Inhibition of neutral sphingomyelinases in skeletal muscle attenuates fatty-acid induced defects in metabolism and stress

Abstract

Background: Chronic metabolic overload leads to insulin resistance in a variety of tissues. It has been shown that exposure to saturated fatty acid palmitate can cause insulin resistance in skeletal muscle cells. Fatty acid induced synthesis of ceramide is considered to be one of the major causes for insulin resistance. Both de novo synthesis and sphingomyelin hydrolysis by sphingomyelinase are implicated for ceramide generation. Aim of this study was to evaluate the impact of neutral sphingomyelinase (nSMase) inhibition on saturated fatty acid induced lipotoxicity and insulin resistance in skeletal muscle myotubes.

Results: Treatment of saturated fatty acid (palmitate) but not unsaturated fatty acid (oleate) caused an up-regulation in expression of various nSMase genes which are associated with ceramide synthesis through the salvage pathway. Inhibition of nSMase by a pharmacological inhibitor (GW4869) partially reverted the palmitate induced insulin resistance in C2C12 myotubes. Inhibition of nSMase improved metabolic functions of myotubes as measured by improved oxidative capacity in terms of increased mitochondrial number, PGC1α expression and ATP levels with concomitant decrease in intramyocellular triglyceride levels. Palmitate induced inflammatory response was also reduced by nSMase inhibitor. GW4869 treatment reduced palmitate induced oxidative and endoplasmic reticulum stress and improved cell survival.

Conclusion: In this study, we provide evidences that inhibition of nSMase can protect skeletal muscles from saturated fatty acid induced insulin resistance, metabolic dysfunction, cellular stress and inflammation.

Keywords: C2C12 myotubes; Cellular stress; Ceramide; Insulin resistance; Oxidative capacity; Sphingomyelinase; T2DM.

Figure 1

Palmitate treatment increases nSMases expression. Differentiated C2C12 myotubes were treated with vehicle control or palmitate or oleate (750 μM) for 16 hrs as described under ‘Methods’ section. After treatments, total RNA was isolated and mRNA levels of nSMase-1 (A), nSMase-2 (B), nSMase-3 (C) and SPTLC1 (D) were quantified by quantitative real time PCR using β-actin as housekeeping gene control. Data are presented as mean + standard deviation. n = 4, **P < 0.01, *P < 0.05, one way ANOVA with Newman-Keuls post test was performed for statistical analyses.

Figure 2

Palmitate induced insulin resistance is alleviated by nSMase inhibition. (A) After palmitate treatment, C2C12 myotubes were starved for 30 min. Myotubes were then treated with or without insulin for 10 min followed by lysing in lysis buffer. Insulin induced Akt phosphorylation was measured in total cell lysate by Western blotting. Unphosphorylated levels of Akt were also measured as control for phosphorylation (Upper panel). Lower panel: quantification of Akt phosphorylation (n = 6, ***P < 0.001, **P < 0.01) (B) C2C12 cells were treated with or without palmitate for 2 hrs in presence or absence of nSMase inhibitor (GW4869). Insulin was also present in the treatments for indicated sets. Gene expression level of PDK4 was analyzed by quantitative real time PCR using β-actin as housekeeping gene control. Data are presented as mean + standard deviation. n = 4, **P < 0.01, *P < 0.05, ns: not significant, one way ANOVA with Newman-Keuls post test was performed for statistical analyses.

Figure 3

Inhibition of nSMase improves oxidative capacity of C2C12 myotubes and reduces TG storage. (A) Analysis of PGC1α gene expression in C2C12 myotubes cultured under palmitate condition in presence or absence of nSMase inhibitor (GW4869). β-actin was used as housekeeping gene control. (B) Mitochondrial DNA copy numbers were estimated by quantitative real time PCR using total DNA isolated from myotubes cultured under control or palmitate condition in presence or absence of nSMase inhibitor (GW4869). Mitochondrial DNA copy number were quantified by assessing ND1 (mitochondrial gene) level which was normalized to HPRT (nuclear gene). Cellular ATP levels (C) and TG storage (D) were quantified after culturing C2C12 myotubes under similar conditions as described under ‘Methods’ section. Data are presented as mean + standard deviation. n = 4, ***P < 0.001, **P < 0.01, *P < 0.05, one way ANOVA with Newman-Keuls post test was performed for statistical analyses.

Figure 4

Reduction in stress signaling, inflammation, oxidative stress and ER stress by nSMase inhibition. (A) After palmitate treatment in presence or absence of nSMase inhibitor (GW4869), C2C12 myotubes were harvested in lysis buffer. Upper panel: Levels of phosphorylated JNK, unphosphorylated JNK, IκB, BiP and CHOP were measured using Western blotting. β-actin was used as a loading control. Lower panels: quantification of Western blotting data. (B) Gene expression of IL6 was measured by quantitative real time PCR using β-actin as housekeeping gene control. (C) After treatment, myotubes were loaded with DCFH-DA ROS indicator fluorescent probe to quantify the amount of cellular ROS. (D) Nitric oxide levels in culture medium were quantified using Griess reagent. (E) Myotubes viability was measured by MTT assay after treatment. Data are presented as mean + standard deviation. n = 4, ***P < 0.001, **P < 0.01, *P < 0.05, one way ANOVA with Newman-Keuls post test was performed for statistical analyses.

Figure 5

Saturated FFA mediated impairment in skeletal muscle and its reversal by nSMase inhibition. Sphingomyelin can be converted into ceramide by nSMases. Palmitate mediated up-regulation of nSMases can expedite this process of ceramide generation. Increased ceramide can lead to an inhibition on insulin signaling pathway thereby reducing glucose oxidation. In this condition PGC1α levels are reduced with a concomitant decrease in muscle oxidative capacity. Because of impaired oxidative function and palmitate overload, fat storage in form of TG is increased. Palmitate overload mediated increased ceramide levels elevate cellular oxidative stress, ER stress and inflammation which can severely affect cell survival. Elevated stress and inflammation can directly inhibit insulin signaling and can impair oxidative capacity (represented as dashed arrows). Palmitate mediated impact on cellular responses is represented by a thick up or down arrow placed just after particular cellular response. Inhibition of nSMase under this background reverses palmitate mediated impact on cellular response.