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.

Viviparity is a remarkable feature in squamate sauropsids and it has evolved multiple times in parallel with the formation of a placenta. One example of this repeated evolution of viviparity and placentation occurs in the species-rich South American genus Liolaemus with at least six independent origins of viviparity. However, evolutionary studies of placentation in this genus are limited by a lack of data on placental morphology. The aim of this study is to describe and compare the microanatomy and vessel diameter (Dv, a function of blood flow) of the placenta using scanning electron microscopy (SEM) and confocal laser scanning microscopy (cLSM) in two sympatric Andean viviparous but highly divergent species, Liolaemus robustus and Liolaemus walkeri. We found interspecific differences in cell types in the chorion, allantois, and omphalopleure that may be explained by divergent phylogenetic history. Time elapsed since divergence may also explain the pronounced interspecific differences in vessel diameter, and within each species, there are strong differences in Dv between tissue locations. Both species show features to improve gas exchange in the chorioallantoic placenta including absence of eggshell, large Dv in the allantois (L. robustus) or embryonic side of the uterus (L. walkeri), and when present, microvillous cells in the allantois (L. walkeri). Both species also show features that suggest transfer of nutrients or water in the omphaloplacenta, including an almost complete reduction of the eggshell, secretive material (L. robustus), or vesicles (L. walkeri) on cell surface uterus, and when present specialized cells in the omphalopleure (L. walkeri). No statistical differences in Dv were found among stages 32-39 in each species, suggesting that a different mechanism, other than enhanced blood flow, might satisfy the increased oxygen demand of the developing embryos in the hypoxic environments of the high Andes.

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).