Numerous lines of evidence over the past 10 years have shown that various inducers of cellular stress such as inflammatory activation, excess saturated fatty acid intake, and chemotherapeutics, result in increased rates of ceramide synthesis. In addition, there is ample evidence demonstrating that the accumulation of cellular ceramides is associated with the pathogenesis of diseases such as obesity, diabetes, atherosclerosis, and cardiomyopathy. For example, studies in mice have correlated endogenous ceramides and glucosylceramides with the antagonism of insulin-stimulated glucose uptake and synthesis. In animal models of obesity, evidence shows that genetic or pharmacological inhibition of ceramide or glucosylceramide biosynthesis leads to increased peripheral insulin sensitivity while at the same time reducing the severity of pathologies associated with insulin resistance including diabetes, atherosclerosis, hepatic steatosis, and/or cardiomyopathy. With respect to overall lipid homeostasis and the role of adipose tissue in disease pathology, studies have revealed roles for the adipokines leptin, adiponectin, and TNFα in the modulation of cellular ceramide levels.
An enhanced systemic inflammatory status as well as cellular stress have both been associated with insulin resistance. With respect to biological lipids, excess lipid intake, especially saturated fatty acids, leads to mitochondrial and endoplasmic reticulum (ER) stress. Increased fat oxidation in mitochondria leads to the production of reactive oxygen species (ROS) which are known to result in insulin resistance. Both mitochondrial and ER stress can result in apoptosis. Excess fatty acid intake also interferes with normal insulin receptor-mediated signal transduction resulting in insulin resistance. Excess saturated fatty acid intake, particularly palmitic acid, results in increased ceramide synthesis which has been shown to be both a cause and effector of pancreatic β-cell stress resulting in impaired insulin secretion. Obesity, which results in insulin resistance and development of type 2 diabetes, has long been associated with low-grade systemic inflammation. The correlation between obesity, ceramide synthesis and insulin resistance is discussed below.
Insulin resistance induced by fatty acids. Model for how excess free fatty acids (FFAs) lead to insulin resistance and enhanced inflammatory responses, predominantly in cells such as skeletal muscle and adipose tissue but also in the liver. Only the major pathways regulated by insulin relative to glucose and lipid homeostasis are shown. Black arrows represent positive actions and red T-lines represent inhibitory actions. JNK: Jun N-terminal kinase. PKC: protein kinase C. IKKβ: inhibitor of nuclear factor kappa B kinase beta. ROS: reactive oxygen species. PI3K: phosphatidylinositol-3 kinase. DAG: diacylglycerol. TAG: triacylglycerides. LCA-CoA: long-chain acyl-CoAs. NFκB: nuclear factor kappa B. PKB (protein kinase B) is a serine/threonine kinase also known as AKT. The role of ceramides in the development of insulin resistance is discussed in the section below.
The ability of ceramides to interfere with insulin receptor signaling is the result of blocking the receptors ability to activate the downstream effector kinase, PKB/AKT. Experiments in cell culture, involving both adipocytes and skeletal muscle cells, have shown that ceramides inhibit insulin-stimulated glucose uptake by blocking translocation of GLUT4 to the plasma membrane as well as interfering with glycogen synthesis. That blockade of PKB/AKT activation is central to the effects of ceramides can be demonstrated by constitutive overexpression of the kinase which negates the effects of ceramides. Thus, far the action of ceramides at blocking activation of PKB/AKT has been shown in all cell types tested.
Several lines of evidence have solidified the model of ceramides leading to insulin resistance as a consequence of blockade of PKB/Akt activation. Administration of ceramide to cells in culture blocks the translocation of PKB/AKT to the plasma membrane. This inhibition of translocation is the result of the phosphorylation of a regulatory site in the PH domain. The phosphorylation leads to reduced affinity of the kinase for phosphoinositides. The kinase responsible for the ceramide-induced phosphorylation of PKB/AKT is likely to be the atypical PKC isoform PKCζ since this kinase is activated by ceramides in vitro. Additional evidence pointing to a link between ceramides and activation of PKCζ is that mutation of a target serine in the kinase, S34, to alanine confers resistance to ceramide action. Also, ceramide addition has been shown to stabilize interactions between PKB/AKT and PKCζ via their recruitment membrane rafts or caveolae. Another mechanism by which ceramides impact the activity of PKB/AKT is by activating protein phosphatase 2A (PP2A) to dephosphorylate the kinase. Experiments that were designed to specifically inhibit PP2A were shown to prevent the effects of ceramide on PKB/AKT in a number of different cell types. In some cell types, both mechanisms are functional, while in other cell culture systems either PKCζ or PP2A is the central mediator of ceramide effects.
Palmitic acid (C16:0) is the most abundant saturated fatty aid in the circulation. The role of saturated fatty acids in increased levels of ceramides has been demonstrated by adding palmitate to cultured muscle cells. In this system the addition of palmitate results in increased ceramide accumulation while simultaneously inhibiting PKB/AKT. Ceramide synthesis was indeed required for the effect of palmitate addition on the activity of PKB/AKT since pharmacological inhibition of ceramide synthesis or siRNA-mediated knockdown of several enzymes required for ceramide biosynthesis (serine palmitoyltransferase, ceramide synthases, or dihydroceramide desaturase) completely blocks the effects of palmitate on insulin signaling.
An alternative means to examine the effects of ceramides on insulin sensitivity is to block the pathways of ceramide metabolism. Treatment of cells with acid ceramidase inhibitors results in increased endogenous ceramide levels while simultaneously blocking insulin-mediated activation of PKB/AKT. Under conditions of ceramidase inhibition there is an exagerated effect of palmitic acid addition on insulin resistance. Conversely, if one overexpresses acid ceramidase, the inhibition of insulin signaling induced by palmitate addition is completely blocked.
The cellular effects of glucosylceramide, although similar to ceramides themselves, does exhibit cell-type specificity. Glucosylceramide is the precursor for a complex family of gangliosides, for example the GM3 ganglioside. Adipocytes are highly sensitive to the insulin inhibitory effects of glucosylated sphingolipids, whereas muscle cells are unaffected. Addition of GM3 ganglioside to adipocytes inhibits insulin activation of the IRS-1. In addition, TNFα treatment induces GM3 accumulation in membrane lipid rafts allowing for association with the insulin receptor through caveolin-1 present in the rafts. The the antagonistic effects of the TNFα can be prevented by depleting cells of glucosylated ceramides. Obesity is associated with adipose tissue enrichment in the complpex gangliosides, GM2, GM1, and GD1a. The significance of the accumulation of these gangliosides has been demonstrated in mice lacking GM3 synthase which generates the major ganglioside precursor. These mice are protected from insulin resistance and glucose intolerance when fed a high-fat diet. Treatment of genetically obese or diet-induced obese mice with highly specific glucosylceramide synthase (GCS) inhibitors results in improved glucose tolerance and increased insulin sensitivity in muscle and liver. Collectively, these studies strongly implicate a role for glucosylated ceramides in increased adipose tissue inflammation, peripheral insulin resistance, and hepatic steatosis.
The most potent reagent used to study the effects of the manipulation of enzymes involved in sphingolipid biosynthesis is the compound myriocin [2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoicos-6-enoic acid]. Myriocin is a highly specific inhibitor of serine palmitoyltransferase (SPT), which is the first and rate-limiting enzyme in the de novo pathway of ceramide synthesis. See the Figure above showing sphingosine and ceramide synthesis. Myriocin (also known as antibiotic ISP-1 and thermozymocidin) was isolated from themophilic fungi such as Mycelia sterilia and Isaria sinclairii. Extracts from these fungi have been used in traditional Chinese medicine as a treatment for numerous conditions including diabetes. Myriocin can be administered chronically to rodents and it appears to be well tolerated. Addition of myriocin to animals that are models of obesity prevents insulin resistance and the development of diabetes, atherosclerosis, and cardiomyopathy. In addition, myriocin improves glucose tolerance, insulin sensitivity and ameliorates hypertension when administered to rodents.
Genetic manipulation of several enzymes in ceramide metabolism has also been shown to insulin sensitizing. In mice heterozygous for the SPT subunit SPTLC2 (serine palmitoyltransferase, long-chain base subunit 2) there is a reduction in peripheral ceramide levels and improved insulin sensitivity when these animals are fed a high-fat diet. Similar results are seen in mice heterozygous for dihydroceramide desaturase-1 (DES1). Both SPT and DES1 are required for ceramide biosynthesis. As described above, a large family of ceramide synthases (CerS) have been identified in mammals. CerS1 is the most abundant isoform expressed in skeletal muscle and is involved primarily in the synthesis of C18:0 ceramides. The level of expression of CerS1 was shown to be significantly elevated in mice fed a high-fat diet. This increase in CerS1 expression was associated with alterations in ceramide levels and reduced glucose tolerance.
Collectively these data demonstrate a complex interrelationship between sphingosine and ceramide metabolism and insulin resistance. As pointed out ceramides can be deacetylated by ceramidases to form sphingosine. As discussed below, sphingosine can be phosphorylated to S1P which is an important biologically active lipid. Ceramides can also be glucosylated by glucosylceramide synthase (GCS) forming glucosylceramides which then serve as the building blocks of complex glycosphingolipids; they can act as substrates for the sphingomyelin synthases yielding sphingomyelins; or they can be phosphorylated by ceramide kinase to yield ceramide-1-phosphate. Thus, it is clear that multiple products of the actions of SPT, CerS, and DES1 could all potentially contribute to the development of insulin resistance and diabetes.
As pointed out earleir, obesity is associated with a low-grade systemic inflammatory state. One of the mechanisms involved in this inflammatory status is the activation of toll-like receptors (TLRs). TLR activation leads to enhanced transcription of pro-inflammatory cytokines such as TNFα and interleukin-6 (IL6). Saturated fatty acids are known to activate TLR4 and this activation is requisite for lipid induction of TNFα and other cytokines. When TLRs are knocked-out in mice the animals are protected from lipid-induced insulin resistance. The signal transduction cascade initiated by TLR activation involves the downstream effectors IKKβ and NFκB. TLR4 activation has been shown to selectively and strongly increase the levels of sphingolipids within cells. Several studies have shown that ceramide is indeed an obligate intermediate linking TLR4 activation to the induction of insulin resistance.