Ceramide accumulation within the hypothalamus contributes to impaired peripheral metabolic health and obesity

The metabolically detrimental effects of ceramide accumulation are not limited to peripheral tissues, as described so far. Indeed, ceramide buildup has also been reported in the hypothalamus of rodents fed a high-fat diet leading to a deterioration of metabolic health^{(Reference Borg, Omran and Weir72)}. Hypothalamic lipotoxicity, and particularly ceramide accumulation, is not without metabolic consequences. In support of this, direct hypothalamic infusion of C6 ceramide, a cell permeable ceramide, induced hypothalamic ER stress and inflammation^{(Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51)}, two pivotal mechanisms responsible for promoting insulin and leptin resistance, posing the pathogenetic basis for obesity and impaired glycaemic control^{(Reference Posey, Clegg and Printz73–Reference Ozcan, Ergin and Lu76)}. As such, animals receiving intracerebroventricular C6 ceramide administration displayed a decrease in insulin sensitivity and developed obesity secondary to decreased energy expenditure and brown adipose tissue thermogenesis^{(Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51)}. Remarkably, these effects were countered by inhibition of hypothalamic ER stress which, not only normalised body weight, but also reversed central ceramide-induced peripheral insulin resistance^{(Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51)}, supporting the notion that ER stress represents an effector of lipotoxicity. These effects were recapitulated in obese Zucker rats which are generally characterised by hypothalamic ER stress and ceramide accumulation. In these animals, inhibition of ER stress was sufficient to improve their metabolic phenotype marked by an improvement in insulin as well as leptin resistance, reduced body weight gain and hepatic steatosis^{(Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51)}. In consideration of this, besides their role in insulin resistance, ceramides may also promote obesity. This was confirmed by the protective effect of ceramide synthase 6 knock out against high-fat diet-induced obesity^{(Reference Turpin, Nicholls and Willmes61)}. Always in support of the metabolically detrimental role of ceramide accumulation in the hypothalamus, inhibition of its synthesis within the hypothalamus resulted in an improvement in insulin sensitivity, an effect that was also confirmed on cultured hypothalamic neurons^{(Reference Campana, Bellini and Rouch77)}. Additionally, the inhibition of ceramide synthesis within the hypothalamus improved glycaemic control by enhancing glucose-stimulated insulin secretion and pancreatic β-cell mass^{(Reference Campana, Bellini and Rouch77)}. Thus, ceramides appear to impair metabolic health and particularly insulin sensitivity by accumulating both in peripheral tissues and in the hypothalamus, with these effects being presumably dependent on the activation of protein kinase Cζ, protein phosphatase 2A, metabolic inflammation and ER stress^{(Reference Chavez and Summers36,Reference Chaurasia, Talbot and Summers40,Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51,Reference Han and Kaufman78)} . Particularly, ER stress may be at the interface between lipotoxicity and metabolic dysfunction. However, ER stress may mediate the deleterious effect of ceramide in concert with inflammation given the cross-talk between these pathophysiological responses^{(Reference Ajoolabady, Lebeaupin and Wu79)} and the ability of ceramides to trigger both^{(Reference Sergi, Morris and Kahn49,Reference Contreras, Gonzalez-Garcia and Martinez-Sanchez51)} . Nevertheless, ER stress may not be responsible for the detrimental effects of ceramide on insulin resistance in in vitro skeletal muscle models. This possibility is supported by the fact that the inhibition of ER stress is not sufficient to rescue palmitic acid-induced impairment in insulin signalling in C2C12 and human myotubes^{(Reference Rieusset, Chauvin and Durand80,Reference Hage Hassan, Hainault and Vilquin81)} .

As described thus far, ceramides represent a particularly strong candidate in linking obesity, dysfunctional lipid metabolism and insulin resistance. In addition, the inhibition of ceramide synthesis has been reported to exert beneficial effects on a full spectrum of obesity and insulin resistance-associated cardiometabolic aberrations, including metabolic dysfunction-associated steatotic liver disease, cardiomyopathy, atherosclerosis and hypertension^{(Reference Chaurasia, Tippetts and Mayoral Monibas39,Reference Holland, Brozinick and Wang57,Reference Glaros, Kim and Quinn82–Reference Kasumov, Li and Li84)} . In light of this, modulating lipid metabolism to counter ceramide synthesis represents a promising therapeutic strategy to improve cardiometabolic health.

Ceramides as a biomarker of insulin resistance and progression to type 2 diabetes mellitus

The deleterious effects of ceramides on skeletal muscle insulin signalling stem beyond their intracellular synthesis and may be dictated by plasma ceramides which are primarily derived from the liver in both rodents and humans^{(Reference Lightle, Tosheva and Lee85)} (Figure 2). These hepatic-synthetised ceramides travel in the blood stream packed in LDL and VLDL^{(Reference Wiesner, Leidl and Boettcher86)} (Figure 2) and have been reported to target the skeletal muscle to elicit insulin resistance^{(Reference Boon, Hoy and Stark50)}. Indeed, infusion of LDL containing C24:0 ceramide in lean mice induced an impairment in insulin signalling via AKT, thereby hampering insulin-mediated glucose uptake in skeletal muscle^{(Reference Boon, Hoy and Stark50)}. The ability of LDL-derived ceramides to elicit insulin resistance may also hold true in humans as the transport of ceramide in LDL is increased in obese and type 2 diabetic individuals and correlated with insulin resistance^{(Reference Boon, Hoy and Stark50)}. This notion is also supported by the fact that insulin resistance, assessed by homoeostasis model assessment: insulin resistance, is positively associated with saturated ceramides^{(Reference Neeland, Singh and McGuire87)}.Thus, an intracellular mismatch between fatty acid supply and oxidation may not be the only driver of ceramide accumulation, but these lipotoxic lipid species may also act in an endocrine fashion to target extra hepatic tissues as demonstrated for skeletal muscle^{(Reference Boon, Hoy and Stark50)}. In consideration of the ability of circulating ceramides to trigger insulin resistance and the positive correlation between circulating ceramides and the onset of insulin resistance^{(Reference Boon, Hoy and Stark50,Reference Haus, Kashyap and Kasumov88)} , these sphingolipids are emerging as a predictive marker of insulin resistance and progression to T2DM. Additionally, total dihydroceramides were reported to be elevated up to 9 years prior to the diagnosis T2DM^{(Reference Wigger, Cruciani-Guglielmacci and Nicolas89)} as well as in obese and type 2 diabetic individuals^{(Reference Zarini, Brozinick and Zemski Berry90)} and are associated with the severity of metabolic dysfunction-associated steatotic liver disease^{(Reference Carlier, Phan and Szpigel91)}, further supporting ceramide and the metabolites in its biosynthetic pathways as potential novel prognostic biomarkers of T2DM. Furthermore, not only serum dihydroceramides correlate with insulin resistance, but they are also able to impair insulin signalling in primary myotubes^{(Reference Zarini, Brozinick and Zemski Berry90)}. However, the ability of dihydroceramides to directly promote insulin resistance remains controversial^{(Reference Chaurasia, Tippetts and Mayoral Monibas39)}. This further confirms the implication of ceramide metabolism, particularly its anabolism, as a pivotal driver of insulin resistance and impaired glycaemic control, suggesting that defects in ceramide homeostasis may prevent the onset of overt T2DM.

The effect of ceramides on lipid metabolism

Ceramide accumulation intracellularly is able to modulate lipid metabolism^{(Reference Summers, Chaurasia and Holland92,Reference Summers93)} . In support of this, ceramides can rewire lipid metabolism to increase fatty acid uptake and promote their storage in the form of triglycerides. In line with this, lowering ceramide levels in the liver or adipose tissue by overexpressing ceramidases in animal models resulted in a down-regulation in CD36^{(Reference Xia, Holland and Kusminski94)}, suggesting ceramide being able to increase fatty acid uptake. Similarly, knocking out dihydroceramide desaturase 1, thereby lowering ceramide content in the liver, was sufficient to dampen hepatic lipid intake^{(Reference Chaurasia, Tippetts and Mayoral Monibas39)}. This effect appears to be dependent upon ceramide-induced protein kinase Cζ activation as the overexpression of a dominant-negative form of this enzyme was sufficient to abrogate the effects of ceramides on fatty acid uptake^{(Reference Xia, Holland and Kusminski94)}. Apart from increasing CD36-mediated fatty acid uptake, ceramides also increase fatty acid esterification via the up-regulation of sterol response element binding proteins and the consequent indication of genes involved in triacylglycerols synthesis^{(Reference Jiang, Xie and Li95)}. Additionally, ceramides decrease the uptake of glucose^{(Reference Wang, O’Brien and Brindley96)} and amino acids^{(Reference Guenther, Peralta and Rosales97,Reference Hyde, Hajduch and Powell98)} , possibly to favour the catabolism of fatty acid as the main energy substrate, even though it is also true that C16:0 ceramide impairs mitochondrial function^{(Reference Raichur, Wang and Chan70)} and may therefore negatively impact fatty acid catabolism. In line with this, ceramides, and particularly C16:0 ceramide accumulation, have been shown to impair mitochondrial respiration and the activity of electron transport chains II^{(Reference Raichur, Wang and Chan70)} and IV^{(Reference Zigdon, Kogot-Levin and Park99)} in vitro, leading to a decrease in β-oxidation as reported in cultured hepatocytes^{(Reference Raichur, Wang and Chan70)}. Always in agreement with their ability to affect lipid metabolism, ceramides inhibit isoproterenol-induced phosphorylation of adipocyte hormone sensitive lipase^{(Reference Chaurasia, Tippetts and Mayoral Monibas39)} with this effect being a potential mechanism by which these sphingolipids may counter further oversupply of NEFA to metabolically active tissues. Nevertheless, despite this appearing a protective mechanism to prevent fatty acid oversupply to liver and skeletal muscle for example, ceramide accumulation within adipocytes may contribute to increase adipocyte size and impair mitochondrial respiration^{(Reference Chaurasia, Kaddai and Lancaster100)}. In agreement with this, genetic approaches aimed at lowering adipocyte ceramide levels led to a decrease in adipocyte size, increased mitochondrial function and enhanced insulin sensitivity^{(Reference Chaurasia, Tippetts and Mayoral Monibas39,Reference Chaurasia, Kaddai and Lancaster100)} . Thus, on one hand ceramides increase fatty acid uptake and intracellular storage and influence adipocyte lipid metabolism possibly to buffer fatty acid excess and the potential perturbation of cell membranes by the detergent-like effects of NEFA; however, on the other hand these mechanisms contribute to increasing intracellular lipid storage and adipocyte hypometabolism, thereby paving the way for the development of insulin resistance^{(Reference Summers93)}.

Long- versus medium-chain saturated fatty acids as the dietary drivers of ceramide synthesis

As already discussed, fatty acid overload is the main driver of ceramide synthesis. However, not all fatty acids promote ceramide synthesis, a paradigm which is in line with the fact that not all fatty acids are metabolically detrimental^{(Reference Luukkonen, Sadevirta and Zhou101,Reference Wanders, Blom and Zock102)} . Indeed, long-chain saturated fatty acids, as opposed to mono- and poly-unsaturated fatty acids, increase circulating ceramide levels^{(Reference Luukkonen, Sadevirta and Zhou101,Reference Rosqvist, Kullberg and Stahlman103,Reference Tuccinardi, Di Mauro and Lattanzi104)} in human subject feeding trials, an effect that was accompanied by insulin resistance^{(Reference Luukkonen, Sadevirta and Zhou101,Reference Tuccinardi, Di Mauro and Lattanzi104)} (Figure 3a). The accumulation of ceramides was also demonstrated in the skeletal muscle and liver of rats upon the infusion of lard oil, mainly composed of long-chain saturated fatty acids, and was paralleled by insulin resistance^{(Reference Holland, Brozinick and Wang57)}. The ability of long-chain saturated fatty acids to promote ceramide buildup may be dependent on an increase in serine palmitoyltransferase and a concomitant decrease in ceramidase activity. The induction of serine palmitoyltransferase activity by long-chain saturated fatty acids is not surprising given that palmitoyl-CoA, the CoA derivate of palmitic acid, along with serine, is a substrate for this enzyme^{(Reference Blachnio-Zabielska, Baranowski and Zabielski105)}. Long-chain saturated fatty acids are also able to trigger inflammatory response in a variety of tissues^{(Reference Sergi, Luscombe-Marsh and Heilbronn106–Reference Williams, Campbell and Drew111)} and inflammation itself has been proposed as one of the triggers of ceramide synthesis, thus representing a further mechanism linking saturated fatty acids and ceramide buildup^{(Reference Chaurasia, Talbot and Summers40)} (Figure 3a). Indeed, the chronic low-grade inflammation typical of obesity not only represents a key pathophysiological mechanism underpinning the cardio-metabolic complications linked with obesity, but also promotes the synthesis of ceramide^{(Reference Chaurasia, Talbot and Summers40)}. This may be at the basis of a vicious cycle in which metabolic inflammation promotes the synthesis of ceramide and ceramide itself fuels inflammation^{(Reference Chaurasia, Talbot and Summers40)}, with both stimuli hampering insulin signal transduction pathway^{(Reference Chavez and Summers36,Reference Hotamisligil112,Reference Hotamisligil113)} . In support of the nexus between inflammation and ceramide synthesis, in cell cultures cytokines stimulate the synthesis of ceramide by promoting the expression of genes involved in de novo ceramide synthesis and sphingomyelin breakdown^{(Reference Xu, Yeh and Chen114–Reference Kolesnick, Haimovitz-Friedman and Fuks116)}. This effect has been further confirmed in vivo as demonstrated by the ability of TNFα to increase ceramide levels in epithelial cells via the activation of neutral sphingomyelinase^{(Reference Mallampalli, Peterson and Carter117)}. Always in line with the ability of inflammation to modulate ceramide metabolism, toll-like receptor 4 activation resulted in increased ceramide synthesis^{(Reference Schilling, Machkovech and He118)} in primary macrophages, while its pharmacological inhibition led to a decrease in skeletal muscle ceramide levels^{(Reference McKenzie, Reidy and Nelson119)}. Particularly, the toll-like receptor 4 appears to mediate the synergistic effects of its cognate ligand, lipopolysaccharide and palmitic acid in mediating ceramide buildup⁽⁹⁴⁾. However, the mechanisms underpinning this synergy between lipopolysaccharide and palmitic acid remain to be fully elucidated, also in consideration of the fact that the ability of palmitic acid to act as a toll-like receptor 4 agonist remains controversial^{(Reference Lancaster, Langley and Berglund120–Reference Erridge and Samani122)}.

Fig. 3. Modulation of ceramide synthesis by long- and medium-chain saturated fatty acids. Long- and medium-chain saturated fatty acids modulated intracellular ceramide accumulation differently. (a) Long-chain saturated fatty acids, and particularly palmitic acid (represented here), promote ceramide synthesis by providing the building blocks for the synthesis of these sphingolipids. Furthermore, Long-chain saturated fatty acids activate pro-inflammatory responses, including the induction of the NFκB signalling, which in turn, has been reported to foster ceramide synthesis. Long-chain saturated fatty acids have also been shown to induce mitochondrial dysfunction, with a consequent impairment of β-oxidation thereby resulting in an increase in intracellular fatty acid available to be funnelled towards ceramide synthesis. (b) Contrarily to Long-chain saturated fatty acids, medium-chain saturated fatty acids such as lauric, capric and caprylic acid (represented here), may prevent intracellular ceramide accumulation by improving mitochondrial oxidative metabolism which, in concert with a decrease in the ATP/AMP ratio, contribute to sustaining β-oxidation thereby decreasing the availability of fatty acids to be directed towards ceramide synthesis. Additionally, as opposed to long-chain saturated fatty acids, medium-chain saturated fatty acids are unable to induce inflammatory responses which, in turn, contribute to preventing intracellular ceramide accumulation. This figure was created using smart.servier.com

As already anticipated, ceramides also impact metabolic health by accumulating in the hypothalamus. In light of this, it is not surprising that diet may also impact hypothalamic ceramide accumulation. Indeed, a high-fat diet rich in long-chain saturated fatty acids promoted ceramide accumulation within the hypothalamus^{(Reference Borg, Omran and Weir72)} with this effect appearing to be sex specific and more marked in male rodents^{(Reference Morselli, Fuente-Martin and Finan123)}. These in vivo data are also supported by in vitro studies in hypothalamic neuronal cell cultures confirming the ability of long-chain saturated fatty acids to promote ceramide accumulation not only in peripheral tissues, but also within the hypothalamus. Saturated fatty acids, and particularly palmitic acid, promoted C16:0 ceramide accumulation within neuronal hypothalamic cell lines with this effect being accompanied by inflammatory responses^{(Reference Sergi, Morris and Kahn121,Reference McFadden, Aja and Li124)} and impaired insulin signalling^{(Reference Campana, Bellini and Rouch77)}. Remarkably, increasing fatty acid oxidation in hypothalamic neurons not only lowered ceramide levels with cultured hypothalamic neurons, but also mitigated inflammation^{(Reference McFadden, Aja and Li124)}. To a similar extend, specifically inhibiting ceramide synthesis using pharmacological or molecular tools was sufficient to rescue hypothalamic insulin signalling^{(Reference Campana, Bellini and Rouch77)}, confirming the role of ceramides as pivotal mediators of the metabolically detrimental role of long-chain saturated fatty acids also with the hypothalamus.

Nevertheless, the effects of saturated fatty acids on ceramide accumulation appear to be chain-length specific as long- but not medium-chain saturated fatty acids are able to increase ceramide synthesis. Indeed, animals fed a high-fat diet, when switched to a high-fat diet supplemented with medium-chain triacylglycerols (C8 and C10), not only experienced an improvement in metabolic health independently of changes in adiposity but also displayed a decrease in hepatic ceramide content^{(Reference Mourad, Abdualkader and Li125)} (Figure 3b). This effect may be dependent upon the ability of medium-chain triglycerides to down-regulate the expression of key enzymes involved in ceramide biosynthesis and sphingomyelin hydrolysis, namely ceramide synthase 6 and sphingomyelin phosphodiesterase 3, respectively^{(Reference Mourad, Abdualkader and Li125)}. Another potential mechanism underpinning these effects may be dependent on the ability of medium-chain saturated fatty acids to modulate mitochondrial oxidative metabolism and the fact that, compared with long-chain saturated fatty acids, they are more effectively β-oxidised^{(Reference DeLany, Windhauser and Champagne126)}. With regard to their impact on mitochondrial oxidative metabolism, lauric acid, a medium-chain saturated fatty acids did not impair mitochondrial membrane potential or promote mitochondrial fission in the primary myotubes of human subjects as opposed to the long-chain saturated fatty acid palmitic acid^{(Reference Sergi, Luscombe-Marsh and Naumovski107)}. Furthermore, in rodents, a high-fat diet enriched in medium, compared with long-chain fatty acids, induced a greater increase in skeletal muscle markers of mitochondrial metabolism with this effect being associated with an improvement in insulin sensitivity in skeletal muscle and adipose tissue^{(Reference Turner, Hariharan and TidAng127)}. Thus, it is tempting to hypothesise that the preserved mitochondrial function in response to medium-chain saturated fatty acids would favour mitochondrial oxidative metabolism, thereby preventing intracellular ceramide buildup. However, this effect was tissue-specific as medium-chain fatty acid supplementation led to hepatic steatosis and liver-selective insulin resistance in rodents while preserving skeletal muscle and adipose tissue insulin sensitivity^{(Reference Turner, Hariharan and TidAng127)}. These effects may also be related to the divergent effect of palmitic and lauric acid on metabolic inflammation, with the former, but not the latter, being able to trigger the activation of the pro-inflammatory NFκB signalling in primary myotubes of human subjects^{(Reference Sergi, Luscombe-Marsh and Naumovski107)} (Figure 3), which, in turn, has been reported to play a key role in disrupting mitochondrial function in myotubes^{(Reference Nisr, Shah and Ganley128)}. Always in line with the effect of medium-chain fatty acid on inflammation, they have been shown to inhibit the pro-inflammatory effect of lipotoxicity by activating the G-protein-coupled receptor 84, as demonstrated on hepatic macrophages^{(Reference Ohue-Kitano, Nonaka and Nishida129)}. Finally, medium chain fatty acids are also able to act as signalling molecules given by their ability to increase intracellular AMP^{(Reference Takikawa, Kumagai and Hirata130)} while lowering ATP levels^{(Reference Debeer, Mannaerts and De Schepper131)} (Figure 3b). The drop in ATP/AMP ratio, in turn, activates AMP-activated protein kinase which is pivotal in switching off anabolic pathways, including ceramide biosynthesis^{(Reference Erickson, Smith and Anthonymuthu132)}, while stimulating ATP-generating pathways such as β-oxidation^{(Reference Herms, Bosch and Reddy133)}. Thus, the activation of intracellular signalling pathways that boost oxidative metabolism while inhibiting anabolic pathways represents a further mechanisms by which medium-chain fatty acids counter ceramide accumulation.