Co-activator binding protein PIMT mediates TNF-α induced insulin resistance in skeletal muscle via the transcriptional down-regulation of MEF2A and GLUT4

Abstract

The mechanisms underlying inflammation induced insulin resistance are poorly understood. Here, we report that the expression of PIMT, a transcriptional co-activator binding protein, was up-regulated in the soleus muscle of high sucrose diet (HSD) induced insulin resistant rats and TNF-α exposed cultured myoblasts. Moreover, TNF-α induced phosphorylation of PIMT at the ERK1/2 target site Ser(298). Wild type (WT) PIMT or phospho-mimic Ser298Asp mutant but not phospho-deficient Ser298Ala PIMT mutant abrogated insulin stimulated glucose uptake by L6 myotubes and neonatal rat skeletal myoblasts. Whereas, PIMT knock down relieved TNF-α inhibited insulin signaling. Mechanistic analysis revealed that PIMT differentially regulated the expression of GLUT4, MEF2A, PGC-1α and HDAC5 in cultured cells and skeletal muscle of Wistar rats. Further characterization showed that PIMT was recruited to GLUT4, MEF2A and HDAC5 promoters and overexpression of PIMT abolished the activity of WT but not MEF2A binding defective mutant GLUT4 promoter. Collectively, we conclude that PIMT mediates TNF-α induced insulin resistance at the skeletal muscle via the transcriptional modulation of GLUT4, MEF2A, PGC-1α and HDAC5 genes.

Figure 1. HSD–induced TNF-α augments PIMT expression in skeletal muscle.

(a) Blood glucose levels during OGTT (2gkg⁻¹) in control diet (CD) and HSD (high sucrose diet) fed rats (n = 6). Values are shown as mean ± SD; **p < 0.01, ***p < 0.005 and ****p < 0.001 for CD vs HSD group using Student’s t-test. (b–d) Serum levels of triglycerides (b), insulin (c) and TNF-α (d) of rats fed with CD and HSD (n = 6). Values are shown as mean ± SD.; *p < 0.05 versus CD using student’s t test. (e,f) mRNA expression of rattus TNF-α (e) and rattus PIMT (f) in soleus muscle of CD and HSD fed rats (n = 3). Values are shown as mean ± SD.; *p < 0.05 versus CD using student’s t test. (g,h) Western blotting for PIMT and GLUT4 in L6 myoblasts (g) and PIMT, GLUT4 and MEF2A in neonatal skeletal myoblasts (h) treated with TNF-α (2 ng/ml) for the indicated time points. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8.

Figure 2. PIMT abrogates insulin stimulated glucose uptake by skeletal muscle cells.

(a,b) Mean basal and insulin stimulated (5 min and 10 min) uptake of 2-NBDG by L6, L6 myotubes (a) and neonatal skeletal myoblasts (b) infected with Ad-PIMT. Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal of control cells which was set to 1; **p < 0.01, ***p < 0.005, ****p < 0.001 versus corresponding control cells (two way ANOVA). (c) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by L6 myoblasts treated with TNF-α and/or PIMT siRNA. Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal of control cells which was set to 1; **p < 0.01, ***p < 0.005 versus corresponding control cells (two way ANOVA); ^@p < 0.01, ^$p < 0.005 versus corresponding TNF-α treated cells (two way ANOVA). (d) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by L6 myoblasts transfected with PIMT siRNA and/or pcDNA3.1 PIMT (hPIMT). Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal control which was set to 1; ***p < 0.005 versus control cells; ^$p < 0.001 versus corresponding PIMT transfected cells (two way ANOVA). (e) Western blotting to detect levels of phosho-IRS1^S307, phospho-IRS1^Y608, total IRS1, pJNK1/2, pAkt, Total Akt and β-actin in siRNA (control or PIMT) tranfected L6 myoblasts cultured with BSA or TNF-α (48 h), treated with or without insulin (100nM) for 30 min. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8.

Figure 3. TNF-α induced ERK mediated phosphorylation of PIMT is required for PIMT dependent inhibition of glucose uptake.

(a) Western blotting to detect phospho-PIMT (using anti-MPM2) and total PIMT treated in TNF-α treated L6 myoblasts. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8. (b) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by PIMT or mutant PIMT transfected L6 myoblasts (left panel) or L6 myotubes infected with Ad-PIMT EGFP or Ad-PIMT mutants (right panel). Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to corresponding control cells which was set to 1; **p < 0.01, ***p < 0.005 versus corresponding control cells, ^@p < 0.01, ^$p < 0.005 versus corresponding PIMT over-expressing cells (two way ANOVA). (c) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by L6 myoblasts transfected with PIMT or its mutants treated with UO126 where indicated. Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal of control cells which was set to 1; ***p < 0.005, versus corresponding control cells, ^$p < 0.005 versus corresponding PIMT transfected cells (two way ANOVA). (d) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by L6 myoblasts treated with TNF-α or UO126 or both. Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal of controls cells which was set to 1; ***p < 0.005, versus corresponding control cells, ^@p < 0.01,^$p < 0.005 versus corresponding TNF-α treated cells (two way ANOVA).

Figure 4. PIMT attenuates GLUT4 expression in Ser²⁹⁸ phosphorylation dependent manner.

(a) mRNA expression of rattus GLUT4 in soleus muscle of CD and HSD fed rats (n = 3). Values are shown as mean ± SD. *p < 0.05 versus CD using Student’s t-test. (b) mRNA expression of rattus GLUT4 in L6 myotubes infected with PIMT (wt and Ser²⁹⁸ mutants). Values are shown as mean ± SD; ***p < 0.005 versus Ad-LacZ infected cells, ^@p < 0.01 versus Ad-PIMT infected cells (two way ANOVA). (c) Western blotting for GLUT4 in wild type and Ser²⁹⁸ mutant PIMT transfected L6 myoblasts. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8. (d) mRNA expression of rattus GLUT4 in L6 myoblasts transfected with PIMT siRNA. Values are shown as the mean ± SD; ***p < 0.005 versus control cells (two way ANOVA). (e) Chromatin immunoprecipitation was performed in lysates of L6 myoblasts, L6myotubes and soleus muscle of Wistar rats using Anti-PIMT or mock Anti-goat IgG on the MEF2A response element (MEF2A_RE) of GLUT4 promoter. The full-length gel pictures are included in Supplemental Fig. 8. (f) Wild-type (GLUT4.Luc.) and MEF2 binding defective mutant (GLUT4.Luc.MEF2 mut) GLUT4 promoter luciferase activity in HEK293T cells transfected with WT or PIMT mutants. Values represent mean ± SD and are expressed relative to the vector transfected cells; **p <  0.01 versus vector transfected cells (two way ANOVA).

Figure 5. Overexpression of PIMT inhibits MEF2A expression in Ser²⁹⁸ phosphorylation dependent manner.

(a–c) mRNA expression of rattus MEF2A (a), MEF2D (b) and MEF2C (c) in soleus muscle of CD and HSD fed rats (n = 3). Values are shown as mean ± SD; *p < 0.05, **p < 0.01 versus CD using Student’s t-test. (d-f) mRNA expression of rattus MEF2A (d), MEF2D (e) and MEF2C (f) in L6 myotubes infected with Ad-PIMT and Ad-PIMT Ser²⁹⁸ mutants. Values are shown as the mean ± SD; ***p < 0.005 versus LacZ infected L6 myotubes, ^$p < 0.05 versus Ad-PIMT infected myotubes (two way ANOVA). (g–i) mRNA expression of rattus MEF2A (g), MEF2D (h) and MEF2C (i) in L6 cells transfected with PIMT siRNA. Values are shown as the mean ± SD; ***p < 0.005 versus control L6 myoblasts (two way ANOVA). (j) Chromatin immunoprecipitation was performed in lysates of L6 myoblasts, L6 myotubes and soleus muscle of Wistar rats using Anti-PIMT or mock Anti-goat IgG of −1 kb upstream region of MEF2A promoter. The full-length gel pictures are included in Supplemental Fig. 8.

Figure 6. TNF-α induced PIMT up-regulates HDAC5 expression in cultured skeletal muscle cells.

(a) mRNA expression of rattus HDAC5 in soleus muscle of CD and HSD fed rats (n = 3). Values are shown as mean ± SD. *p < 0.05 versus CD using Student’s t test. (b) mRNA expression of rattus HDAC5 and PIMT in TNF-α treated L6 myoblasts. Values are shown as mean ± SD; **p < 0.01 versus control treated using Student’s t test. (c) mRNA expression of rattus HDAC5 in L6 myotubes infected with Ad-PIMT or Ad-PIMT Ser²⁹⁸ mutants. Values are shown as the mean ± SD; ***p < 0.005 versus Ad-LacZ infected L6 myotubes, ^$p < 0.05 versus PIMT (WT) infected myotubes (two way ANOVA). (d) mRNA expression of rattus HDAC5 and PIMT transfected with PIMT siRNA in L6 myoblasts. Values are shown as mean ± SD; ***p < 0.005 versus control L6 myoblasts (two way ANOVA). (e,f) Chromatin immunoprecipitation was performed in lysates of L6 myoblasts, L6 myotubes and soleus muscle using Anti-PIMT or mock Anti-goat IgG of HDAC5 promoter (−1 kb (e) and −10 kb (f) upstream). The full-length gel pictures are included in Supplemental Fig. 8.

Figure 7. PIMT mediated inhibition of glucose uptake involves Med1 and PGC-1α.

(a) Co-immunoprecipiation and western blotting to detect the phosphorylation of PIMT (MPM2), total PIMT, Med1, MEF2A and PGC-1α in TNF-α treated L6 myoblasts. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8. (b) mRNA levels of PGC-1α in Ad-PIMT and Ad-PIMT Ser²⁹⁸ mutant infected L6 myotubes. Values are shown as mean ± SD; ****p < 0.001 versus Ad-LacZ infected cells, ^$p < 0.005 versus PIMT (WT) infected cells (two way ANOVA). (c) Western blotting for PGC-1α in WT and PIMT mutant transfected L6 myoblasts. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplemental Fig. 8. (d) mRNA levels of PGC-1α in PIMT siRNA transfected L6 myoblasts. Values are shown as mean ± SD; **p < 0.01 versus control transfected cells (two way ANOVA). (e,f) Basal and insulin stimulated (5 and 10 min) uptake of 2-NBDG by L6 myoblasts transfected with PGC-1α (e) and Med1 (f). Values are shown as mean ± SD after normalizing with the corresponding protein content and expressed relative to basal of control cells which was set to 1; **p < 0.001versus corresponding control cells (two way ANOVA).

Figure 8. Overexpression of PIMT abrogates GLUT4 and MEF2A expression in vivo in Ser²⁹⁸ phosphorylation dependent manner.

(a–f) mRNA expression of GLUT4 (a), PGC-1α (b), HDAC5 (c), MEF2A (d), MEF2D (e) and MEF2C (f) in Ad/PIMT (WT and mutant) injected rat skeletal muscle tissues. Values are shown as mean ± SD; **p < 0.01, versus Ad-LacZ injected rat skeletal muscle tissue, ^@p < 0.01 versus Ad-PIMT infected rat skeletal muscle tissue (two way ANOVA).