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. 2016 Nov 11;291(46):23978-23988.
doi: 10.1074/jbc.M116.737684. Epub 2016 Oct 4.

A Role for Ceramides, but Not Sphingomyelins, as Antagonists of Insulin Signaling and Mitochondrial Metabolism in C2C12 Myotubes

Min Park  1 , Vincent Kaddai  2 , Jianhong Ching  1 , Kevin T Fridianto  1 , Ryan J Sieli  3 , Shigeki Sugii  1   4 , Scott A Summers  5   3
Affiliations

A Role for Ceramides, but Not Sphingomyelins, as Antagonists of Insulin Signaling and Mitochondrial Metabolism in C2C12 Myotubes

Min Park et al. J Biol Chem. .

Abstract

The accumulation of sphingolipids in obesity leads to impairments in insulin sensitivity and mitochondrial metabolism, but the precise species driving these defects is unclear. We have modeled these obesity-induced effects in cultured C2C12 myotubes, using BSA-conjugated palmitate to increase synthesis of endogenous sphingolipids and to inhibit insulin signaling and oxidative phosphorylation. Palmitate (a) induced the accumulation of sphingomyelin (SM) precursors such as sphinganine, dihydroceramide, and ceramide; (b) inhibited insulin stimulation of a central modulator of anabolic metabolism, Akt/PKB; (c) inhibited insulin-stimulated glycogen synthesis; and (d) decreased oxygen consumption and ATP synthesis. Under these conditions, palmitate failed to alter levels of SMs, which are the most abundant sphingolipids, suggesting that they are not the primary intermediates accounting for the deleterious palmitate effects. Treating cells with a pharmacological inhibitor of SM synthase or using CRISPR to knock out the Sms2 gene recapitulated the palmitate effects by inducing the accumulation of SM precursors and impairing insulin signaling and mitochondrial metabolism. To profile the sphingolipids that accumulate in obesity, we performed lipidomics on quadriceps muscles from obese mice with impaired glucose tolerance. Like the cultured myotubes, these tissues accumulated ceramides but not SMs. Collectively, these data suggest that SM precursors such as ceramides, rather than SMs, are likely nutritional antagonists of metabolic function in skeletal muscle.

Keywords: Akt PKB; ceramide; insulin resistance; lipotoxicity; mitochondria; skeletal muscle; sphingolipid.

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Figures

FIGURE 1.
FIGURE 1.
Palmitate and D609 induce SM precursors in C2C12 myotubes. C2C12 myotubes were treated with BSA (control), BSA-conjugated palmitate (palmitate, 500 μm), and/or D609 (300 μm) for 16 h. Lipids were extracted and quantified by targeted lipidomics. A–E, data shown are the sum of SM or SM precursors with various acyl chain lengths present within detection limits (n ≥5). F, efficacy of D609 treatment in SMS activity was measured by accessing the amount of NBD-SM converted from NBD-C6-ceramide (n ≥3); * and # denote significance of palmitate or D609, respectively, at p < 0.05.
FIGURE 2.
FIGURE 2.
Palmitate and D609 inhibit insulin signaling in C2C12 myotubes. A, C2C12 myotubes were treated with D609 or palmitate as in Fig. 1. Selected samples were additionally treated with the sphingomyelinase inhibitor GW4869 (20 μm, 16 h). Following this incubation, cells were stimulated with insulin (1 nm, 15 min). Proteins were resolved by SDS-PAGE and Western blotted with antibodies recognizing total or phosphorylated (Ser-473) Akt. A and D display representative Western blots, and B and E display quantification of the extent of phosphorylation (fold over basal, n ≥3). C shows the effect of D609 and palmitate in insulin (100 nm, 1 h)-stimulated glycogen synthesis in C2C12 myotubes (fold over basal, n ≥3). * and # denote significance of D609 and GW4869, respectively, with a p value of <0.05.
FIGURE 3.
FIGURE 3.
Palmitate and D609 impair mitochondrial function in C2C12 myotubes. C2C12 myotubes were treated with BSA (control), BSA-conjugated palmitate (palmitate, 500 μm), and/or D609 (300 μm). Basal (A), maximal (B), and coupled (C) oxygen consumption rates were measured by a Seahorse XF24 extracellular analyzer as described under “Experimental Procedures.” n = 4; * and # denote palmitate and D609 (as compared with controls), respectively; p < 0.05.
FIGURE 4.
FIGURE 4.
SMS2 deficiency induces accumulation of SM precursors. C2C12 myotubes exposed to control or the CRISPR/Cas9 knock-out plasmids (Sgms2 KO) were treated with or without BSA-conjugated palmitate. A and B, transcripts encoding sphingomyelin synthase (i.e. SGMS1 and SGMS2), sphingomyelinase (i.e. SMPD1, SMPD2, and SMPD4), ceramide synthases (CerS1–6), dihydroceramide desaturase 1 (DEGS1), and serine palmitoyltransferase (SPTLC1 and -2) were measured by qPCR (n = 3). The mRNA expressions of SMPD3 and DEGS2 were minimal with a Cq value of >35. C, data represent the effect of Sms2 KO in total SMS activity by accessing the amount of NBD-SM converted from NBD-C-ceramide (n ≥3). D and E, LC-MS/MS analysis of sphingolipids in cells treated as in A, above. D, data represent detected sphinganine and the sum of intracellular levels of ceramides, dihydroceramides, or SMs with various acyl chain lengths present within the detection limits. E, displays the levels of individual ceramide species with different acyl chain lengths. n = 5; * and # denote significance of palmitate and SMS2KO treatments, respectively, with a p value of <0.05.
FIGURE 5.
FIGURE 5.
Sgms2 deficiency inhibits insulin signaling and impairs mitochondrial function in C2C12 myotubes. Control and SMS2KO cells were treated as in Fig. 4. A, representative Western blots show the effects of SMS2KO on insulin-stimulated phosphorylation of Akt (Ser-473) and GSK3β (Ser-9) (n = 3). B and C, quantification (n = 3) of the extent of Akt and GSK phosphorylation, expressed as a fold response to basal, untreated cells. D shows that SMS2KO attenuates insulin (100 nm, 1 h)-stimulated glycogen synthesis in C2C12 myotubes (fold over basal, n ≥3). # and * denote significance of palmitate treatment and SMS2KO, respectively, with p < 0.05. E, oxygen consumption rates were measured in wild type and SMS2KO C2C12 myotubes by Seahorse XF24 extracellular analyzer (n = 4). * denotes significance of palmitate treatment and/or SMS2KO with a p value of less than 0.05.
FIGURE 6.
FIGURE 6.
Sgms1 ablation does not impact insulin signaling. C2C12 myotubes exposed to control or the CRISPR/Cas9 knock-out plasmids (SMS1KO) were treated with BSA (control) or BSA-conjugated palmitate (500 μm). A, transcripts encoding Sgms1 and Sgms2 were quantified by qPCR (n = 4). B, data represent the effect of Sgms1 KO on total SMS activity as determined by quantifying the amount of NBD-C6-ceramide converted from NBD-SM (n = 3). * denotes significance of Sgms1 KO with p value <0.05. C and D, cell lysates were resolved by SDS-PAGE and Western blotted with antibodies recognizing phosphorylated Akt (Ser-473), total Akt, or β-actin (n = 3). * denotes significance of palmitate treatment with a p value of <0.05.
FIGURE 7.
FIGURE 7.
Ceramides, but not sphingomyelins, accumulate in skeletal muscle of insulin-resistant obese mice. Mice were fed either a normal chow diet or high fat diet for 12 weeks. Glucose (2 g/kg of lean mass) (A) or insulin (B) (0.75 IU/kg lean mass) tolerance tests were conducted. Lipids were extracted from quadriceps muscle samples and quantified LC-MS/MS (C and D). The most abundant ceramide, Cer(d18:1/18:0), was found to accumulate in quadriceps, in contrast to the two most abundant sphingomyelins (SM36:1 and SM34:1). Values are expressed as means ± S.E., *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus chow diet and n = 4 animals per group.
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