AICAR inhibits ceramide biosynthesis in skeletal muscle

Abstract

Background: The worldwide prevalence of obesity has lead to increased efforts to find therapies to treat obesity-related pathologies. Ceramide is a well-established mediator of several health problems that arise from adipose tissue expansion. The purpose of this study was to determine whether AICAR, an AMPK-activating drug, selectively reduces skeletal muscle ceramide synthesis.

Methods: Murine myotubes and rats were challenged with palmitate and high-fat diet, respectively, to induce ceramide accrual, in the absence or presence of AICAR. Transcript levels of the rate-limiting enzyme in ceramide biosynthesis, serine palmitoyltransferase 2 (SPT2) were measured, in addition to lipid analysis. Student's t-test and ANOVA were used to assess the association between outcomes and groups.

Results: Palmitate alone induced an increase in serine palmitoyltransferase 2 (SPT2) expression and an elevation of ceramide levels in myotubes. Co-incubation with palmitate and AICAR prevented both effects. However, ceramide and SPT2 increased with the addition of compound C, an AMPK inhibitor. In rats fed a high-fat diet (HFD), soleus SPT2 expression increased compared with normal chow-fed littermates. Moreover, rats on HFD that received daily AICAR injections had lower SPT2 levels and reduced muscle ceramide content compared with those on HFD only.

Conclusions: These results suggest that AICAR reduces ceramide synthesis by targeting SPT2 transcription, likely via AMPK activation as AMPK inhibition prevented the AICAR-induced improvements. Given the role of skeletal muscle ceramide in insulin resistance, it is tempting to speculate that interventions that activate AMPK may lead to long-term ceramide reduction and improved metabolic function.

Figure 1

Palmitate-induced sphingolipid accumulation is prevented with AICAR. C2C12 myotubes were incubated with palmitate (PA) for 16 h at 0.75 mM. PA treatment increased all measured lipids. However, while ceramides (C) and glucosylceramides (D) were reduced with AICAR inclusion (2 mM), triacylglycerols (A) and diacylglycerols (B) were not significantly affected. Similarly, the inclusion of Compound C (20 μM) with AICAR (PA+AIC+CC) increased lipid levels over PA+AIC in ceramides (C) and glucosylceramides (D). *p<0.05 for PA vs. BSA. +p<0.05 for PA+AIC vs PA. §p<0.05 for PA+AIC+CC vs. PA+AIC (n=5).

Figure 2

Palmitate increases transcription of multiple enzymes of de novo sphingolipid synthesis. C2C12 myotubes were treated with palmitate (PA; 16 h, 0.75 mM) in the absence (PA) or presence of AICAR (PA+AIC; 2 mM). Levels of serinepalmitoyl transferase 2 (SPT2) and ceramide synthase 2 and 6 (CerS 2 and 6) were elevated with PA, but only SPT2 was significantly affected by inclusion of AICAR. Moreover, addition of Compound C (20 μM) caused a robust increase in SPT2 and, to a lesser degree, CerS6 transcript levels. SPT1-2: serine palmitoyltransferase 1–2; CerS 1–6: ceramide synthase 1–6; Des1: dihydroceramide desaturase 1; GCS: glucosylceramide synthase. *p<0.05 for PA vs BSA. +p<0.05 for PA+AIC vs PA. §p<0.05 for PA+AIC+CC vs. PA+AIC (n=4).

Figure 3

AICAR inhibits SPT2 induction. Male Wistar rats were fed a high-fat diet (HFD) and received daily injections of vehicle (saline) or AICAR (AIC; 0.5 mg/g body weight) for 6 wk. Daily AICAR injections prevented the high-fat diet-induced increase in SPT2 transcription (A) and resulted in decreased SPT2 protein levels (B). *p<0.05 for HFD vs Chow. +p<0.05 for HFD+AICAR vs HFD (n=9-11).

Figure 4

AICAR prevents high-fat diet-induced ceramide accumulation in muscle. Male Wistar rats were fed a high-fat diet (HFD) and received daily injections of vehicle (saline) or AICAR (AIC; 0.5 mg/g body weight) for 6 wk. HFD alone did not significantly increase ceramides compared with the Chow-fed group, but HFD+AIC resulted in a significant reduction in ceramides compared with HFD alone. +p<0.05 for HFD+AICAR vs HFD (n=9-11).