It has been hypothesized that consumption of a diet rich in saturated animal fats leads to generation of a lipid metabolite that in turn triggers insulin resistance associated with the increasing prevalence of obesity and Type 2 diabetes (noninsulin-dependent diabetes mellitus).1, 4, 40, 41, 42
The nature of this lipid species has been extensively investigated over the past decade, and considerable recent focus has been placed on the sphingolipid ceramide.2, 18, 21, 41, 43
However, there are little data linking elevations in plasma levels of ceramides to the development of insulin resistance in humans or non-human primates. Here we have performed an extensive analysis of various plasma sphingolipids in rhesus monkeys, which we utilized because of the closer similarity in their sphingolipid profile to humans than in cynomologus monkeys, another extensively studied species.19, 20
This analysis was performed on animals placed on normal chow or on a defined HFFD ‘western' diet for various periods of time and characterized as pre-diabetic or diabetic based on measures of insulin sensitivity.
A primary finding of the present investigation is the significant increase in plasma ceramides due to HFFD feeding, and its correlation with markers of in vivo
insulin sensitivity. Total ceramide mass as well as a number of individual ceramide species were increased in both pre-diabetic and diabetics as compared with control animals. In parallel with these changes, reductions in insulin sensitivity were also noted, as fasting insulin, HOMA-IR, glucose AUC and insulin AUC were all increased in animals from these groups. Indicative of induction of metabolic syndrome by the HFFD, %BF, body mass as well as plasma triglycerides and cholesterol were all also increased in these animals. It is also noteworthy that the levels of four of the seven ceramide species measured, as well as total ceramide levels, were further increased in the diabetic animals as compared with pre-diabetics, indicating that the decline in markers of insulin sensitivity correlates with further increase in plasma ceramides. Spearman's correlation analysis () indicated that four of the species of ceramide (14:0, 16:0, 22:0 and 24:0) as well as total ceramides were significantly correlated with HOMA-IR. This relationship is also driven by both obesity (%BF) and the length of time the animals were on the diet, as removal of either variable from the analysis resulted in the loss of correlation between HOMA-IR and the shorter chain ceramide species (C14:0, C16:0 and C20:0), which are believed to have a larger role in disruption of insulin signaling.17, 41, 43, 44
These results differ from a previously published report in spontaneously diabetic cynomolgus monkeys that indicated that plasma ceramides in diabetic monkeys declined when compared with control animals.22
However, we believe that both the HFFD feeding paradigm utilized in the present study and the similarity between the sphingolipid profiles of rhesus and humans make these results more relevant to the etiology of human disease.
It is also significant that differences exist in the responses of the various ceramide species to the HFFD. Plasma levels of two of the shorter ceramide species (C14:0 and C16:0) showed some the largest changes (2.6- and 2.0-fold, respectively) compared with control animals, likely because of these animals consuming a diet enriched in animal fat and comprising largely C16:0 fatty acids (palmitic acid).24
Additionally, these two species of ceramide were significantly correlated with the largest number of in vivo
parameters (%BF, fasting insulin, fasting glucose and HOMA-IR; ). Ceramides are concentrated in microdomains within the plasma membrane (lipid rafts) that serve as platforms for complexes of signaling molecules.13, 45, 46
Interestingly, it has been shown that shorter chain ceramide species are more disruptive to membrane architecture and organization of signaling complexes within these domains.17, 41, 43, 44
Therefore, although the shorter chain ceramides are less abundant based on their total mass, the increases in these species are especially significant in that they play a larger role in disruption of insulin signaling.
Ceramides are also further metabolized to create additional biologically active sphingolipids ().28
These include ceramide-1-phosphate, SM, Sph, Sa and S-1-P, a potent agonist at EDG1 receptors that is involved in atherosclerosis.47, 48
In the present results, SM levels were not altered in HFFD animals, indicating that pathway flux through SM synthase does not appear to be a major disposal pathway for ceramide in primates. This is further supported by the elevation in plasma PC levels, which indicates a reduction in PC utilization by SM synthase. Together with the elevations in Sph (diabetics only) and S-1-P, these data indicate that the major pathway for metabolism of de novo
generated ceramides is through ceramidase. We hypothesize that this is a compensatory reaction in order to minimize the apoptotic effects of an increase in intracellular ceramide levels (, ‘Sphingosine Rheostat'); however, it will require analysis of tissues from these animals to determine if this hypothesis is correct.
It has been proposed that a ceramide metabolite rather than ceramide itself is the causative agent in lipid-induced insulin resistance. Most notably, previously published animal, human and in vitro
studies have implicated increases in GM-3 rather than ceramide in insulin resistance.31, 34, 49, 50
Here we show that although both C16:0 and C18:0 GM-3 were elevated in pre-diabetics, neither of these lipids showed a significant correlation with any insulin sensitivity parameter (). Although it is possible that GM-3 participates in HFFD-induced insulin resistance, ceramides appear to be a more robust biomarker of this condition.
A second key finding of the present paper is the increase in deoxySa, which occurs because of the condensation of alanine rather than serine with palmitoyl-CoA by SPT. This metabolite lacks the C1 hydroxyl group of Sa and cannot be further metabolized to form complex sphingolipids.37
This mis-incorporation occurs infrequently in vivo
; however, the frequency of this event is increased by mutations in the SPT1 subunit of SPT that cause hereditary sensory neuropathies (HSAN1),30, 35, 36
a disease characterized by loss of sensory neurons and chronic ulceration in the lower extremities.51
Furthermore, the etiology and symptoms of this disease are similar to those of diabetic neuropathy and human diabetics show significant increases in C18:1 deoxySa.30, 38
Here we show for the first time that increased consumption of a HFFD increases plasma C18:0 deoxySa. Although a direct causative role for these lipids in insulin resistance is unclear, we hypothesize that they are a biomarker of insulin resistance and diabetic complications, such as neuropathy. This is supported by the fact that when length of time on diet is removed from the analysis, the correlation between C18:0 deoxySa and HOMA-IR is lost (Supplementary Table 3
). In addition, this lipid species was significantly correlated with fasting insulin, insulin AUC and HOMA-IR () as well as %BF, providing further support for the value of deoxySa as a biomarker of insulin resistance.38
It was previously assumed that the increases in deoxySa resulted from an elevation in SPT activity due to increased dietary substrate provision. However, recent studies have suggested that SPT exists in a complex with other proteins that regulate activity of the enzyme.52, 53
Thus, an alternative hypothesis is that intake of a HFDD diet causes deregulation of the SPT enzyme that consequently increases the frequency of alanine incorporation. This hypothesis is further supported by the increases in dihydroceramides (Supplementary Figure 1
), also indicative of increased SPT activity and substrate flux.
The increase in plasma C18:0 deoxySa is paralleled by an increase in plasma serine levels in the diabetic animals. Serine is synthesized from the glycolytic precursor 3-phosphoglycerate, and therefore this could be attributed to increased synthesis via glycolytic metabolism, but may also reflect a reduction in serine catabolism. De novo
sphingolipid synthesis via SPT represents an intersection between lipid (palmitoyl-CoA) and amino acid (serine) metabolism, and is a major catabolic sink for serine. We hypothesize that the increase in plasma serine represents a reduction in catabolism of serine by SPT because of increased alanine utilization.30
Moreover, this may also indicate that plasma serine levels are potentially an additional biomarker of lipotoxic insulin resistance.
A potential caveat of the present work is that we are measuring plasma ceramides as surrogate biomarkers of tissue insulin resistance, but have not measured ceramides in insulin-sensitive tissues. Although it is assumed that liver is responsible for generation of the majority of plasma ceramides,30, 54
other cell types can contribute significant amounts of ceramide to the plasma in the form of exosomes, vesicles secreted from cells in response to the fusion of multivesicular bodies with the plasma membrane.55, 56
These vesicles are purported to function in cell–cell communication by merging with and releasing their contents into other cells.56, 57
This represents a mechanism whereby de novo
ceramide production in one tissue can induce insulin resistance in other peripheral tissues. Nevertheless, the differential biological effects of plasma versus intracellular ceramide on insulin-sensitive tissues is an area that will require further investigation.17, 46, 58
In conclusion, these data have shown that provision of a HFFD ‘Western' diet to nonhuman primates increased de novo ceramide biosynthesis and resulted in elevated plasma levels of ceramides. Although these data do not provide a direct causal relationship between ceramides and insulin resistance, the increases in ceramides significantly correlated with reductions in insulin sensitivity in the pre-diabetic and diabetic animals. Furthermore, the increases in deoxySa support that SPT activity is deregulated, and indicate that these sphingolipids are an additional marker of insulin resistance. Although the identity of the tissue(s) responsible for the increase in plasma ceramides remains unknown, this will require further analysis of sphingolipid levels in insulin-sensitive tissues from these animals, and perhaps analysis of the sphingolipid content of the raft domain to definitively answer this question. Clearly, this should remain an area of vigorous research for the foreseeable future.