**5.3. Regulation of GLUT4 protein levels in fish**

To date, studies on the regulation of GLUT4 protein levels in fish are limited to salmonid species. The availability of a polyclonal antibody against okGLUT4 made possible the locali‐ zation and the quantification of GLUT4 in trout skeletal muscle. By performing immunoloc‐ alization studies of GLUT4 in trout skeletal muscle cells in culture, an increase in the amount of total GLUT4 protein was observed during the differentiation of myoblasts into myotubes [32]. Subsequent studies showed that the total content of GLUT4 differs between the two types of skeletal muscle in trout, with red muscle containing a higher amount of GLUT4 than white muscle [70]. In agreement with changes in the expression of GLUT4 at the mRNA level, the total amount of GLUT4 protein in red muscle increased in trout stimulated with insulin *in vivo* and decreased after fasting. On the other hand, insulin administration failed to increase GLUT4 protein content in trout white muscle [32], supporting the lack of changes of GLUT4 mRNA levels previously described [60]. Interestingly, nutritional factors regulate GLUT4 mRNA and protein levels in white muscle in different manners. In trout, fasting decreased the amount of GLUT4 protein in white muscle [32], whereas no changes in mRNA levels were observed in the same condition [60], suggesting that post-transcriptional regulation of GLUT4 expression may take place in white skeletal muscle in fish. Therefore, it appears that insulin plasma levels may regulate the amount of GLUT4 present in red skeletal muscle in fish and strongly suggest that insulin may stimulate the de novo synthesis of GLUT4, at least in red skeletal muscle, by increasing the mRNA levels of GLUT4. The lack of effects after insulin administration *in vivo* on GLUT4 mRNA and protein levels in white muscle in trout are puzzling in the light of data showing that glucose uptake increases in white muscle after a glucose load in trout and that this tissue contributes about five times more than red muscle to the total glucose uptake when expressed as percent of the total body mass [23]. Further studies are required to understand the factors and mechanisms involved in the regulation of glucose uptake in white skeletal muscle in fish.

As part of the complex regulation of GLUT4, the translocation of this glucose transporter to the PM from intracellular vesicles is highly dynamic and is regulated by a number of factors [71], representing an efficient mechanism that allows a fast equilibration of glucose levels at either side of the PM in response to a hypoglycemic stimulus. In fish, insulin has been shown to increase the PM levels of GLUT4 in *in vitro* stimulated trout muscle cells in culture [32], demonstrating that insulin stimulates glucose uptake in fish skeletal muscle cells by increasing the levels of the GLUT4 protein at the PM, as in mammals. Other stimuli that have been shown to increase the uptake of glucose by trout myocytes and that also increase the cell surface levels of GLUT4 are AMPK activators (i.e. AICAR and metformin) [66] and the pro-inflammatory cytokine tumor necrosis factor α (TNFα) [72]. These results indicate that the regulation of the total amount of GLUT4 protein in skeletal muscle and, more importantly, the cell surface levels of GLUT4 in skeletal muscle cells are similar between fish and mammals, evidencing a remarkable degree of conservation of the mechanism(s) by which insulin exerts its hypogly‐ cemic effects on skeletal muscle.
