Role of Ceramides in the Pathogenesis of Diabetes Mellitus and its Complications

Summary/Abstract

Diabetes mellitus (DM) is a systemic metabolic disease that affects 463 million adults worldwide and is a leading cause of cardiovascular disease, blindness, nephropathy, peripheral neuropathy, and amputation. Lipids have long been recognized as contributors to the pathogenesis and pathophysiology of DM and its complications, but recent discoveries have highlighted ceramides, a class of bioactive sphingolipids with cell signaling and second messenger capabilities, as particularly important contributors to insulin resistance and the underlying mechanisms of DM complications. Besides their association with insulin resistance and pathophysiology of type 2 diabetes, evidence is emerging that certain species of ceramides are mediators of cellular mechanisms involved in the initiation and progression of microvascular and macrovascular complications of DM. Advances in our understanding of these associations provides unique opportunities for exploring ceramide species as potential novel therapeutic targets and biomarkers. This review discusses the links between ceramides and the pathogenesis of DM and diabetic complications and identifies opportunities for novel discoveries and applications.

Introduction

Diabetes mellitus (DM), a global epidemic that affects 463 million adults worldwide, now ranks among the leading non-communicable public health challenges of the present era^1–3. The number of people living with diabetes has increased by more than three-fold over the past two decades^1–4. As the leading cause of blindness, amputation, and chronic kidney disease, and a major contributor to myocardial infarction (Ml), heart failure, stroke, and peripheral vascular disease (PVD), diabetes imposes a high toll on morbidity and mortality^5–8. Type 2 diabetes (T2D) accounts for approximately 90-95% of the diabetes burden, whereas type 1 diabetes (T1D; typically seen in children) accounts for 5-10% of the disease burden. Insulin deficiency, usually triggered by autoimmune mechanisms in genetically susceptible persons, is the proximate cause of T1D. In contrast, the pathophysiology of T2D involves a complex interaction among genetic predisposition, insulin resistance, and β-cell dysfunction, among other contributory factors. Activation of pro-inflammatory and oxidative stress pathways is involved in the pathophysiology of both forms of diabetes as well as diabetes complications⁹. Approximately 7.3 million of the 34 million people affected with diabetes in the U.S. are undiagnosed and half of the 463 million people with diabetes worldwide also are undiagnosed^1,10. Sadly, individuals with undiagnosed diabetes still are at risk of developing diabetes complications: in the United Kingdom Prospective Diabetes Study, nearly one-quarter of people with newly diagnosed T2D already had developed one or more complications⁹.

Sphingolipids are a group of specialized lipids containing an amino alcohol in their backbone and are present in all mammalian cells (Figure 1). They were originally assumed to only have structural roles, but certain types of bioactive sphingolipids have been found to be involved in cell signaling, growth/survival, differentiation, death, inflammation, and other processes¹¹. Sphingolipids are structurally diverse and can be divided into different classes based on major variations in molecular structure¹². Sphingolipid classes are further divided into different species by smaller differences in parameters such as chain length, allowing for the existence of tens of thousands of unique sphingolipid molecules in every cell¹³. Due to their ubiquity and widespread bioactivity, sphingolipid metabolic abnormalities and dysregulation have been implicated as potential driving forces in many pathologic conditions, including DM and DM-associated complications.

Figure 1: Diversity and Synthesis of Ceramides.

Synthesis of ceramide occurs via three main pathways, each of which utilizes enzymes with multiple isoforms, pathway-specific localization, and/or reactants with variable R-groups to yield a diverse array of Ceramide species with unique bioactivity. The De Novo Pathway occurs in the endoplasmic reticulum and begins with rate-limiting Serine Palmitoyltransferase-catalyzed condensation of Palmitoyl-CoA and L-Serine to form 3-keto-Sphinganine, which is then reduced to Sphinganine by 3-keto-Sphinganine Reductase. Ceramide Synthases 1-6 add a fatty acid chain (blue) of variable length (R) to form Dihydroceramide, which is then is converted to Ceramide by Dihydroceramide Desaturase and transported to the Golgi¹⁸². The Sphingomyelinase Pathway hydrolyzes Sphingomyelin to Ceramide via acid or neutral Sphingomyelinases 1-5, which are found in the plasma membrane, cytosol, mitochondria, endosomes, and lysosomes^{183, 184}. The Salvage Pathway breaks down complex sphingolipids such as gangliosides in endo-lysosomal compartments to Sphingosine, which may then be transported to the ER and reacylated to Ceramide by Ceramide Synthases 1-5. Alternatively, Sphingosine (R1 = H) may be phosphorylated at R1 to Sphingosine-1-Phosphate by Sphingosine Kinase^185–187. Ceramide may be phosphorylated at R1 by Ceramide Kinase to generate Ceramide-1-Phosphate, or it may be glycosylated to form Cerebrosides (Glucosylceramides and Galactosylceramides). Addition of a Galactose residue to a Glucosylceramide yields a Lactosylceramide, which may be further glycosylated or acquire sialic acid residues at various sites (Gn, Gn+1...) to form Gangliosides¹⁸⁸.

At the center of the sphingolipid metabolic labyrinth is ceramide, which may be formed by three main pathways: de novo synthesis, hydrolysis of sphingomyelin (SM), and salvage of complex sphingolipids (Figure 1). The different routes of ceramide synthesis determine the initial localization and species of ceramide generated, and hence its bioactivity and downstream products¹³. Several decades of research have established the involvement of various forms of ceramide, and even systemic or plasma ratios of ceramide species, in important biological processes central to human diseases such as DM and its complications. While the involvement of ceramide in these diabetic complications is already evident and supported by literature, many new studies further implicate ceramide and help elaborate ceramide-mediated mechanisms underlying their pathophysiology. The emergence of ceramides as key mediators of the onset and progression of insulin resistance and diabetic complications provides unique opportunities to explore their potential utility as novel therapeutic targets and disease biomarkers. This review briefly discusses past research and recent findings regarding ceramide’s roles in insulin resistance and some of the major complications of diabetes. Currently, information on the role of ceramides in diabetes is disseminated in scores of publications in numerous biomedical journals. Some review articles published in the past decade have diligently assembled and discussed studies on the roles of ceramides in diabetes and its complications^14–17. Some focus heavily on sphingolipid metabolism and signaling, while others emphasize mechanisms of pathogenesis. This review attempts to aggregate current mechanistic knowledge while also providing a comprehensive narrative regarding the roles of ceramides in various diabetic complications in a format that will be useful for clinicians and researchers.

Ceramide and Insulin Resistance

Metabolic syndrome refers to a group of pathological disorders including obesity, hypertension, dyslipidemia, and hyperglycemia that acts as compounding risk factors for diabetes, cardiovascular disease, and other conditions¹⁸. Metabolic syndrome is closely related to insulin resistance, a condition characterized by impaired insulin-stimulated metabolic activities in certain tissues (skeletal muscle, adipocytes, liver, etc.)¹⁹. Insulin is produced by the pancreas to maintain glucose homeostasis, but in insulin resistance, insulin levels may be elevated due to lack of glucose uptake and feedback. Excess caloric intake causes expansion of adipose tissue, which increases adipocyte lipolysis and leads to ectopic deposition of lipids in non-adipose tissue²⁰. Transport of sarcolemmal fatty acids²¹ and delivery of non-esterified fatty acids and triacylglycerols to peripheral tissues is also increased²². These fatty acids are esterified to diacylglycerol and triacylglycerol and stored in the liver, skeletal muscle, heart, and pancreas²³, where they participate in ceramide biosynthesis and cause ceramide accumulation^{24, 25}. Emerging evidence has implicated the accumulation of ceramide in metabolic tissues as contributing to impairment of insulin sensitivity and glucose homeostasis^{14, 26–29}.

Obesity is a strong risk factor for diabetes³⁰. Plasma analyses of obese individuals show significantly higher levels of ceramide³¹ and palmitate³², which, as the source of palmitoyl-CoA in the de novo pathway, is a precursor of ceramide biosynthesis (Figure 1). A multicohort study of diabetic patients showed that dysfunctional visceral adiposity and insulin resistance are associated with higher plasma ceramides³³, suggesting that plasma ceramides could act as DM biomarkers^{34, 35}. Elevated ceramides were also reported in the skeletal muscle, liver, and hypothalamus of obese humans and experimental obese rodents³⁶. In another genetically obese animal model, the levels of hepatic and plasma C16:0 ceramide increased significantly. However, use of antisense oligonucleotides of the C16:0 ceramide biosynthetic enzyme ceramide synthase 6 (CerS6) in this model significantly reduced hepatic and plasma C16:0 ceramide while also increasing insulin sensitivity³⁷. Similarly, in experimental high fat diet animals, increased levels of C18:0 ceramide were observed in skeletal muscle. Knocking out the CerS1 gene, which synthesizes C18:0 ceramide, leads to improved glucose homeostasis and insulin sensitivity³⁸. High-fat diet in a Wistar rat model correlated with increased ceramide levels and decreased insulin sensitivity³⁹. Other obese animal models showed increased levels of serine palmitoyl transferase (SPT) and dihydroceramide desaturase (DES), indicating increased activity of the de novo ceramide synthesis pathway (Figure 1)⁴⁰. This is further supported by experiments showing that ablation of dihydroceramide desaturase 1 (DES1) in mice resolves hepatic steatosis and insulin resistance caused by obesogenic diet and leptin deficiency⁴¹. Leptin regulates lipid metabolism in the skeletal muscle and is responsible for dampening hyperlipidemia and lipotoxicity. Higher levels of ceramide detected in leptin-deficient mice suggests a relationship between ceramide synthesis and hyperlipidemia leading to decreased insulin action. Collectively, these results indicate that tissue-specific ceramide accumulation is dependent upon distinct ceramide species generated by the de novo and salvage pathways.

Ceramide-regulated insulin resistance occurs through multiple pathways (Figure 2). In one mechanism, ceramide activates PKCζ, which phosphorylates and inhibits the translocation of AKT/protein kinase B (PKB) to the plasma membrane⁴². Inhibiting translocation of AKT/PKB interferes with insulin-dependent GLUT glucose transporter proteins, blocking glucose uptake and impairing nutrient storage¹⁴. Ceramide also stimulates activation of cytosolic protein phosphatase 2A (PP2A), which is the primary phosphatase for AKT/PKB dephosphorylation^{43, 44}. Increased accumulation of ceramide in the endoplasmic reticulum and/or mitochondria impairs insulin action and causes cellular stress ^26,28.

Figure 2: Ceramides and Insulin Resistance.

Ceramide plays a central role in insulin resistance and β-cell death. Obesity and hyperglycemia both increase circulating shorter-chain saturated free fatty acids (FFA) that serve as substrates for and induce de novo ceramide synthesis^{24, 25}. Chronic inflammation in obesity is associated with TLR4 activation, which in turn increases cytokines and ceramide levels¹⁸⁹. Diabetes is associated with decreased adiponectin, which is a known inhibitor of ceramide synthesis¹⁶⁴. Ceramide activates PP2A and PKCζ, which inhibit AKT/PKB and decrease insulin sensitivity^{58, 59}. Ceramide also activates pro-inflammatory and pro-apoptotic pathways via caspase, PP1, PKc, and cathepsin D^{15, 57}, leading to pancreatic β-cell death⁵³.

The chronic inflammation associated with obesity is due in part to accumulation of ceramides¹⁹. In blood samples of diabetic patients, increased levels of proinflammatory cytokines are correlated with dysregulated lipid metabolism and ceramide production. Obesity in leptin-deficient, genetically obese mice (ob/ob), is associated with elevated TNFα and hyperinsulinemia. The adipose tissue of these transgenic animals also show increased gene expression of the ceramide biosynthetic enzymes neutral sphingomyelinase (nSMase), acidic sphingomyelinase (aSMase), and SPT⁴⁵. Ceramide elevation in adipose tissue increases NLRP3-dependent IL-1β⁴⁶, and knock-out of NLRP3 in mouse models protects them from ceramide-mediated glucose intolerance, insulin resistance, and inflammation due to diet-induced obesity⁴⁷. Similarly, silencing of proinflammatory genes (i.e., TNF, IL-1α, and stress-induced kinase JNK-1) reduces peripheral insulin resistance⁴⁸, suggesting correlations between inflammation and insulin resistance. The ceramide-mediated inflammatory pathway is regulated by toll like receptors (TLRs). TLRs such as TLR4 can induce insulin resistance⁴⁹ via IκKβ and NFκB signaling pathways⁵⁰. As well as modulating insulin sensitivity, adiponectin plays an important role in controlling ceramide-mediated inflammation in metabolic disorders through AMPK, a serine threonine kinase⁵¹.

Lipotoxicity and hyperglycemia cause ceramide to accumulate in pancreatic β-cells⁵² and β-cell destruction can be prevented by blocking ceramide production⁵³. Unlike T2D, in which cells do not respond properly to insulin, T1D is caused by inadequate production, secretion, and release of insulin due to loss of pancreatic β-cells. In Zucker diabetic fatty (ZDF) rats, inhibition of de novo ceramide biosynthesis by cycloserine attenuates Langerhans islet apoptosis and helps prevent hyperglycemia⁵⁴. In ceramide-mediated β-cell dysfunction and cell death, the fatty acid transporter/cluster determinant 36 (FAT/CD36) plays a significant role. During obesity and hyperlipidemia, CD36 induces adipose tissue inflammation by enhancing macrophage activation and impairing insulin signaling⁵⁵, and inhibition of CD36 is shown to prevent ceramide-induced thioredoxin-interacting protein activation by inhibiting NFκB and in turn protects β-cells from apoptosis⁵⁶. In addition to interfering in signaling mechanisms, disrupting glucose transport and storage, and aggravating inflammation, ceramide enhances cell death by stimulating caspase, PKC, serine/threonine protein phosphatase 1 (PP1), and cathepsin D^{15, 57}. In islets of Langerhans and pancreatic β-cell lines, it has been reported that ceramide dephosphorylates ERK1/2 by PP2A, which in turn inhibits insulin expression^58,59. Ceramide-mediated activation of PKCζ not only inactivates AKT/PKB pathway but also phosphorylates and inactivates pancreatic and duodenal homeobox gene-1 (PDX-1), which is a transcription factor of insulin gene expression⁶⁰. Hence, targeting ceramide synthesis as a therapeutic strategy to prevent insulin resistance could be an effective strategy, although global inhibition of ceramide production could be harmful due to its involvement in diverse physiologic mechanisms.

Ceramide in Liver and Pancreatic Diseases

There is a strong connection between metabolic syndrome and dyslipidemia, insulin resistance, and dysglycemia. The liver and pancreas are two vital organs that regulate systemic lipid and glucose metabolism, but are also affected by their dysregulation. In fact, the pathophysiology of the most common metabolic disorder of the liver, non-alcoholic fatty liver disease (NAFLD), involves dyslipidemia, insulin resistance, hyperglycemia, inflammation, and their interplay^61,62. Accumulation of cytotoxic lipids, especially ceramide, has been shown in metabolic NAFLD^{63,64, 65}. Elevated levels of C16:0 and C18:0 dihydroceramide in human livers, indicating de novo ceramide biosynthesis elevation, were associated with NFALD and insulin resistance⁶⁶. This finding was supported by animal studies wherein CerS6 deficient mice exhibited lower levels of C16:0 ceramide and are safeguarded from high fat-induced obesity, insulin resistance, and adipose tissue inflammation⁶⁷. Further, Sprague-Dawley rats were protected from developing hepatic steatosis and fibrosis with NAFLD through inhibition of de novo ceramide biosynthesis by myriocin⁶⁸. Involvement of acidic SMase and dihydroceramide desaturase has been reported in generation and accumulation of ceramide during NAFLD^{41, 69} (Figure 1). Like in NAFLD, blood samples from an experimental mouse model of non-alcoholic steatohepatitis (NASH) and liver biopsies from NASH patients show significant accumulation of C16:0 ceramide⁷⁰. In addition to hepatic dihydroceramides, lactosylceramide levels were also found to be increased in insulin-resistant NASH patients⁶⁴. Oxidative stress is one of the pathological hallmarks of NASH⁷¹, and increased levels of ceramides generate oxidative stress and inflammation in metabolic syndrome, which initiates fibrosis in NASH, leading to NAFLD⁷². In particular, C16:0 ceramide in the mitochondrial outer membrane is especially known to activate mitophagy and oxidative stress⁷³.

Similar to the liver, the pancreas is another region where hyperglycemia and insulin resistance influence cytotoxic lipid deposition⁷⁴. Pancreatitis susceptibility is linked to diabetes through metabolic changes which induce inflammation and generation of reactive oxygen species^{75, 76}. Plasma sphingolipid metabolism is affected during acute pancreatitis, where significant increases in the ceramide levels and decreases in S1P have been reported in severe acute pancreatitis patients during early stage of the disease⁷⁷. As discussed in the previous section, increased accumulation of ceramides influence secretion of insulin from pancreatic β-cells. The genetic deletion of sphingomyelin synthase 1 (SMS1) leads to accumulation of ceramide in islets of Langerhans, which affects mitochondrial function and subsequently inhibits insulin secretion⁷⁸. Similar to inhibition of SMS1, siRNA knockdown of ceramide transporter protein (CERT) leads to accumulation of intracellular ceramide and pancreatic β-cell dysfunction during exposure with palmitic acid⁷⁹. Palmitic acid induces de novo ceramide synthesis and this pathway appears to be involved in ceramide-induced pancreatic β-cell death and dysfunction (Figure 1). This information supports association of ceramide not only with insulin resistance and hyperglycemia, but also with liver and pancreatic diseases.

Ceramides in Diabetic Complications

The long-term complications of DM are classified into microvascular/neuropathic and macrovascular subgroups. The recognized microvascular and neuropathic complications of diabetes mellitus are diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy. Diabetic retinopathy is the leading cause of blindness; diabetic nephropathy may progress to end-stage kidney disease (ESKD); and neuropathy is a major risk factor for lower extremity amputation. The development of microvascular complications of diabetes is a function of both the duration of the disease and the state of metabolic control. The pathogenesis of microvascular complications, though incompletely understood, involves mechanisms that include genetic predisposition; hyperglycemia-induced activation of the polyol, hexosamine, and PKC pathways; generation of advanced glycosylated end products; glomerular hyperfiltration; growth factor expression; oxidative stress; free radical damage; and abnormal endothelial, pericyte, and mesangial structure and/or function^{2, 3}. Diabetic nephropathy and severe retinopathy also tend to cluster in families⁸⁰.

The macrovascular complications of diabetes include cardiovascular disease (CVD), stroke, and PVD. Coronary artery disease, MI, and congestive heart failure are major components of CVD. Unlike microvascular complications, hyperglycemia is not the sole or major driver in the etiology of macrovascular complications. The risk factors for macrovascular disease include insulin resistance, hypertension, dyslipidemia, hyperglycemia, increased coagulation, impaired fibrinolysis, inflammation, oxidative stress, and lifestyle factors (cigarette smoking, obesity, physical deconditioning). These CVD risks frequently co-exist in people with DM. CVD is the leading cause of death in patients with diabetes and is 2-4 times more prevalent among people with diabetes compared to nondiabetic patients^{5, 8}. Besides the traditional hyperglycemic and multifactorial landscape of diabetic complications, emerging evidence has implicated the involvement of ceramides in various mechanisms leading to diabetic complications in target tissues (heart, blood vessels, eyes, kidney, and peripheral nerves).

Ceramide in Diabetes-Induced Vascular Diseases

Cardiovascular disease can be a devastating complication of diabetes and obesity and continues to be a leading cause of death in developed countries⁸¹. Sphingolipids have been implicated in cardiovascular disease pathogenesis⁸², and even though it is unclear which lipid metabolites might directly cause cardiomyopathy, ceramide accumulation in multiple models correlates with these pathophysiological events and cardiac dysfunction^83–85. Conventionally, cardiac dysfunction in T2D relates to generation of toxic lipid species by oxidation from excessive fatty acid uptake⁸⁶. Studies of fenofibrate-induced lipid-lowering effects on cardiac function revealed that fenofibrate lowers plasma TG, cholesterol, and the ratio of ceramide C24:0/C16:0, and also minimally alters oxidative stress markers⁸⁷, indicating that certain ceramide species may have independent or coordinated roles in cardiac dysfunction in diabetic patients. In recent years, several studies have found associations between circulating ceramide levels and adverse cardiovascular events, such as MI and stroke^{29, 88}. These studies consistently showed that several long-chain ceramide species were associated with an increased risk of cardiac death and deleterious outcomes in patients with cardiovascular complications⁸⁹. Studies have reported that elevated ceramide levels are associated with major adverse cardiovascular events⁹⁰. In the same vein, several studies have also reported multiple ceramide species and their ratios as strong indicators of those events. C18:1 ceramide has been reported to be a key indicator of necrosis after coronary angiography procedures, as are C24:1 ceramide and sphingomyelin for cardiovascular mortality, while C22:0 and C24:0 ceramides strongly associate with reduced improvement in verbal memory in patients with coronary artery disease in response to exercise⁹¹.

One of the key features of heart failure is apoptotic cell loss⁹², and saturated free fatty acids (FFAs) have been shown to induce cardiomyocyte apoptosis and damage myofibrils^93–97. Interestingly, inhibition of CerS was found to reverse this effect⁹⁴. It has also been proposed that generation of ROS by ceramide could play a role in cardiomyocyte apoptosis^97–99. In addition, mitochondrial fission stimulated by ceramide is also associated with early activation of cardiomyocyte apoptosis, further cementing the role of ceramide in this complication¹⁰⁰. Ceramide has also been implicated in the process of atherosclerosis; degradation of sphingomyelin to ceramide by aSMase on low density lipoprotein (LDL) surfaces promotes their aggregation by ceramide-ceramide interactions, promoting atherosclerosis initiation^{101, 102}. In addition, inhibition of DES1, the ultimate enzyme in the ceramide biosynthesis pathway that converts dihydroceramide to ceramide, has been shown to reverse pathogenesis of atherosclerosis and cardiomyopathy in rodent models¹⁰³. Plasma ceramide levels are reported to be more precise than currently used lipid prediction markers, thus highlighting their potential use as biomarkers to identify patients in need of more aggressive treatment. Indeed, a plasma ceramide test kit for evaluation of cardiac risk was commercially released in 2016 by Mayo Medical Laboratories¹⁰⁴. With ceramide being a strong biomarker for cardiac conditions like atherosclerosis, it may be interesting to further investigate it in fenofibrate-treated patients¹⁰⁵.

In general, interventions such as inhibiting any of the de novo ceramide synthesis enzymes (namely SPT, CerS6 and DES1) in mouse models reportedly reduce cardiovascular complications such as atherosclerosis and lipotoxic cardiomyopathy¹⁷. Studies have shown that Adiponectin increases cellular ceramidase activity, thereby reducing ceramide levels, and exhibits cardioprotective properties¹⁴. Though the heart needs essential lipids, the heart is likely to have lipid accumulation resulting in severe complications in diabetes. Even though several studies using animal models have implicated ceramides in the pathogenesis of cardiovascular diseases, further research is necessary to delineate the exact nature of its toxicity based on structural variability.

Ceramide in Diabetic Nephropathy

Diabetes is the primary cause of chronic kidney disease (CKD) and ESKD worldwide¹⁰⁶. Diabetic nephropathy is a highly complex disease which involves multiple pathogenic mechanisms including glomerular damage, hemodynamic dysfunction, inflammation, and fibrosis¹⁰⁷. Ceramides are highly abundant in the kidney and several studies have shown that ceramide is involved in the pathogenesis of acute kidney injury caused by ischemic reperfusion, toxic insults, and oxidative stress^{14, 108}. A recent study reported elevated levels of SPT, the rate-limiting enzyme in the de novo ceramide biosynthetic pathway, in renal tubular epithelial cells isolated from diabetic patients. The same study also reported prevention of tubular epithelial cell death by inhibition of ceramide synthesis¹⁰⁹, implying a critical role of ceramide in diabetic nephropathy. Over the years, multiple studies have linked ceramides and elevated expression of SPT to apoptosis of mesangial, renal tubular epithelial, and microvascular endothelial cells in diabetic patients¹⁴.

High levels of glycosphingolipids in the kidney and urinary tract have been shown to contribute to the pathophysiological basis of several other renal diseases, including Fabry’s disease, hemolytic uremic syndrome, and bacterial adhesion in urinary tract infections¹¹⁰. The activation of biosynthetic pathways for glycosphingolipid production plays an important role in glomerulonephritis, acute kidney injury, kidney cancer, and diabetic nephropathy¹¹¹. In addition to playing a role in renal hypertrophy in type 1 diabetic kidney disease¹¹², glycosphingolipids have recently been shown to induce fibrosis in the early stages of type 2 diabetic kidney disease¹¹³. It has been hypothesized that excessive glucose increases glycosphingolipid production by increasing the availability of substrates namely ceramide and UDP-glucose¹¹³. It has been shown that reduced plasma levels of long- and very long-chain species of ceramides have predictive value for progression of macroalbuminuria (MA) in diabetic nephropathy¹¹⁴. This same study also showed that reduced levels of lactosylceramides and hexosylceramides (C18:1-H), are significantly associated with higher risk of progression to MA and CKD, respectively. Furthermore, it has been reported that elevated levels of advanced glycation end-products (AGE), usually found in diabetic conditions, are responsible for increased levels of several gangliosides in tissues, especially GM3. GM3 was shown to compromise renal pericyte and mesangial cell regeneration via inactivation of VEGF receptors and the receptor-associated AKT signaling pathway¹¹⁵. Mitochondrial damage in podocytes by reactive oxygen species (ROS) produced by ceramide accumulation was reported in a new study using Otsuka Long Evans Tokushima Fatty (OLETF) rats and mice on a high-fat diet (HFD)¹¹⁶. Their results also show albuminuria and histologic features characteristic of diabetic nephropathy and podocyte injury, which were reversed by treatment with myriocin, an inhibitor of SPT. An increase in ceramide accumulation and ROS generation in mitochondrial podocytes was also observed when cultured podocytes were exposed to high glucose, high free fatty acid, and angiotensin II in combination (GFA). However, pretreatment with myriocin protected them from GFA-induced damage of mitochondrial integrity. It has also been reported that in glomerular endothelial cells (GECs) and podocytes, lowering cellular ceramide levels by activation of acid ceramidase reduces palmitate-induced lipotoxicity, oxidative stress, and apoptosis¹¹⁷. In addition, other studies have shown that the expression of sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b), a homologue of aSMase responsible for elevation of ceramide levels, is increased in diabetic kidney disease and targeting it could protect podocytes from damage^{118, 119}.

In a study of bi-racial healthy adults with parental history of T2DM, adiponectin was shown to be a powerful risk marker of incident prediabetes¹²⁰. Since it has been reported that adiponectin stimulates ceramidase activity via its receptors¹²¹, this study underscores the importance of ceramide as a biomarker for diabetes and its complications. It has also been reported that AdipoRon, a synthetic adiponectin agonist, can decrease ceramide-induced lipotoxicity in DKD by decreasing ceramide, oxidative stress, and apoptosis in glomerular endothelial cells and podocytes in db/db mouse model¹²². The study also showed that AdipoRon was able to reduce both albuminuria and lipid accumulation in the kidneys¹²². Recently, it was shown that administration of exogenous C1P was associated with protection from albuminuria and mesangial expansion in db/db mice in vivo¹²³. Also, it was reported that the use of Fingolimod (FTY720), an FDA-approved drug for treatment of multiple sclerosis, renders nephroprotection in streptozotocin-induced diabetic nephropathy rat models¹²⁴. As FTY720 inhibits ceramide synthase and reduces ceramide levels¹²⁵, these studies highlight the potential of ceramide and ceramide-focused pharmacologic intervention in diabetic nephropathy.

The results of these experiments strongly imply that alteration of ceramide metabolic pathways is a factor in the pathophysiology of DKD. Considering the severity of renal dysfunction and failure as complications of diabetes, it is enormously important to further explore the role of ceramide in DKD pathogenesis.

Ceramide in Diabetic Retinopathy

Diabetic retinopathy (DR) is characterized by progressive retinal degeneration, neovascularization, and eventual blindness, and is the principal cause of blindness in people aged 20-65¹²⁶. Although proper glycemic control is the mainstay preventative measure for DR, it has been shown that DR may develop and progress even in normoglycemic conditions^127–129. Increased levels of sphingolipids, particularly ceramide, have been associated with various aspects of retinopathy. Analysis of sphingolipid composition of type 2 diabetic and non-diabetic post-mortem human tissue showed a relative increase in total ceramide, lactosylceramide (LacCer), and SM in diabetic vitreous samples. This study not only indicates that changes in sphingolipid composition in the vitreous as a result of T2D could be connected to pathologies of the retina, retinal vessels, vitreous, and surrounding tissues, but also underscores the importance of ceramide in complications resulting from DM¹³⁰. In the pathogenesis of DR, apoptosis of pericytes (PCs) is an early event. This is generally thought to be a consequence of sustained hyperglycemia, which increases the concentration of the saturated free fatty acid palmitate. In an in-vitro study of cultured PCs, incubation with palmitate increased the cellular content of ceramide, leading to apoptosis. Inhibition of ceramide synthase and overexpression of N-acylsphingosine amidohydrolase 1 (ASAH1), an enzyme responsible for conversion of ceramide to sphingosine, reversed the proapoptotic effect of palmitate, underscoring the role of ceramide in the early pathogenesis of DR¹³¹. Animal models of streptozotocin-induced diabetes have shown significantly increased glucosylceramide levels in the retina. This study speculated that accumulation of glycosphingolipids leads to activation of the endoplasmic reticulum stress response, leading to insulin resistance and neuronal apoptosis and thereby contributing to the pathogenesis of DR¹³². In addition, several studies have reported an increase in ceramide due to activation of the sphingomyelinase pathway (Figure 1) to be critical to the pathogenesis of DR. aSMase is shown to be highly activated in diabetic retinas, particularly in the retinal endothelium. Increased ceramide levels due to conversion of sphingomyelin (SM) to ceramide by aSMase significantly contribute to retinal inflammation¹³³. It has also been reported that the aSMase vascular isoform specifically increases in the retinas of diabetic animals during the vaso-degenerative stage¹³³. Importantly, DHA, which is decreased in diabetic retinas¹³⁴, downregulates the expression of aSMase in human retinal endothelial cells, leading to decrease in ceramide levels and protecting the retina from vaso-degeneration^135–138. In addition, downregulation of aSMase with DHA prevents capillary formation and cytokine production^{135, 137–139}. Supplementation with a DHA-rich diet has been shown to be protective against capillary loss in diabetic retinopathy in-vivo¹⁴⁰. DHA supplementation was also observed to normalize impaired b-wave amplitude and latency time in diabetic rats¹⁴⁰. Furthermore, increasing the retinal levels of n3-PUFAs, which were shown to be reduced in the retinas of human diabetic eyes¹⁴¹, reduced pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy¹⁴⁰. These studies advocate for the beneficial role of lowering ceramide levels in DR pathology and also highlights the importance of ceramide in DR. In DR, mitochondrial damage leading to cell breakdown in the blood-retinal barrier (BRB) causes pathological abnormalities¹⁴². In-vivo studies of diabetic rats have demonstrated a relationship between ceramide and mitochondrial function¹⁴³; aSMase-dependent accumulation of ceramide was observed in the mitochondria of STZ-induced diabetic rat retinas. Increases in ceramide were shown to be responsible for induction of proinflammatory cytokines like IL-1β and IL-6, and impaired mitochondrial function in-vitro. Increase in very long-chain (VLC) ceramides have been shown to stabilize tight junctions and prevent blood-retinal barrier dysregulation in in-vitro models. Elongation of very long-chain fatty acids protein 4 (ELOVL4) is responsible for production of VLC fatty acids that are incorporated in VLC ceramides. ELOVL4 is reduced to a great extent in STZ-induced diabetic rat retinas¹³⁴, and overexpression of retinal ELOVL4 decreased basal, IL-1β-induced, and vascular endothelial growth factor (VEGF)-induced permeability in a Bovine retinal endothelial cell (BREC) model¹⁴⁴.

Thus, increase in ceramide levels as a consequence of altered sphingolipid metabolism appear to be relevant to the pathogenesis of diabetic retinopathy. Given ceramide’s central role in sphingolipid metabolism, this also underscores the importance of modulating sphingolipid metabolism in general as an important aspect of treating this complication.

Ceramide in Diabetic Neuropathy

While hyperglycemia is a well-established contributor to nerve injury, multiple other factors play important roles in diabetic neuropathy (DN)¹⁴⁵. Recent developments suggest modulation of sphingolipid metabolism significantly contributes to the development and progression of DN¹⁴⁵ and multiple studies provide evidence that dysregulated lipid metabolism, both at cellular and macro levels, aggravates DN in diabetic animal models¹⁴⁶. Abnormalities of axonal protein transport are thought to have an important role in the pathogenesis of DN, and earlier reports showing a positive effect of exogenous gangliosides on the axonal transport of structural proteins in the sensory fibers of streptozotocin-induced diabetic rats highlight the possible role of sphingolipids in DN¹⁴⁷. Also, in-vitro studies of Schwann cell apoptosis by palmitate was reported to be significantly suppressed by ceramide synthase inhibitors, further supporting a role of ceramide¹⁴⁸. Atypical deoxysphingolipids, which are toxic to neurons and pancreatic β-cells^149,150, are formed when SPT metabolizes alanine or glycine instead of serine. Deoxysphingolipid levels are reported to be elevated in hereditary sensory and autonomic neuropathy type 1 and in type 2 DM¹⁷. It has also been reported that plasma levels of deoxysphingolipids are elevated in individuals with T1D, though not as much as in type 2 diabetes^151–153. One study showed that 1-deoxydihydroceramide is a particularly important mediator for cytotoxicity towards insulin-producing pancreatic β-cells, and highlights the importance of targeting deoxysphingolipid biosynthesis with fenofibrate as a promising therapeutic approach for diabetic neuropathy ¹⁷. The fact that disproportionate increases in certain deoxysphingolipids are seen in diabetic individuals with neuropathy reinforces their importance in this complication. A recent pilot study of type I diabetic patients further suggests an important role of ceramide in neuropathy. The study was conducted in the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) type 1 diabetes sub-cohort with the goal to assess the associations between multiple sphingolipid species, deoxysphingolipids, and free amino acids, and the presence of symptomatic neuropathy. Using mass spectroscopy, plasma levels of DSL and free amino acids were measured in participants with and without symptoms of neuropathy. The study showed significant associations between increased levels of ceramides, plasma long-chain deoxy-ceramides, and patients reporting neuropathy. This study also highlights the potential of plasma ceramide and deoxy-ceramide species as diagnostic and prognostic biomarkers in diabetic neuropathy¹⁵². Altered sphingolipid metabolism is a developing area of study in the context of neuropathy and further research might lead to new therapies targeting sphingolipid metabolism.

Ceramides, Adiponectin, and FGF Interactions: Clinical Implications

Adiponectin, the most abundant product secreted from adipocytes, lowers hepatic and muscle triglycerides¹⁵⁴ and is associated with improved insulin sensitivity, decreased cardiometabolic risk, and decreased risk of progression from prediabetes to J2D^155–159. Indeed, a longitudinal cohort study showed that adiponectin levels could act as a biomarker for diabetes risk¹⁵⁹. Furthermore, higher levels of adiponectin are associated with decreased risk of progression from normoglycemia to prediabetes in African American and European American offspring of parents with T2D¹⁶⁰. Besides improvements in insulin sensitivity, other mechanisms of adiponectin’s favorable cardiometabolic profile include anti-inflammatory effects, decreased apoptosis and preservation of β-cell function, and reversal of lipotoxicity^161–165. These numerous pro-cardiometabolic mechanisms appear to be unified by interactions among adiponectin, fibroblast growth factor 21 (FGF-21; a potent regulator of metabolism and energy utilization), and ceramides. Studies in rodent models show significant effects of FGF-21 in regulating blood glucose levels and increasing insulin sensitivity in mice, mediated by upregulation of adiponectin expression^{154, 164}. FGF-21 accelerates the clearance of toxic ceramides in tissues. The observation that adiponectin-knockout mice are refractory to the ceramide-clearing effects of FGF-21 directly implicates adiponectin in the mechanism of ceramide clearance¹⁶⁴. These interactions have some clinical relevance: the well-known insulin-sensitizing effect of PPAR-γ agonists used for diabetes treatment has been shown to be mediated, at least in part, by increased adiponectin expression¹⁶⁶. Several other interventions that improve cardiometabolic risk (including exercise, weight loss, smoking cessation and treatment with PPAR-γ agonists, ACE inhibitors, and angiotensin II receptor blockers) are known to also increase adiponectin levels^{165,167–171}, and, through that mechanism, could potentially decrease ceramide burden in metabolically active tissues. Adiponectin is believed to modulate ceramide levels via the AdipoRI and AdipoR2 receptors, which help regulate ceramidase activity in tissues¹⁷².

Conclusions and Future Directions: Ceramides as biomarkers and therapeutic targets

The advances in sphingolipid and diabetes research over the past few years provide additional support to establish ceramides as key actors in the pathophysiology of diabetes and associated complications. By further developing our understanding of the numerous roles of sphingolipids in mediating the processes underlying diabetes, foundations are laid for future breakthroughs and development of therapeutic treatments and disease biomarkers. Important advances in the field of sphingolipid dysregulation are by no means limited to ceramide or sphingolipids involved in diabetic complications. Indeed, important recent advances also include other sphingolipid classes involved in many other diseases but are beyond the scope of this review. Nevertheless, it should be emphasized that the ubiquity and widespread bioactivity of certain sphingolipids virtually ensures their involvement in the pathophysiology of a wide range of human diseases.

Targeted research into applications of sphingolipid screening are already beginning to yield useful insights which may be clinically translatable. For instance, a recent project analyzed ceramide species in participants of the Framingham Heart Study and the Study of Health in Pomerania, finding evidence that the ratio of C24:0/C16:0 ceramides isolated from peripheral blood may have utility as a biomarker for congestive heart failure¹⁷³. Another study very recently published a significant association between incident T2D and elevated levels of saturated SM species in a cohort selected from participants in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL)¹⁷⁴. Some experimental models have shown elevations in free fatty acids and triglycerides before anomalie s in plasma glucose become detectable^{17, 175}, and the ratio of ceramide C18:0/016:0 has been shown to have predictive value for diabetes and prediabetes up to 10 years prior to onset¹⁷⁶. The detection of sphingolipid perturbations in other diabetic tissues, such as the vitreous¹⁷⁷, further supports their involvement in diabetic complications and suggests that unique biomarkers could be utilized for early detection or prognosis of specific diabetic complications. As the onset of these complications is generally quite insidious, effective biomarkers for screening could have a substantial impact on diabetes complication incidence and/or severity by widening the window for instituting early interventions.

Given what is known about the bioactive potential of ceramides, the changes in sphingolipid metabolism and regulation in diabetes as suggested by these studies may in fact contribute to diabetes pathology and could therefore eventually help identify pharmacologic targets. The validity of targeting sphingolipid metabolism in disease states is supported in various disease models, including retinal degeneration¹⁷⁸, cancer¹⁷⁹, atherosclerosis¹⁸⁰, and inflammation¹⁸¹, to name a few. Identifying exploitable sphingolipid targets in diabetic complications could prove valuable in developing new therapeutic drugs. Currently, much of the data on the roles of ceramides in diabetes pathophysiology and complications have been derived from animal models and limited cross-sectional human studies. Longitudinal investigation of ceramide expression among people with prediabetes who progress to type 2 diabetes would provide valuable insights into the chronology and causal interactions between ceramides and incident diabetes. Similarly, exploring the role of ceramide in the initial transition from normoglycemia to prediabetes would extend such insight to an even more proximal stage of early dysglycemia. Longitudinal studies among people with diabetes that analyze repeated measurements of ceramide burden in relation to the development of microvascular and macrovascular complications over extended follow-up periods would be highly informative. Novel findings regarding ceramide involvement in diabetes are likely to yield additional clinically useful revelations as the relevant mechanisms of sphingolipid bioactivity are further unraveled. Building upon these findings will be critical to the long-term goals of eventually developing preventative and curative therapies for diabetic complications and other human diseases.

Highlights:

Our paper highlights classical concepts and recent developments regarding the role of ceramides, a class of bioactive sphingolipids with cell signaling and second messenger capabilities, in mechanisms involving insulin action, glucoregulatory, pathogenesis of diabetes and its complications. The emerging understanding of cross-talk among ceramides, adiponectin and FGF21 in mediating cardiometabolic risk and responses to interventions (thiazolidinediones, angiotensin inhibitors, etc.) is discussed and amplified in the sub-section of our review devoted to clinical translation.

Abbreviations

DM

Diabetes mellitus

MI

Myocardial infarction

PVD

Peripheral vascular disease

T2D

Type 2 diabetes

T1D

Type 1 diabetes

SM

Sphingomyelin

CerS

Ceramide synthase

SPT

Serine palmitoyl transferase

DES

Dihydroceramide desaturase

SMS1

Sphingomyelin synthase 1

PKCζ

Protein kinase C zeta

AKT/PKB

Protein kinase B

PP2A

Protein phosphatase 2A

Smase

Sphingomyelinase

TNFα

Tumor necrosis factor alpha

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

IL-1β

Interleukin-1 Beta

JNK-1

c-Jun N-terminal kinase-1

TLR

Toll-like receptor

IκKβ

Inhibitor of kappa B kinase beta

NFκB

Nuclear factor kappa B

AMPK

AMP-activated protein kinase

ZDF

Zukar diabetic fatty rat

FAT

Fatty acid transporter

CD36

Cluster determinant 36

PP1

Protein phosphatase 1

ERK1/2

Extracellular-signal-regulated kinase 1/2

PDX-1

Pancreatic and duodenal homeobox gene-1

ESKD

End-stage kidney disease

NAFLD

Non-alcoholic fatty liver disease

NASH

Non-alcoholic steatohepatitis

CVD

Cardiovascular disease

TG

Triglyceride

FFA

Free fatty acid

ROS

Reactive oxygen species

LDL

Low density lipoprotein

UDP

Uridine diphosphate

CKD

Chronic kidney disease

MA

Macroalbuminuria

AGE

Advanced glycation end-products

GM3

Monosialodihexosylganglioside

VEGF

Vascular endothelial growth factor

OLETF

Otsuka Long Evans Tokushima Fatty

HFD

High-fat diet

ROS

Reactive oxygen species

GFA

Glial fibrillary acidic

GECs

Glomerular endothelial cells

SMPDL3b

Sphingomyelin phosphodiesterase acid-like 3B

C1P

Cermaide-1-phosphate

AdipoRon

Adiponectin receptor agonist

FTY720

Fingolimod

DKD

Diabetic Kidney Disease

LacCer

Lactosylceramide

PCs

Pericytes

ASAH1

N-acylsphingosine amidohydrolase 1

DHA

Docosahexaenoic acid

n3-PUFAs

OMEGA-3-Polyunsaturated Fatty-Acids

DR

Diabetic Retinopathy

BRB

Blood-retinal barrier

aSMase

Acid SphingoMyelinase

VLC

Very Long Chain

STZ

Streptozotocin

ELOVL4

Elongation of very long-chain fatty acids protein 4

BREC

Bovine Retinal Endothelial Cell

DN

Diabetic Neuropathy

EDIC

Epidemiology of Diabetes Interventions and Complications

DCCT

Diabetes Control and Complications Trial

DSL

Deoxy-sphingolipid

FGF-21

Fibroblast growth factor 21

ACE

Angiotensin converting enzyme

PPAR-γ

Peroxisome proliferator-activated receptor gamma

AdipoR

Adiponectin receptor