Ceramides, mitochondrial fission, and reactive oxygen species a novel mechanism of obesity‐induced insulin resistance (LB167)

Abstract

Reactive oxidative species (ROS) is a known byproduct of mitochondrial dysfunction and is believed to contribute to inflammation signaling commonly found in insulin resistance. Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress‐related pathways. Ceramide has long been known to have morphological and physiological effects on mitochondria, which could potentially alter levels of ROS. The focus of this study is to determine whether increased ceramide levels result in ROS generation and decreased mitochondrial respiration in myotubes via altered mitochondrial morphology. C2C12 Skeletal muscle cells were used to assess the effect of ceramide on ROS generation and electron microsopy was used to image the morphological changes. Ceramide synthesis can be stimulated by treatment with exogenous fatty acid, thus myotubes were simultaneously treated with exogenous fatty acid following SPT2 knockdown (SPT is the rate limiting enzyme in ceramide synthesis), ROS increased within the myotubes and cellular respiration dropped. To verify the disruptive effect of ceramide on respiration caused by a change of mitochondrial morphology, cells were treated with a fission inhibitor (Mdivi‐1) prior to the addition of exogenous fatty acid. The resulting inhibition of mitochondrial fission via Mdivi‐1 helped protect the myotubes from ceramide‐induced metabolic disruption, including maintained mitochondrial respiration, and reduced ROS levels. Images taken with the confocal microscope verify that cells with increased levels of ceramide undergo mitochondrial fission while cells treated with Mdivi‐1 are protected from morphological changes. Altogether, these data suggest a critical role for mitochondrial fission as a mediator of ceramide‐induced metabolic disruption.