Physiological Role of Akt in Insulin-Stimulated Translocation of GLUT4 in Transfected Rat Adipose Cells

Li-Na Cong, Hui Chen, Yunhua Li, Lixin Zhou, Margaret A. McGibbon, Simeon I. Taylor and Michael J. Quon

Hypertension-Endocrine Branch National Heart, Lung, and Blood Institute (L.-N.C., H.C., Y.L., M.J.Q.) and Diabetes Branch National Institute of Diabetes and Digestive and Kidney Diseases (L.Z., M.A.M., S.I.T.) National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of glucose transport is among the most important metabolic actions of insulin. Studies in adipose cells have demonstrated that insulin stimulates its receptor to phosphorylate tyrosine residues in IRS-1, leading to activation of phosphatidylinositol 3-kinase, which plays a necessary role in mediating the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase recently identified as a direct downstream target of phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed that overexpression of a constitutively active mutant of Akt is sufficient to recruit GLUT4 to the cell surface. Since effects of overexpression of signaling molecules in tissue culture models do not always reflect physiological function, we have overexpressed a dominant inhibitory mutant of Akt in rat adipose cells to investigate the effects of inhibiting endogenous Akt in a physiologically relevant insulin target cell. Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin is an important regulator of growth, differentiation, and metabolism. The ability of insulin to recruit the insulin-responsive glucose transporter GLUT4 from an intracellular compartment to the cell surface in muscle and adipose tissue accounts for the majority of insulin’s effect to stimulate glucose uptake in the body. We, and others, have previously shown that the insulin receptor tyrosine kinase, IRS-1, and phosphatidylinositol 3-kinase (PI3K) are key elements of the signal transduction pathway responsible for insulin-stimulated translocation of GLUT4 (Refs. 1–9 and references contained therein).

Akt is a protooncogene encoding a serine-threonine kinase (also known as PKB or RAC-PK). Recently, Akt has been identified as a downstream target of PI3K that mediates mitogenic actions and antiapoptotic effects of growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor, insulin-like growth factor I, and insulin that are initiated by their cognate receptor tyrosine kinases (for review see Ref.10). Interestingly, overexpression of constitutively active mutants of Akt in 3T3-L1 cells results in spontaneous differentiation of these preadipose cells into an adipocyte-like phenotype and also results in increased glucose uptake and elevated levels of cell surface GLUT4 in the absence of insulin in the differentiated cells (11, 12). Furthermore, glycogen synthase kinase-3 (GSK3, an enzyme involved with the regulation of glycogen synthesis by insulin) has been identified as a physiological substrate for Akt (13). These studies suggest that Akt may participate in metabolic signaling pathways for insulin (in addition to its mitogenic functions). However, overexpression of constitutively active mutants in tissue culture cells may result in effects that do not reflect what occurs under physiological conditions. In this study, we have overexpressed wild-type, constitutively active, or dominant inhibitory forms of Akt in primary cultures of rat adipose cells. Overexpression of wild type and constitutively active Akt resulted in increased levels of cell surface GLUT4 in the absence of insulin. More importantly, overexpression of a dominant inhibitory mutant of Akt resulted in inhibition of insulin-stimulated translocation of GLUT4. The dominant inhibitory mutant of Akt that we use in this study is a kinase-deficient Akt that results from the substitution of alanine for lysine at position 179 in the canonical ATP-binding domain. This mutant is not only catalytically inactive but has been shown to inhibit the activity and actions of endogenous Akt (presumably by competing with endogenous Akt for other upstream or downstream molecules) (14, 15, 16). Our results, in a bona fide insulin target cell, suggest a physiological role for Akt in insulin-stimulated glucose transport that may also apply to other metabolic actions of insulin.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Overexpression of Akt Constructs
To directly evaluate the role of Akt in insulin-stimulated translocation of GLUT4, we transfected wild type Akt (Akt-WT), Akt-Myr, Akt-K179A, or Myr-K179A into primary cultures of rat adipose cells. We confirmed overexpression of all four Akt constructs by immunoblotting cell extracts isolated from transfected cells with an anti-Akt antibody (Fig. 1Go). In the lane containing cell extracts from control cells transfected with the empty expression vector pCIS2, there is a faint band representing endogenous rat Akt. Lanes containing extracts from groups of cells transfected with recombinant Akt constructs show overexpression of recombinant Akt at levels that are much higher than the endogenous Akt levels. Since only 5% of the adipose cells that have undergone electroporation are actually transfected (1), we estimate that there is at least 100-fold overexpression of the recombinant Akt constructs relative to endogenous Akt in the transfected cells. The constructs with a mutation in the ATP-binding site (Akt-K179A and Myr-K179A) are predicted to act in a dominant inhibitory manner that depends on the level of overexpression. Therefore, we tested the effect of increasing the concentration of plasmid DNA used for transfection on expression levels for these constructs. As expected, increasing the concentration of DNA from 4 µg/cuvette to 7 µg/cuvette significantly increased the levels of overexpression for Akt-K179A (Fig. 1Go, compare lanes 4 and 5).



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Figure 1. Overexpression of Recombinant Mouse Akt Constructs in Transfected Rat Adipose Cells

Whole cell homogenates derived from cells transfected with either pCIS2, Akt-WT, Akt-K179A, Akt-myr, or Myr-K179A were subjected to immunoblotting with an anti-Akt antibody. In lane 1, containing extract from cells transfected with the empty expression vector pCIS2, a band representing endogenous rat Akt can be seen. Overexpression of recombinant Akt constructs can be seen in lanes 2, 3, and 4 containing extracts from cells transfected with Akt-WT (4 µg/cuvette), Akt-myr (4 µg/cuvette), or Akt-K179A (4 µg/cuvette). Lanes 5 and 6 contain extracts from cells transfected with Akt-K179A or Myr-K179A at higher DNA concentrations (7 µg/cuvette). By scanning densitometry, the relative intensities of the bands representing Akt in lanes 1–6 were 420, 4510, 3790, 2920, 4220, and 4150, respectively. It is possible that the Akt antibody we used does not detect rat and mouse Akt with equal efficiency. A representative blot is shown from an experiment that was repeated independently three times.

 
To confirm that the overexpressed Akt constructs were functional, we assessed Akt kinase activity in adipose cells overexpressing either Akt-WT or Akt-myr (Fig. 2Go). Whole cell homogenates derived from groups of transfected cells treated without or with insulin were subjected to immunoprecipitation with an Akt antibody, and the ability of the immunoprecipitates to stimulate incorporation of [32P]ATP into the substrate histone 2B was measured. In control cells transfected with the empty expression vector pCIS2, there was a small increase in endogenous Akt activity caused by insulin stimulation. Cells overexpressing Akt-WT showed a marked increase in detectable Akt activity upon insulin stimulation (compared with control cells) while cells overexpressing the constitutively active Akt-myr had high levels of Akt activity in both the basal and insulin-stimulated states as expected. We did not measure Akt activity in cells transfected with the kinase-inactive mutants because complete inhibition of Akt activity in the 5% of cells that are transiently transfected would be difficult to detect against the background of 95% untransfected cells.



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Figure 2. Akt Kinase Activity in Adipose Cells Overexpressing Akt-WT or Akt-myr

Whole cell homogenates derived from cells transfected with pCIS2, Akt-WT, or Akt-myr and treated without or with insulin (60 nM) were subjected to immunoprecipitation with an antibody against Akt, and the kinase activity in the immunoprecipitates was assessed by incorporation of [32P]ATP into histone 2B. Relative intensities of bands representing phosphorylated histone 2B in lanes 1–6 were 149, 178, 109, 308, 438, and 537, respectively, as assessed by PhosphorImager.

 
Effects of Overexpression of Akt-WT and Akt-K179A on Translocation of GLUT4
After confirming overexpression and activity of the recombinant Akt constructs in transfected adipose cells, we next determined their effects on the ability of insulin to recruit a cotransfected epitope-tagged GLUT4 to the cell surface. The insulin dose-response curve for control cells cotransfected with the empty expression vector pCIS2 and GLUT4-HA showed a 3-fold increase in cell surface GLUT4-HA upon maximal insulin stimulation (60 nM) with an ED50 of 0.06 nM (Fig. 3AGo). Interestingly, in the absence of insulin, overexpression of Akt-WT resulted in significant translocation of the cotransfected GLUT4-HA to the cell surface to levels that were approximately 80% of that seen in the control cells treated with a maximally stimulating dose of insulin. Treatment of cells overexpressing Akt-WT with insulin increased the amount of GLUT4-HA at the cell surface to levels that were comparable to levels observed in the control cells treated with 60 nM insulin. These results demonstrate that overexpression of wild type Akt is sufficient to cause translocation of GLUT4 in adipose cells.



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Figure 3. Insulin-Stimulated Recruitment of GLUT4-HA to the Cell Surface of Adipose Cells Overexpressing Akt-WT or Akt-K179A

The amount of each plasmid transfected is presented in Table 1Go. Data are expressed as a percentage of cell surface GLUT4 in the presence of a maximally effective insulin concentration for the control group (pCIS2/GLUT4-HA). A, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with either Akt-WT/GLUT4-HA (•) or pCIS2/GLUT4-HA ({circ}). Results are the mean ± SEM of five independent experiments. The actual value for the specific cell-associated radioactivity for the control group at 60 nM insulin was 1082 ± 250 cpm. The best-fit curve generated from the mean data for the control group had an ED50 = 0.06 nM. The level of cell surface GLUT4-HA in the Akt-WT group in the absence of insulin was approximately 80% of the level seen in the control group maximally stimulated with insulin. At maximally stimulating concentrations of insulin (60 nM), there was no significant difference in the level of cell surface GLUT4-HA between the two groups. The two curves are significantly different when analyzed by multivariate ANOVA (F = 12.4, P < 0.001). B, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with either Akt-K179A/GLUT4-HA ({blacktriangleup}) or pCIS2/GLUT4-HA ({circ}). Results are the mean ± SEM of five independent experiments. The actual value for the specific cell-associated radioactivity for the control group at 60 nM insulin was 1240 ± 251 cpm. The best-fit curve generated from the mean data for the control group had an ED50 = 0.05 nM. The best-fit curve generated from the mean data for the control group had an ED50 = 0.13 nM. At every insulin dose tested, the level of cell surface GLUT4-HA in cells overexpressing Akt-K179A was significantly less than that of the control cells (20% decrease at 60 nM insulin). In addition, the two curves are significantly different when analyzed by multivariate ANOVA (F = 51, P < 1 x 10-8).

 
To determine whether Akt plays a necessary physiological role in insulin-stimulated translocation of GLUT4, we overexpressed Akt-K179A, a point mutant of Akt that does not bind ATP and thus has no kinase activity. This mutant is predicted to behave in a dominant inhibitory fashion. When we transfected cells with Akt-K179A at the same DNA concentration as we used for Akt-WT (4 µg/cuvette), the insulin dose-response curve for translocation of GLUT4-HA was similar to that of the control cells (data not shown). These results suggest that kinase activity is important for the effect of overexpressed Akt-WT on translocation of GLUT4. More importantly, when we transfected higher concentrations of Akt-K179A (7 µg/cuvette), we observed a statistically significant 20% decrease in insulin responsiveness and 2.5-fold decrease in insulin sensitivity with respect to translocation of GLUT4 (Fig. 3BGo). These results are consistent with the idea that endogenous Akt contributes significantly to insulin-stimulated glucose transport in a bona fide insulin target tissue under physiological conditions.

To help rule out the possibility that differences we observed in the insulin dose-response curves for cells overexpressing the various Akt constructs are due to effects of these constructs on expression of GLUT4-HA, we evaluated total levels of GLUT4-HA in cells cotransfected with GLUT4-HA and the various Akt constructs. Total membrane fractions derived from each group of transfected cells were immunoblotted with an anti-HA antibody (Fig. 4Go). The results of this experiment demonstrate that there is no detectable effect of overexpressing the various Akt constructs on the total level of GLUT4-HA in transfected cells. Thus, any differences in the insulin dose-response curves of cells overexpressing the Akt constructs are most likely due to effects of these constructs on signal transduction pathways related to translocation of GLUT4.



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Figure 4. Cotransfected Cells Express Comparable Levels of GLUT4-HA

Total membrane fractions prepared from cells co-transfected with GLUT4-HA (2 µg/cuvette) and either pCIS2, Akt-WT, or Akt-myr (4 µg/cuvette), or Akt-K179A or Myr-K179A (7 µg/cuvette) were subjected to immunoblotting with the anti-HA antibody HA-11. Cells transfected with pCIS2 alone represent a negative control since these cells do not express GLUT4-HA (lane 1). Comparable levels of GLUT4-HA are seen for cells cotransfected with GLUT4-HA and either pCIS2, Akt-WT, Akt-myr, Akt-K179A, or Myr-K179A (lanes 2–6, density in arbitrary units = 1310, 1160, 954, 1089, and 1120, respectively). A representative blot is shown from an experiment that was repeated independently twice.

 
Effects of Overexpression of Akt-myr and Myr-K179A on Translocation of GLUT4
Since localization to the cell membrane is thought to play an important role in the activation of Akt, we also investigated effects of overexpressing Akt constructs that are targeted to the cell membrane as a result of having a myristoylation sequence fused in-frame with the N terminus of Akt. In the absence of insulin, overexpression of Akt-myr in adipose cells resulted in a dramatic translocation of the cotransfected GLUT4-HA to the cell surface to levels that were approximately 150% of the levels observed in the control cells treated with a maximally stimulating dose of insulin (Fig. 5AGo). Interestingly, this effect was not influenced significantly by insulin treatment. These results demonstrate that targeting wild type Akt to the cell membrane is sufficient to generate a signal for translocation of GLUT4 that exceeds that produced by maximal insulin stimulation under physiological conditions.



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Figure 5. Insulin-Stimulated Recruitment of GLUT4-HA to the Cell Surface of Adipose Cells Overexpressing Akt-myr or Myr-K179A

The amount of each plasmid transfected is presented in Table 1Go. Data are expressed as a percentage of cell surface GLUT4 in the presence of a maximally effective insulin concentration for the control group (pCIS2/GLUT4-HA). A, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with either Akt-myr/GLUT4-HA (•) or pCIS2/GLUT4-HA ({circ}). Results are the mean ± SEM of three independent experiments. The actual value for the specific cell-associated radioactivity for the control group at 60 nM insulin was 1290 ± 215 cpm. The best-fit curve generated from the mean data for the control group had an ED50 = 0.06 nM. In the absence of insulin, and at every insulin dose tested, the level of cell surface GLUT4-HA in cells overexpressing Akt-myr was approximately 150% of the level seen in the control group maximally stimulated with insulin. The difference in the two curves is statistically significant by multivariate ANOVA (F = 190, P < 5 x 10-12). B, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with either Myr-K179A/GLUT4-HA ({blacktriangleup}) or pCIS2/GLUT4-HA ({circ}). Results are the mean ± SEM of five independent experiments. The actual value for the specific cell-associated radioactivity for the control group at 60 nM insulin was 998 ± 220 cpm. The best-fit curve generated from the mean data for the control group had an ED50 = 0.06 nM. The two curves are statistically different by multivariate analysis of variance (F = 19, P < 1 x 10-4).

 
To determine whether Akt kinase activity is important for mediating the effects of the membrane-targeted Akt on translocation of GLUT4, we overexpressed a point mutant of the Akt-myr construct (Myr-K179A) that does not bind ATP and therefore has no kinase activity. Transfection of adipose cells with high concentrations of Myr-K179A (7 µg DNA/cuvette) resulted in an inhibition of insulin-stimulated translocation of GLUT4 that was similar to that observed with Akt-K179A (Fig. 5BGo). These results demonstrate that targeting of Akt to the cell membrane is not sufficient to mediate translocation of GLUT4 in the absence of kinase activity. Furthermore, the inhibitory effect of overexpression of Myr-K179A is consistent with a physiological role for endogenous Akt in insulin-stimulated translocation of GLUT4. As with Akt-WT and Akt-K179A, the fact that overexpression of Akt-myr and Myr-K179A did not significantly affect expression of GLUT4-HA (Fig. 4Go) suggests that any differences observed in the insulin dose-response curves for cells overexpressing Akt-myr or Myr-K179A are due to effects of these Akt constructs on signaling pathways.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor belongs to a large family of ligand-activated tyrosine kinases that includes receptors for growth factors such as insulin-like growth factor I, PDGF, epidermal growth factor, and fibroblast growth factor. Although insulin and these other growth factors mediate a diverse array of biological effects, the signal transduction pathways for their receptors share many common elements. For example, PI3K is activated when the SH2 domains of the p85-regulatory subunit of PI3K bind to specific phosphorylated tyrosine motifs located either on the growth factor receptors themselves or on substrates of the receptors such as IRS-1 (for review see Refs. 4 and 17). PI3K is involved with both metabolic signaling by insulin (3) and mitogenic signaling by other growth factors (18). However, activation of PI3K by growth factors such as PDGF is not sufficient to mimic metabolic effects of insulin in adipose cells (19, 20). Thus, specificity in insulin signaling must be generated by other cell type-specific mechanisms such as subcellular localization of specific PI3K-signaling complexes (21), the use of multiple isoforms of PI3K (18), integration of multiple upstream signals (22), or divergent downstream pathways.

Recently, Akt has been identified as a direct downstream target of PI3K that plays an important role in mediating the mitogenic and antiapoptotic effects of several growth factors including insulin (10, 14, 15, 16, 23, 24, 25, 26, 27). Products of PI3K such as phosphatidylinositol-3,4-diphosphate bind to the N-terminal PH domain of Akt and may serve to recruit Akt to the cell membrane and activate its serine-threonine kinase activity (28, 29). The oncogenic form of Akt (v-Akt) contains a Gag sequence that is myristoylated and results in targeting of v-Akt to the cell membrane and constitutive activation (30). Using a myristoylated form of Akt lacking the PH domain, Kohn et al. (12) recently showed that overexpression of this constitutively active mutant resulted in recruitment of GLUT4 to the cell surface in differentiated 3T3-L1 cells. Although these experiments suggest activated Akt is sufficient to recruit GLUT4, they did not determine whether Akt is a necessary factor in insulin-stimulated translocation of GLUT4.

The 3T3-L1 cell line is a tissue culture model derived from primitive mouse mesodermal cells that can differentiate into an adipocyte-like phenotype under the appropriate conditions (31). These tissue culture cells have been extremely useful for understanding the regulation and control of adipocyte differentiation (32, 33). However, these cells do not always differentiate completely or uniformly. Furthermore, they do not display the full repertoire of genes expressed in primary adipose cells. For example, ob gene mRNA levels in differentiated 3T3-L1 cells are <1% of the levels observed in freshly isolated rat adipose cells. Finally, these tissue culture cells are much less responsive than isolated rat adipose cells with respect to the effects of insulin and other hormones on glucose transport and metabolism.

To address the physiological role of Akt in insulin-stimulated glucose transport in a bona fide insulin target cell, we have overexpressed a dominant inhibitory mutant of Akt in primary cultures of rat adipose cells to determine whether endogenous Akt is necessary for insulin-stimulated translocation of GLUT4.

Overexpression of Akt Constructs in Adipose Cells
Transfection of rat adipose cells with the various Akt constructs resulted in high levels of overexpression similar to what we previously observed with other recombinant genes in our system (1, 3, 34, 35). In addition, we were able to demonstrate that increasing the concentration of DNA during transfection for the K179A construct led to a comparable increase in the expression of these constructs. Interestingly, Akt-WT had somewhat higher levels of expression than Akt-K179A when the same concentrations of DNA were used for transfection. This finding is similar to a previous report in transfected 293 cells overexpressing wild type and kinase-dead forms of Akt (15). In the case of Akt-WT and Akt-myr, we were also able to demonstrate that overexpression of these recombinant proteins resulted in Akt activity that was consistent with the level of expression and expected kinase activity. That is, the ability of Akt-WT to phosphorylate histone 2B was increased in response to insulin while Akt-myr showed high constitutive kinase activity. Unfortunately, the 5% transfection efficiency of our adipose cell transfection system (1) limits our ability to directly measure decreases in Akt activity expected with the kinase-inactive mutants Akt-K179A and Myr-K179A because of the background activity of 95% of the untransfected cells.

As discussed previously (35), the use of epitope-tagged GLUT4 allows us to distinguish and study transfected cells without interference from nontransfected cells. The fact that levels of GLUT4-HA were comparable in all groups of transfected cells suggests that changes in cell surface GLUT4 caused by overexpression of the various Akt constructs are due to effects on insulin signal transduction pathways rather than effects on the total level of expression of GLUT4. Although highly unlikely, it is formally possible that effects of the Akt constructs on translocation of GLUT4 are due, in part, to effects of Akt on levels of expression of upstream signaling molecules such as the insulin receptor or IRS-1. We do not believe this is the case, however, because we have previously demonstrated that 20-fold overexpression of these upstream molecules does not have as large an effect on translocation of GLUT4 as overexpression of the constitutively active Akt (1, 2). Nevertheless, it is difficult for us to directly rule out an effect of Akt on expression level of these upstream signaling molecules because it is not possible for us to assess the amounts of endogenous insulin receptor and IRS-1 exclusively in the 5% of cells that are transfected with the Akt constructs. In our cotransfection experiments, we used at least twice as much DNA for the Akt constructs as we did for GLUT4-HA to increase the likelihood that cells transfected with GLUT4-HA were also transfected with the vector of interest. If some fraction of cells were transfected only with GLUT4-HA, our results would underestimate the differences between control and experimental groups. We estimate that at least 95% of cells expressing GLUT4-HA also express the cotransfected second plasmid under our experimental conditions (3).

Recruitment of GLUT4 by Akt-WT and Akt-myr
Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface in the absence of insulin. These results are similar to what we previously observed with overexpression of either wild type insulin receptors or IRS-1 in adipose cells (1, 2). Presumably, there is a small signal present even in the absence of insulin that can be amplified by an excess of Akt-WT. The existence of this basal level of signaling is also supported by our recent demonstration that overexpression of the protein tyrosine phosphatase PTP1B in adipose cells led to a decrease in the amount of cell surface GLUT4 present in both the absence and presence of insulin (35). As with overexpression of the insulin receptor or IRS-1, insulin stimulation of adipose cells overexpressing Akt-WT resulted in a further increase in the amount of cell surface GLUT4 to a level that was comparable to that observed in control cells treated with a maximally stimulating concentration of insulin. Thus, although overexpression of Akt-WT was sufficient to recruit GLUT4, this did not result in a larger effect than insulin alone could elicit. In contrast, overexpression of the constitutively active mutant Akt-myr resulted in dramatic translocation of GLUT4 to the cell surface at levels that significantly exceeded those achievable by insulin stimulation of the control cells. Furthermore, this effect was independent of insulin. It is likely that overexpression of Akt-myr results in a higher level of Akt activity at the cell membrane than is achievable by insulin stimulation of endogenous Akt in the control cells. However, this is not merely a function of the amount of Akt present because comparable overexpression of Akt-WT did not have as large an effect as Akt-myr (even with insulin stimulation). It is also possible that targeting of overexpressed Akt to the cell membrane stimulates pathways for signaling recruitment of GLUT4 that may not be operative under physiological conditions. For example, even though Ras probably does not contribute to insulin-stimulated translocation of GLUT4 under physiological conditions, we previously reported that overexpression of constitutively active mutants of Ras results in a similarly large effect to recruit GLUT4 in adipose cells (although in this case, insulin treatment results in a further increase in cell surface GLUT4) (3). Our results with Akt-myr are consistent with those of Kohn et al. (12), who showed that overexpression of a myristoylated mutant of Akt lacking its PH domain had similar effects on translocation of GLUT4 in differentiated 3T3-L1 adipocytes.

Akt-K179A and Myr-K179A Inhibit Translocation of GLUT4
Because overexpression of a signaling protein can lead to events that are not related to the physiological functions of that protein, it is useful to assess the effects of inhibiting endogenous Akt on insulin-stimulated translocation of GLUT4. Toward this end we used Akt constructs that have a mutation in the ATP-binding domain rendering the kinase catalytically inactive. Importantly, the PH domain and other regions of the molecule are intact so that overexpressed Akt-K179A or Myr-K179A can presumably compete with endogenous Akt for upstream or downstream factors. Indeed, the catalytically inactive mutant Akt-K179A has been extensively characterized (15, 25, 26, 36) and has been shown to have dominant inhibitory effects in other contexts (23, 24, 28).

When Akt-K179A was transfected into adipose cells using the same concentration of DNA as was used for Akt-WT and Akt-Myr, the resulting insulin dose-response curve was similar to that of the control cells. This suggests that intact kinase activity is important for mediating the effect of Akt-WT on translocation of GLUT4. When higher concentrations of Akt-K179A were used, we observed inhibition of insulin-stimulated translocation of GLUT4 with significant decreases in both insulin sensitivity and responsiveness. Thus, endogenous Akt is likely to contribute importantly to the physiological regulation of GLUT4 by insulin. The significance of these results is highlighted by comparison with results from our previous study on the role of Ras in insulin-stimulated translocation of GLUT4 (3). In that study, we showed that while overexpression of constitutively active mutants of Ras leads to recruitment of GLUT4, overexpression of a dominant inhibitory mutant had no effect on insulin-stimulated translocation of GLUT4, leading us to conclude that Ras does not play a physiological role in insulin-stimulated glucose transport. Our observation of an expression level-dependent effect of Akt-K179A on inhibition of GLUT4 recruitment is consistent with the putative dominant inhibitory mechanism of Akt-K179A (i.e. competition with endogenous Akt for limiting factors). Using a dominant inhibitory mutant of PI3K, we previously demonstrated nearly complete inhibition of insulin-stimulated translocation of GLUT4 in adipose cells (3). Interestingly, even though Akt is a direct downstream target of PI3K, overexpression of Akt-K179A did not have as large an effect as the dominant inhibitory mutant of PI3K. It is possible that a further increase in the level of expression of Akt-K179A would result in further inhibition of GLUT4 recruitment. Because of limitations on the total amounts of DNA that we can use in our system (37), we were not able to test this possibility. It is also likely that there are multiple downstream effectors of PI3K or other signaling molecules that contribute to the effect of insulin on recruitment of GLUT4. For example, using a catalytically inactive mutant of Syp we recently demonstrated a small role for Syp in insulin-stimulated translocation of GLUT4 in adipose cells (35). Thus, maximal inhibition of endogenous Akt may lead to only partial inhibition of translocation of GLUT4 because there are other effectors of PI3K that contribute to this effect.

Overexpression of Myr-K179A had an inhibitory effect on recruitment of GLUT4 that was similar to that seen with Akt-K179A. This suggests that localization of Akt to the membrane is not sufficient to mediate effects on recruitment of GLUT4 in the absence of kinase activity. Furthermore, even when localized to the cell membrane, Myr-K179A is presumably able to compete with endogenous Akt for limiting factors and results in inhibition of insulin-stimulated translocation of GLUT4. In addition, the magnitude of inhibition caused by both Akt-K179A and Myr-K179A was similar and supports the idea that endogenous Akt is not the only effector of PI3K in this action of insulin. These results further support a physiological role for Akt in insulin-stimulated glucose transport.

In summary, overexpression of a constitutively active Akt has a greater effect on recruitment of GLUT4 than overexpression of wild-type Akt. More importantly, overexpression of catalytically inactive mutants of Akt in primary cultures of rat adipose cells inhibits insulin-stimulated translocation of GLUT4. Our results strongly suggest a physiological role for Akt in insulin-stimulated glucose transport in insulin target tissues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Vector Constructions
pCIS2

An expression vector that generates high expression levels in transfected rat adipose cells (37) was used as the parent vector for subsequent constructions.

GLUT4-HA
Complementary DNA coding for human GLUT4 with the influenza hemagglutinin epitope (HA1) inserted in the first exofacial loop of GLUT4 was subcloned into the pCIS2 vector (1).

Akt-WT
A 1.4-kb XbaI/BamHI fragment containing the cDNA for mouse Akt-1 [the generous gift of Drs. P. N. Tsichlis and K. Datta (27)] was blunt-ended and ligated in the sense orientation into the HpaI site in the multiple cloning region of pCIS2.

Akt-myr
BglII/BamHI fragment containing the cDNA for mouse Akt-1 with a myristoylation sequence from pp60 c-src (38) fused in-frame with the N terminus of Akt (a generous gift from Drs. P.N. Tsichlis and K. Datta) was blunt-ended and ligated in the sense orientation into the HpaI site in the multiple cloning region of pCIS2.

Akt-K179A
Point mutant of Akt-WT with a substitution of alanine for lysine at position 179 was constructed using a mutagenic oligonucleotide 5'-GC TAC TAT GCC ATG GCG ATC CTC AAG AAG G-3' and the MORPH site-specific plasmid DNA mutagenesis kit according to the manufacturers instructions (5 prime 3 prime, Inc., Boulder, CO). The creation of the mutation introduced a new NcoI site. In addition, the mutation was confirmed by direct sequencing.

Myr-K179A
Point mutant of Akt-myr with a substitution of alanine for lysine at position 179 was constructed exactly as described above for Akt-K179A.

Isolated Rat Adipose Cell Preparation
Isolated adipose cells were prepared from the epididymal fat pads of male rats (170–200 g, CD strain, Charles River Breeding Laboratories, Wilmington, MA) by collagenase digestion as described (37, 39).

Electroporation
Isolated adipose cells were transfected by electroporation as described (35, 37). Cells from multiple cuvettes were pooled to obtain the necessary volume of cells for each experiment as described (see Table 1Go for number of cuvettes and amount of DNA used).


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Table 1. Transfection of Akt Constructs in Rat Adipose Cells

 
Assay for Cell Surface Epitope-Tagged GLUT4
Twenty hours after electroporation, adipose cells were processed as described (1, 2, 3, 35) and treated with insulin at final concentrations of 0, 0.024, 0.072, 0.3, or 60 nM at 37 C for 30 min. Cell surface epitope-tagged GLUT4 was determined by using the anti-HA1 mouse monoclonal antibody HA-11 (Berkeley Antibody Company, Richmond, CA) at a final dilution of 1:2000 in conjunction with [125I]-sheep anti-mouse IgG as described (35). Cells transfected with the empty expression vector pCIS2 were used to determine nonspecific binding of the antibodies. Typically, the nonspecific binding was 20% of the total binding to cells transfected with GLUT4-HA and maximally stimulated with insulin. The actual specific counts were comparable from experiment to experiment (see figure legends). The lipid weight from a 200-µl aliquot of cells was determined as described (40) and used to normalize the data for each sample.

Immunoblotting of Akt and GLUT4-HA
Expression of recombinant Akt-WT, Akt-myr, Akt-K179A, Myr-K179A, and GLUT4-HA was confirmed by immunoblotting extracts of cells that were prepared at the same time and had undergone transfection in parallel with the cells used for the translocation assay described above. Cells from 12 cuvettes were pooled for each group. Whole cell homogenates were prepared from cells cotransfected with GLUT4-HA (2 µg/cuvette) and either pCIS2, Akt-WT, Akt-myr, (4 µg/cuvette) or Akt-K179A or Myr-K179A (7 µg/cuvette). To determine relative levels of GLUT4-HA in each group of transfected cells, total membrane fractions were prepared from the whole cell homogenate by centrifuging 30 min at 400,000 x g at 4 C. The pellet containing the total membrane fraction was resuspended in 300 µl TES buffer (20 mM Tris, 1 mM EDTA, 8.73% sucrose, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml BSA, 1 mM phenylmethylsulfonyl fluoride, pH 7.4, 4 C). The protein concentration in each sample was measured and aliquots containing 60 µg of protein were subjected to SDS-PAGE. The contents of the gel were transferred to nitrocellulose and immunblotted with the monoclonal anti-HA antibody HA-11 (final concentration, 5 µg/ml). Immunolabeled bands were visualized using an antibody against mouse IgG in conjunction with an enhanced chemiluminescent detection system (ECL, Amersham, Arlington Heights, IL).

For immunodetection of Akt constructs, whole cell homogenates containing equal amounts of protein (80 µg) were solubilized in Laemmli sample buffer and subjected to SDS-PAGE. The contents of the gel were transferred to nitrocellulose, and the Akt protein was detected with a polyclonal anti-Akt antibody (Upstate Biotechnology Inc., Lake Placid, NY) at a final concentration of 1 µg/ml. Bands were visualized using an antibody against mouse IgG in conjunction with an ECL detection system (Amersham).

Akt Kinase Assay
To assess the kinase activity of the recombinant Akt-WT and Akt-myr in transfected adipose cells, cells were transfected with either the empty expression vector pCIS2, Akt-WT, or Akt-myr (4 µg DNA/cuvette, 15 cuvettes per group) and treated without or with insulin (60 nM) for 2 min, and whole cell homogenates of each group were prepared as described above. Samples containing 200 µg protein from each group were subjected to immunoprecipitation with an antibody against Akt, and kinase activity in the immunoprecipitates was assessed by incorporation of [32P]ATP into the substrate histone 2B as described (27). Samples were separated on a 12% SDS-PAGE, and the phosphorylation of histone 2B in each sample was quantified using PhosphorImager analysis of the gel (Molecular Dynamics, Sunnyvale, CA).

Statistical Analysis
Insulin dose-response curves were compared using multivariate ANOVA. Paired t tests were used to compare individual points where appropriate. P values of less than 0.05 were considered statistically significant. The insulin dose-response curves were fit to the equation y = a + b [x/(x + k)] using a Marquardt-Levenberg nonlinear least squares algorithm. When plotted on linear-log axes, this equation gives a sigmoidal curve where the parameters are associated with the following properties: a = basal response; a + b = maximal response; k = half-maximal dose (ED50); and x = concentration of insulin.


    ACKNOWLEDGMENTS
 
We thank Dr. G.I. Bell for supplying the human GLUT4 cDNA, Dr. C. Gorman for the pCIS2 expression vector, and Drs. T. N. Tsichlis and K. Datta for the mouse Akt cDNA constructs. We thank Dr. L. Kohn for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Michael J. Quon, M.D., Ph.D., Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-103, 10 Center Drive MSC 1754, Bethesda, Maryland 20892-1754.

This work was supported in part by a Research Award from the American Diabetes Association (to M.J.Q.).

Received for publication June 3, 1997. Revision received August 20, 1997. Accepted for publication September 5, 1997.


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 MATERIALS AND METHODS
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