabolism for energetic purposes increasing nitrogen excretion, as previously reported (Cho and Kaushik, 1990). When insufficient energy is available in a diet from non-protein sources, protein may be catabolized to meet the energy requirements at the cost of nutrient supply somatic growth (Capuzzo & Lancaster 1979). In a previous study, Peragón et al. (1999) demonstrated that absence of carbohydrates in diet of rainbow trout (Oncorhynchus mykiss) reduces significant growth and daily weight gain due to muscle mass loss. At day 60, fasted fish lost weight. Weight loss results from energy mobilization from different body reserves to maintain vital processes during fasting (Pérez-Jiménez et al., 2012). In our study, pacu used glycogen and mesenteric fat, however, pacu juveniles did not lose the ability to re-grow. Re-fed fish grew although they had lower WG compared to continuously-fed fish. Fasted fish weighed 38.9 ± 6.0 g at the end of fasting against 78.9 ± 23.4 g of continuously-fed fish, but both groups had similar SGRs at day 90, the final sampling. We did not observe an effect of dietary CHO levels on blood glucose. However, at day 30, HK and GK activity in fish fed 45% CHO was higher. Soon after glucose enters cells, it is phosphorylated to glucose 6-phosphate by HK and GK and enters one of three pathways; glycolysis, glycogenesis or pentose-phosphate. At day 30, all fish groups had similar glycogen levels. In the common carp (Cyprinus carpio), an omnivorous fish, the hepatic glycogen contents were higher in the fish fed digestible starch than in those on a CHO free diet and the activity of HK remained almost constant after ingestion of the carbohydrate diets, while the activity in fish on the CHO free diet declined 24 h after feeding (Capilla et al., 2004). Together, our findings indicate that, 32 with high CHO levels, fish may have increased energy generation through the glycolysis and pentose-phosphate pathways. In this mode, protein sources are spared and directed towards growth, whereas NADPH is directed towards lipid synthesis, as supported by the higher MFI observed in these fish. At day 60, immediately after fasting, HK and GK were similarly reduced regardless of diet, evidently as a result of reduced substrate. HK did not change in the acute response to re-feeding (day 62), but GK showed a large increase in fish fed the 45% CHO diet. In gilthead sea bream juveniles, dietary glucose was more effective in inducing the response of GK than starch. These higher GK activity may be related to the higher glycaemia observed in fish fed the glucose diet, which may increase glucose uptake by the liver. Accordingly, both HSI and liver glycogen content were far higher in fish fed the glucose diet than the starch diet (Enes et al., 2008). Thus, reduced blood glucose and liver glycogen levels, and the prompt recovery of these values after one day of re-feeding indicate the importance of glucose homeostasis and liver glycogen as important energy reserves in pacu juveniles. The higher triglyceride and cholesterol levels observed at day 30 in fish fed 45% CHO seem to reflect nutritional status. A previous study with the same species reported unchanged triglycerides with different dietary CHO levels (Abimorad et al., 2007). Differently from our study, these authors used ad libitum feeding and observed a tendency towards reduced consumption with increasing CHO concentrations. In our study, we controlled feeding and differences in CHO intake. Thus, fish indeed consumed more CHO, and excess CHO may have been converted to triglycerides in the liver and stored in adipocytes, as previously reported (White, 2009). Values of MFI at day 30 support this view. After fasting, triglycerides and cholesterol levels had different profiles. Fasted fish on both diets had reduced triglycerides and increased cholesterol levels. Under long-term fasting, glycerol derived from the hydrolysis of triglycerides is actively used as a substrate for the gluconeogenic process (Pérez-Jiménez et al., 2012). The reduction of MFI in fasted fish reflects the important role of lipid reserves during long- term fasting. In previous studies with pacu, cholesterol levels either reduced (Takahashi et al., 2011) or increased (Fávero et al., unpublished data) after fasting. In our study, increased cholesterol levels may have resulted from stimulation, during 33 prolonged fasting, of the cholesterol synthesis pathway by acetyl Co-A provided from the β-oxidation of fatty acids (Maita et al., 2006). According to Fávero et al. (unpublished data), the increase in cholesterol levels in fasted fish may be associated with the stress induced by food deprivation, because cholesterol represents a precursor of cortisol. The importance of cortisol in the regulation of energy metabolism and immune response in fasted pacu has been described (Gimbo et al., 2015). In fasted fish, lipid synthesis was reduced as evidenced by a decrease in G6PDH activity. This reduction, observed at days 60 and 62 after fasting, correlates well with reduced MFI. Low G6PDH activity, in turn, may be associated with reduced HK activity in the absence of glucose during fasting. Glucose 6-phosphate, resulting from HK activity, may follow the pentose-phosphate pathway, involving G6PDH, a key enzyme catalyzing the first step of the pathway which generates NADPH for anabolic pathways, including lipid synthesis, and protection systems in various organisms, including fish (Hu et al., 2013).. The lack of alterations in HK and G6PDH activities between days 60 and 62 with ensuing normalization at day 90 may reflect the time lapse necessary for up-regulation of gene transcription and mRNA translation. During prolonged fasting, fish previously fed 25% CHO used glycogen, mesenteric fat and protein as energy sources. Total serum protein did not differ between fasted and fed fish, but increased AST values indicate increased protein catabolism in fish fed 25% CHO. Unchanged AST activity in fish fed 45% CHO suggests prevention of protein catabolism during long-term fasting. In Dentex dentex, plasma protein concentrations decreased after prolonged fasting for 5 weeks (Pérez- Jiménez, et al., 2012), and similar results were observed in carp (Shimeno et al., 1997). However, even with elevated protein catabolism, the unchanged total serum protein observed in this study indicates that a protein protection mechanism may exist in pacu. One day after re-feeding started, previously fasted fish on both diets had lower AST activity than continuously-fed fish. This response reduces protein catabolism in order to restore blood metabolites, energy reserves and to promote a return to normal growth conditions. The hormonal control of metabolic pathways by insulin is well known. In salmonids, plasma insulin titers increased after a high carbohydrate meal or a glucose load (Mommsen and Plisetskaya, 1991). This rise in insulin may affect metabolism in 34 hepatocytes because of the anabolic and anti-catabolic effects of this hormone in fish. Although we did not measure insulin, our results suggest an effect of this hormone as metabolic mediator in pacu. High CHO levels, supposedly resulting in high insulin levels, increased HK activity and decreased AST activity. During fasting, a period of low insulin, AST activity increased, whereas glycogen reserves, MFI, HK and G6PDH activity decreased. In conclusion, we show that pacu efficiently utilizes high dietary CHO levels reducing protein catabolism to promote growth. This fish endures prolonged fasting, adjusting enzyme activities to spare energy during food deprivation, and to quickly replenish energy reserves after the reestablishment of an appropriate feeding schedule. Acknowledgments The authors would like to thank the Centro de Aquicultura of UNESP (CAUNESP) for supplying the fish for this research, and Ms. Damares Perecim and Ms. Mayara Moura for the technical support. This research was funded by the CNPq (National Council for the Scientific and Technological Development) Q2 . References ABIMORAD, E.G.; CARNEIRO, D.J.; URBINATI, E.C. Growth and metabolism of pacu (Piaractus mesopotamicus Holmberg 1887) juveniles fed diets containing different protein, lipid and carbohydrate levels. Aquaculture Research, v. 38, p. 36-44, 2007. BLIGH, E.G.; DYER, W.J. A rapid method for total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, v. 37, p. 911-917, 1959. BOND, C.E. Nervous and endocrine systems. In: Bond CE (ed) Biology of fishes. Saunders College Publishing, FortWorth, pp 241-258, 1996 BRADFORD, M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye-binding. Analytical Biochemistry, v.72, p. 248-254, 1976. CAPILLA, E., MÉDALE, F., PANSERAT, S., VACHOT, C., REMA, P., GOMES, E., KAUSHIK, S., NAVARRO, I., GUTIÉRREZ, J. Response of hexokinase enzymes and the insulin system to dietary carbohydrates in the common carp, Cyprinus carpio. Reproduction and Nutrition Development, v. 44, p. 233–242, 2004. 35 CAPUZZO, J.M. & LANCASTER, B.A. The effects of dietary carbohydrate levels on protein utilization in the American lobster (Homarus americanus). Proceedings World Mariculture Society, v. 10, p. 689–700, 1979 CHO, C.Y.; KAUSHIK, S.J. Nutritional energetics in fish: energy and protein utilization in rainbow trout (Salmo gairdneri). World Review of Nutrition and Dietetics, v. 61, p.132–172, 1990. DENG, L.; ZHANG, W.M.; LIN, H.R.; CHENG, C.H.K. Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli. Comparative Biochemistry and Physiology Part B, v. 137, p. 421-432, 2004 EINEN, O.; ROEN, A.J. Dietary protein/energy ratios for Atlantic salmon in relation to fish size: growth, feed utilization and slaughter quality. Aquaculture Nutrition, v. 3, p. 115-126, 1997. ENES, P., PANSERAT, S., KAUSHIK, S., OLIVA-TELES, A. Hepatic glucokinase and glucose-6-phosphatase responses to dietary glucose and starch in gilthead sea bream (Sparus aurata) juveniles reared at two temperatures. Comparative Biochemistry and Physiology, Part A, v.149, p.80–86, 2009. FU, S.J., XIE, X.J. Nutritional homeostasis in carnivorous southern catfish (Silurus meridionalis): is there a mechanism for increased energy expenditure during carbohydrate overfeeding? Comparative Biochemistry and Physiology, Part A, v.139, p.359– 363, 2004. GAO, W., LIU, Y-J., TIAN, L-X, MAI, K.S., LIANG, G-Y., YANG, H-J., HUAI, M-Y, LUO, W-J. Effects of dietary carbohydrate-to-lipid ratios on growth performance, body composition, nutrient utilization and hepatic enzymes activities of herbivorous grass carp (Ctenopharyngodon idella). Aquaculture Nutrition, v.16, p.327-333, 2010. HEMRE, G-I., MOMMSEN, T.P., KROGDAHL, A. Carbohydrates in fish nutrition: effects on growth, glucose metabolism and hepatic enzymes. Aquaculture Nutrition, v.8, p.175-194, 2002. HORIO, Y.; TANAKA, T.; TAKETOSHI, M.; UNO, T.; WADA, H. Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis. Journal of Biochemistry, v. 103, p. 805-808, 1988. HU, W., ZHI, L., ZHUO, M-Q., ZHU, Q-L., ZHENG, J-L., CHEN, Q-L., GONG, Y., LIU, C-X. Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro. Fish Physiology and Biochemistry, v.39, p.637–647, 2013. KALDERON, B.; MAYOREK, N.; BERRY, E.; ZEVIT, N.; BAR-TANA, J. Fatty acid cycling in the fasting rat. American Journal of Physiology, v. 279, p. E221–E227, 2000. 36 KAUSHIK, S.J.; MÉDALE, F. Energy requirements, utilization and dietary supply to salmonids. Aquaculture, v. 124, p. 81-97, 1994. KUMAR, V.; SAHU, N.P.; PAL, A.K.; KUMAR, S.; SINHA, A.K.; RAJAN, J.; BARUAH, K. Modulation of key enzymes of glycolysis, gluconeogenesis, amino acid catabolism, and TCA cycle of the tropical freshwater fish Labeo rohita fed gelatinized and non- gelatinized starch diet. Fish Physiology and Biochemistry, v. 36, p. 491-499, 2010. LOCHMANN, R., CHEN, R., CHU-KOO, F.W., CAMARGO, W.N., KOHLER, C.C. Effects of carbohydrate-rich alternative feedstuffs on growth, survival, body composition, hematology, and nonspecific immune response of black pacu, Colossoma macropomum, and red pacu, Piaractus brachypomus. Journal of the World Aquaculture Society, v.40, p.33-44, 2009. LUNDSTEDT, L.M.; MELO, J.F.B.; MORAES, G. Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition. Comparative Biochemistry and Physiology Part B, v. 137, p. 331-339, 2004. MAITA, M.; MAEKAWA, J.; SATOH, K.; FUTAMI, K.; SATOH, S. Disease resistance and hypocholesterolemia in yellowtail Seriola quinqueradiata fed a non-fishmeal diet. Fisheries Science, v. 72, p. 513 – 519, 2006. MOON, T.W. Glucose intolerance in teleost fish: fact or fiction? Comparative Physiology and Biochemistry Part B, v.129, p.243-249, 2001. MOON, T.W.; FOSTER, G.D.; PLISETSKAYA E.M. Changes in peptide hormones and liver enzymes in the rainbow trout deprived of food 6 weeks. Canadian Journal of Zoology, v. 67, n. 9, p. 2189-2193, 1989. MOMMSEN, T.P.; PLISETSKAYA, E.M. Insulin in fish and agnathans: history, structure and metabolic regulation. Reviews in Aquatic Science, v. 4, p. 225-259, 1991. MORALES, A.E.; GARCÍA-REJÓN, L.; DE LA HIGUERA, M. Influence of handling and/or anaesthesia on stress response in rainbow trout. Effects on liver primary metabolism. Comparative Biochemistry and Physiology Part A, v. 95, p. 87-93, 1990. PAVE-PREUX, M.; FERRY, N.; BOUGUET, J.; HANOUNE, J.; BAROUKI, R. Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase. Journal of Biology and Chemistry, v. 263, p. 17459-17464, 1988. PERAGÓN J., BARROSO J.B., GARCÍA-SALGUERO L., HIGUERA M.D.L. & LUPIÁÑEZ J.A. Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss). Aquaculture, 179, 425–437, 1999. 37 PÉREZ-JIMÉNEZ, A.; CARDENETE, G.; HIDALGO, M.C.; GRACÍA-ALCÁSAR, A.; ABELLÁN, E.; MORALES, A.E. Metabolic adjustments of Dentex dentex to prolonged starvation and refeeding. Fish Physiology and Biochemistry, v.38, p.1145-1157, 2012 PÉREZ-JIMÉNEZ, A.; HIDALGO, M.C.; MORALES, A.E.; ARIZCUN, M.; ABELLÁN, E.; CARDENETE, G. Use of different combinations of macronutrients in diets for dentex (Dentex dentex) Effects on intermediary metabolism. Comparative Biochemistry and Physiology Part A, v. 152, p. 314-321, 2009. POLAKOF, S.; ÁVEREZ, R.; SOENGAS, J.L. Gut glucose metabolism in rainbow trout: implications in glucose homeostasis and glucosensing capacity. American Journal of Physiology Regulatory Integrative and Comparative Physiology, v. 299, p. R19–R32, 2010. SINGER, T.D.; MAHADEVAPPA, V.G.; BALLANTYNE, J.S. Aspects of the energy metabolism of lake sturgeon, Acipenser fulvescens, with special emphasis on lipid and ketone body metabolism. Canadian Journal of Fisheries and Aquatic Sciences, v. 47, p. 873–881, 1990 SHIMENO, S.; SHIKATA, T.; HOSOKAWA, H.; MASUMOTO, T.; KHEYYALI, D. Metabolic response to feeding rates in common carp, Cyprinus carpio. Aquaculture, v. 151, p. 371-377, 1997. TAKAHASHI, L.S; BILLER, J.D.; CRISCUOLO-URBINATI, E.; URBINATI, E.C. Feeding strategy with alternate fasting and refeeding: effects on farmed pacu production. Journal of Animal Physiology and Animal Nutrition, v. 95, p. 259-266, 2011. VIJAYAN, M.M., BALLANTYNE, J.S., LEATHERLAND, J.F. High stocking density alters the energy metabolism of brook charr, Salvelinus fontinalis. Aquaculture, v. 88, p. 371-381, 1990. METÓN, I.; MEDIAVILLA, D.; CASERAS, A.; CANTO, E.; FERNÁNDEZ, F.; BAANANTE, I.V. Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, the pentose phosphate pathway and amino acid metabolism in liver of gilthead sea bream (Sparus aurata). British Journal of Nutrition, v. 82, p. 223-232, 1999 Stone, D.A.J. Dietary carbohydrate utilization by fish. Reviews in Fisheries Science, v. 11, p. 337-369, 2003 WALTON, M.J.; COWEY, C.B. Aspects of intermediary metabolism in salmonid fish. Comparative Biochemistry and Physiology Part B, v. 73, p. 59-79, 1982. WHITE, B.A. Hormonal Regulation of Energy Metabolism. In: B.M. Koeppen; B.A. Stanton 6th ed Berne and Levy Physiology. Rio de Janeiro: Elsevier, 2009. p. 669-700. WILSON, R.P. Utilization of dietary carbohydrate by fish. Aquaculture, v. 124, p. 67- 80,1994. 38 Figure 1. Effects of carbohydrates (CHO) and feeding strategy on weight gain (WG), specific growth rate (SGR) and protein efficiency rate (PER). Pacu fish fed diets containing 25% or 45% carbohydrates (CHO) were submitted to a sequence of feeding, fasting and re-feeding periods of 30 days each (Fasted), or were continuously fed. Fish were collected and analyzed after each period – days 30, 60 and 90 of the experimental protocol. Uppercase letters indicate differences between fish continuously fed and lowercase letters indicate differences between fasted fish analyzed by Tukey test (P<0.05). 39 Figure 2. Effects of carbohydrates (CHO) and feeding strategy on blood glucose, triglycerides, cholesterol and non-esterified fatty acids (NEFA) levels. Pacu fish fed diets containing 25% or 45% carbohydrates (CHO) were submitted to a sequence of feeding, fasting and re-feeding periods of 30 days each (Fasted), or were continuously fed. Fish were collected and analyzed after each period – days 30, 60 and 90 of the experimental protocol, and at day 62 for the evaluation of the acute response to re-feeding. Uppercase letters indicate differences between feeding strategies, lowercase letters (a, b) indicate differences between diets, and lowercase letters (w, x, y) indicate differences between time points analyzed by Tukey test (P<0.05). 40 Figure 3. Effects of carbohydrates (CHO) and feeding strategy on liver glycogen, liver lipids, hepatossomatic index (HSI) and mesenteric fat index (MFI). Pacu fish fed diets containing 25% or 45% carbohydrates (CHO) were submitted to a sequence of feeding, fasting and re-feeding periods of 30 days each (Fasted), or were continuously fed. Fish were collected and analyzed after each period – days 30, 60 and 90 of the experimental protocol, and at day 62 for the evaluation of the acute response to re-feeding. Uppercase letters indicate differences between feeding strategies, lowercase letters (a, b) indicate differences between diets and lowercase letters (w, x, y) indicate differences among time points analyzed by Tukey test (P<0.05). 41 Figure 4. Effects of carbohydrates (CHO) and feeding strategy on the activities of hexokinase (HK), glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PDH) and aspartate amino transferase (AST). Pacu fish fed diets containing 25% or 45% carbohydrates (CHO) were submitted to a sequence of feeding, fasting and re-feeding periods of 30 days each (Fasted), or were continuously fed. Fish were collected and analyzed after each period – days 30, 60 and 90 of the experimental protocol, and at day 62 for the evaluation of the acute response to re-feeding. Uppercase letters indicate differences between feeding strategies, lowercase letters (a, b) indicate differences between diets, lowercase letters (w, x, y) indicate differences among time points analyzed by Tukey test (P<0.05). 42 CAPÍTULO 3. Serum ammonia as indicator of unbalanced diet in pacu (Piaractus mesopotamicus) Short communication Introduction Plasma ammonia is usually used as indicator of stress in fish. Stress results in increased levels of cortisol (Wendelaar Bonga, 1997), stimulating both glycogenesis and gluconeogenesis, by increased protein catabolism (Mommsen et al., 1999) and ammonia production (Randall and Tsui, 2002). Ammonia and urea are the major nitrogenous end products in fishes, with ammonia comprising at least 80% of nitrogen excretion in most teleosts (Wright and Wood, 2012). The production of ammonia occurs mainly through the transamination of various amino acids (Forster and Goldstein 1969; Watts and Watts, 1974). The primary site for ammonia production is probably the liver (Randall and Tsui, 2002), but the necessary enzymes have also be