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Endocrinology Vol. 143, No. 1 37-46
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

Ceramide and Glucosamine Antagonism of Alternate Signaling Pathways Regulating Insulin- and Osmotic Shock-Induced Glucose Transporter 4 Translocation

Steve F. Kralik1, Ping Liu1, Brian J. Leffler and Jeffrey S. Elmendorf

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Center for Diabetes Research, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Jeffrey S. Elmendorf, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Center for Diabetes Research, Indianapolis, Indiana 46202-5120. E-mail: jelmendo{at}iupui.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to insulin, hyperosmolarity induces glucose transporter 4 (GLUT4) translocation in 3T3-L1 adipocytes. However, in contrast to insulin this stimulation is independent of PI3K/Akt. In this study we assessed whether ceramide and/or glucosamine, two known insulin-signaling antagonists, also affected the PI3K/Akt-independent signal. Insulin, but not hyperosmolarity, clearly increased the activities of PI3K and Akt. C2-ceramide did not alter insulin-stimulated PI3K activity, but did decrease the ability of insulin to activate Akt and GLUT4 translocation. Consistent with osmotic shock- mediated GLUT4 translocation being independent of PI3K/Akt, GLUT4 translocation induced by hyperosmolarity was not altered by C2-ceramide. In contrast to the specific C2-ceramide-induced attenuation of insulin-stimulated GLUT4 translocation, overexpression of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme in the synthesis of UDP-N-acetylglucosamine, and/or pretreatment of cells with glucosamine, a precursor of UDP-N-acetylglucosamine, inhibited both insulin- and hyperosmolarity-stimulated GLUT4 translocation. Glucosamine did not alter any of the known proximal insulin signal transduction events. These data suggest that although the hyperosmolarity-induced signal bypasses the initial insulin signal transduction steps, it is likely to induce GLUT4 translocation through activation of a common convergent signal transduction step, targeted by UDP-N-acetylglucosamine, downstream of and/or in parallel to PI3K/Akt.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDER NORMAL physiological conditions, excess glucose in the circulation is transported into muscle and fat cells by an insulin-stimulated process. This process requires transduction of an insulin signal(s), trafficking of the glucose transporter 4 (GLUT4) vesicle and a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated docking and fusion event (1, 2, 3, 4). With regard to signal transduction, it is well documented that insulin binding to the insulin receptor results in tyrosine autophosphorylation of the ß-subunit and activation of its intrinsic tyrosine kinase (4, 5, 6). Subsequently, the insulin receptor tyrosine kinase phosphorylates several intracellular proteins on tyrosine residues. In particular, tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins provides docking sites for p85, the regulatory subunit of type I PI3K, resulting in activation of the catalytic p110 subunit (4, 5, 6). It is well documented that activation of PI3K is a necessary step for the stimulation of GLUT4 translocation by insulin (7, 8, 9, 10, 11, 12, 13). The distal targets of PI3K, which mediate GLUT4 translocation, remain unclear, but Akt serine/threonine kinase isoforms 1 and 2 are candidate proteins that could be the downstream targets mediating the process (14, 15, 16, 17).

Although the exact molecular mechanisms linking the insulin receptor to GLUT4 translocation are uncertain, several pathophysiological events are well recognized to be potential causative factors that can induce insulin resistance by causing postreceptor defects in the insulin signaling pathway (18, 19, 20). Two events in particular are obesity and hyperglycemia. With regard to obesity, it has been postulated that adipose tissue secretes factors that impair insulin action. For example, TNF{alpha} is synthesized and secreted from adipocytes. TNF{alpha} stimulates sphingomyelinase activity to produce ceramides in cells, and it appears to be the ceramides that decrease insulin sensitivity. Although chronic TNF{alpha} treatment periods (2–4 d) decrease the tyrosine phosphorylation of IRS1, the decrease in insulin-stimulated glucose uptake observed at 2–24 h after treatment with cell-permeable C2-ceramides occurs without a decrease in tyrosine phosphorylation of the insulin receptor or IRS1 or a decrease in the interaction of IRS1 with PI3K (21, 22, 23). It has been demonstrated that C2-ceramide inhibits Akt activation independently of any effect on the insulin receptor, IRS1, and PI3K (24). Recent studies demonstrate that C2-ceramide prevented the membrane localization of the pleckstrin homology (PH) domains for Akt and the general receptor for phosphoinositides-1 (Grp1), which bind PI(3, 4, 5)P3, but not the PH domain for PLC{delta}, which binds PI(4, 5)P2 (25). The accumulation of 3'-phosphoinositides was not altered by C2-ceramide, indicating that this inhibition occurs via a previously undefined mechanism unrelated to 3'-phosphoinositide generation. In addition, the results indicate that cells lacking the PI(3, 4, 5)P3 phosphatase PTEN retained sensitivity to ceramide, indicating that the sphingolipid is unlikely to mediate its effects by targeting PTEN to a selected pool of PI(3, 4, 5)P3 (25). Together the data suggest a general mechanism by which ceramide antagonizes PI3K-dependent signaling at the level of Akt activation.

Hyperglycemia-induced insulin resistance is postulated to result from an increased flux of glucose through the hexosamine biosynthesis pathway, which, in turn, increases the tissue concentration of a hexosamine metabolite, UDP-N-acetylglucosamine (UDP-GlcNAc) (26, 27). Recent observations indicate that although UDP-GlcNAc induces defects in proximal insulin signaling, the variable times required for these effects could be compatible with more than one UDP-GlcNAc-induced defect in the insulin effector cascade (28, 29). A consideration, however, is that direct administration of glucosamine (GlcN; a precursor of UDP-GlcNAc) can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-GlcNAc (30). However, data argue against the possibility that ATP depletion (to the extent that it impairs insulin receptor function) is the major mechanism for GlcN-induced insulin resistance in 3T3-L1 adipocytes (31, 32, 33). Although the exact molecular mechanisms inducing insulin resistance are not defined, increases in the hexosamine biosynthetic pathway appear to render insulin-stimulated GLUT4 translocation ineffective at a distal site(s) in the signaling cascade.

It is now clear that GLUT4 translocation can be activated by a variety of agents by a mechanism not involving the proximal steps of the insulin signaling cascade. For example, exercise/contraction, hyperosmolarity, guanosine 5'-[{gamma}-thio]triphosphate (GTP{gamma}S; a nonhydrolyzable GTP analog), endothelin-1, expression of constitutively active Gq (Gq/Q209L), and sphingolipids all have been reported to stimulate GLUT4 translocation and glucose transport by a novel mechanism independent of PI3K activation (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Even though these alternative signaling pathways leading to GLUT4 translocation apparently bypass the initial insulin signal transduction steps, they are likely to induce GLUT4 translocation through activation of a common convergent signal transduction step downstream of and/or in parallel to PI3K. To further address the relationship between insulin- and hyperosmolarity-stimulated GLUT4 translocation, this study examined the effects of exposure of cells to either C2-ceramide or GlcN and also the effects of overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the synthesis of UDP-GlcNAc, on insulin- and hyperosmolarity-induced GLUT4 translocation. We found that GLUT4 translocation induced by hyperosmolarity was not affected by C2-ceramide, whereas increases in the hexosamine biosynthetic pathway blocked this insulinomimetic action of hyperosmolarity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Murine 3T3-L1 preadipocytes were obtained from American Type Culture Collection (Manassas, VA) and were cultured in DMEM containing 25 mM glucose and 10% calf serum at 37 C in an 8% CO2 atmosphere. Confluent cultures were induced to differentiate into adipocytes as previously described (39). All studies were performed on adipocytes, between 8–12 d after differentiation. Before all experimental treatments, the differentiated adipocytes were serum-starved in DMEM containing 25 mM glucose or 5.5 mM glucose (GlcN studies) and with 0.5% fatty acid-free BSA (C2-ceramide studies) for 3 h at 37 C.

Induction of insulin resistance
Ceramide treatment. The effects of ceramide on insulin- and hyperosmolarity-mediated events were investigated as described by Summers et al. (24). Briefly, cells were pretreated with either 0.1% dimethylsulfoxide (DMSO) vehicle control or 100 µM of the short-chain ceramide analog C2-ceramide for 2 h at 37 C. This concentration and treatment duration of C2-ceramide were documented to induce insulin resistance without a decrease in the tyrosine phosphorylation of the insulin receptor or IRS1 or a decrease in the interaction of IRS1 with PI3K or the enzyme’s in vitro activity (21, 22, 23, 24, 45). Consistent with these previous reports, preliminary experiments established that the concentration and treatment duration of C2-ceramide induced insulin resistance independently of any effect on the insulin receptor and IRS1 (data not shown). Thirty minutes before the end of C2-ceramide exposure, the cells were left untreated or were treated with insulin or sorbitol as described in the figure legends.

Transfection of GFAT. To first directly compare the effects of hexosamine metabolism on insulin- and hyperosmolarity-induced GLUT4 translocation, we performed cotransfection experiments with either empty vector control or GFAT plasmid cDNA (provided by Dr. Donald A. McClain, University of Utah) with GLUT4-enhanced green fluorescent protein (EGFP) plasmid cDNA as described below under Transient transfection. After transfection, the cells were allowed to recover for 16 h in the presence of 6 nM insulin and 25 mM glucose before experimental treatments.

GlcN treatment. As insulin resistance induced by GlcN exposure has been suggested to be secondary to depletion of ATP pools (30), we used a GlcN-induced model described by Ross et al. (31) that induces insulin resistance without affecting either the cellular ATP levels or the early insulin-regulated phosphorylation of substrates. Briefly, before induction of insulin resistance, adipocytes were treated with DMEM/5.5 mM glucose medium containing 10% FBS for 2 d. Cells were then treated with DMEM containing 10% FBS and 2 mM GlcN and lacking glucose, glutamine, and insulin for 12–14 h. The culture medium contained 1 mM pyruvate as an additional energy source. Also, 2 mM L-glucose was added to adjust the osmolarity of sugars in control groups.

Transient transfection
Differentiated adipocytes were electroporated (0.16 kV and 960 µF) as previously described (46). As indicated in the figure legends, transfection or cotransfected experiments were performed with 50 µg EGFP-tagged plasmid DNA or with 50 µg EGFP-tagged plasmid DNA plus 200 µg of additional plasmid DNA for analysis of EGFP fluorescence, respectively. After electroporation, the adipocytes were replated on glass coverslips and allowed to recover for 16–18 h before use.

Plasma membrane sheet assay
The preparation of plasma membrane sheets from the adipocytes was performed essentially by the method of Robinson et al. (41) with minor modifications as previously described (39). After the isolation of plasma membrane sheets, these purified membranes were used for indirect immunofluorescence. The plasma membrane sheets were fixed for 20 min at 25 C in a solution containing 2% paraformaldehyde, 70 mM KCl, 30 mM HEPES (pH 7.5), 5 mM MgCl2, and 3 mM EGTA as previously described (39). The membrane sheets were then blocked in 5% donkey serum for 60 min at 25 C and incubated for 60 min at 25 C with a 1:1000 dilution of polyclonal rabbit GLUT4 antibody (provided by Dr. Jeffrey E Pessin, University of Iowa, Iowa City, IA), followed by incubation with a 1:50 dilution of rhodamine red-X-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min at 25 C.

Preparation of total cell extracts and immunoprecipitation
Total cell extracts were prepared from 100-mm plates of 3T3-L1 adipocytes after the appropriate treatment. Cells from each plate were washed twice with ice-cold PBS and scraped into 1 ml lysis buffer [25 mM Tris (pH 7.4), 50 mM NaF, 10 mM Na3P2O7, 137 mM NaCl, 10% glycerol, and 1% Nonidet P-40] containing 1.0 mM phenylmethylsulfonylfluoride, 2 mM Na3VO4, 1 µg/ml aprotinin, 10 mM leupeptin, and 1 mM pepstatin A by rotation for 15 min at 4 C. Insoluble material was separated from the soluble extract by microcentrifugation for 15 min at 4 C. Protein concentration was determined, and samples were either directly subjected to SDS-PAGE (as described below) or immunoprecipitated for Cbl. Briefly, 3–5 mg cellular protein were immunoprecipitated with 5 µg Cbl polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h at 4 C. Immune complexes were recovered by the addition of protein A-Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL) and subjected to SDS-PAGE (as described below).

PI3K activity assay
Whole-cell detergent lysates were immunoprecipitated overnight (12–16 h) at 4 C with phosphotyrosine antibody (PT-66) conjugated to agarose (Sigma, St. Louis, MO). The immunoprecipitated lipid kinase activity was determined as previously described (47, 48). Briefly, the immunoprecipitates were incubated with 40 µCi [{gamma}-32P]ATP plus 20 µg phosphatidylinositol (Avanti Polar Lipids, Birmingham, AL) for 15 min at room temperature. The radiolabeled phospholipid product was spotted onto silica plates (Analtech, Newark, DE), subjected to TLC, and visualized by autoradiography.

Electrophoresis and immunoblotting
Whole cell lysates were separated by 7.5% SDS-PAGE. The resolved proteins were transferred to an Immobilon P membrane (Millipore Corp., Bedford, MA) and immunoblotted with a monoclonal phosphotyrosine antibody (PY20:HRPO; Transduction Laboratories, Inc., Lexington, KY), the phosphoserine-specific Akt antibody (New England Biolabs, Inc., Beverley, MA), or an anti-Akt2-specific antibody (provided by Dr. Morris J. Birnbaum, University of Pennsylvania, Philadelphia, PA). All immunoblots were subjected to enhanced chemiluminescence detection (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin-stimulated, but not hyperosmolarity-stimulated, GLUT4 translocation is inhibited by C2-ceramide
The involvement of ceramides in modulating insulin signaling is well documented (24, 45, 49, 50). As typically observed, both insulin- and sorbitol-induced osmotic shock resulted in the translocation of GLUT4 to the cell surface membrane, as detected by increased GLUT4 immunofluorescence in isolated plasma membrane sheets (Fig. 1Go, panels 1–3). Exposure of 3T3-L1 adipocytes with 100 µM C2- ceramide for 2 h had no effect on the basal state level of plasma membrane-localized GLUT4 (Fig. 1Go, panel 4), but inhibited insulin-stimulated GLUT4 translocation (Fig. 1Go, panel 5). In contrast, 100 µM C2-ceramide for 2 h had no effect on sorbitol-induced GLUT4 translocation (Fig. 1Go, panel 6). It is noteworthy that the insulin-stimulated level of plasma membrane GLUT4 immunofluorescence was not completely abolished in the presence of C2-ceramide.



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  		Figure 1. C2-ceramide decreases insulin-stimulated, but not hyperosmolarity-stimulated, GLUT4 translocation. Cells were incubated in the presence of vehicle control (V; 0.1% DMSO; panels 1–3) or 100 µM C2-ceramide (panels 4–6) for 2 h. The cells were then left untreated (C; control; panels 1 and 4) or were treated with 1 nM insulin (I; panels 2 and 5) or with 600 mM sorbitol (S; panels 3 and 6) during the final 30 min of C2-ceramide exposure. Plasma membrane sheets were prepared and subjected to immunofluorescence microscopy as described in Materials and Methods. These data are representative observations from three independent experiments.

 
To further investigate the apparent specificity of C2- ceramide on insulin, but not osmotic shock, signaling, we took advantage of the fact that expressed GLUT4-EGFP displays identical subcellular distribution and insulin-stimulated translocation as the endogenous GLUT4 protein (51). As typically observed for the endogenous GLUT4 protein, in the basal state GLUT4-EGFP was localized to the perinuclear region and small vesicles scattered throughout the cytoplasm (Fig. 2AGo, panel 1). This distribution was identical to that in cells pretreated with 100 µM C2-ceramide for 2 h (Fig. 2AGo, panel 4). As expected, both insulin stimulation and osmotic shock resulted in the translocation of the GLUT4-EGFP protein to the plasma membrane detected as a continuous rim of cell surface fluorescence (Fig. 2AGo, panels 2 and 3). Consistent with the effect of C2-ceramide treatment on GLUT4 translocation elicited by insulin and osmotic shock, as assessed by the plasma membrane sheet assay (Fig. 1Go), insulin-induced, but not osmotic shock-induced, GLUT4-EGFP translocation was inhibited (Fig. 2Go, panels 5 and 6). Quantitation of these data by scoring the transfected cells for GLUT4-EGFP plasma membrane rim fluorescence is presented in Fig. 2BGo. As previously reported (38), sorbitol treatment was not as effective as insulin in stimulating GLUT4 translocation (Figs. 1Go and 2Go). In agreement with the incomplete inhibition observed by the plasma membrane sheet assay (Fig. 1Go), in the presence of C2-ceramide, insulin mediates GLUT4 translocation to a magnitude similar to that induced by hyperosmolarity.



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  		Figure 2. C2-ceramide decreases insulin-stimulated, but not hyperosmolarity-stimulated, translocation of GLUT4-EGFP. A, Differentiated 3T3-L1 adipocytes were electroporated with 50 µg GLUT4-EGFP cDNA (panels 1–6). The cells were allowed to recover for 16 h, then were incubated in serum-free medium for 2 h and incubated in the presence of vehicle control (V; 0.1% DMSO; panels 1–3) or 100 µM C2-ceramide (panels 4–6) for 2 h. Cells were subsequently left untreated (C; control; panels 1 and 4) or were treated with 1 nM insulin (I; panels 2 and 5) or 600 mM sorbitol (S; panels 3 and 6) during the final 30 min of C2-ceramide exposure. Cells were fixed and subjected to confocal fluorescence microscopy, and representative images were obtained. B, Quantitation of the number of GLUT4-EGFP-expressing cells displaying rim fluorescence indicative of GLUT4 translocation. These data represent scoring of 50 cells from 3 independent experiments. Each bar represents the average number of cells displaying cell surface fluorescence ± SEM.

 
C2-ceramide reduces insulin-stimulated Akt activity
It is well documented that the antagonistic actions of ceramides on insulin action occur without a decrease in the tyrosine phosphorylation of the insulin receptor or IRS1 or a decrease in the interaction of IRS1 with PI3K or the activity of PI3K (21, 22, 23, 24, 45). In full agreement, we observed that cells treated with 100 µM C2-ceramide for 2 h displayed no decrease in the autophosphorylation of the insulin receptor ß-subunit or the protein tyrosine phosphorylation of IRS (data not shown). Furthermore, C2-ceramide did not affect the amount of insulin-stimulated PI3K activity in antiphosphotyrosine immunoprecipitates (Fig. 3Go). To confirm whether C2-ceramide inhibits the phosphorylation of Akt, we used an antiphospho-Ser473 antibody specific for the serine-phosphorylated activated Akt1 isoform and an Akt2-specific antibody that specifically allows detection of the mobility of that isoform indicative of a phosphorylation activation state (24, 52). C2-ceramide’s effect on insulin-stimulated Akt1 activity was apparent by a marked inhibition of the phosphorylation of serine 473, a regulatory phosphorylation site on Akt (Fig. 4AGo, lanes 3 and 4). Moreover, an insulin-induced mobility shift in the Akt2 isoform protein was decreased by C2-ceramide (Fig. 4BGo, lanes 3 and 4). As we previously reported (38), sorbitol-induced osmotic shock did not result in phosphorylation of either Akt isoform (Figs. 4AGo and 3BGo, lane 5). C2-ceramide had no apparent effect on the phosphorylation and mobility of Akt1 and Akt2, respectively, in nontreated and sorbitol-treated cells (Fig. 4Go, A and B, lanes 1 and 2, and 5 and 6).



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  		Figure 3. C2-ceramide does not antagonize insulin-stimulated immunoprecipitable PI3K activity. 3T3-L1 adipocytes were incubated in the presence of vehicle control (V; 0.1% DMSO; lanes 1, 3, and 5) or 100 µM C2-ceramide (lanes 2, 4, and 6) for 2 h. Cells were subsequently left untreated (C; control; lanes 1 and 2) or were treated with 1 nM insulin (I; lanes 3 and 4) or 600 mM sorbitol (S; lanes 5 and 6) during the final 30 min of C2-ceramide exposure. Whole cell detergent lysates were prepared, immunoprecipitated with the PT66 phosphotyrosine antibody, and assayed for the presence of PI3K activity as described in Materials and Methods. The radioactivity incorporated into phosphatidylinositol 3-phosphate was visualized by autoradiography (upper panel). The spot corresponding to phosphatidylinositol 3-phosphate was scraped, and its radioactivity was quantified by liquid scintillation counting (lower panel). Each bar is expressed as a percentage of control activity in the absence of C2-ceramide pretreatment and represents the mean ± SEM of five determinations.

 


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  		Figure 4. C2-ceramide inhibits insulin-stimulated phosphorylation of Akt. 3T3L1 adipocytes were incubated in the presence of vehicle control (-; 0.1% DMSO; lanes 1, 3, and 5) or 100 µM C2-ceramide (+; lanes 2, 4, and 6) for 2 h. Cells were subsequently left untreated (C; control; lanes 1 and 2) or were treated with 1 nM insulin (I; lanes 3 and 4) or 600 mM sorbitol (S; lanes 5 and 6) during the final 30 min of C2-ceramide exposure. Western blots of total cell detergent lysates were probed with antiphosphoserine 473-specific Akt antibody (A; pSer-Akt) or anti-Akt-2 antibody (B). These are representative immunoblots independently performed three times.

 
As the ability of C2-ceramide to decrease Akt activation has been reported to result from a specific prevention of the recruitment of the PI(3, 4, 5)P3-binding proteins Akt and Grp1 to the plasma membrane, we next assessed the in vivo localization of the Grp1 PH domain with PI(3, 4, 5)P3. This was accomplished by generating a fusion protein consisting of EGFP fused to this PH domain (EGFP-PH/Grp1) as previously described (48). In the absence of insulin, expression of EGFP-PH/Grp1 resulted in a predominant nuclear localization, with a smaller amount distributed throughout the cell cytoplasm in the absence or presence of C2-ceramide (Fig. 5AGo, panels 1 and 4). The accumulation of the EGFP-PH/Grp1 fusion protein in the nucleus is a property of EGFP in 3T3-L1 adipocytes, as expression of EGFP alone also results in predominant nuclear localization (data not shown). In any case, insulin stimulation resulted in accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane, indicative of PI(3, 4, 5)P3 formation at the plasma membrane (Fig. 5AGo, panel 2). Consistent with osmotic shock eliciting a PI3K-independent signaling pathway, sorbitol incubation did not result in the formation of PI(3, 4, 5)P3 (Fig. 5AGo, panel 3). In agreement with Stratford et al. (25), C2-ceramide clearly reduced the stimulated translocation of EGFP-PH/Grp1 to the cell surface (Fig. 5AGo, panel 5). Quantification of these data demonstrated that insulin-stimulated cell surface EGFP-PH/Grp1 fluorescence was markedly reduced in cells pretreated with C2-ceramide (Fig. 5BGo).



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  		Figure 5. C2-ceramide prevented the insulin-stimulated translocation of insulin-stimulated EGFP-PH/Grp1 to the plasma membrane. 3T3-L1 adipocytes were transfected with 50 µg EGFP-PH/Grp1 cDNA and allowed to recover for 16 h. The cells were then incubated in the presence of vehicle control (V; 0.1% DMSO; panels 1–3) or 100 µM C2- ceramide (C; panels 4–6) for 2 h. Cells were subsequently left untreated (C; control; panels 1 and 4) or were treated with 1 nM insulin (I; panels 2 and 5) or 600 mM sorbitol (S; panels 3 and 6) during the final 30 min of C2-ceramide exposure and then fixed in 2% paraformaldehyde. A, The distribution of the EGFP-PH/Grp1 fusion protein was visualized by confocal fluorescence microscopy as described under Materials and Methods. B, Quantitation of the number of cells displaying EGFP-PH/Grp1 cell surface fluorescence was determined from counting of 50 cells from 3 independent experiments. Each point represents the average number of cells displaying cell surface fluorescence ± SEM.

 
Increases in the hexosamine biosynthesis pathway inhibit both insulin- and osmotic shock-stimulated GLUT4 translocation
Work by several laboratories has demonstrated that increases in the hexosamine biosynthesis pathway inhibit insulin-stimulated GLUT4 translocation and glucose transport (28, 30, 31, 32, 33). This insulin-resistant state can be induced experimentally by expression of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme in the synthesis of UDP-GlcNAc and/or pretreatment of cells with GlcN, a precursor of UDP-GlcNAc (Fig. 6AGo). Recent data are consistent with the concept that the mechanism by which increased hexosamine biosynthesis pathway flux inhibits insulin-stimulated GLUT4 translocation and glucose transport occurs at a target site(s) distal to PI 3-Kinase/Akt (29, 31, 32). As hyperosmolarity appears to induce GLUT4 translocation by a signaling mechanism distal and/or parallel to PI3K/Akt, we determined whether increased hexosamine biosynthesis renders its signal ineffective. As expected, both insulin stimulation and osmotic shock resulted in translocation of the GLUT4-EGFP protein to the plasma membrane, detected as a continuous rim of cell surface fluorescence (Fig. 6BGo). In contrast, the ability of insulin and sorbitol treatment to stimulate the translocation of GLUT4-EGFP in cells coexpressing GFAT was decreased (Fig. 6BGo).



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  		Figure 6. Effects of the hexosamine biosynthesis pathway on GLUT4 translocation elicited by insulin and hyperosmolarity. A, In isolated adipocytes, 2–3% of incoming glucose and all GlcN are routed through the hexosamine biosynthetic pathway, culminating in the production of UDP-GlcNAc (61 ). The enzyme GFAT mediates the rate-limiting step in converting fructose 6-phosphate to GlcN 6-phosphate. B, Differentiated 3T3-L1 adipocytes were electroporated with 50 µg GLUT4-EGFP cDNA (bars 1–6) and 200 µg GFAT cDNA empty vector (CONT; {square}) or GFAT cDNA (GFAT; {blacksquare}). The cells were allowed to recover for 16 h in the presence of 6 nM insulin and 25 mM glucose. Cells were subsequently incubated in serum-free medium without insulin for 2 h and then left untreated (C; control) or treated with 100 nM insulin (I) or 600 mM sorbitol (S) for 30 min. Cells were fixed and subjected to confocal fluorescence microscopy, and representative images obtained. Quantitation of the number of GLUT4-EGFP-expressing cells displaying rim fluorescence indicative of GLUT4 translocation was obtained by scoring 50 cells from 3 independent experiments. Each bar represents the average number of cells displaying cell surface fluorescence ± SEM.

 
The incomplete inhibition observed was not an entirely unexpected result, because GFAT-stimulated production of UDP-GlcNAc is dependent on substrate and insulin availability. For this reason we allowed our electroporated 3T3-L1 adipocytes to recover in the presence of 6 nM insulin and 25 mM glucose for 16 h before serum starvation and treatments. Rather than obtaining a more significant inhibition with GFAT expression by modifying the incubation conditions, we tested the effects of providing a direct precursor of UDP-GlcNAc, GlcN (Fig. 7Go). Insulin and sorbitol treatment resulted in translocation of GLUT4 to the cell surface membrane, as detected by increased GLUT4 immunofluorescence in isolated plasma membrane sheets (Fig. 7AGo, panels 1–3). Pretreatment of 3T3-L1 adipocytes with 2 mM GlcN for 12 h had no effect on the basal state level of plasma membrane-localized GLUT4 (Fig. 7AGo, panel 4), but markedly inhibited both insulin- and sorbitol-induced GLUT4 translocation (Fig. 7AGo, panels 5 and 6). We also assessed the effects of GlcN on the distribution of GLUT4-EGFP. In the basal state GLUT4-EGFP was localized to the perinuclear region and small vesicles scattered throughout the cytoplasm. This distribution was identical to that in cells pretreated with GlcN. As expected, both insulin stimulation and sorbitol resulted in translocation of the GLUT4-EGFP protein to the plasma membrane, detected as a continuous rim of cell surface fluorescence. Consistent with the effect of GlcN pretreatment on insulin- and sorbitol-induced GLUT4 translocation, as assessed by the plasma membrane sheet assay (Fig. 7AGo), insulin- and sorbitol-induced GLUT4-EGFP translocation was markedly inhibited. Quantitation of these data by scoring the transfected cells for GLUT4-EGFP plasma membrane rim fluorescence is presented in Fig. 7BGo.



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  		Figure 7. GlcN exposure decreases both insulin- and hyperosmolarity-stimulated GLUT4 translocation. As described in Materials and Methods, cells were incubated overnight in the absence (-) or presence (+) of 2 mM GlcN. After GlcN treatment, nontransfected cells (A) or cells electroporated with 50 µg GLUT4-EGFP cDNA (B) were left untreated (C; control) or were treated with 100 nM insulin (I) or 600 mM sorbitol (S) for 30 min. A, Plasma membrane sheets were prepared and subjected to immunofluorescence microscopy. Representative observations from 3 independent experiments are shown. B, Transfected cells were fixed and subjected to confocal fluorescence microscopy, and representative images were obtained and counted for the number of GLUT4-EGFP-expressing cells displaying rim fluorescence indicative of GLUT4 translocation. These data represent scoring of 50 cells from 3 independent experiments. Each bar represents the average number of cells displaying cell surface fluorescence ± SEM.

 
Insulin receptor autophosphorylation, IRS1, and Cbl phosphorylation
Based on the above observations, we next examined the effect of GlcN on the early insulin receptor signal transduction events. There was no significant difference in insulin receptor ß-subunit autophosphorylation in the absence or presence of GlcN (Fig. 8Go, lanes 3 and 4). Similarly, the extent of IRS1 tyrosine phosphorylation was not significantly affected (Fig. 8Go, lanes 3 and 4). Osmotic shock has been reported to stimulate GLUT4 translocation by a tyrosine kinase- dependent mechanism (38). As previously reported, osmotic shock induced the tyrosine phosphorylation of several proteins in the molecular mass ranges of 55–75, 120–130, and 160–180 kDa (38) (Fig. 8Go, compare lanes 1 and 5). The extent of tyrosine phosphorylation of these proteins was also not significantly affected by GlcN pretreatment (Fig. 8Go, lane 6).



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  		Figure 8. GlcN treatment did not alter tyrosine phosphorylation events elicited by insulin or hyperosmolarity. After the overnight treatment without (-) or with (+) GlcN, performed as described in Materials and Methods, cells were left untreated (C; control) or were treated with 100 nM insulin (I) or 600 mM sorbitol (S) for 5 min. Whole cell detergent extracts were prepared, resolved on a 7.5% polyacrylamide gel, and subjected to Western blotting with the PY20 monoclonal phosphotyrosine antibody. This is a representative immunoblot from three independent experiments.

 
In addition to IRS proteins, in adipocytes the insulin receptor and the osmotic shock-induced signal also tyrosine phosphorylate Cbl (38, 53, 54, 55). This can readily be detected by the immunoprecipitation of Cbl, followed by phosphotyrosine immunoblotting (Fig. 9Go). There was no significant difference in insulin- and osmotic shock-stimulated tyrosine phosphorylation of Cbl in the absence or presence of GlcN (Fig. 9AGo). Cbl immunoblotting of that same membrane demonstrated the presence of equal amounts of Cbl protein (Fig. 9BGo, lanes 1–6). Although the signaling events distal to the Cbl cascade remain unclear, as previously discussed a likely downstream candidate molecule of the IRS cascade involved in GLUT4 translocation is the Akt serine/threonine kinase. In contrast to the inhibitory effect of C2-ceramide on Akt1/2, there was no apparent GlcN-induced effect on insulin-stimulated Akt1 and/or Akt2 activity, as assessed by the enzyme’s serine 473 phosphorylation state (Fig. 10AGo, lanes 3 and 4) and/or its electrophoretic mobility (Fig. 10BGo, lanes 3 and 4), respectively. As depicted in Fig. 4Go, sorbitol-induced osmotic shock did not result in the activation of either Akt isoform (Fig. 10Go, A and B, lane 5). Glucosamine had no apparent effect on the phosphorylation and/or the mobility of Akt from nontreated and sorbitol-treated cells (Fig. 10Go, A and B, lanes 1 and 2, and 5 and 6). Furthermore, GlcN treatment did not affect insulin-stimulated PI(3, 4, 5)P3 formation, as assessed by accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane, in EGFP-PH/Grp1-expressing cells (data not shown).



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  		Figure 9. Insulin- and hyperosmolarity-stimulated Cbl tyrosine phosphorylations were not affected by GlcN treatment. 3T3L1 adipocytes were treated without (-) or with (+) GlcN as described in Materials and Methods and then left untreated (C; control; lanes 1 and 2) or were treated with 100 nM insulin (I; lanes 3 and 4) or 600 mM sorbitol (S; lanes 5 and 6) for 5 min. Whole cell detergent extracts were prepared and immunoprecipitated with a Cbl antibody as described in Materials and Methods. The samples were then resolved by SDS-PAGE and subjected to PY20 phosphotyrosine (A) and Cbl (B) immunoblotting. This is a representative immunoblot from three independent experiments.

 


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  		Figure 10. Insulin-stimulated phosphorylation of Akt was not altered by GlcN treatment. 3T3L1 adipocytes were treated without (-) or with (+) GlcN as described in Materials and Methods and then left untreated (C; control; lanes 1 and 2) or treated with 100 nM insulin (I; lanes 3 and 4) or 600 mM sorbitol (S; lanes 5 and 6) for 5 min. Western blots of total cell detergent lysates were probed with antiphosphoserine 473-specific Akt antibody (A; pSer-Akt) or anti-Akt-2 antibody (B). These are representative immunoblots independently performed three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study used two models of insulin resistance as molecular probes to better understand the relationship between insulin- and osmotic shock-induced GLUT4 translocation. The main finding of this work is that although insulin-stimulated GLUT4 translocation is attenuated in cells exposed to C2-ceramide and GlcN and in cells overexpressing GFAT, antagonism of GLUT4 translocation elicited by hyperosmotic incubation conditions is only present in cells exposed to GlcN or in cells overexpressing GFAT, not in cells treated with C2-ceramide. This finding confirms that the hyperosmolarity-induced signaling pathway bypasses initial insulin signal transduction steps and suggests that it probably induces GLUT4 translocation through activation of a common convergent signal transduction step downstream of and/or in parallel to PI3K/Akt.

Our C2-ceramide results are consistent with those from the work of Summer et al. (24), and demonstrate that C2- ceramide inhibits insulin-stimulated GLUT4 translocation and Akt phosphorylation by similar extents. It was also reported that C2-ceramide, at concentrations that antagonized the activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of IRS1 or the levels of p85 protein and PI3K activity (24). Moreover, C2-ceramide inhibited stimulation of Akt by platelet-derived growth factor (PDGF), an event that is IRS1 independent. In agreement with these observations, we demonstrate in this work that C2-ceramide did not inhibit insulin-stimulated events such as autophosphorylation of the insulin receptor ß-subunit, tyrosine phosphorylation of IRS1, or PI3K activation. Interestingly, we observed that C2-ceramide markedly inhibited the insulin-stimulated localization of EGFP-PH/Grp1 to the cell surface. This finding is in full agreement with a recent study by Stratford et al. (25), who reported that exposure of 3T3-L1 adipocytes to C2-ceramide inhibits PDGF-stimulated translocation of full-length Akt as well as truncated proteins encoding only the PH domains of Akt/PKB or Grp1. Also, the membrane localization of the PH domain for PLC{delta}, which preferentially binds PI(4, 5)P2, or the PDGF-stimulated production of PI(3, 4)P2 or PI(3, 4, 5)P3 was found not to be affected by C2-ceramide. Although our work did not assess the effect of C2-ceramide on PI(3, 4, 5)P3 levels, our data support the concept that C2-ceramide affects the ability of insulin to activate GLUT4 translocation at the level of Akt, and the mechanism for that inhibition appears to involve a defect in some functional aspect of PH domain/PI(3, 4, 5)P3-directed plasma membrane targeting.

In any case, C2-ceramide was clearly ineffective in blocking osmotic shock-induced GLUT4 translocation. This is consistent with a PI3K/Akt-independent pathway regulating GLUT4 translocation. In this regard, it is clear that a variety of stimuli display insulinomimetic properties and can induce the trafficking of GLUT4 to the plasma membrane by a mechanism not involving PI3K/Akt. For example, in addition to hyperosmolarity, exercise/contraction, GTP{gamma}S (a nonhydrolyzable GTP analog), and endothelin-1 all have been reported to stimulate GLUT4 translocation and glucose transport by a novel mechanism independent of PI3K/Akt activation (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44). Interestingly, the PI3K/Akt-independent mechanism initiated by hyperosmolarity and GTP{gamma}S requires tyrosine kinase activity (38, 39, 43). Studies by Wu-Wong et al. (44) are in full agreement and demonstrate that the PI3K-independent insulinomimetic actions of endothelin-1 are also tyrosine kinase dependent. Studies by Standaert et al. (56) and Imamura et al. (57) also observed the insulinomimetic actions of GTP{gamma}S and endothelin-1. Although their results are in agreement with the ability of these agents to stimulate GLUT4 translocation, their data indicate that GTP{gamma}S and endothelin-1 are upstream activators of PI3K signaling. Although the basis for this difference is not apparent, and it remains unclear whether the signaling pathway used by hyperosmolarity, GTP{gamma}S, and endothelin-1 are the same, this study clearly lends support to a PI3K/Akt-independent signal mediating osmotic shock-induced GLUT4 translocation.

Even though the signaling pathway elicited by hyperosmolarity presumably bypasses the initial insulin signal transduction steps, it is likely to induce GLUT4 translocation through activation of a common convergent signal transduction step downstream of and/or in parallel to PI3K/Akt. In particular, the docking/fusion of GLUT4 vesicles with the plasma membrane appear to use a similar mechanism, as expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation (38). Analogously, several lines of evidence have suggested such a signaling scenario by which the GTP-binding protein Gq and/or G11 couple to GLUT4 translocation. For example, introduction of GDPßS into adipocytes, microinjection of a Gq/G11-specific antibody, and/or expression of RGS proteins inhibit insulin-stimulated GLUT4 translocation, thus suggesting that Gq/G11 plays a necessary role in insulin-stimulated glucose transport (39, 57, 58). The fact that in the presence of C2-ceramide insulin-stimulated GLUT4 translocation was reduced to a magnitude similar to that induced by hyperosmolarity provides suggestive evidence that insulin stimulates both a ceramide-sensitive and a ceramide-insensitive pool of GLUT4 vesicles. The data could thus be interpreted that hyperosmolarity-induced GLUT4 translocation is mediated by an insulin-stimulated PI3K/Akt-independent pathway, such as the recently described Cbl/CAP signaling pathway (59, 60). However, this interpretation is complicated by the fact that the inhibitory effect of C2-ceramide on Akt is not complete, and it is unclear how much Akt activation is required for insulin-stimulated GLUT4 translocation.

In an alternative approach to examine the relationship between insulin- and hyperosmolarity-activated signals, we assessed the insulin resistance-inducing effects of the hexosamine biosynthesis pathway. The involvement of this pathway in the regulation of insulin action was originally discovered by Marshell et al. (61). That work demonstrated the induction of insulin resistance in isolated primary rat adipocytes with insulin, glucose, and glutamine. They postulated and confirmed that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance. Although both in vitro and in vivo experimental evidence agree that increases in the hexosamine biosynthesis pathway are accompanied by inhibition of the insulin-stimulated GLUT4 translocation process (32, 33, 62), how the metabolic end products of this pathway inhibit insulin-stimulated GLUT4 translocation remains unresolved. The current studies strongly suggest that the GlcN-induced defect in the insulin effector cascade occurs at a site(s) distal and/or parallel to Akt and/or Cbl. The disabling effect of GlcN on the PI3K/Akt-independent signal elicited by osmotic shock provides further support for a GlcN-induced defect in a cellular target(s) distal to those enzymes. Although this may imply that the osmotic shock-elicited signal regulating GLUT4 translocation shares common insulin signal transduction steps downstream of and/or in parallel to PI3K/Akt and/or Cbl, it is possible that GlcN-induced resistance may involve the existence of defects at more than one site.

The present data indicate that the ability of insulin to activate Akt and GLUT4 translocation is attenuated in the presence of C2-ceramide, whereas hyperosmolarity-induced GLUT4 translocation is intact under similar exposure to C2-ceramide. As experimentally induced increases in the hexosamine biosynthetic pathway in 3T3-L1 cells rendered both the PI3K/Akt-dependent and the PI3K/Akt-independent pathways ineffective, the GLUT4 translocation defect lies distal and/or parallel to PI3K/Akt. Presumably, increased flux into the GlcN pathway could alter some aspect of the newly described Cbl/CAP signaling pathway (59, 60). We found that both insulin- and osmotic shock-elicited tyrosine phosphorylations of Cbl are intact with GlcN exposure. Taken together, the data suggest that ceramide-induced defects in GLUT4 translocation result from a specific alteration early in the insulin signal transduction cascade at the level of Akt activation, whereas end products of the hexosamine biosynthetic pathway may target more distant and/or parallel insulin signaling cascade events. Interestingly, this work clearly demonstrates that increases in the hexosamine biosynthetic pathway rendered both insulin and osmotic shock signals ineffective. Although it remains to be determined whether hyperosmolarity-induced GLUT4 translocation uses the Cbl/CAP pathway, it is documented that insulin- and hyperosmolarity-activated signals involve common SNARE proteins. Whether increases in the hexosamine biosynthetic pathway influences the Cbl/CAP pathway and/or any SNARE proteins involved in GLUT4 vesicular trafficking awaits further experimentation.


   Acknowledgments
 
We are grateful to Drs. Morris J. Birnbaum, Donald A. McClain, and Jeffrey E. Pessin for generously providing us with GLUT4 and Akt2-specific antibodies and GLUT4-EGFP, EGFP-PH/Grp1, and GFAT plasmid cDNAs. We thank Drs. Robert V. Considine, and Scott A. Summers for helpful discussion of the data.


   Footnotes
 
This work was supported by an American Diabetes Foundation Career Development Award.

1 S.K. and P.L. contributed equally to this study. Back

Abbreviations: DMSO, Dimethylsulfoxide; EGFP, enhanced green fluorescent protein; GFAT, glutamine:fructose-6-phosphate amidotransferase; GlcN, glucosamine; GLUT4, the insulin-responsive glucose transporter; GTP{gamma}S, guanosine 5'-[{gamma}-thio]triphosphate; IRS, insulin receptor substrate; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI(3 4 5 )P3, phosphaditylinositol-3,4,5-trisphosphate; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; UDP-GlcNAc, UDP-N-acetylglucosamine.

Received February 26, 2001.

Accepted for publication September 27, 2001.


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