Abstract
Metabolic dysfunction, dysregulated differentiation, and atrophy of skeletal muscle occur as part of a cluster of abnormalities associated with the development of Type 2 diabetes mellitus (T2DM). Recent interest has turned to the attention of the role of 1-deoxysphingolipids (1-DSL), atypical class of sphingolipids which are found significantly elevated in patients diagnosed with T2DM but also in the asymptomatic population who later develop T2DM. In vitro studies demonstrated that 1-DSL have cytotoxic properties and compromise the secretion of insulin from pancreatic beta cells. However, the role of 1-DSL on the functionality of skeletal muscle cells in the pathophysiology of T2DM still remains unclear. This study aimed to investigate whether 1-DSL are cytotoxic and disrupt the cellular processes of skeletal muscle precursors (myoblasts) and differentiated cells (myotubes) by performing a battery of in vitro assays including cell viability adenosine triphosphate assay, migration assay, myoblast fusion assay, glucose uptake assay, and immunocytochemistry. Our results demonstrated that 1-DSL significantly reduced the viability of myoblasts in a concentration and time-dependent manner, and induced apoptosis as well as cellular necrosis. Importantly, myoblasts were more sensitive to the cytotoxic effects induced by 1-DSL rather than by saturated fatty acids, such as palmitate, which are critical mediators of skeletal muscle dysfunction in T2DM. Additionally, 1-DSL significantly reduced the migration ability of myoblasts and the differentiation process of myoblasts into myotubes. 1-DSL also triggered autophagy in myoblasts and significantly reduced insulin-stimulated glucose uptake in myotubes. These findings demonstrate that 1-DSL directly compromise the functionality of skeletal muscle cells and suggest that increased levels of 1-DSL observed during the development of T2DM are likely to contribute to the pathophysiology of muscle dysfunction detected in this disease.
Highlights
The first step in de novo sphingolipid synthesis occurs in the endoplasmic reticulum (ER) where the enzyme serine palmitoyltransferase (SPT) catalyses the condensation of serine and palmitoyl Co-A to form sphinganine (SA) which is metabolised to ceramide and complex sphingolipids [6]
Given the cytotoxic properties associated with atypical lipids, the aim of this study was to investigate whether 1-DSL compromises the functionality of skeletal myoblasts and their differentiation into mature myotubes contributing to the pathophysiology of Type 2 diabetes mellitus (T2DM)
Data are presented as mean ± Standard Error of Mean (SEM) (n=3). *p < 0.05, SA vs. 1-DSA; one-way ANOVA followed by post hoc tests. (F) 1-DSA induced autophagy as shown by immunostaining with autophagic marker P62/SQSTM1, FITC-phalloidin, and DAPI
Summary
The first step in de novo sphingolipid synthesis occurs in the endoplasmic reticulum (ER) where the enzyme serine palmitoyltransferase (SPT) catalyses the condensation of serine and palmitoyl Co-A to form sphinganine (SA) which is metabolised to ceramide and complex sphingolipids (sphingomyelins, glycosphingolipids) [6]. Recent in vitro studies have demonstrated accumulation of 1-DSL promote death of pancreatic beta-cells and interfere with insulin secretion [8] and may be involved in the mechanisms of insulin resistance [5]. A reduction in skeletal muscle’s ability to respond to insulin and the subsequent development of insulin resistance are key events in the establishment of high blood glucose levels and impaired glycaemic control. In addition to these metabolic dysfunctions, muscle loss or atrophy is associated with diminished strength, quality of life, and early mortality [11, 12]. Given the cytotoxic properties associated with atypical lipids, the aim of this study was to investigate whether 1-DSL compromises the functionality of skeletal myoblasts and their differentiation into mature myotubes contributing to the pathophysiology of T2DM
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