The Role of Acyl-CoA Synthetase 1 in Bioactive Lipid Accumulation and the Development of Hepatic Insulin Resistance.

The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study was to explore the involvement of acyl-CoA synthetase 1 (ACSL1) in bioactive lipid accumulation and the induction of liver insulin resistance (InsR) in animals fed an HFD. The experiments were performed on male C57BL/6 mice divided into the following experimental groups: 1. Animals fed a control diet; 2. animals fed HFD; and 3. HFD-fed animals with the hepatic ACSL1 gene silenced through a hydrodynamic gene delivery technique. Long-chain acyl-CoAs, sphingolipids, and diacylglycerols were measured by LC/MS/MS. Glycogen was measured by means of a commercially available kit. The protein expression and phosphorylation state of the insulin pathway was estimated by Western blot. HFD-fed mice developed InsR, manifested as an increase in fasting blood glucose levels (202.5 mg/dL vs. 130.5 mg/dL in the control group) and inhibition of the insulin pathway, which resulted in an increase in the rate of gluconeogenesis (0.420 vs. 0.208 in the control group) and a decrease in the hepatic glycogen content (1.17 µg/mg vs. 2.32 µg/mg in the control group). Hepatic ACSL1 silencing resulted in decreased lipid content and improved insulin sensitivity, accounting for the decreased rate of gluconeogenesis (0.348 vs. 0.420 in HFD(+/+)) and the increased glycogen content (4.3 µg/mg vs. 1.17 µg/mg in HFD(+/+)). The elevation of gluconeogenesis and the decrease in glycogenesis in the hepatic tissue of HFD-fed mice resulted from cellular lipid accumulation. Inhibition of lipid synthesis through silencing ACSL1 alleviated HFD-induced hepatic InsR.

Serine Palmitoyltransferase Gene Silencing Prevents Ceramide Accumulation and Insulin Resistance in Muscles in Mice Fed a High-Fat Diet.

Skeletal muscles account for ~80% of insulin-stimulated glucose uptake and play a key role in lipid metabolism. Consumption of a high-fat diet (HFD) contributes to metabolic changes in muscles, including the development of insulin resistance. The studies carried out to date indicate that the accumulation of biologically active lipids, such as long-chain acyl-CoA, diacylglycerols and ceramides, play an important role in the development of insulin resistance in skeletal muscles. Unfortunately, it has not yet been clarified which of these lipid groups plays the dominant role in inducing these disorders. In order to explore this topic further, we locally silenced the gene encoding serine palmitoyltransferase (SPT) in the gastrocnemius muscle of animals with HFD-induced insulin resistance. This enzyme is primarily responsible for the first step of de novo ceramide biosynthesis. The obtained results confirm that the HFD induces the development of whole-body insulin resistance, which results in inhibition of the insulin pathway. This is associated with an increased level of biologically active lipids in the muscles. Our results also demonstrate that silencing the SPT gene with the shRNA plasmid reduces the accumulation of ceramides in gastrocnemius muscle, which, in turn, boosts the activity of the insulin signaling pathway. Furthermore, inhibition of ceramide synthesis does not significantly affect the content of other lipids, which suggests the leading role of ceramide in the lipid-related induction of skeletal muscle insulin resistance.

CerS1 but Not CerS5 Gene Silencing, Improves Insulin Sensitivity and Glucose Uptake in Skeletal Muscle.

Skeletal muscle is perceived as a major tissue in glucose and lipid metabolism. High fat diet (HFD) lead to the accumulation of intramuscular lipids, including: long chain acyl-CoA, diacylglycerols, and ceramides. Ceramides are considered to be one of the most important lipid groups in the generation of skeletal muscle insulin resistance. So far, it has not been clearly established whether all ceramides adversely affect the functioning of the insulin pathway, or whether there are certain ceramide species that play a pivotal role in the induction of insulin resistance. Therefore, we designed a study in which the expression of CerS1 and CerS5 genes responsible for the synthesis of C18:0-Cer and C16:0-Cer, respectively, was locally silenced in the gastrocnemius muscle of HFD-fed mice through in vivo electroporation-mediated shRNA plasmids. Our study indicates that HFD feeding induced both, the systemic and skeletal muscle insulin resistance, which was accompanied by an increase in the intramuscular lipid levels, decreased activation of the insulin pathway and, consequently, a decrease in the skeletal muscle glucose uptake. CerS1 silencing leads to a reduction in C18:0-Cer content, with a subsequent increase in the activity of the insulin pathway, and an improvement in skeletal muscle glucose uptake. Such effects were not visible in case of CerS5 silencing, which indicates that the accumulation of C18:0-Cer plays a decisive role in the induction of skeletal muscle insulin resistance.

Sphingolipids as a Culprit of Mitochondrial Dysfunction in Insulin Resistance and Type 2 Diabetes.

Insulin resistance is defined as a complex pathological condition of abnormal cellular and metabolic response to insulin. Obesity and consumption of high-fat diet lead to ectopic accumulation of bioactive lipids in insulin-sensitive tissues. Intracellular lipid accumulation is regarded as one of the major factors in the induction of insulin resistance and type 2 diabetes (T2D). A significant number of studies have described the involvement of ceramides and other sphingolipids in the inhibition of insulin-signaling pathway in both skeletal muscles and the liver. Adverse effects of sphingolipid accumulation have recently been linked to the activation of protein kinase Cζ (PKCζ) and protein phosphatase 2A (PP2A), which, in turn, negatively affect phosphorylation of serine/threonine kinase Akt [also known as protein kinase B (PKB)], leading to decreased glucose uptake in skeletal muscles as well as increased gluconeogenesis and glycogenolysis in the liver. Sphingolipids, in addition to their direct impact on the insulin signaling pathway, may be responsible for other negative aspects of diabetes, namely mitochondrial dysfunction and deficiency. Mitochondrial health, which is characterized by appropriate mitochondrial quantity, oxidative capacity, controlled oxidative stress, undisturbed respiratory chain function, adenosine triphosphate (ATP) production and mitochondrial proliferation through fission and fusion, is impaired in the skeletal muscles and liver of T2D subjects. Recent findings suggest that impaired mitochondrial function may play a key role in the development of insulin resistance. Mitochondria stay in contact with the endoplasmic reticulum (ER), Golgi membranes and mitochondria-associated membranes (MAM) that are the main places of sphingolipid synthesis. Moreover, mitochondria are capable of synthesizing ceramide though ceramide synthase (CerS) activity. Recently, ceramides have been demonstrated to negatively affect mitochondrial respiratory chain function and fission/fusion activity, which is also a hallmark of T2D. Despite a significant correlation between sphingolipids, mitochondrial dysfunction, insulin resistance and T2D, this subject has not received much attention compared to the direct effect of sphingolipids on the insulin signaling pathway. In this review, we focus on the current state of scientific knowledge regarding the involvement of sphingolipids in the induction of insulin resistance by inhibiting mitochondrial function.

GPAT Gene Silencing in Muscle Reduces Diacylglycerols Content and Improves Insulin Action in Diet-Induced Insulin Resistance.

Skeletal muscle is an important tissue responsible for glucose and lipid metabolism. High-fat diet (HFD) consumption is associated with the accumulation of bioactive lipids: long chain acyl-CoA, diacylglycerols (DAG) and ceramides. This leads to impaired insulin signaling in skeletal muscle. There is little data on the involvement of DAG in the development of these disorders. Therefore, to clarify this enigma, the gene encoding glycerol-3-phosphate acyltransferase enzyme (GPAT, responsible for DAG synthesis) was silenced through shRNA interference in the gastrocnemius muscle of animals with diet-induced insulin resistance. This work shows that HFD induces insulin resistance, which is accompanied by an increase in the concentration of plasma fatty acids and the level of bioactive lipids in muscle. The increase in these lipids inhibits the insulin pathway and reduces muscle glucose uptake. GPAT silencing through electroporation with shRNA plasmid leads to a reduction in DAG and triacylglycerol (TAG) content, an increase in the activity of the insulin pathway and glucose uptake without a significant effect on ceramide content. This work clearly shows that DAG accumulation has a significant effect on the induction of muscle insulin resistance and that inhibition of DAG synthesis through GPAT modulation may be a potential target in the treatment of insulin resistance.