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Endocrinology Vol. 148, No. 3 1359-1366
Copyright © 2007 by The Endocrine Society

Ceramide-Activated Protein Phosphatase Involvement in Insulin Resistance via Akt, Serine/Arginine-Rich Protein 40, and Ribonucleic Acid Splicing in L6 Skeletal Muscle Cells

Nilanjan Ghosh, Niketa Patel, Kun Jiang, James E. Watson, Jin Cheng, Charles E. Chalfant and Denise R. Cooper

Department of Molecular Medicine (N.G., N.P., K.J., D.R.C.), College of Medicine, University of South Florida, Tampa, Florida 33612; The Research Service (J.E.W., D.R.C.), James A. Haley Veterans Hospital, Tampa, Florida 33612; Moffitt Cancer Center (J.C.), Tampa, Florida 33612; and Department of Biochemistry (C.E.C.), Virginia Commonwealth University and Hunter Holmes McGuire Veterans Medical Center, Richmond, Virginia 23298

Address all correspondence and requests for reprints to: Denise R. Cooper, Ph.D., J. A. Haley Veterans Hospital VAR 151, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: dcooper{at}health.usf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Elevated TNF{alpha} levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of protein kinase CßII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNF{alpha} attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase. Western blot analysis of L6 cell lysates demonstrated impaired insulin-stimulated phosphorylation of Akt, serine/arginine-rich protein 40, and glycogen synthase kinase 3ß in response to TNF{alpha} and the short chain C6 ceramide analog. TNF{alpha} increased serine/threonine phosphatase activity of protein phosphatase 1 (PP1) in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by liquid chromatography-mass spectrometry showed consistent increases in C24:1 and C16 ceramides. Effects of TNF{alpha} and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3ß were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNF{alpha} abolished insulin-mediated inclusion of the protein kinase CßII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNF{alpha} effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL LINES OF evidence indicate that TNF{alpha}, a proinflammatory cytokine, plays a role in insulin resistance. TNF{alpha} expression is increased in insulin-resistant individuals, and infusing humans with TNF{alpha} induces insulin resistance (1, 2, 3). Most animal models of insulin resistance produce significantly higher levels of TNF{alpha}, and the TNF{alpha} knockout mouse demonstrated increased insulin sensitivity and improved lipid metabolism (2, 4). Furthermore, cells treated with TNF{alpha} also present with impaired insulin signaling: decreased insulin receptor tyrosine kinase activity, increased serine phosphorylation of insulin receptor substrate (IRS) 1, decreased GLUT4 translocation, and abolishment of insulin-mediated Akt phosphorylation (4, 5, 6, 7).

Ceramide, a sphingolipid second messenger, is also linked to insulin resistance (2). Ceramide is generated via hydrolysis of sphingomyelin by sphingomyelinase or by the de novo pathway via condensation of palmitoyl CoA and serine (8). Ceramide is elevated in insulin-responsive tissues of diabetic animals (9) and inhibits a number of kinases that are stimulated by insulin, including protein kinase B (PKBß/Akt) (10, 11, 12, 13) and protein kinase C (PKC) (7, 14, 15). It is also known to activate atypical PKCs (16, 17). TNF{alpha} activates both de novo and hydrolysis pathways of ceramide generation (18), and ceramide mimics the effects of TNF{alpha} on insulin signaling (19, 20, 21, 22) and apoptosis (23). Furthermore, when ceramide production is suppressed, TNF{alpha}-induced insulin resistance is reversed (24). However, the molecular mechanisms defining ceramide-induced alterations in insulin signaling remain unclear. Ceramide-activated protein phosphatases (CAPPs), including protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), are allosterically activated by ceramide (25, 26). CAPPs have been shown to inhibit Akt by dephosphorylation of serines (27) and to modulate key cellular processes including exocytosis, alternative pre-mRNA splicing, and glycogen metabolism (28, 29, 30). Hence, we investigated whether a CAPP could act on Akt and subsequent downstream substrates including serine/arginine-rich (SR) proteins involved in alternative splicing of PKCßII mRNA in skeletal muscle cells.

PKCß is a component of the serine/threonine kinase superfamily with roles in insulin signaling related to glucose transport and glycogen metabolism (31, 32, 33). It is a conventional PKC with two splice variants, ßI and ßII, that have distinct cellular functions. Insulin regulates alternative splicing of the PKCßII pre-mRNA (34, 35, 36). The regulation occurs by the phosphorylation of SR proteins under the control of Akt (37). The regulation is impaired in insulin-resistant states and diabetes (35).

This study was designed to define the molecular link between acute inflammatory cytokines, the activation of CAPPs, the ceramide species involved, phosphorylation status of Akt and SR proteins, and TNF{alpha} effects on insulin-stimulated PKCßII splicing. To investigate this, L6 rat myotubes were treated with TNF{alpha} and C6 ceramide, a short chain ceramide analog, and examined for insulin-dependent phosphorylation of Akt, serine/arginine-rich protein 40 (SRp40), and glycogen synthase kinase 3ß (GSK3ß). Inhibition of de novo ceramide generation blocked PP1, but not PP2A activation by TNF{alpha}. C16 and C24:1 ceramides were the major species increased by TNF{alpha}, and there was a significant reduction in insulin-mediated PKCßII exon inclusion in cells treated with the cytokine. Collectively, the results suggested that TNF{alpha} caused alterations in insulin signaling in a ceramide-dependent mechanism by activating CAPPs, which resulted in Akt and SRp40 dephosphorylation and decreased PKCßII splicing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
L6 rat skeletal muscle cells (obtained from Dr. Amira Klip, Hospital for Sick Children, Toronto, Canada) were grown in {alpha}-MEM supplemented with 10% fetal bovine serum (FBS) (Cellgro) to 50–60% confluency. Penicillin G and streptomycin sulfate (100 U/100 µg) were added and the cells were incubated at 5% CO2, 37 C, and 95% humidity. Cells were induced to differentiate by changing medium to {alpha}-MEM supplemented with 2% FBS for approximately 8 d. The extent of cellular differentiation was established by observation of multinucleation of more than 70% of cells. For phosphatase assays, cells were placed in HEPES-buffered saline with 0.1% BSA before treatments. For Western blot analysis, cells remained in {alpha}-MEM. TNF{alpha} was added to a final concentration of 15–150 ng/ml for 30 min. C6 ceramide (20 µM) was added for 2 h. Insulin was added at a final concentration of 10 nM for 30 min. Myriocin (5 nM), an inhibitor of de novo ceramide synthesis, was added for 1 h before TNF{alpha} treatment. Tautomycin (10 nM), an inhibitor of PP1, was added 1 h before TNF{alpha} treatment.

Immunoprecipitation
Culture dishes with differentiated cells (100 mm) were washed with Ca2+/Mg2+-free PBS, and cells were mechanically detached in buffer (Tris HCl, pH 7.4, 50 mM; NaCl, 150 mM; EDTA 1 mM; NaF, 10 mM; Triton x 100, 1% SDS, 0.1%; Na deoxycholate, 1%) with a mixture of antiproteases [leupeptin, 20 µg/ml; aprotinin, 10 µg/ml; phenylmethylsulfonyl fluoride (PMSF), 0.1 mM; dithiothreitol, 1 mM]. The lysate was centrifuged at 17,000 x g for 20 min at 4 C. The supernatant (0.5 ml) was used for immunoprecipitation by adding 25 µl A/G Sepharose, rotating the suspension 30 min at 4 C. SRp40 antibody was then added and samples rotated overnight at 4 C. The suspension was centrifuged at 2000 x g for 1 min, and the pellet was washed twice in Tris-buffered saline with Tween (TBST). After addition of Laemmli buffer, samples were separated on SDS-PAGE (38). Proteins were detected using antirabbit or antimouse IgG antibodies conjugated to horseradish peroxidase and enhanced chemiluminescense reagents. Controls include protein A and IgG (preimmune sera).

Western blotting
Culture dishes with differentiated cells were washed with Ca2+/Mg2+-free PBS, and cells were detached mechanically in buffer (Tris HCL, pH 7.4, 50 mM; NaCl, 150 mM; EDTA 1 mM; NaF, 10 mM; Triton X-100, 1%; SDS, 0.1%; Na deoxycholate, 1%) with a mixture of antiproteases (leupeptin, 20 µg/ml; aprotinin, 10 µg/ml; PMSF, 0.1 mM; DTT, 1 mM). The lysate was centrifuged at 17,000 x g for 20 min at 4 C. Cell lysates in Laemmli buffer (50 µg) were separated via 12.5% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose membranes and blocked with PBS with 0.1% Tween 20 and 4% nonfat dried milk. Membranes were washed and incubated with the following antibodies: phospho-Akt (serine 473; Cell Signaling, Danvers, MA); phospho-GSK3ß (serine 9; Cell Signaling); mAb104, a monoclonal antibody against the phosphoepitope (RS domain) of SR proteins; phospho-Akt substrate, detects phosphorylation of Akt substrates at their Akt motifs (R-X-R-X-X-S/T), SRp40, phospho-IRS-1 (serine 616; Cell Signaling) or IRS-1 (Cell Signaling). Equal loading or identification of SR proteins was verified by stripping and reprobing membranes with ß-actin or SRp40. After incubation with secondary antirabbit or antimouse IgG-HRP, detection was performed using enhanced chemiluminesence (Pierce, Rockford, IL). Results were verified in two to six separate experiments.

Phosphatase assays
Cells washed three times in ice-cold HEPES-buffered saline were scraped into 1 ml lysis buffer (50 mmol/liter Tris, pH 7.4, 150 mmol/liter NaCl, 1 mmol/liter PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and sonicated. Lysates were centrifuged at 400,000 x g for 10 min at 4 C. Up to 250 µl of supernatants were passed through 10-ml Sephadex G-25 columns to eliminate ATP and free phosphate. Serine/threonine phosphatase activities in these fractions were determined by measuring generation of free phosphate from phosphopeptide RRA(pT)VA using the molybdate-malachite green-phosphate complex assay (Promega, Madison, WI). The peptide substrate was used in the absence of Ca2+ and Mg2+, which are required for the activity of PP2B and 2C. Assays were carried out using 10 µl of lysate in 10 µl of a Ca2+- and Mg2+-deficient reaction buffer [final concentration 50 mmol/liter imidazole (pH 7.2), 0.2 mmol/liter EGTA, 0.02% 2-mercaptoethanol, 0.1 mg/ml BSA, and 100 µmol/liter phosphopeptide substrate], 5 µl phosphopeptide and 25 µl phosphate-free water with the addition of either okadaic acid (20 or 0.2 nm) to distinguish between PP1 and PP2A activities. Reactions were carried out for 15 min in a 37 C shaker water bath. To stop the reaction, molybdate dye was added and free phosphate was measured by OD at 630 nm. A standard curve was generated to determine free phosphate concentrations. Results were corrected for total protein in cell fractions by the Bradford method using a BSA standard curve, and specific activity was calculated. Experiments were repeated a minimum of two times, and reactions were carried out in duplicate or triplicate. Buffer plus peptide was used as a blank control.

Ceramide mass spectrometry
Cells were treated and collected for phosphatase assay, and approximately half of the total cell pellet was reserved for ceramide mass spectrometer analysis. The pellet was stored at –80 C. Ceramide molecular species analyses were performed by the Lipidomics Core Facility at the Medical University of South Carolina. Briefly, cell pellets were extracted into a one-phase neutral organic solvent system and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of ceramides was performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of ceramides (39). The results are presented as mean ± SD of picomoles of ceramide species per micrograms of protein in the sample.

Alternative splicing of PKCßII
L6 cells were plated at 40–50% confluency in six-well plates, and differentiation to myotubes was initiated for 24 h before transfection of the PKCßII reporter minigene (2 µg) mixed with 10 µl of Lipofectamine (Invitrogen, Carlsbad, CA) reagent for 4 h (35). After 36 h, fused myotubes (>70%) were pretreated for 30 min with TNF{alpha} or inhibitors, then insulin was added for 30 min. The minigene was evaluated for exon inclusion as follows. Total RNA was obtained using TRIzol reagent (Invitrogen). One microgram total RNA was reverse transcribed using oligo dT and the Superscript II preamplification kit. One microliter of the RT reaction was amplified using splice site donor and splice site acceptor-specific primers and Platinum Taq DNA polymerase. After 25 cycles (95 C for 30 sec melt and 68 C for 1 min anneal/extension), the PCR is examined on PAGE followed by silver staining. As a control, ß-actin was amplified (35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} and ceramide altered insulin-dependent phosphorylation cascades
The activation of Akt via phosphatidylinositol 3 (PI3) kinase is a central step in insulin action. Data from our lab have demonstrated that SRp40 is a direct target for phosphorylation and activation by Akt (37). SRp40 phosphorylation by PI3-kinase-dependent pathways is required for the splicing of PKCßII in insulin-dependent cells (35, 36). It has not been demonstrated whether TNF{alpha} via de novo ceramide synthesis activates a serine/threonine protein phosphatase activity that targets Akt and SRp40. This could represent another mechanism for TNF{alpha} to attenuate insulin action in skeletal muscle in addition to other known pathways that inhibit IRS-1 function (40). To examine the potential role of the ceramide pathway, L6 myotubes were pretreated for 30 min with TNF{alpha} or the short chain ceramide analog, D-e-C6 ceramide, and the phosphorylation states of Akt and SRp40 were analyzed by Western immunoblot. Insulin treatment increased Akt phosphorylation on serine 473, whereas TNF{alpha} pretreatment attenuated the action of insulin and Akt was less phosphorylated. C6 ceramide mimicked the effects of TNF{alpha} but had a much more potent effect in attenuating insulin effects (Fig. 1AGo). Similar results were obtained when mAb104 was used to detect phosphorylation at the RS domain of SRp40 immunoprecipitated from L6 myotubes (Fig. 1BGo). Because SRp40 is also an Akt substrate (37), insulin regulated phosphorylation patterns at the Akt substrate motifs of SRp40 were also analyzed in L6 myotubes (Fig. 1CGo). TNF{alpha} and C6 ceramide reduced insulin-stimulated phosphorylation of SRp40 at its Akt motifs. This effect was not observed with dihydro-C6 ceramide, an inactive ceramide analog, suggesting that the ceramide effects are not due to nonspecific effects of lipid treatment. Therefore, both ceramide and TNF{alpha} treatment resulted in the decreased phosphorylation of Akt and SRp40.


Figure 1
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  		FIG. 1. TNF{alpha} and C6 ceramide blocked insulin-mediated phosphorylation of Akt and SRp40. A, L6 cells were pretreated with TNF{alpha} (150 ng/ml, 30 min) or C6 ceramide (20 µM, 2 h) before insulin (Ins) (10 nM, 30 min) addition as indicated. Western blot analysis was conducted with phospho-Akt antibody (serine 473). The membrane was stripped and re-probed with ß-actin to ensure equal protein loading. B, L6 cells underwent similar treatments as above except that TNF{alpha} was 15 ng/ml. Lysates were immunoprecipitated with SRp40 antibody, proteins were separated, and membranes were probed first with mAb104 followed by SRp40 antibody. C, L6 cells were treated as above with the addition of a dihydroC6 treatment (20 µM, 2 h) or as a pretreatment followed by insulin as a control. Membrane was probed with phosphoAkt substrate antibody, stripped, and reprobed with SRp40 antibody to confirm identity of the protein. Each experiment was repeated a minimum of three times with similar results. The bar graphs summarize results from three experiments. The asterisk indicates a significant difference (P < 0.05, t test compared with insulin effect).

 
TNF{alpha} blocked PKCßII exon inclusion
Previous work in our lab has demonstrated that phosphorylated SRp40 is required for PKCßII exon inclusion (36), and insulin is responsible for this phosphorylation via Akt activity. To test the functional relevance of TNF{alpha}-mediated decrease in phosphorylation of Akt and SRp40, alterations in insulin-induced PKCßII exon inclusion were assessed using a heterologous splicing minigene. Our data demonstrate that insulin promoted inclusion of the PKCßII exon. Two splice sites were activated. Activation of splice site II (SSII) was blocked by TNF{alpha} and the PI3-kinase inhibitors. Splice site I usage was partially blocked, which may reflect the stronger role of SRp40 on SSII selection (35). Importantly, TNF{alpha} pretreatment blocked insulin effects on PKCßII SSII activation to the same extent as blocking Akt activation via the PI3-kinase/PDK pathway with LY294002 or wortmannin (Fig. 2Go).


Figure 2
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  		FIG. 2. TNF{alpha} blocked insulin-induced PKCßII exon inclusion. A heterologous minigene constructed by inserting the PKCßII genomic fragment into a multicloning site between the splice site donor (SD) and splice site acceptor (SA) in pSPL3 vector was used as a reporter for alternative splicing as described (35 ). PCR primers corresponding to the arrows in the upper diagram were used to detect insulin-mediated inclusion of the PKCßII exon at both 5' splice sites within 30 min; both splice variants encode PKCßII. Insulin effects were blocked by TNF{alpha} (15 ng/ml) similar to the two PI3-kinase/Akt inhibitors: Wortmannin (100 nM) and LY294002 (10 µM). The densitometric scan summarizes three separate experiments.

 
TNF{alpha} increased ceramide activated phosphatase activity in skeletal muscle cells
TNF{alpha} increases ceramide production via the de novo and sphingomyelinase pathways (2, 18). PP1 and PP2A are CAPPs (25). Although increases in ceramides are associated with insulin resistance (10, 19, 20, 21, 24), the activation of serine/threonine protein phosphatases in skeletal muscle by cytokines has not been examined. To further determine whether the aberrant phosphorylation patterns noted in the immunoblot analyses above were consistent with CAPP activity, we examined L6 myotubes for protein phosphatase activity after TNF{alpha}, insulin, and C6 ceramide treatment along with an inhibitor of de novo ceramide synthesis. Short-term TNF{alpha} treatment increased protein phosphatase-specific activity approximately 2-fold (P < 0.05, Student’s t test). C6-ceramide treatment mimicked this within 2 h, whereas insulin treatment showed no increase in protein phosphatase activity. To distinguish between PP1-type and PP2A-type activities, okadaic acid was added to the phosphatase assay reaction with concentrations that inhibit both (20 nM) or PP2A specifically (0.2 nM). In L6 cell lysates, high-dose okadaic acid led to nearly complete abolishment of phosphatase activity, whereas the lower dose led to little or no inhibition, indicating that the predominant phosphatase activated by TNF{alpha} and C6 ceramide was PP1 (P < 0.05) (Fig. 3Go). Exposure of L6 cells to myriocin, an inhibitor of serine palmitoyltransferase in the de novo pathway (41), significantly suppressed activation of the phosphatase activity induced by TNF{alpha} to levels comparable to controls in L6 cells, suggesting that TNF{alpha} activated de novo ceramide synthesis that, in turn, activated PP1 activity in L6 cells.


Figure 3
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  		FIG. 3. TNF{alpha} and C6 ceramide stimulated a PP1-like serine/threonine phosphatase activity. L6 cells were treated with TNF{alpha} (15 or 150 ng/nl) for 30 min, C6 ceramide (20 µM) for 2 h, insulin (10 nM) for 30 min, or myriocin (M) (5 nM) for 30 min. In the phosphatase assay wells, samples were treated with no inhibitor, 20 nM okadaic acid, or 0.2 nM okadaic acid. Free phosphate was measured by optical density at 630 nm. Results are mean ± SEM of specific activity from two to six separate experiments assayed in duplicate. The asterisk indicates a significant difference from control (P < 0.05, t test).

 
TNF{alpha} increased ceramide levels via the de novo sphingolipid pathway in L6 cells
To determine whether intracellular ceramide levels were increased as a result of treatment with TNF{alpha}, cell pellets were assayed for ceramide content by liquid chromatography-mass spectrometry. In L6 cells, C16 and C24:1 ceramides were the predominant species detected (Fig. 4AGo). Both species were increased by TNF{alpha} treatment, but only elevations of C24:1 ceramide (35–40%) were noted in the presence of insulin. This increase was blunted by pretreatment with myriocin. Insulin treatment alone did not significantly increase C24:1 ceramide levels (Fig. 4BGo). The levels of ceramide noted correspond to the decrease in protein phosphorylation noted in Fig. 1Go and increase in protein phosphatase activity.


Figure 4
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  		FIG. 4. TNF{alpha} stimulated de novo synthesis of ceramide. A, L6 cell pellets were analyzed via liquid chromatography-mass spectrometry to determine all the known cellular ceramide species. The predominant species detected in six different experiments were C16 and C24:1 ceramide. B, The change from control for C16 and C24:1 ceramide after TNF{alpha} (T) pretreatment followed by insulin (I) or in the presence of myriocin (M) as determined by liquid chromatography-mass spectrometry. Shown is the mean of two representative experiments.

 
Ceramide impaired phosphorylation of glycogen synthase kinase 3-ß and Akt
To further characterize the mechanisms of TNF{alpha}-induced insulin resistance, we examined L6 cells for phosphorylation/dephosphorylation of GSK3ß. GSK3ß is also an Akt substrate, which would make it a potential target of CAPPs activated by TNF{alpha}. As expected, insulin increased the phosphorylation of GSK3ß and C6 ceramide attenuated this (Fig. 5AGo). The attunation was not as great as noted with Akt in Fig. 1Go, and this may be due to the subcellular partitioning of C6 and its ability to interact with phosphatases. The phosphorylation of GSK3ß is an inactivating phosphorylation that leads to downstream glycogen production. When phosphoGSK3ß was evaluated in the presence of TNF{alpha}, the effect of insulin on phosphorylation was blunted. When de novo ceramide synthesis was blocked by myriocin or when PP1 was inhibited by tautomycin, TNF{alpha} inhibition of insulin-stimulated GSK3ß phosphorylation was largely reversed (Fig. 5BGo). The effect of C6 ceramide was more robust than TNF{alpha} in this case and may reflect the short time course used in these experiments to demonstrate activation of CAPPs. A similar reversal of TNF{alpha} activity was also noted on pAkt by myriocin and tautomycin (Fig. 5BGo). IRS-1 phosphorylation was not significantly increased by TNF{alpha} and neither myriocin nor tautomycin treatment had any effect suggesting IRS-1 phosphorylation associated with insulin resistance was not occurring in this scenario (42, 43).


Figure 5
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  		FIG. 5. Ceramide-activated protein phosphatase inhibits GSK3ß phosphorylation and behaves in a de novo ceramide-dependent, PP1 manner. A, L6 cells were treated as previously described. Membrane was probed with phospho-GSK3ß antibody (serine 9) and reprobed with ß-actin to ensure equal loading. B, L6 cells were treated with myriocin (50 nM) for 1 h, myriocin 1 h then TNF{alpha} (150 ng/ml) for 30 min, tautomycin (10 nM) for 1 h, or tautomycin for 1 h then TNF{alpha} for 30 min before insulin (10 nM) for 30 min. Membrane was probed with phospho-GSK3ß antibody before reprobing with phospho-Akt and ß-actin antibodies. A separate membrane was probed for phospho-IRS and IRS. Densitometric scans summarize data from three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is an abundance of correlative evidence indicating that TNF{alpha} causes insulin resistance. The molecular mechanisms are not well defined because TNF{alpha} has multiple effects on the insulin receptor and IRS-1 phosphorylation (4, 6, 43, 44). It also alters the inhibitory nuclear factor {kappa}B kinase/nuclear factor {kappa}B/inhibitory nuclear factor {kappa}B complex (45). There are also reports of impaired PP1, glycogen synthase activity, and insulin-stimulated glucose uptake in L6 cells after TNF{alpha} treatment (46, 47). The molecular details of these pathways have not been demonstrated. The current studies expand the pathways involved in TNF{alpha} signaling to CAPPs, which, in this case, are PP1-like in nature.

We first investigated the effects of TNF{alpha} and ceramide on the insulin-induced phosphorylation state of Akt, a central controller of insulin action, and SRp40, an Akt2 target important for insulin regulation of alternative splicing of the PKCß gene. Ceramide analogs have been shown to diminish Akt phosphorylation in brown adipocytes and skeletal muscle (12, 48). However, whether this was due to a serine/threonine protein phosphatase activity was not shown, nor were the increases in endogenous ceramide species. The effects of C6 ceramide treatment on Akt phosphorylation strongly suggest that its change in phosphorylation might be due to a protein phosphatase activity. To evaluate this, the substrate system was extended to other potential targets in the insulin signaling cascade, SRp40, also a substrate for Akt. Its phosphorylation was evaluated using the monoclonal antibody, mAb104, which recognizes phosphoepitopes in "RS," arginine-serine domains (49), as well as with the Akt substrate antibody. TNF{alpha} and C6 ceramide modulated insulin effects on SRp40 phosphorylation. C6 ceramide pretreatment attenuated the effect of insulin on Akt, SRp40, and GSK3ß phosphorylation.

We linked the effects of TNF{alpha} in skeletal muscle to PKCßII alternative splicing because aberrant mRNA splicing is associated with many disease states including type 2 diabetes (37). PKCßII splicing is regulated by insulin via PI3-kinase/Akt phosphorylation of SRp40 (35, 36, 37). TNF{alpha} abolished insulin-stimulated PKCßII exon inclusion in L6 myotubes. This is consistent with the inhibition of Akt activation and decreased SRp40 phosphorylation anticipated by LY294002 (36).

We characterized the protein phosphatase activity that TNF{alpha} stimulated presuming that it could be modified by ceramides. Previous studies suggested that protein phosphatases could be mediators of TNF{alpha}-induced insulin resistance (47), and TNF{alpha} is known to be increased in skeletal muscle of diabetic subjects (50). Earlier studies identified a PP2A activity and showed that ceramide analogs mimicked the TNF{alpha} effect on MAPK inhibition and PP2A activation (46, 47). The phosphatase substrate presented to the enzyme in this study is different and thought to be more specific for PP2A; however, using differential doses of okadaic acid to distinguish between the two activities in the assay, it is clear that a PP1 activity was modified by TNF{alpha} in a manner consistent with CAPPs activation. Recently, a PP2A activity was found to dephosphorylate Akt (48, 51). However, ceramides allosterically activate both PP1 and PP2A (25, 28, 52). In the present case, a PP1-type of activity seems most likely because no inhibitory effects on CAPPs were observed with insulin but TNF{alpha} activation was blocked with myriocin treatment, suggesting a specific de novo ceramide pathway distinct from other phosphatases reported. The inhibition of alternative splicing via TNF{alpha} also supports our belief that a PP1 activity is relevant because PP1, not PP2A, has been shown to be involved in alternative splicing (53, 54). Nuclear inhibitor of PP1, a major nuclear PP1-binding protein, colocalizes with splicing factors in the nucleus, and only PP1 activity has been shown to dephosphorylate SR proteins (28).

We were interested in determining the specific ceramide species responsible for the insulin-resistant state induced by TNF{alpha}. TNF{alpha} treatment was associated with an increase in C16 and C24:1 ceramides, the major species detected in L6 myotubes, suggesting that these species regulated the PP1 activity. However, only C24:1 ceramide levels but not C16 ceramide levels remained elevated in the presence of insulin. Insulin resistance is induced via de novo ceramide synthesis by treating C2C12 skeletal muscle and primary rat islets cells with palmitate (10, 55); however, the species was not identified. In addition, treating 3T3-L1 adipocytes with both C2 and C6 ceramide analogs causes increases in various markers of deficient insulin signaling such as insulin-stimulated glucose uptake (56). These studies do not rule out the additional involvement of the sphingomyelinase pathway because this also increases ceramide levels, and it has been demonstrated that TNF{alpha} inhibits insulin signaling in 3T3L1 adipocytes and myeloid 32D cells via activation of sphingomyelinase (19).

Previous work in L6 myotubes demonstrates that insulin increases a PP1 activity while concomitantly decreasing PP2A (57, 58). The difference noted may be explained by evidence of combinatorial control among serine/threonine phosphatases that confers functional specificity (30). It is not clear specifically which PP1 heterodimer is activated by insulin and whether this PP1 is ceramide-activated (46). Furthermore, the allosteric modulation of PP1 by ceramide may change its substrate specificity, subcellular localization, and metabolic consequences.

Finally, the ability of tautomycin and myriocin to inhibit a PP1 phosphatase and ceramide synthesis, respectively, clearly reversed the phosphorylation state of GSK3ß and Akt. It could be argued that TNF{alpha} induced phosphorylation of IRS-1 at Ser312 via PKC{zeta} activation could be the cause for inhibition of Akt phosphorylation (59), and this possibility is not ruled out. However, we saw no effect of TNF{alpha} or myriocin and tautomycin on IRS-1 Ser616, another phosphorylation site associated with the development of peripheral insulin resistance (42), suggesting that this sight is not a target for CAPP or TNF{alpha} phosphorylation and the effect described here is different.

In summary, this study has identified an additional molecular mechanism for cellular TNF{alpha}-induced insulin resistance that involves the de novo generation of ceramide that activates a PP1 activity capable of dephosphorylating both kinases and splicing factors. Although further characterization of the phosphatase binding protein are in order, this study has identified the species of ceramides increased by TNF{alpha} and an additional signaling pathway relevant to the cytokine in skeletal muscle cells that explains some of the actions of TNF{alpha} in insulin resistance.


   Acknowledgments
 
We thank Karen D. Corbin for assistance with experiments and manuscript preparation.


   Footnotes
 
This work was supported by National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases 54393 (to D.R.C.) and the Department of Veteran’s Affairs Medical Research Service (to D.R.C. and N.P.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 7, 2006

Abbreviations: CAPP, Ceramide-activated protein phosphatase; GSK3ß, glycogen synthase kinase 3ß; IRS, insulin receptor substrate; PI3, phosphatidylinositol 3; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; SR, serine/arginine rich; SRp40, serine/arginine-rich protein 40; SSII, splice site II.

Received June 6, 2006.

Accepted for publication November 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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