Docosahexaenoic acid counteracts palmitate-induced endoplasmic reticulum stress in C2C12 myotubes: Impact on muscle atrophy

Lipid accumulation in skeletal muscle results in dysregulation of protein meta- bolism and muscle atrophy. We previously reported that treating C2C12 myo- tubes with palmitate (PA), a saturated fatty acid, increases the overall rate of proteolysis via activation of the ubiquitin-proteasome and autophagy systems; co-treatment with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents the PA-induced responses. Others have reported that PA induces endoplasmic reticulum (ER) stress which initiates the unfolded pro- tein response (UPR), a collective group of responses that can lead to activa- tion of caspase-mediated proteolysis and autophagy. Presently, we tested the hypothesis that DHA protects against PA-induced ER stress/UPR and its atro- phy-related responses in muscle cells. C2C12 myotubes were treated with 500 lmol/L PA and/or 100 lmol/L DHA for 24 h. Proteins and mRNA asso- ciated with ER stress/UPR, autophagy, and caspase-3 activation were evalu- ated. PA robustly increased the phosphorylation of protein kinase R (PKR)- like ER kinase (PERK) and eukaryotic initiation factor 2a (eIF2a). It also increased the mRNAs encoding activating transcription factor 4 (ATF4), spliced X-box binding protein 1 (XBP1s), C/EBP homologous protein (CHOP), and autophagy-related 5 (Atg5) as well as the protein levels of the PERK target nuclear factor erythroid 2-related factor (Nrf2), CHOP, and cleaved (i.e., activated) caspase-3. Co-treatment with DHA prevented all of the PA-induced responses. Our results indicate that DHA prevents PA- induced muscle cell atrophy, in part, by preventing ER stress/UPR, a process that leads to activation of caspase-mediated proteolysis and an increase in expression of autophagy-related genes

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional condi- tions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho- PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein syn- thesis in skeletal muscle in part by induction of ER stress

Calcineurin-NFAT signaling regulates atrogin-1 and MuRF1 via microRNA-23a (miR-23a) during muscle atrophy

Muscle atrophy is prevalent in chronic kidney disease (CKD) patients. MicroRNAs play a critical role in biological processes including muscle atrophy. MicroRNA-23a (miR-23a) negatively regulates the expression of two atrophy-related ubiquitin ligases, atrogin-1 and MuRF1; it is reduced in muscle during atrophy. Although miR-23a expression was recently shown to be positively regulated by NFATc3, the underlying mechanism of miR-23a suppression during atrophy remains unknown. We previously reported that the activity of calcineurin (Cn), the calcium-activated phosphatase that regulates NFATc proteins, is decreased when insulin signaling is decreased. Since CKD causes muscle atrophy, and glucocorticoids are required for the response, we investigated how dexamethasone (DEX) affects Cn activity, NFATc3 signaling, and miR-23a expression. C2C12 or L6 myotubes were treated with 100 uM DEX to induce atrophy. Within 1 h, Cn activity was reduced and less NFATc3 was located in the nucleus. Further, miR-23a was also decreased within 30 minutes. After 48 h, expression of the NFATC3 target gene, MCIP1.4, and miR-23a were decreased. Expression of atrogin-1 and MuRF1 were also increased 48 h after DEX. Collectively, these findings indicate the Cn-NFAT signaling pathway may play an important role in the regulation of atrogin-1 and MuRF1 by suppressing miR23a during CKD and glucocorticoid-related muscle atrophy. Support: NIH DK007656; AHA GRNT766002

Docosahexaenoic acid counteracts palmitate-induced endoplasmic reticulum stress in C2C12 myotubes: Impact on muscle atrophy

Lipid accumulation in skeletal muscle results in dysregulation of protein meta- bolism and muscle atrophy. We previously reported that treating C2C12 myo- tubes with palmitate (PA), a saturated fatty acid, increases the overall rate of proteolysis via activation of the ubiquitin-proteasome and autophagy systems; co-treatment with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents the PA-induced responses. Others have reported that PA induces endoplasmic reticulum (ER) stress which initiates the unfolded pro- tein response (UPR), a collective group of responses that can lead to activa- tion of caspase-mediated proteolysis and autophagy. Presently, we tested the hypothesis that DHA protects against PA-induced ER stress/UPR and its atro- phy-related responses in muscle cells. C2C12 myotubes were treated with 500 lmol/L PA and/or 100 lmol/L DHA for 24 h. Proteins and mRNA asso- ciated with ER stress/UPR, autophagy, and caspase-3 activation were evalu- ated. PA robustly increased the phosphorylation of protein kinase R (PKR)- like ER kinase (PERK) and eukaryotic initiation factor 2a (eIF2a). It also increased the mRNAs encoding activating transcription factor 4 (ATF4), spliced X-box binding protein 1 (XBP1s), C/EBP homologous protein (CHOP), and autophagy-related 5 (Atg5) as well as the protein levels of the PERK target nuclear factor erythroid 2-related factor (Nrf2), CHOP, and cleaved (i.e., activated) caspase-3. Co-treatment with DHA prevented all of the PA-induced responses. Our results indicate that DHA prevents PA- induced muscle cell atrophy, in part, by preventing ER stress/UPR, a process that leads to activation of caspase-mediated proteolysis and an increase in expression of autophagy-related genes

Dexamethasone decreases CRTC1 and CRTC2 nuclear localization.

<p>Treatment of L6 myotubes with Dex (100nM, 48hrs) decreased (A) total CRTC1 (p = 0.004 and CRTC2 protein (p = 0.0007). n = 9/treatment from 3 experiments. Representative western blots and corresponding sections of the Ponceau S-stained membranes are shown. Dex decreased (B) nuclear CRTC1 (p = 0.04) and nuclear CRTC2 (p = 0.03) protein and (C) nuclear CnA protein (p = 0.04); no differences in the cytosolic levels of these proteins were detected. Dex increased (D) nuclear SIK1 protein (p = 0.005) with no detectable difference in cytosolic protein. n = 3-7/treatment from 3–5 experiments. (E) Representative western blots for cytosolic and nuclear CRTC1, CRTC2, SIK, and CnA proteins are shown; controls for the nuclear (Histone) and cytosolic (GAPDH) fractions are also shown. (F) IBMX (250uM, 15 min) increased nuclear CRTC2 protein. A representative western blot image and corresponding Ponceau S-stained membrane from a single experiment run on the same gel are shown; the experiment was repeated one other time with similar results. In panels A-D, data are expressed as mean percent of control ± s.d and were analyzed by Student’s <i>t</i>-test. Asterisks indicate a value that is significantly different from control: p<0.05 = *, p<0.01 = ** and p<0.001 = ***.</p

Dexamethasone, CRTC2, and atrogene expression.

<p>Two-way ANOVA was used to evaluate whether CRTC2 alters atrogene mRNA responses to Dex. Treatment with Dex increased (A) MuRF-1 mRNA (p<0.0001) and (B) Atrogin-1/MAFbx mRNA (p<0.0001). Overexpression of CRTC2 did not affect MuRF-1 or Atrogin/MAFbx mRNA expression (p≥0.1) or effect the response to Dex treatment (p≥0.1). n = 11-12/treatment from 4 experiments. Data are expressed as mean percent of control ± s.d. Asterisks denote significant effects of Dex compared to respective controls as indicated by <i>post hoc</i> analysis: p<0.01 = **, p<0.001 = *** and p<0.0001 = ****.</p

Dexamethasone decreases PGC-1α protein expression and transcription.

<p>(A) Treatment of L6 myotubes with Dex (100nM, 48hrs) decreased PGC-1α protein expression (p = 0.01). n = 3/treatment from 3 experiments. A representative western blot image and corresponding section of the Ponceau S—stained membrane are shown. (B) Dex decreased the mRNAs for PGC-1α (p<0.0001) and its target genes Tfam (p = 0.03) and CytC (p = 0.03); Dex also increased MuRF-1 (p = 0.003) and Atrogin-1/MAFbx mRNAs (p<0.0001). n = 10-14/treatment from 4–5 experiments. (C) In L6 cells transfected with a pPGC-1α-Luc, Dex decreased luciferase activity (p = 0.001). n = 6/treatment from 6 experiments. (D) In separate experiments with cells transfected with a pPGC-1α-ΔCRE-Luc, Dex increased luciferase activity (p = 0.0005). n = 3/treatment from 3 experiments. Data are expressed as mean values ± s.d. and were analyzed by Student’s <i>t</i>-test. Asterisks indicate values that are significantly different from control: p<0.05 = *, p<0.01 = **, p<0.001 = *** and p<0.0001 = ****.</p

Overexpression of CRTC proteins increase PGC-1α transcription.

<p>L6 myoblasts were transfected with either pPGC-1α-Luc or pPGC-1α-ΔCRE-Luc. After 24 h, cells were infected with adenoviruses to express GFP, human CRTC1 or human CRTC2 and harvested 48 h later to measure luciferase activity. A two-way ANOVA was used to evaluate the significance of CRTC protein overexpression and the mutation of the CRE site on pPGC-1α-Luc activity. Overexpressing either CRTC1 (A) or CRTC2 (B) increased luciferase activity (CRTC1, p = 0.0001, CRTC2, p = 0.0002). In both sets of experiments, luciferase activity is reduced in cells transfected with pPGC-1α-ΔCRE-Luc (p<0.0001 in each panel). The CRTC1 or CRTC2-induced increase in luciferase activity was prevented by mutation of the CRE site in pPGC-1α-Luc (CRTC1, p = 0.003; CRTC2, p = 0.01). n = 5-6/treatment from 5–6 experiments. Data are expressed as the mean value ± s.d of normalized (i.e., firefly:renilla ratio) luciferase activity. The # indicates that the effects of CRTC1 or CRTC2 on luciferase activity are different at p<0.01 when CRE site is mutated in the PGC-1α promoter. Asterisks denote significant effects of CRTC1 or CRTC2 compared to GFP controls as indicated by <i>post hoc</i> analysis: p<0.001 = ***.</p

24 h of palmitate treatment increased XBP1 s and CHOP protein expression and were not attenuated by inhibition of TLR4 signaling in C2C12 myotubes.

<p>(<b>A</b>) 24 h of palmitate (500 μM) or palmitate with the TLR inhibitor TAK-242 (1 μM) increased XBP1 s protein (n = 4) with thapsigargin (Tg) used as a positive control. The fainter, lower band in the representative blot was confirmed as non-specific binding by the manufacturer. (<b>B</b>) 24 h of palmitate (500 μM) or palmitate with the TLR inhibitor TAK-242 (1 μM) induced CHOP protein expression (n = 4) with thapsigargin (Tg) used as a positive control. CHOP was not quantifiable in the control or LPS treated samples. (<b>C</b>) 24 h of palmitate (500 μM) or palmitate with the TLR inhibitor TAK-242 (1 μM) increased XBP1 s mRNA expression similarly compared to controls (n = 4). (<b>D</b>) 24 h of palmitate (500 μM) or palmitate with the TLR inhibitor TAK-242 (1 μM) increased CHOP mRNA expression compared to controls, while combined palmitate and TAK-242 treatment elevated CHOP mRNA to levels higher than palmitate (n = 4). (<b>E</b>) 24 h of palmitate (500 μM) or palmitate with TAK-242 (1 μM) increased ATF mRNA expression similarly compared to controls (n = 4). (<b>F-H</b>) TAK-242 alone does not independently change XBP1 s, ATF4 or CHOP mRNA levels compared to control (n = 3). Values shown are Mean ± SD. C = control, L = LPS (100 ng/ml), P = palmitate (500 μM), PT = Palmitate (500 μM) and TAK-242 (1 μM). Values shown are mean ± SD. * Higher than control (p<0.05), # Higher than PA (p<0.05).</p