Ceramide-activated protein phosphatase involvement in insulin resistance via Akt, serine/arginine-rich protein 40, and ribonucleic acid splicing in L6 skeletal muscle cells

Abstract

Elevated TNFalpha 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 CbetaII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFalpha 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 3beta in response to TNFalpha and the short chain C6 ceramide analog. TNFalpha 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 TNFalpha and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3beta were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFalpha abolished insulin-mediated inclusion of the protein kinase CbetaII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFalpha effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.

Figure 1. TNFα and C6 ceramide blocked insulin-mediated phosphorylation of Akt and SRp40

(a) L6 cells were pre-treated with TNFα (150ng/ml, 30 minutes) or C6 ceramide (20uM, 2 hours) prior to insulin (I) (10nM, 30 minutes) addition as indicated. Western blot analysis was conducted with phospho-Akt antibody (serine 473). The membrane was stripped and re-probed with β-actin to ensure equal protein loading. (b) L6 cells underwent similar treatments as above except that TNFα was 15 ng/ml. Lysates were immunoprecipitated with SRp40 antibody, proteins 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 (20uM, 2 hours) or as a pre-treatment followed by insulin as a control. Membrane was probed with phosphoAkt substrate antibody, stripped and re-probed 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 3 experiments. The asterisk indicates a significant difference (p < 0.05, t-test compared to insulin effect.)

Figure 2. TNFα blocked insulin induced PKCβII exon inclusion

A heterologous minigene constructed by inserting the PKCβII genomic fragment into a multi-cloning 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 PKCβII exon at both 5′ splice sites within 30 minutes; both splice variants encode PKCβII. Insulin effects were blocked by TNFα (15 ng/ml) similar to the two PI3Kinase/Akt inhibitors: Wortmannin (100 nM) and LY294002 (10 μM). The densitometric scan summarizes of three separate experiments.

Figure 3. TNFα and C6 ceramide stimulated a PP1-like serine/threonine phosphatase activity

L6 cells were treated with TNFα (15 or 150 ng/nl) for 30 minutes, C6 ceramide (20 μM) for 2 hours insulin (10 nM) for 30 minutes or myriocin (M) (5nM) for 30 minutes. In the phosphatase assay wells, samples were treated with no inhibitor, okadaic acid 20 nM or okadaic acid 0.2 nM. Free phosphate was measured by optical density at 630 nm. Results are mean ±SEM of specific activity from 2–6 separate experiments assayed in duplicate. The asterisk indicates a significant difference from control (p < 0.05, t-test).

Figure 4. TNFα 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 6 different experiments were C16 and C24:1 ceramide. (B) The change from control for C16 and C24:1 ceramide following TNFα (T) pretreatment followed by insulin (I) or in the presence of myriocin (M) as determined by LC-MS. Shown is the mean of 2 representative experiments.

Figure 5. Ceramide Activated Protein Phosphatase inhibits GSK3β phosphorylation and behaves in a de novo ceramide-dependent, PP1 manner

(A) L6 cells were treated as previously described. Membrane was probed with phospho-GSK3β antibody (serine 9) and reprobed with β-actin to ensure equal loading. (B) L6 cells were treated with myriocin (50 nM) for 1 hour, myriocin 1 hour then TNFα (150 ng/ml) for 30 minutes, tautomycin (10 nM) for 1 hour, or tautomycin for 1 hour then TNFα for 30 minutes prior to insulin (10 nM) for 30 minutes. Membrane was probed with phospho-GSK3β antibody before re-probing with phospho-Akt and β actin antibodies. A separate membrane was probed or phospho-IRS and IRS. Densitometric scans summarize data from three separate experiments.