Where is glut4 found

More recently, it was shown that GLUT4 expression in white skeletal muscle of trout fed a diet rich in carbohydrates was not affected [ 57 ].

Therefore, there is strong evidence suggesting that GLUT4 mRNA levels in red skeletal muscle may be regulated in vivo by circulating insulin in trout, as in mammals. However, these observations raised the question as to whether the expression of GLUT4 could be regulated in white skeletal muscle, given that it accounts for the bulk of glucose taken up by skeletal muscle.

In contrast to trout, GLUT4 mRNA levels in the white muscle of Atlantic cod increased after fasting and decreased after refeeding [ 56 ], suggesting the possibility of species-specific differences in the regulation of GLUT4 in this tissue.

Since insulin is known to stimulate the uptake of glucose by trout skeletal muscle cells in vitro [ 32 ], it has been hypothesized that this effect of insulin may have been due, at least in part, to its effects on GLUT4 expression.

Therefore, it appears that the hypoglycemic effects of insulin in fish, as in mammals, may involve the stimulation of GLUT4 mRNA expression in skeletal muscle. In mammals, exercise is known to increase the transcription of the GLUT4 gene and, consequently, to increase glucose utilization in skeletal muscle [ 61 , 62 ].

In trout, swimming-induced exercise was also recently shown to promote glucose uptake and utilization in skeletal muscle [ 64 ]. Importantly, swimming-induced exercise increased the mRNA levels of GLUT4 in red and white skeletal muscle in trout, as in mammals [ 65 ], supporting the notion that the increase in GLUT4 in skeletal muscle may have been responsible, at least in part, for the decrease in circulating glucose levels and increased uptake and utilization of glucose by skeletal muscle of exercised trout [ 64 ].

Given that swimming-induced exercise increased AMPK activity in red and white skeletal muscle in trout Magnoni and Planas, unpublished observations , there is strong evidence to believe that swimming-induced exercise increases GLUT4 mRNA levels in skeletal muscle through the induction of AMPK activity.

In mammals, the cis-regulatory region of the GLUT4 gene is relatively well characterized and is known to contain motifs that are important for the tissue-specific expression of the GLUT4 gene and its regulation. As indicated above section 5. However, the regulation of the transcription of the GLUT4 gene by insulin in mammals is not well understood, particularly in the light of published data indicating that, paradoxically, insulin inhibits the transcription of the GLUT4 gene [ 67 , 68 ].

Interestingly, a recent study reported that the activity of a fish i. Although the mechanism by which insulin represses the activity of the GLUT4 gene is not known in mammals, deletion analyses of the Fugu GLUT4 promoter have indicated that the region of the Fugu GLUT4 gene that is downstream of the main transcription start site may be sufficient for mediating the inhibitory effects of insulin on GLUT4 transcription [ 30 ].

Further studies are clearly needed to resolve the question of the paradoxical effects of insulin on GLUT4 gene transcription. Given the recent demonstration that swimming-induced skeletal muscle activity in trout increased the mRNA levels of GLUT4 in trout skeletal muscle [ 65 ], these results suggest that induction of contractile activity in skeletal muscle cells results in the transcriptional activation of the GLUT4 gene, resulting in increased GLUT4 mRNA levels that, in turn, may increase the amount of GLUT4 and, consequently, the entry and utilization of glucose in skeletal muscle in fish.

To date, studies on the regulation of GLUT4 protein levels in fish are limited to salmonid species. By performing immunolocalization 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 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.

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 hypoglycemic effects on skeletal muscle.

In mammals, the main feature that characterizes GLUT4 in skeletal muscle and adipose tissue and makes it unique is its ability to translocate to the PM in response to insulin [ 15 , 73 ]. This greatly increases the capacity of the cells to uptake glucose during the postprandial state, which is crucial to properly maintain glucose homeostasis. Notwithstanding, evidence in mammalian cells clearly indicates that in the basal state GLUT4 is not static; instead, GLUT4 circulates among numerous intracellular compartments, such as the trans-Golgi network TGN , early and late endosomes, a specialized insulin responsive compartment IRC , as well as the PM [ 71 , 74 - 75 ].

The intracellular trafficking characteristics of the two glucose transporters identified in salmonids btGLUT4 and okGLUT4 have been studied in comparison with mammalian GLUT4 mainly when expressed in heterologous systems mammalian adipocytic or myoblastic cell lines , but also as the endogenous GLUT4 in primary cultured trout myocytes.

Importantly, the basal localization of endogenous GLUT4 at the PM in trout myocytes in culture was also relatively high [ 32 ]. Therefore, under basal or unstimulated conditions fish GLUT4 appears to be less efficiently retained in the cytosol in adipocytes and myocytes than mammalian GLUT4, suggesting differences in the mechanisms responsible for the intracellular retention of GLUT4 between fish and mammals see below.

Furthermore, based on the observed differences in PM localization between fish GLUT4s under basal conditions, with okGLUT4 being more similar to its mammalian counterparts than btGLUT4, it has been suggested that the different traffic behavior of these two fish GLUT4 protein variants may be related to differences in characteristic regulatory features in the GLUT4 protein sequence i. N-and C-terminal protein motifs see section 3; [ 79 ].

Moreover, the ability of fish GLUT4s to respond to insulin has been also evaluated. The first studies trying to demonstrate that a fish GLUT4 translocates to the PM upon insulin stimulation were performed in Xenopus oocytes [ 31 ].

Nevertheless, the system was not appropriate to study the translocation of GLUT4 and oocytes expressing okGLUT4 or a rat GLUT4 did not show differences in transporter localization within the cell in response to insulin [ 31 ]. Instead, the 3T3-L1 adipocyte cell system was used successfully to demonstrate that both okGLUT4 and btGLUT4 were able to significantly translocate to the PM after insulin treatment [ 31 , 79 ], as it occurs in mammals.

Therefore, the fish homologs of GLUT4 were shown to be insulin responsive like their mammalian counterpart, despite their higher PM localization at steady-state. As previously mentioned, GLUT4 in mammals is distributed inside the cells in two major storage compartments, the IRC and the endosomal system [ 75 , 80 ]. This observation, together with the fact that btGLUT4 showed lower levels of retention in intracellular compartments during basal conditions although it still responded to insulin stimulation in both cell types [ 32 , 79 ], suggested that btGLUT4 is equally distributed between the specialized IRC and the endosomal compartment, from where it cycles continuously with the PM.

Moreover, both in 3T3-L1 adipocytes and L6 muscle cells, the higher PM levels observed for btGLUT4 were shown to be due to a faster externalization rate rather than to a decrease in the rate of endocytosis [ 32 , 79 ]. In mammals, several proteins have been described to interact with GLUT4 to regulate its intracellular traffic and to maintain the proteins sequestered in the IRC.

In this step, sortilin has been described also to have a key role, as GLUT4 does not contain the specific targeting motif to be recognized by GGA as a cargo molecule [ 82 ].

The possible roles of several GLUT4-interacting proteins in the regulation of the traffic of the fish GLUT4 isoforms have been explored in 3T3-L1 adipocytes expressing the corresponding mammalian orthologs. When a plasmid coding rat GLUT4 is transfected into 3T3-L1 adipocytes, the cells require 6 to 9 hours to produce the new protein and to target it to the IRC [ 73 , 85 ]. In contrast, both okGLUT4 and btGLUT4, when expressed in the same cellular system, undergo insulin-stimulated translocation only 3 hours after transfection [ 79 ], suggesting that fish GLUT4 undergoes faster synthesis, processing or traffic.

ER: endoplasmic reticulum. The possibility that these sequence differences were able to account for the increased basal cell surface levels observed for btGLUT4 was investigated Capilla and Planas, unpublished data. The box encloses the important trafficking motif F 5 QQI 8 partially conserved in the fish species. Thus, in order to identify the protein domains in trout GLUT4 that confer its particular traffic characteristics i.

These constructs were then stably expressed in 3T3-L1 cells and their capacity to be retained in the cytosol under basal conditions and to respond to insulin were analyzed Simoes, Planas and Camps, unpublished results. The results obtained indicated that all constructs were able to translocate to the PM in response to insulin but with certain differences among them Figure 9. Cell surface levels of various GLUT4 constructs in the presence of insulin. Differentiated 3T3-L1 adipocytes expressing the various GLUT4 constructs were incubated in the absence or presence of insulin nM for 30 min and the determination of surface GLUT4 levels was performed as described in [ 32 ].

Cell surface GLUT4 is expressed relative to the unstimulated control for each cell line. Following insulin stimulation, mammalian GLUT4 traffics and fuses with the PM, increasing its presence in the cell surface up to fold; thus, supporting the increase in glucose uptake observed after feeding.

Insulin increases the number of transporters at the PM not only by enhancing exocytosis but also by decreasing the rate of endocytosis [ 90 - 92 ]. Insulin exerts its effects through two different intracellular signaling pathways [ 15 , 93 ]. The second pathway is that including the Cbl associated protein CAP , which binds the insulin receptor and activates a small GTPase from the Rho family named TC10, and that was described in adipocytes [ 97 ].

However, the TC10 pathway appears not to be involved in muscle cells, in which another Akt-independent input was shown to contribute to the cytoskeleton remodeling required for complete GLUT4 translocation [ 98 , 99 ]. Downstream of Akt, a protein named Akt substrate of KDa AS or TCB1D4, has been found to be the key to communicate the phosphorylation cascade initiated by insulin with the vesicle trafficking machinery [ , ].

When co-expressed in a cellular system together with rat GLUT4, the translocation of this molecule to the PM was blocked, as well as the increase in glucose uptake observed after insulin incubation [ 40 , ]. All the evidence accumulated to date on the function and regulation of GLUT4 in fish indicates that the various molecular and cellular mechanisms regulating the amount of GLUT4 that is present at the cell surface in skeletal muscle and adipose tissue cells and that determine the amount of glucose uptake have been relatively well conserved during evolution from fish to mammals.

Glucose Transporter Type 4 GLUT4 is an insulin-regulated membrane protein responsible for decreasing blood glucose concentration found in both adipose tissues as well as striated muscle [1]. Activity of this protein is primarily regulated by the signal transduction pathway and several transcriptional factors found in the tissues where GLUT4 is located [2] [3] [4]. GLUT4 is responsible for returning blood glucose to a physiological concentration of mM after the ingestion of carbohydrates, as well as increasing the rate of glycogen synthesis in skeletal muscle following glycogen depleting exercise [5] [6].

During periods in which insulin is low, GLUT4 is found in intracellular vesicles among adipose and muscle cells. When blood glucose increases and insulin spikes, insulin binds to insulin receptors on the plasma membrane. This activation starts a protein activation cascade leading to the translocation of GLUT4, starting with the phosphorylation of Insulin Receptor Substrate, which then binds PI-3 kinase.

PI-3 kinase is responsible for the conversion of membrane lipid phosphatidylinositol 4,5-bisphosphate PIP2 to phosphatidylinositol 3,4,5 -triphosphate PIP3. In total homogenates of hippocampi, cortex and cerebelli from adult CD-1 mice and Sprague-Dawley rats, Glut4 was dramatically enriched in the latter preparation. Glut4 protein was undetectable in cerebelli of newborn animals and was dramatically up-regulated with age.

Immunohistochemistry of mouse brain sections showed that Glut4 expression was limited to the granular layer of the cerebellum. Immunofluorescence analysis of cultured cerebelli neurons demonstrated that Glut4 was localized predominantly in the perinuclear region similar to its localization in insulin-sensitive cells. In order to further study the intracellular localization of Glut4 in neurons, cerebelli extracts were fractionated in sucrose velocity and equilibrium density gradients.

Glut4 was found in an individual vesicular population, which was separated from both small synaptic vesicles and large dense core granules and had the same sedimentation coefficient and the buoyant density as insulin-responsive Glut4-vesicles from fat and skeletal muscle tissues. In addition, Glut4-vesicles from cerebellum neurons contained IRAP and sortilin, the two marker proteins of the insulin-responsive Glut4-vesicles from fat and skeletal muscle. Treatment of cultured cerebellum neurons with insulin caused marked translocation of Glut4 to the plasma membrane.