Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 metabolic homeostasis and energy balance, nutrient sensing mechanisms could be opted for a role in the control of proliferation. Growth cycle arrest of salmon adipocytes was in parallel with the up-regulation of the fatty acid binding protein 3 (FABP3) that participates in the uptake, intracellular metabolism and transport of long chain fatty acids. FABP3 seems like a good candidate that could be involved in this type of regula- tion since its growth arrest activity was previously demonstrated in mammalian epithelial cells [50]. Further, minimizing protein synthesis may also control proliferation as cell cycle arrest is a well established consequence of the general translational arrest, implied from day 15 onwards. Microarray analyses suggested changes in nutrient metabolism in the late phase of adipogenesis (day 30). As a consequence of the up-regulation of pyruvate dehy- drogenase kinase that inactivates pyruvate dehydrogen- ase, the Krebbs cycle most likely relied on the preferential use of acetyl-CoA from FA oxidation rather than pyruvate to cover the cellular energy requirements. Also, the expression of genes involved in FA b-oxida- tion, acyl-Coenzyme A dehydrogenase and acyl-Coen- zyme A binding protein, steadily increased towards the end of adipogenesis. The up-regulation of G6PD and PGD pointed to the importance of the PPP, which gen- erates NADPH required for accumulation of lipids. The PPP can be seen as an alternative to glycolysis, because in addition to generating NADPH and R5P, it can also provide cells with G3P, the phosphorylated glycerol backbone suitable for TAG synthesis. Induction of gly- colytic PFK was also in line with the increased produc- tion of G3P and TAG biosynthesis. Biosynthesis of carbohydrates in carnivourous fish species could be important due to the very low carbohydrate dietary level in their natural diets [51]. Indeed, we have seen regula- tions in all major glucose metabolic pathways in our model (Fig. 8); however, differentiation of adipocytes did not involve induction of HK, one of the key enzymes of glucose metabolism that only showed slight up-regula- tion at day 30. This indirectly suggested that the trans- formation of gluconeogenic amino acids into glucose is important in adipocytes of Atlantic salmon, at least in vitro. In addition, late activity of two key glyceroneo- genic genes, MDHC and PEPCKC implied that glycero- neogenesis, which can be fed with mitochondrial intermediates derived from lactate and gluconeogenic amino acids, in addition to pyruvate, was active in devel- oping fish adipocytes. To conclude, these findings illu- minate potentially novel aspects of nutrient metabolism in adipocytes, which might take advantage of the cou- pling of glycolysis, the PPP and glyceroneogenesis, in order to fulfil shifting demands for the three major pro- ducts of these pathways: G3P, R5P and NADPH. Page 11 of 17 NADPH produced by PPP is also crucially important for the cellular antioxidant defence since it is required for the regeneration of oxidized glutathione by glu- tathione reductase (GSR). Up-regulation of GSR and a suite of genes involved in metabolism of glutathione, thioredoxin and iron was a prominent feature of late salmon adipocyte differentiation (days 15-30). Indeed, a recently introduced concept [52-54,4] clearly demon- strated ROS as anti-adipogenic molecules that inhibit preadipocyte proliferation and differentiation. A reduced redox state in WAT is now recognised as a distinct characteristic of visceral obesity in mammals, with over- expression of GPX peroxidases and high content of hydrophilic antioxidant gluthathione associated with pro-adipogenic processes. Induction of the number of GPXs, known to be sensitive to lipid peroxidation, implied ROS-provoked response in our culture and the need to maintain highly reduced state of the intracellu- lar environment. However, contrary to what could be expected in situations of high ROS production, neither gene expression nor activity of SOD increased. In fact, the activity of this important ROS scavenger progres- sively declined during adipocyte differentiation. Thus, our data corroborated the indespensible role of glu- tathione-based antioxidant system in the maintenance of the reduced intracellular state in white adipocytes of fish. Accumulation of lipids in adipocytes coincided with the activation of genes coding for secreted proteins, such as adipokines adipsin and visfatin. Together with the previous finding of leptin secretion by Atlantic sal- mon adipocytes [10], this supports the notion of WAT as an active endocrine organ in fish. Enhanced secretion may impose an additional load on the ER actively engaged in lipid droplet synthesis [55]. The ER compart- ment is suspected to exert a great deal of control over adipogenesis and association of obesity with ER-stress is firmly established [56]. Up-regulation of XBP1 and ATF6 at day 15 in salmon adipocytes implied activation of UPR in response to perturbations in the ER homeos- tasis [57]. Moreover, endoplasmic GRP78, which is located downstream from ATF6 in the UPR signalling cascade was down-regulated throughout the whole stu- died period but increased in expression at day 30. A number of changes that are typical of UPR [35] was a hallmark of the latest stages of adipogenesis in our model. UPR as a collection of pathways aimed at restor- ing ER function may serve different adaptive roles and is observed both under pathological and normal physiolo- gical situations [58]; for example, it is part of the devel- opmental program in highly specialized secretory B and T cells [59] and ß-pancreatic cells [60]. The revealed UPR in developing fish adipocytes appears as a highly tailored homeostatic mechanism that is activated when