Rainbow trout as a model for nutrition and nutrient metabolism studies

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The importance for studying AA metabolism in fish

Based on traditional approaches (e.g., digestibility trials, nitrogen balance, assessments of growth, feed utilization and reproductive performance, isotope tracer techniques, as well as northern and western blots), significant developments have been already made for studying gross protein requirements, qualitative AA requirements, AA utilization and deposition, substitution of fish meal with alternative protein sources in the last half century (Guillaume et al., 2001; Halver and Halver, 2002; Kaushik and Seiliez, 2010; Wilson and Halver, 1986). However, due to the complexities and handicaps in studying fish nutrition, i.e. difficulties in study linked to the environment and the nutritional characteristics of fish, the large number of fish species and currently poorly applied advanced techniques in fish nutrition studies, our knowledge regarding the roles of AAs in regulating gene expression, cellular signaling pathways and intermediary metabolism remains limited (Kaushik and Seiliez, 2010). Therefore, it would be desirable to go on exploring the physiological and metabolic importance of protein/AAs at molecular and mechanism levels so as to expand our basic knowledge of AA biochemistry and nutrition in fish. Furthermore, the emergence of advanced bio-molecular method applied to fish (e.g., high-throughput functional genomics, microarray, metabolomics and proteomics) offers us new opportunities to do so. From the practical aspect, how to effectively improve the utilization of protein/AAs, especially the protein from non-fishmeal sources in fish is the key to go on improving feed efficiency, reducing the inclusion rates of FM in aquafeeds and cutting the rising costs for the feed enterprises. Thus, understanding the mechanisms underlying the regulation of intermediary metabolism, gene expression and cellular signaling pathways by protein/AAs may have both scientific and practical significance.

Overview of insulin signaling pathway and some critical nodes

Growth factors such as insulin activate the insulin receptor (IR) tyrosine kinase, which phosphorylates and recruits different substrate adaptors such as the IRS family of proteins (Siddle, 2011). Tyrosine phosphorylated insulin receptor substrates (IRS) then displays binding sites for numerous signaling partners (Taniguchi et al., 2006). Among them, phosphoinositide 3-kinase (PI3K) has a major role in insulin function, mainly via the activation of the Akt cascades (Manning and Cantley, 2007). Activated Akt induces glycogen synthesis through inhibition of glycogen synthase kinase 3 (GSK-3), protein synthesis via the activation of the mechanistic target of rapamycin (mTOR) pathway, and cell survival through– inhibition of several pro-apoptotic agents (Forkhead box protein O (FoxO), mammalian sterile 20 like kinase-1(MST1) and GSK-3) (Siddle, 2011). Akt phosphorylates FoxO and directly inhibits transcription factors, which regulates metabolism and autophagy (Figure 1.7). Insulin stimulates glucose uptake in muscle and adipocytes via translocation of glucose transporter 4 (GLUT4) vesicles to the plasma membrane (Rowland et al., 2011). In addition, insulin signaling inhibits gluconeogenesis in the liver, through disruption of CREB/CBP/mTORC2 binding (Manning and Cantley, 2007). Insulin signaling induces fatty acid and cholesterol synthesis via the regulation of SREBP transcription factor (Wong and Sul, 2010). Insulin signaling also promotes fatty acid synthesis through activation of upstream transcription factor 1 (USF1) and liver X receptor (LXR) (Shao and Espenshade, 2012).

Insulin stimulates lipid synthesis but inhibits lipid degradation

Insulin signaling also plays an important role in the regulation of fatty acids (FAs) metabolism, underscoring the close relation between lipid and glucose metabolism (Bechmann et al., 2012). Insulin stimulates fatty acids and triglyceride synthesis mainly by increasing the mRNA and the processed nuclear form of sterol regulatory element-binding protein-1c (SREBP-1c) (Jeon and Osborne, 2012; Shao and Espenshade, 2012), a transcription factor that activates all the genes needed to produce fatty acids and triglycerides in liver (Horton et al., 2002). Furthermore, insulin positively regulates Akt-mediated production of very low-density lipoproteins (VLDLs) (Savage and Semple, 2010). Moreover, insulin, along with other mediators (e.g., calpain-1), represses autophagy and thus the associated lipophagy within the hepatocyte, and thus induces lipogenesis and represses lipid degradation in the fed state as well (Rautou et al., 2010).
In adipocytes, glucose is stored primarily as lipid, owing to increased uptake of glucose and activation of lipid synthetic enzymes, including pyruvate dehydrogenase, fatty acid synthase and acetyl-CoA carboxylase (Saltiel and Kahn, 2001). Insulin also profoundly inhibits lipolysis in adipocytes, primarily through inhibition of the enzyme hormone-sensitive lipase (Anthonsen et al., 1998). This enzyme is acutely regulated by control of its phosphorylation state, which is activated by protein kinase A (PKA)-dependent phosphorylation, and inhibited as a result of a combination of kinase inhibition and phosphatase activation. Insulin inhibits the activity of the lipase primarily through reductions in cAMP levels, owing to the activation of a cAMP-specific phosphodiesterase in fat cells (Kitamura et al., 1999).

Impaired insulin action and insulin resistance

Defective insulin secretion or impaired insulin action may lead to multiple metabolic abnormalities (Muoio and Newgard, 2008). Insulin resistance, which is defined as the inability of insulin to promote efficient glucose uptake by peripheral tissues, is a metabolic condition associated with obesity, type 2 diabetes (T2D), dyslipidemia, and cardiovascular diseases (Laplante and Sabatini, 2010). Under insulin-resistant status, impaired insulin signaling leads to hyperglycaemia due to impaired insulin-stimulated glucose uptake in the skeletal muscle, uncontrolled hepatic glucose production in the liver and hypertriglyceridaemia due to misdelivered glucose, enhanced lipogenesis in the liver, and increased lipolysis in the adipose tissue (Figure 1.10) (Muoio and Newgard, 2008; Samuel and Shulman, 2012). Chronically, these increases in circulating glucose and lipid levels can further impair insulin secretion and action, and cause other forms of tissue damage, thereby inducing or worsening obesity, T2D and nonalcoholic fatty liver disease (NAFLD) (Lynch and Adams, 2014; Newgard, 2012; Zoncu et al., 2011).

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mTORC1 controls protein synthesis

Protein synthesis is by far the best-characterized process controlled by mTORC1 (Laplante and Sabatini, 2012; Shimobayashi and Hall, 2014). When activated, mTORC1 stimulates both an acute increase in the translation of specific mRNAs and a broader increase in the protein synthetic capacity of the cell (Dibble and Manning, 2013). mTORC1 promotes protein synthesis by phosphorylating the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase 1 (S6K1) (Figure 1.14 A) (Laplante and Sabatini, 2009). The phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E at the 5 -cap of mRNAs and blocking assembly of the translation initiation complex to promote cap-dependent translation (Richter and Sonenberg, 2005). Activated S6K1 by mTORC1 increases in mRNA biogenesis, cap-dependent translation and elongation, and the translation of ribosomal proteins via regulating the activity of many proteins, such as S6K1 aly/REF-like target (SKAR), programmed cell death 4 (PDCD4), eukaryotic elongation factor 2 kinase (eEF2K) and ribosomal protein S6 (Ma and Blenis, 2009). Additionally, the activation of mTORC1 also can promote ribosome biogenesis by stimulating the transcription of ribosomal RNA through a process involving the protein phosphatase 2A (PP2A) and the transcription initiation factor IA (TIF-IA), which promotes its interaction with RNA Polymerase I (Pol I) and the expression of ribosomal RNA (rRNA) (Mayer et al., 2004). Therefore, the acute translational control over this class of mRNAs allows mTORC1 signaling to globally enhance cellular protein synthesis.

mTORC1 controls lipid metabolism

mTORC1 plays a central role in the control of lipid metabolism (Caron et al., 2015; Lamming and Sabatini, 2013; Ricoult and Manning, 2013). Activated mTORC1 can stimulate lipid synthesis, as well as promote glucose uptake, glycolysis and NADPH production to support this anabolic process (Dibble and Manning, 2013). mTORC1 promotes lipogenesis by activating SREBPs through multiple manners (Figure 1.14 B and 1.15) (Caron et al., 2015; Han et al., 2015). Using a small molecule inhibitor of S6K, Owen et al. (Owen et al., 2012) find that the transcriptional regulation of SREBP1c by insulin is not dependent on S6K, whereas posttranscriptional processing of SREBP1c is S6K dependent. mTORC1 also regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase that down-regulates SREBP activity (Peterson et al., 2011). Phosphorylation of lipin1 by mTORC1 regulates its subcellular localization, with phosphorylated lipin1 residing in the cytoplasm and dephosphorylated lipin1 accumulating in the nucleus. Nuclear lipin1 represses SREBP-dependent gene transcription by reducing the nuclear SREBP protein levels (Peterson et al., 2011). Moreover, latest study demonstrated that activated mTORC1 promotes the COPII-dependent SREBP1 maturation through the transcriptional coactivator CREB regulated transcription coactivator 2 (CRTC2) (Han et al., 2015). Han et al. (Han et al., 2015) demonstrated that CRTC2 competes with Sec23A, a subunit of the COPII complex, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport from ER to the Golgi (Figure 1.15 B). During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. Therefore, CRTC2 functions as a mediator of mTOR signaling to modulate COPII-dependent SREBP1 processing.

Table of contents :

1.1 Aquaculture development: current status, trends and future prospects
􀍳.􀍳.􀍳 Aquaculture􀇯s role in the global good system, trends and prospects
1.1.2 Towards sustainable aquaculture
1.1.3 The importance of protein/amino acids research for aquaculture
1.2 Cellular signaling
1.2.1 Insulin signaling in mammals Overview of insulin signaling pathway and some critical nodes Insulin regulates glucose metabolism Insulin stimulates lipid synthesis but inhibits lipid degradation Insulin signaling regulates mitochondrial metabolism Impaired insulin action and insulin resistance
1.2.2 AA signaling pathways: mTOR & AAR pathway in mammals Overview of mTOR signaling pathway in mammals mTORC1 signaling pathway in mammals mTORC2 signaling pathway in mammals AAR pathway in mammals
1.2.3 AAs & insulin secretion, insulin action and insulin resistance in mammals
1.2.4 AAs and insulin/mTOR signaling pathways in fish
1.3 The regulation of hepatic intermediary metabolism in mammals and fish
1.3.1 The regulation of hepatic glucose metabolism Hepatocyte glucose uptake and phosphorylation Glycolysis and glycogen synthesis Glycogenolysis and gluconeogenesis
1.3.2 The regulation of hepatic lipid metabolism in mammals and fish Hepatic lipogenesis FA catabolism/β-Oxidation
1.3.3 The regulation of hepatic amino acid catabolism in mammals and fish
1.4 Rainbow trout as a model for nutrition and nutrient metabolism studies
1.5 Hypothesis and objectives of the thesis
2.1 Experimental trials
2.1.1 Ethics statement
2.1.2 In vivo experimental trial Experimental and sampling procedures Sampling procedure
2.1.3 In vitro experiments Animals Primary cell culture of rainbow trout hepatocytes Stimulations and cell recovery
2.2 Analytical methods
2.2.1 Plasma metabolites analysis
2.2.2 Western blot analysis
2.2.3 Gene expression analysis: real time RT-PCR
2.2.4 Enzyme activity analysis
2.2.5 Statistical analysis
Chapter 3 RESULTS
4.1 mTOR activation, overactivation and inhibition
4.2 mTORC1 activation regulates hepatic intermediary metabolism
4.3 Nutritional regulation of hepatic fatty acid biosynthesis in rainbow trout
4.4 The regulation of hepatic gluconeogenesis
4.5 The regulation of AA catabolism


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