Gen He

Differential regulation of taurine biosynthesis in rainbow trout and Japanese flounder

Animals have varied taurine biosynthesis capability, which was determined by activities of key enzymes including cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD). However, whether CDO and CSD are differentially regulated across species remains unexplored. In the present study, we examined the regulations of CDO and CSD in rainbow trout and Japanese flounder, the two fish species with high and low taurine biosynthesis ability respectively. Our results showed that the expression of CDO was lower in rainbow trout but more responsive to cysteine stimulation compared to that in Japanese flounder. On the other hand, both the expression and catalytic efficiency (kcat) of CSD were higher in rainbow trout than those of Japanese flounder. A three-residue substrate recognition motif in rainbow trout CSD with sequence of F126/S146/Y148 was identified to be responsible for high kcat, while that with sequence of F88/N108/F110 in Japanese flounder led to low kcat, as suggested by site-directed mutagenesis studies. In summary, our results determined new aspects of taurine biosynthesis regulation across species.

Chronic rapamycin treatment on the nutrient utilization and metabolism of juvenile turbot (Psetta maxima)

High dietary protein inclusion is necessary in fish feeds and also represents a major cost in the aquaculture industry, which demands improved dietary conversion into body proteins in fish. In mammals, the target of rapamycin (TOR) is a key nutritionally responsive molecule governing postprandial anabolism. However, its physiological significance in teleosts has not been fully examined. In the present study, we examined the nutritional physiology of turbot after chronic rapamycin inhibition. Our results showed that a 6-week inhibition of TOR using dietary rapamycin inclusion (30 mg/kg diet) reduced growth performance and feed utilization. The rapamycin treatment inhibited TOR signaling and reduced expression of key enzymes in glycolysis, lipogenesis, cholesterol biosynthesis, while increasing the expression of enzymes involved in gluconeogenesis. Furthermore, rapamycin treatment increased intestinal goblet cell number in turbot, while the expressions of Notch and Hes1 were down regulated. It was possible that stimulated goblet cell differentiation by rapamycin was mediated through Notch-Hes1 pathway. Therefore, our results demonstrate the important role of TOR signaling in fish nutritional physiology.

Soybean saponin modulates nutrient sensing pathways and metabolism in zebrafish

Soybean saponin (SA) is known as a major anti-nutritional factor that causes metabolic disturbances and growth reduction in fish. However, the mechanisms underlying these effects were far from fully understood. In particular, the influences of SA on nutrient sensing and downstream metabolic pathways remain largely unexplored. Using zebrafish as an animal model, this study was conducted to examine the phenotypic and molecular responses after dietary SA treatment for 2 weeks. SA at both 5 and 10 g/kg diet levels significantly reduced growth performance and feed efficiency, and damaged the morphology of the intestinal mucosa. SA stimulated AMP-activated protein kinase but reduced target of rapamycin (TOR) activities in both feeding trial and cellular studies. Furthermore, SA increased the mRNA expressions of growth axis genes including growth hormone, insulin-like growth factor 1, growth hormone receptor A, and growth hormone receptor B, but decreased insulin-like growth factor-binding protein 2 at both mRNA and protein levels. SA also increased the expressions of key metabolic enzymes involved in glutamine synthetase, glutamate dehydrogenase and lipolysis, hormone-sensitive lipase and lipoprotein lipase. Our results demonstrated that SA modulated nutrient sensing pathways and metabolism, thus provide new aspects on the explanation of the physiological effects of SA.

Development of a Whole Organism Platform for Phenotype-Based Analysis of IGF1R-PI3K-Akt-Tor Action

Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.

Ca2+ concentration–dependent premature death of igfbp5a−/− fish reveals a critical role of IGF signaling in adaptive epithelial growth

Igfbp5a enables an epithelial cell population in zebrafish to respond to low environmental Ca2+ conditions. Stressing out about low Ca2+ Under low Ca2+ conditions, an epithelial cell population in zebrafish known as NaR cells proliferate so that they can take up more Ca2+. Liu et al. found that Igf-binding protein 5A (Igfbp5a) was required for this adaptive proliferation and for survival under low Ca2+ conditions. The defect in NaR adaptive proliferation in igfbp5a−/− zebrafish was rescued by expression of wild-type human or zebrafish IGFBP5 but not two cancer-associated IGFBP5 mutants. Mice deficient in the various IGFBPs lack obvious phenotypes, and these results reveal the importance of using the appropriate physiological conditions to determine the function of IGFBPs. The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in igfbp5a did not show overt phenotypes when raised in Ca2+-rich solutions, they died prematurely in low Ca2+ conditions. A group of epithelial cells expressing igfbp5a take up Ca2+ and proliferate under low Ca2+ conditions because of activation of IGF signaling. Deletion of igfbp5a blunted low Ca2+ stress–induced IGF signaling and impaired adaptive proliferation. Reintroducing zebrafish Igfbp5a, but not its ligand binding–deficient mutant, restored adaptive proliferation. Similarly, adaptive proliferation was restored in zebrafish lacking igfbp5a by expression of human IGFBP5, but not two cancer-associated IGFBP5 mutants. Knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. These results reveal a conserved mechanism by which a locally expressed Igfbp regulates organismal Ca2+ homeostasis and survival by activating IGF signaling in epithelial cells and promoting their proliferation in Ca2+-deficient states. These findings underscore the importance of physiological context when analyzing loss-of-function phenotypes of endocrine factors.

The Mitotic and Metabolic Effects of Phosphatidic Acid in the Primary Muscle Cells of Turbot (Scophthalmus maximus)

Searching for nutraceuticals and understanding the underlying mechanism that promote fish growth is at high demand for aquaculture industry. In this study, the modulatory effects of soy phosphatidic acids (PA) on cell proliferation, nutrient sensing, and metabolic pathways were systematically examined in primary muscle cells of turbot (Scophthalmus maximus). PA was found to stimulate cell proliferation and promote G1/S phase transition through activation of target of rapamycin signaling pathway. The expression of myogenic regulatory factors, including myoD and follistatin, was upregulated, while that of myogenin and myostatin was downregulated by PA. Furthermore, PA increased intracellular free amino acid levels and enhanced protein synthesis, lipogenesis, and glycolysis, while suppressed amino acid degradation and lipolysis. PA also was found to increased cellular energy production through stimulated tricarboxylic acid cycle and oxidative phosphorylation. Our results identified PA as a potential nutraceutical that stimulates muscle cell proliferation and anabolism in fish.