ABSTRACT
For the first time in skeletal muscle, the sphingolipid profile and key enzymes involved in the generation of ceramide in cells were investigated in simulated microgravity. It was found that, in C57B1/6 mice, the 4-day hindlimb unloading, in addition to reducing the mass of m. soleus, leads to the ceramide accumulation (3-fold) and the decrease of sphingomyelin content in this muscle (7.2-fold), as well as to the increase (2.7-fold) of protein level of acid sphingomyelinase. In a loaded m. biceps brachii the amount of ceramide is also enhanced, but both the amount of sphingomyelin and sphingomyelinase, as well as the muscle mass do not change, while the level of serine palmitoyltranspherase becomes significantly lower than in control mice. Taking into account the negative effects of ceramide in skeletal muscle (insulin resistance, inhibition of protein synthesis and increase of its decay) we can assume that sphingolipid mechanisms may be involved in the development of structural and functional abnormalities of skeletal muscle under conditions of weightlessness.
Subject(s)
Ceramides/metabolism , Muscle, Skeletal/chemistry , Sphingomyelins/metabolism , Weightlessness Simulation , Animals , Ceramides/analysis , Forelimb , Hindlimb , Hindlimb Suspension , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/physiology , Serine C-Palmitoyltransferase/analysis , Serine C-Palmitoyltransferase/metabolism , Sphingomyelin Phosphodiesterase/analysis , Sphingomyelin Phosphodiesterase/metabolism , Sphingomyelins/analysisABSTRACT
Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.
Subject(s)
Aerobiosis/physiology , Ceramidases/metabolism , Muscle, Skeletal/metabolism , Physical Conditioning, Animal/physiology , Serine C-Palmitoyltransferase/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Animals , Blood Glucose/analysis , Blood Glucose/metabolism , Ceramidases/analysis , Ceramides/analysis , Ceramides/blood , Chromatography, High Pressure Liquid , Enzyme Activation , Fatty Acids, Nonesterified/analysis , Fatty Acids, Nonesterified/blood , Fatty Acids, Nonesterified/metabolism , Insulin/analysis , Insulin/blood , Male , Muscle, Skeletal/chemistry , Rats , Rats, Wistar , Serine C-Palmitoyltransferase/analysis , Sphingomyelin Phosphodiesterase/analysis , Sphingosine/analysis , Sphingosine/bloodABSTRACT
CHO-LY-B cells have been useful in studies of sphingolipid metabolism and function because they lack serine palmitoyltransferase (SPT) activity. Cloning and sequencing of the SPT1 transcript of LY-B cells identified the mutation as a guanine to adenine change at nucleotide 738, causing a G246R transformation. Western blots revealed low expression of the mutant SPT1 peptide, but activity was not detectable by mass spectrometric analysis of [(13)C]-palmitate incorporation into sphinganine, sphingosine, 1-deoxysphinganine, or 1-desoxymethylsphinganine. Treatment of LY-B cells with chemical chaperones (DMSO or glycerol) increased the amounts of mutant SPT1 as well as SPT2, but SPT activity was not restored. This study has established that G246R mutation in hamster SPT1 results in the loss of SPT activity.
Subject(s)
Cell Line , Serine C-Palmitoyltransferase/chemistry , Serine C-Palmitoyltransferase/genetics , Amino Acid Sequence , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Cricetulus , Models, Molecular , Mutant Proteins/analysis , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Stability , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Serine C-Palmitoyltransferase/analysisABSTRACT
Serine palmitoyltransferase (SPT) catalyzes the condensation of l-serine and palmitoyl-CoA, which is the rate-limiting step in the de novo synthesis of sphingolipids. SPT activity is commonly measured by monitoring the incorporation of radiolabeled l-serine into 3-ketodihydrosphingosine. In this article, we introduce several adaptations of the established protocol to improve sensitivity, reproducibility, and practicability of the assay. A significant improvement of this new protocol is the possibility to measure SPT activity in total cell lysate instead of microsomes. The assay is furthermore extended by the introduction of a nonradioactive, HPLC-based detection protocol. The suggested HPLC method offers several advantages, most importantly, a 20-fold lower detection limit compared with the radioactive assay and the possibility to use an internal standard to correct for variation in the extraction.
