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Inhibition of Protein Kinase Cß_II Increases Glucose Uptake in 3T3-L1 Adipocytes through Elevated Expression of Glucose Transporter 1 at the Plasma Membrane

Department of Chemical Endocrinology (R.R.B., M.M.W., P.N.S., A.J.O., C.G.J.S.), Department of Endocrinology (R.R.B., A.R.M.M.H.), and Department of Biochemistry (P.H.G.M.W.), University Medical Centre, 6500 HB Nijmegen, The Netherlands; Department of Molecular Cell Biology (M.B., J.A.M.), Leiden University Medical Center, 2333 AL Leiden, The Netherlands; and Department of Pharmacology (A.S.), German Institute of Human Nutrition, 14588 Potsdam-Rehbrücke, Germany

Address all correspondence and requests for reprints to: R. R. Bosch, Ph.D., Department of Chemical Endocrinology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: R.Bosch{at}ace.umcn.nl.

	ABSTRACT

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The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCß_II during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCß_II with a specific myristoylated PKCßC2–4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCßC2–4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCß_II, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCß_II influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

	INTRODUCTION

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GLUCOSE TRANSPORT IN insulin-sensitive tissues is a major regulatory process in the homeostatic control of blood glucose levels. Insulin stimulates, via a complex network of signaling molecules, the translocation of glucose transporters [predominantly glucose transporter (GLUT)-4 and to a lesser extent GLUT-1] from an intracellular pool to the cell surface membrane (1). Diminished insulin-stimulated GLUT-4 translocation is thought to be a major mechanism in the development of type 2 diabetes mellitus. The involvement of other glucose transporters, such as GLUT-1, in the regulation of glucose uptake or in the pathogenesis of type 2 diabetes mellitus is less clear.

The enzyme protein kinase C (PKC; EC 2.7.1.37) is involved in cell signaling by catalyzing the phosphorylation of specific serine and threonine residues on various cellular proteins, thereby modulating their function. The PKC family consists of at least 11 isoforms that can be divided into three classes; the classical or conventional (c) PKC-isotypes (, ß_I, ß_II, and ), which are stimulated by both Ca²⁺ and diacylglycerol (DAG); the novel (n) isoforms (, , , and ), which are stimulated by DAG but are Ca²⁺-insensitive; and the atypical (a) isoforms (, and ), which are stimulated by phosphatidic acid and phosphatidylinositol 3,4,5-trisphosphate (2, 3, 4, 5). In adipocytes it has been shown that PKC and/or - are required for insulin-stimulated glucose transport (5, 6, 7, 8, 9).

In addition to aPKC, 3T3-L1 adipocytes also contain cPKC, -ß_I, and -ß_II and nPKC and - (6, 9, 10). Exposure of these cells to the phorbol ester phorbol-12-myristate-13-acetate (PMA), a potent activator of conventional and novel PKCs, increases the rate of glucose transport (11, 12). The mechanism via which DAG-sensitive PKCs stimulate glucose transport is poorly defined. Phorbol esters stimulate the translocation of GLUT-1 and GLUT-4 to the plasma membrane in 3T3-L1 adipocytes, although PMA stimulates the translocation of both glucose transporters much less effectively compared with insulin (11). Inhibition of phosphatidylinositol-3-kinase by wortmannin and LY294002 effectively prevented PMA-stimulated, as well as insulin-stimulated, glucose transport in these cells (12). This suggests that PMA and insulin share a common pathway to increase the rate of glucose transport. However, others have shown that PMA-stimulated GLUT-4 translocation in Chinese hamster ovary cells occurred in a phosphatidylinositol-3-kinase-independent manner (13).

Persistent activation of PKC by PMA causes prolonged exposure of PKC sites sensitive for proteolytic cleavage (14). Consequently, chronic phorbol ester treatment results in the proteolytic degradation, termed down-regulation, of DAG-sensitive PKCs (15, 16). Although short-term PMA treatment (1 h maximum) increased the rate of glucose uptake, it was found that chronic PMA treatment (overnight incubation) had no stimulatory effect (11, 17, 18). These observations suggest that activation of one of the DAG-sensitive PKC isoforms stimulates glucose uptake. The goal of this study was to identify this PKC isoform. To our surprise, we observed that during PMA-stimulated glucose uptake in 3T3-L1 cells, PKCß_II was rapidly down-regulated. In addition, inhibition of PKCß_II with a specific myristoylated PKCßC2–4 pseudosubstrate increased the rate of glucose transport. Western blot analysis demonstrated that this was accompanied by increased levels of GLUT-1 and not GLUT-4 at the plasma membrane. To our knowledge, we are the first to show that through inactivation of PKC, most likely PKCß_II, glucose uptake in 3T3-L1 adipocytes is enhanced by increasing the amount of GLUT-1 at the plasma membrane. The observation that PKCß_II is involved in regulating GLUT-1-mediated glucose uptake might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

	RESULTS

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Analysis of PKC Isoform Expression in 3T3-L1 Adipocytes
To study the role of DAG-sensitive PKC isoforms in glucose uptake, we first examined the presence of several cPKCs and nPKCs in 3T3-L1 adipocytes. Untreated cells expressed PKCß_I, -ß_II, -, -, and - (Fig. 1, left lanes). We did not observe the presence of PKC or - in 3T3-L1 adipocytes, albeit the PKC isotype-specific antibodies showed a positive staining of PKC and - in rabbit brain homogenate (data not shown). Treatment with 0.1 µM PMA for 24 h down-regulated PKCß_II almost completely and appeared to slightly reduce the amount of PKC (middle lanes). The level of the other three isoforms remained unaffected after 24 h of 0.1 µM PMA treatment. The specificity of the antibodies was demonstrated either by coincubating with the corresponding PKC isoform-specific peptides in the case of PKCß_II, - and, - or by the selective disappearance (down-regulation) of a single band of expected size after treatment with a high (≥1 µM) concentration of PMA in the case of PKCß_I and - (right lanes).

View larger version (45K): [in this window] [in a new window]   Figure 1. Expression of Different PKC Isoforms in Control and PMA-Treated 3T3-L1 Adipocytes Cells were incubated without (left lane) or with (middle lane) 0.1 µM PMA for 24 h. After washing, cells were lysed and subjected to SDS-PAGE followed by Western blotting. Membranes were incubated with PKC isotype-specific antibodies. The specificity of the antibodies was demonstrated by incubating the membranes of the Western blot with PKC isotype-specific antibodies (i.e. PKCßII, -, and -) in the presence of corresponding PKC isoform-specific peptide (right lanes). In Western blot analysis of PKCßI and -, the specificity of the antibodies was demonstrated by the selective disappearance (down-regulation) of a single band of expected size after treatment with a high (≥1 µM) concentration of PMA (right lanes). The results shown are a representative from two (for PKCßI, -, and -) and three (for PKCßII and -) experiments.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of Treatment with 0.1 and 5 µM PMA on the Expression Level of PKCßII in 3T3-L1 Adipocytes Cells were treated for 30 min and 1, 2, 4, and 7 h with 0.1 or 5 µM PMA. After washing, cells were lysed and subjected to SDS-PAGE followed by Western blotting. Membranes were incubated with a PKCßII-specific antibody. The results are one representative from three experiments.

View larger version (15K): [in this window] [in a new window]   Figure 3. Time-Dependent Effect of 0.1 and 5 µM PMA on Glucose Uptake in 3T3-L1 Adipocytes Cells were incubated with either 0.1 (open circles) or 5 (closed circles) µM PMA for different time periods. After washing, the rate of glucose uptake was determined by addition of [3H]-2-DOG uptake for 10 min. In untreated cells, glucose uptake amounted to 632 ± 122 pmol/min/well (n = 8) in incubations with 0.1 µM PMA, and in incubations with 5 µM PMA, basal glucose uptake in untreated cells was 618 ± 176 pmol/min/well (n = 5). The basal glucose uptake was set at 100% to which all other values were related. The values presented are the mean + SEM of three to eight experiments. *, Significantly different from untreated cells. #, Significantly different from 7 h of 0.1 µM PMA-treated cells.

View larger version (19K): [in this window] [in a new window]   Figure 4. Insulin-Stimulated Glucose Uptake in Short- and Long-Term PMA-Treated 3T3-L1 Adipocytes Cells were incubated with 0.1 µM PMA for 10 min, 7 h, and 24 h. After washing, 0.1 µM insulin was added, and after 10 min of incubation, the rate of glucose uptake was determined upon addition of [3H]-2-DOG for 10 min. In untreated cells, insulin elevated the rate of glucose uptake with 2603 ± 421 pmol/min/well (n = 3), which was set at 100%, to which all other values were related. The values presented are the mean ± SEM of three individual experiments.

The Myristoylated PKCßC2–4 Peptide Inhibitor Mimics PMA-Increased Glucose Uptake
To confirm that the increase in glucose transport after PMA treatment was due to depletion of PKCß_II, and not because of activation of other PKC isoforms, we inhibited PKCß_II with the myristoylated PKCßC2–4 peptide inhibitor, a peptide that prevents the interaction between cPKCs (including PKCß_II) with their endogenous substrates. Figure 5 shows that this pseudosubstrate inhibitor increased the rate of glucose uptake in 3T3-L1 adipocytes approximately 5-fold, after 90-min incubation with 100 µM of the myristoylated PKCßC2–4 peptide inhibitor. In contrast, inhibition of the atypical PKC isoforms with the myristoylated PKC/ peptide inhibitor did not elevate the rate of glucose uptake. Incubation with PMA (0.1 µM) for 7 h, to down-regulate PKCß_II, in combination with the myristoylated PKCßC2–4 peptide inhibitor during the last 90 min, most effectively increased the rate of glucose uptake, although this combination did not increase glucose uptake in an additive manner. In contrast, incubation with PMA (0.1 µM) for 7 h in combination with the myristoylated PKC/ peptide inhibitor during the last 90 min did not increase the rate of glucose uptake.

View larger version (15K): [in this window] [in a new window]   Figure 5. Incubation of the Myristoylated PKCßC2–4 Inhibitory Peptide Increases Glucose Uptake in 3T3-L1 Adipocytes Untreated and PMA-treated cells were incubated for 90 min with either the myristoylated PKCßC2–4 or the PKC/ inhibitor (100 µM). After washing, the rate of glucose uptake was determined upon addition of [14C]-2-DOG for 5 min. Basal glucose uptake in untreated cells (368 ± 28 fmol/min/well; n = 10) was set at 100%, to which all other values were related. The values presented are the mean ± SEM of five experiments. *, Significantly different from untreated control cells. #, Significantly different from cells exposed for 7 h to 0.1 µM PMA and for the last 90 min to the myristoylated PKCßC2–4 inhibitor.

View larger version (37K): [in this window] [in a new window]   Figure 6. Long-Term PMA Treatment and Incubation with the Myristoylated PKCßC2–4 Inhibitor Increases GLUT-1 Expression in 3T3-L1 Adipocytes Cells were incubated either for 7 h with 0.1 µM PMA (upper row), for 90 min with the myristoylated PKCßC2–4 inhibitor (middle row), or for 20 min with 0.1 µM insulin (bottom row). After washing, homogenates (Hom) of the cells were subjected to differential centrifugation to yield plasma membrane (PM) and low density membrane (LDM) fractions. Proteins (20 µg) of these fractions were separated by means of SDS-PAGE and subjected to Western blotting. The membranes were incubated with glucose transporter isotype-specific antibodies. Untreated cells (- lanes) are depicted for comparison adjacent to treated cells (+ lanes). The results are one representative from three individual experiments.

View larger version (17K): [in this window] [in a new window]   Figure 7. The Effect of Staurosporine and Ro-31–8220 on Basal and PMA-Stimulated Glucose Uptake in 3T3-L1 Adipocytes Staurosporine (0.1 µM) and Ro-31–8220 (1 and 10 µM) were added to control cells and to cells incubated for 7 h with 0.1 µM PMA. After a 30-min incubation, the amount of [3H]-2-DOG uptake during another 10 min was measured. Basal glucose uptake in untreated cells was set at 100% to which all other values were related. The values presented are the mean + SEM of three experiments. *, Significantly different from untreated control cells. #, Significantly different from control cells exposed for 7 h to 0.1 µM PMA.

	DISCUSSION

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At present, it is believed that PMA increases the rate of glucose transport by activating DAG-sensitive PKC isoforms in 3T3-L1 adipocytes (11, 12, 18). This idea is based on the observation that, upon addition of PMA for 20 min, glucose transport was profoundly increased. After 24 h of PMA treatment, only a slight increase in the rate of glucose uptake was observed, suggesting that chronic treatment with PMA down-regulated DAG-sensitive PKC isoforms involved in glucose transport. However, we observed that PKCß_II was rapidly down-regulated when 3T3-L1 adipocytes were exposed to the phorbol ester. Incubation with 5 µM PMA resulted in a complete down-regulation of PKCß_II within 1–2 h. Treatment with 0.1 µM PMA required approximately 4–7 h to remove PKCß_II. In parallel, the rate of glucose transport was significantly increased after 2 h of 5 µM PMA and 7 h of 0.1 µM PMA treatment. Given this good correlation between the absence of PKCß_II and the increased rate of glucose transport, it is reasonable to speculate that this PKC isoform plays a prominent role in glucose transport. In the case of 5 µM PMA, however, the rate of glucose uptake increased until 7 h of incubation of 3T3-L1 adipocytes, whereas PKCß_II was down-regulated after 2 h of incubation. It is possible that, in addition to the down-regulation of PKCß_II, PMA promotes the expression of genes involved in glucose transport. In support of this possibility, we observed an increase in the total amount of GLUT-1 after 7 h with 0.1 µM PMA. The latter may very well contribute to the PMA-stimulated glucose uptake in 3T3-L1 adipocytes.

Addition of 0.1 µM PMA for 24 h affected predominantly the level of PKCß_II, to a lesser extent PKC, and had no down-regulatory effect on PKCß_I, -, and -. This means that at 7 and 24 h PMA treatment, only PKCß_II was lacking, although the PMA-stimulated glucose uptake after 24 h of treatment was less than at 7 h of treatment. It is therefore unlikely that the decrease in glucose transport measured at 24 h was due to the down-regulation of DAG-sensitive PKC isoforms. In addition, insulin equipotently stimulated glucose uptake in 24-h PMA-treated cells compared with untreated cells, demonstrating that the glucose uptake mechanism was still fully functional. At present, we can only speculate why the initial increase (0–7 h) in glucose uptake was followed by a decrease (24 h).

To investigate whether part of the increase in the rate of glucose uptake by PMA was due to loss of function of PKCß_II and not to activation of the remaining PKC isoforms, we incubated the cells with the myristoylated PKCßC2–4 peptide inhibitor. This peptide is a sequence that is conserved within the C2 domain of all cPKCs (19). The receptor for activated C kinase protein binds the activated PKC at the C2 domain and, in doing so, anchors PKC near its protein substrates. The myristoylated pseudosubstrate prevents interaction of receptor for activated C kinase protein with the conventional PKCs to inhibit their translocation and function (20). The myristoylated PKCßC2–4 peptide inhibitor prevented translocation of PKC in pancreatic ß cells (21) and translocation of PKCß in Xenopus oocytes (22). In our fully differentiated cells we did not detect PKC, which is in agreement with earlier results which found that the expression of the latter PKC isoform decreases during adipogenesis of 3T3-L1 adipocytes (10), leaving the remaining three cPKCs as possible targets for the inhibitory peptide. Thus similar to long-term PMA treatment, the myristoylated PKCßC2–4 peptide inhibitor will generate cells in which the endogenous substrates for PKCß_II can no longer interact with this kinase. We measured a significant increase in the rate of glucose transport upon addition of the myristoylated PKCßC2–4 peptide inhibitor for 90 min. Moreover, the rate of glucose transport was only slightly further increased when the myristoylated PKCßC2–4 peptide inhibitor was added to cells in which PKCß_II was depleted by 7 h of PMA treatment. The latter observation is a strong indication that 7 h PMA and the myristoylated PKCßC2–4 peptide inhibitor stimulate the rate of glucose transport through an identical mechanism. Importantly, the myristoylated PKC/ peptide inhibitor did not affect the rate of glucose uptake, demonstrating the specificity of the myristoylated PKCßC2–4 peptide inhibitor. Moreover, it completely prevented PMA-stimulated glucose uptake. The latter observation reveals the importance of PKC, reported as the only aPKC isoform present in 3T3-L1 adipocytes (8), in PMA-mediated glucose uptake and further supports the idea that aPKC isoforms are critical for activating glucose transport responses (5).

Neither staurosporine nor Ro-31–8220 increased the rate of glucose uptake in 3T3-L1 adipocytes, as observed after long-term PMA treatment or incubation with the myristoylated PKCßC2–4 inhibitor. Because the latter two methods prevent interaction of PKCß_II with its substrates and staurosporine and Ro-31–8220 act by inhibiting the kinase activity of DAG-sensitive PKCs (including PKCß_II), these data suggest that PKCß_II inhibits the rate of glucose uptake through binding, rather than phosphorylation, of a yet unidentified factor. The observation that 0.1 µM staurosporine and 1 µM Ro-31–8220 did not reduce the increase in the rate of glucose uptake in cells after 7 h of PMA treatment is in keeping with the idea that the stimulatory effect on the rate of glucose transport was due to depletion of PKCß_II and not to activation of the other conventional or novel PKCs. The rate of glucose transport decreased in 7-h PMA-treated cells when Ro-31–8220 was added at a concentration of 10 µM. At this concentration, however, Ro-31–8220 inhibits the kinase activity of PKC and - (23). Similar to the myristoylated PKC/ peptide inhibitor, which prevented the increase in glucose transport of 7-h PMA-treated cells, this observation demonstrates the importance of PKC in PMA-mediated glucose transport.

By means of differential centrifugation, we demonstrated that 7 h PMA (0.1 µM) treatment resulted in an elevated expression of GLUT-1, and not GLUT-4, at the plasma membrane. The nearly 2-fold increase in the amount of GLUT-1 at the plasma membrane did not result in a concomitant decrease of this transporter in the intracellular vesicles as the amount of GLUT-1 also doubled in the LDM fraction. Taken together, this implicates that the total GLUT-1 content within the 3T3-L1 adipocytes increased during the 7-h PMA treatment. This is confirmed by the observation that the amount of GLUT-1 in the total homogenate increased 1.60-fold. Others have demonstrated that the amount of GLUT-1 at the plasma membrane increases as early as 60 min after 1 µM PMA treatment (11). However, in contrast to our observation, these authors detected a small but significant decrease in the amount of GLUT-1 at the intracellular membranes, suggesting that, during the 60 min of PMA treatment, translocation, and not de novo synthesis, of GLUT-1 is stimulated.

Similar to the effects of long-term PMA treatment, incubation with the myristoylated PKCßC2–4 peptide inhibitor increased the amount of GLUT-1 and not GLUT-4 at the plasma membrane. However, the amount of GLUT-1 in the LDM fraction did not increase as we observed a small, but consistent, decrease of 20%, suggesting increased translocation of GLUT-1. In agreement, we did not detect an increase of GLUT-1 in the total homogenate fraction. Of note, incubation for 90 min with the myristoylated PKCßC2–4 peptide inhibitor might have been too short to detect increased expression of GLUT-1 by means of Western blotting. Unfortunately, incubation with the myristoylated PKCßC2–4 peptide inhibitor was not possible in culture medium, and a nutrients-deprived buffer was used instead. As a result, in order to avoid damage to the adipocytes, the maximal incubation time was limited to 90 min.

The role of PKCß_II in insulin-stimulated glucose uptake is controversial. Although some observed that overexpression of kinase-negative PKCß_II in L6 myotubes inhibits insulin-stimulated glucose uptake (24), others did not find evidence for the involvement of this PKC isoform in insulin-stimulated glucose transport in L6 myotubes (25). Of note, these kinase-negative mutants did not increase the rate of basal glucose uptake, similar to our observations with staurosporine and Ro-31–8220. Moreover, in accordance with Bandyopadhyay et al. (25), we observed that the insulin-stimulated glucose transport was identical in PKCß_II-depleted 3T3-L1 adipocytes compared with control, PKCß_II-containing cells. In adipocytes from PKCß knockout mice, however, insulin-stimulated glucose transport was enhanced by 50–100% compared with adipocytes from control mice (26). In addition, muscle cells from these PKCß knockout mice had a higher basal glucose uptake than the same cells from control mice. In keeping with these findings is the observation that the serum glucose levels were approximately 10% lower in homozygous PKCß^-/- offspring compared with wild-type PKCß^+/+ offspring in both the fasting state and after ip glucose injection. Given our observations that PKCß_II inactivation or depletion in 3T3-L1 cells results in enhanced glucose uptake by elevation of GLUT-1 at the plasma membrane level, it is tempting to speculate that this mechanism is the underlying principle of the enhanced glucose disposal in the PKCß knockout mice. Although Standaert et al. (26) observed no differences in total GLUT-1 and GLUT-4 levels, they did not determine the amount of the two glucose transporters specifically present in the plasma membrane in insulin-sensitive tissues from the PKCß knockout mice.

In many cells, including rat adipocytes, BC3H-1 myocytes, and 3T3-L1 adipocytes, phorbol esters activate glucose transport (11, 12, 18, 27, 28). It has not been exclusively demonstrated that phorbol esters activate glucose transport by provoking an increase in the activity of DAG-sensitive PKCs rather than by down-regulation of DAG-sensitive PKCs. Our data suggest that the PMA-stimulated glucose transport in 3T3-L1 adipocytes is not due to activation of PKC, as 0.1 µM staurosporine and 1 µM Ro-31–8220 did not lower PMA-stimulated glucose transport. In agreement with these findings is the observation that the myristoylated PKCßC2–4 peptide inhibitor also effectively increases the rate of glucose uptake, which occurs in the absence of any PKC stimulus. However, we do not observe translocation of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes after 7 h of PMA treatment. In contrast, others showed that shorter incubation periods with PMA resulted in the translocation of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes (11) and rat adipocytes (29). Given this difference, we cannot exclude that during ongoing PMA treatment of 3T3-L1 adipocytes, the initial increase in glucose transport by GLUT-4 is due to activation of PKCs, which is followed by a second phase in which the increase in glucose transport by GLUT-1 is due to down-regulation of PKCß_II.

In summary, our data demonstrate that PKCß_II controls GLUT-1 expression at the plasma membrane and, as such, influences glucose uptake. To our knowledge, we are the first to demonstrate that, in the case of 3T3-L1 adipocytes, through inactivation or depletion of PKCß_II the rate of glucose transport is enhanced. These observations might reveal a yet-unidentified pathway involved in the cellular regulation of glucose uptake. Therefore, we are currently introducing short interfering RNAs in both insulin-sensitive and insulin-insensitive cells, to functionally down-regulate PKCß_II, and subsequently, study GLUT-1-mediated glucose uptake. These studies will help to further determine the physiological relevance of the role of PKCß_II in the regulation of glucose homeostasis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
DMEM was purchased from ICN Biomedicals, Inc. (Aurora, OH) and fetal calf serum (FCS) was purchased from PPA Laboratories (Linz, Austria). All other culture reagents and the antibodies directed against PKC, -, -, -, and - were purchased from Life Technologies, Inc. (Paisley, UK). The other isotype-specific PKC antibodies (directed against PKCß_I and -ß_II) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The myristoylated PKCßC2–4 and PKC/ peptide inhibitors were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). BSA, 3-isobutyl-1-methylxanthine (IBMX), 2-deoxyglucose (2-DOG), goat antirabbit IgG peroxidase conjugate, PMA, and Ro-31–8220 were obtained from Sigma (St. Louis, MO). Staurosporine was obtained from Roche Molecular Chemicals (Mannheim, Germany). Recombinant human insulin was from Eli Lilly \|[amp ]\| Co. (Nieuwegein, The Netherlands), and I-Block Reagent was obtained from Tropix (Bedford, MA). Dithiothreitol was purchased from Research Organics (Santa Cruz, CA), and 1-[³H]-2-DOG (spec. act. 481 GBq/mmol) and 1-[¹⁴C]-2-DOG (specific activity 2.0 GBq/mmol) were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). All other chemicals were of analytical grade.

Adipogenesis of 3T3-L1 Adipocytes
The 3T3-L1 cells (ATCC, Manassas, VA) were seeded in T₇₅-flasks, 6- and 12-well plates, and grown to confluence in DMEM with 25 mM glucose and 10% (vol/vol) FCS for 10 d. Subsequently, insulin (1.7 µM), IBMX (1 µM), and dexamethasone (25 pM) were added. After 48 h, IBMX and dexamethasone were omitted from the culture medium and, after an additional 6 d of culture, insulin was also omitted. After another 7 d of culture, the 3T3-L1 cells were fully differentiated to adipocytes (>90% of the cells contained lipid droplets) and used for analysis. In the case of PMA-treated cells, the phorbol ester was added to the culture medium to reach an end concentration of 0.1 or 5 µM for different time periods before assessment of glucose uptake or Western blot analysis.

Glucose Uptake Assay
Fully differentiated 3T3-L1 adipocytes, subcultured in 12-well plates, were incubated in DMEM without FCS for 3 h. Subsequently, cells were washed twice with PBS and incubated at 37 C for 45 min in reaction buffer containing 138 mM NaCl, 1.85 mM CaCl₂, 1.3 mM MgSO₄, 4.8 mM KCl, 0.2% (wt/vol) BSA, and 50 mM HEPES adjusted to pH 7.4. In the case of PMA-treated cells, the phorbol ester was added to reach a final concentration of 0.1 or 5 µM for 10, 30, or 45 min. To assess the rate of glucose uptake, cells were refreshed with 500 µl reaction buffer and after an additional 10 min, reaction buffer (250 µl) containing 27.8 kBq 1-[³H]-2-DOG and 1.5 mM 2-DOG was added. After 10 min of incubation at 37 C, glucose uptake was terminated upon washing three times with ice-cold PBS containing 10 mM glucose. In the case of the myristoylated PKCßC2–4 or PKC/ peptide inhibitors, cells were incubated with 100 µM of the peptide inhibitor for 90 min in reaction buffer. The adipocytes were refreshed with 760 µl reaction buffer containing 333 kBq 1-[¹⁴C]-2-DOG and 40 µM 2-DOG. After 5 min of incubation at 37 C, glucose uptake was terminated upon washing three times with ice-cold PBS containing 10 mM glucose. Subsequently, cells from both treatments were lysed in 1% (wt/vol) sodium dodecyl sulfate and 0.2 M NaOH. Incorporated radioactivity was measured by liquid scintillation spectrometry.

Western Blot Analysis of PKC Isoforms
Fully differentiated 3T3-L1 adipocytes, subcultured in six-well plates, were washed twice with PBS. The adipocytes in each well were scraped in 500 µl homogenization buffer containing 2 mM EDTA, 10 mM EGTA, 0.25 M sucrose, 5 mM dithiothreitol, 1 tablet/10 ml complete miniprotease inhibitor cocktail tablet (Roche, Mannheim, Germany), and 20 mM Tris adjusted to pH 7.4. The cell suspensions were sonicated 10 times for 5 sec (Branson Ultrasonics Corp., Danbury, CT; model 250 sonifier) on ice and subsequently, total protein content was determined (Bio-Rad Laboratories, Inc., Hertfordshire, UK). Proteins were precipitated with 5% trichloroacetic acid and after 10 min incubation on ice, the homogenate was centrifuged for 10 min at 16,000 x g. The pellets were resuspended in sample buffer to give a final protein concentration of 10 mg/ml. Subsequently, 75 µg of total protein was subjected to SDS-PAGE, after which the proteins were transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore Corp., Bedford, MA) by Western blotting. Membranes were washed with PBS and blocked for 1 h at room temperature (RT) in PBS containing 0.2% (wt/vol) I-Block and 0.1% (vol/vol) Tween 20. Finally, membranes were incubated overnight at RT in PBS containing 0.1% (wt/vol) I-Block and 0.2% (vol/vol) Tween 20 with PKC isotype-specific antibodies, diluted 1:500 for the detection of PKC, -ß_I, -ß_II, -, -, and -, and diluted 1:100 for the detection of PKC. Subsequently, membranes were washed with PBS containing 0.2% (wt/vol) I-Block and 0.1% (vol/vol) Tween 20 and incubated at RT for 1 h with goat antirabbit IgG antibodies conjugated to horseradish peroxidase diluted 1:1000 in PBS containing 0.1% (wt/vol) I-Block and 0.2% (vol/vol) Tween 20. After further wash steps, bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Preparation of Cell Fractions
Differentiated 3T3-L1 adipocytes, grown in T₇₅-flasks, were treated for 7 h with 0.1 µM PMA in serum-free medium or 90 min with 100 µM myristoylated PKCßC2–4 pseudosubstrate in reaction buffer from the glucose uptake assay (see above). After washing with PBS, adipocytes from a T₇₅-flask were scraped in ice-cold HES buffer containing 5 mM EDTA, 250 mM sucrose, 1 tablet/15 ml complete miniprotease inhibitor cocktail tablet, and 10 mM HEPES adjusted to pH 7.4. Adipocytes from two flasks were pooled and homogenized at 4 C using a Potter-Braun (Braun Biotech International GmbH, Melsungen, Germany) (type S) homogenizer (glass-teflon) by 15 strokes (manually). All subsequent procedures were also performed at 4 C. The homogenate was centrifuged for 15 min at 8000 x g. The pellet was resuspended (25 strokes in a 1-ml glass-glass potter) in 1 ml HES buffer and layered on a 1.12 M sucrose cushion. After a 60-min centrifugation at 100,000 x g in a swing-out rotor, the top layer was taken off. To this top layer HES buffer was added until the volume was 10 ml and the mixture was centrifuged for another 60 min at 100,000 x g to pellet the plasma membrane (PM) fraction. The supernatant from the first centrifugation (15 min at 8,000 x g) step was centrifuged for 20 min at 41,000 x g. To pellet the low density membrane (LDM) fraction, the resulting supernatant was centrifuged for 75 min at 180,000 x g. The PM and LDM fractions were resuspended in 200 µl HES buffer and total protein content was determined (BCA; Pierce Chemical Co., Rockford, IL). Finally, the fractions were stored at -80 C until further use.

Western Blot Analysis of GLUT-1 and GLUT-4 Glucose Transporters
Proteins (20 µg) from the different fractions, yielded as described above, were precipitated with 5% trichloroacetic acid and after 10 min incubation on ice, the fractions were centrifuged for 10 min at 16,000 x g. The pellets were resuspended in sample buffer to give a final protein concentration of 2 mg/ml. Subsequently, samples were subjected to SDS-PAGE, after which the proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech) by Western blotting. Membranes were washed twice with PBS and blocked for 2 h at RT in Tris-buffered saline (TBS) buffer containing 0.1% (vol/vol) Tween 20, 150 mM NaCl, and 50 mM Tris adjusted to pH 7.4. Subsequently, membranes were incubated for 1 h at RT in TBS buffer containing the rabbit glucose transporter isotype-specific antibodies (30). The antibodies were diluted 1:2000 for the detection of GLUT-1 and 1:5000 for detection of GLUT-4. After washing three times with TBS buffer, the membranes were incubated for 1 h at RT in TBS buffer containing goat antirabbit IgG antibodies conjugated to horseradish peroxidase (1:1000). After washing three times with TBS buffer, bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). The bands were scanned using the documentation and analysis system AlphaImager 1220 (Biozym, Landgraaf, The Netherlands), and the integrated density values were calculated with the computer program AlphaEase version 5.1 (Biozym).

Data Analysis
The results presented are the mean ± SEM of at least three individual experiments, and statistical significance was determined by ANOVA. Comparison of significance (P < 0.05) among individual groups was performed according to Tukey-Kramer.

	ACKNOWLEDGMENTS

 
We thank Sjenet van Emst-de Vries (Department of Biochemistry, University Medical Center Nijmegen) for her help in detecting the PKC isoforms by means of Western blotting. We thank Helga van Rennes (Department of Chemical Endocrinology, University Medical Center Nijmegen) for preparation of the 3T3-L1 adipocytes.

	FOOTNOTES

 
Abbreviations: aPKC, Atypical PKC; cPKC, classical PKC; DAG, diacylglycerol; 2-DOG, 2-deoxyglucose; FCS, fetal calf serum; GLUT, glucose transporter; IBMX, 3-isobutyl-1-methylxanthine; LDM, low-density membrane; nPKC, novel PKC; PKC, protein kinase C; PM, plasma membrane; PMA, phorbol-12-myristate-13-acetate; RT, room temperature; TBS, Tris-buffered saline.

Received for publication February 17, 2003. Accepted for publication April 11, 2003.

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