Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. We sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely to be a result of increased Drp1 (dynamin-related protein 1) action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e. respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fibre bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB (protein kinase B) phosphorylation in myotubes. However, inhibition of mitochondrial fission via Drp1 knockdown completely protected the myotubes and fibre bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels and unaffected insulin signalling. These data suggest that the forced and sustained mitochondrial fission that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.

BackgroundThe 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.MethodsMurine 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.ResultsPalmitate 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.ConclusionsThese 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.

Fat gain is associated with ceramide accrual in tissues of high‐metabolic rate, such as liver, brain and skeletal muscle. We predict that ceramide accumulation in skeletal muscle affects mitochondrial morphology and physiology. We found that ceramide treatment on murine myoblasts decreased viable mitochondrial density and induced mitochondrial fission, possibly via dynamin‐related protein 1 (Drp1), which was increased with ceramide accrual. Inhibition of endogenous ceramide synthesis via myriocin and serine palmitoyltransferase 2 (SPT2) siRNA prevented fatty acid‐induced mitochondrial fission in myoblasts. Moreover, inhibition of ceramide biosynthesis prevented the reduction in mitochondrial O2 consumption in myoblasts treated with fatty acids. Altogether, these findings suggest a critical role for ceramide in altering mitochondrial dynamics and function. Ongoing work will determine the extent to which mitochondrial fission mediates the adverse alterations in mitochondrial function. This work was supported by a BYU Graduate Research Fellowship (MES) and a BYU Mentoring Environment Grant (BTB).

Environmental toxins are increasingly implicated in adverse health outcomes. Considering the lung's apposition with the environment, we sought to test the hypothesis that ceramide functions as an intermediate between smoking and systemic insulin resistance. To determine the effect of cigarette smoke on lung ceramide metabolism, common lung cell lines including A‐549 and Beas2B were exposed to cigarette smoke extract (CSE). CSE treatment elicited a robust increase in ceramide and elevated expression of several enzymes involved with de novo ceramide synthesis. Moreover, myoblasts incubated with conditioned medium from CSE‐treated lung cells became completely insensitive to insulin stimulation, suggesting the presence of an insulin antagonist in the medium of CSE‐treated lung cells. Lastly, we measured systemic lipids in mice following nasal instillation of CSE and found an increase in muscle and hepatic ceramides; however, such findings were not observed in lung‐RAGE KO mice. This work identifies ceramide as a possible intermediate between the lung and peripheral tissues. Future work will determine the role of lung‐secreted ceramides on systemic effects in vivo, such as cardiovascular function and insulin signaling. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI; PRR) and a BYU Mentoring Environment Grant (BTB).