The current study represents an extension of previous work demonstrating that in various disease states of peripheral insulin resistance, including diet-induced obesity and nitrosamine exposure, the expression of several genes regulating ceramide production via de novo
biosynthesis or sphingomyelin degradation pathways was increased in liver, and ceramide levels (immunoreactivity) were increased in liver and/or blood [2
]. Importantly, these abnormalities were associated with brain insulin resistance and mild neurodegeneration [43
]. For example, in experimental diet-induced obesity with T2DM and NASH, ceramide gene expression was shown to be increased in liver, and accompanied by mild neurodegeneration with brain insulin/IGF resistance [43
]. With alcoholor nitrosamine-induced steatohepatitis, pro-ceramide gene expression in liver was correlated with hepatic and brain insulin/IGF resistance and tissue injury [2
]. Finally, in vitro
experiments demonstrated that direct exposure to cytotoxic ceramides impairs liver and brain cell viability, mitochondrial function, and insulin/IGF signaling mechanisms [51
], consistent with previous reports [10
The main objective of this study was to demonstrate the potential role of cytotoxic ceramides originating from the periphery as mediators of neurodegeneration. We and others have shown that in peripheral insulin resistance diseases associated with brain insulin resistance and neurodegeneration, serum and hepatic levels of ceramides, and other toxic lipids are increased [2
]. Moreover, we demonstrated that in vitro
cytotoxic ceramide exposure causes neurodegeneration with impaired viability, energy metabolism, and insulin/IGF signaling in neuronal cells [51
]. Although generally longer chain ceramides have been detected in insulin-resistance disease models and humans [27
], in our studies we used relatively short-chain synthetic ceramides because this approach has been validated in a number of experimental models, and the compounds are known to be cell permeable, impair signaling, and promote inflammation, mitochondrial dysfunction, and cell death [39
], all of which are features of insulin resistance diseases. Future studies will employ longer chain synthetic ceramides, once their bio-distributions and cell permeability characteristics have been determined.
In the current study, we did not regenerate models of obesity, T2DM, or NASH, and instead focused on testing the hypothesis that peripherally administered cytotoxic ceramides can cause brain insulin resistance with impairments of cognitive and motor functions. Therefore, we examined the degree to which limited early-life bioactive ceramide exposure leads to both hepatic and CNS insulin/IGF resistance and neurodegeneration. This study is novel because it directly examines the role of extra-CNS ceramides in relation to neurobehavioral deficits and brain insulin resistance in the absence of confounders produced by chronic high dietary fat intake, exposures to alcohol or toxins, and aging. The expectation was that ceramide exposure would impair insulin/IGF-1 signaling mechanisms in liver and brain, irrespective of peripheral blood indices of insulin resistance because the toxic lipids were deemed the culprits rather than hyperglycemia or hyperlipidemia per se. Correspondingly, in this study, C2Cer-induced hyperglycemia and hyperlipidemia were modest, and triglyceride levels were in fact reduced in serum, yet serum, liver, and brain ceramide levels were significantly increased. The significance of this work is that it helps establish mechanistic links among hepatic insulin/IGF resistance, lipotoxicity states, cognitive impairment, and neurodegeneration associated with deficits in brain insulin/IGF signaling. Moreover, the findings suggest that examining peripheral blood levels of ceramides may aid in identifying individuals at risk for developing cognitive impairment in the setting of obesity, irrespective of traditional biomarkers of type 2-diabetes and peripheral insulin resistance.
The over-arching hypothesis is that ceramides, which are recognized mediators of insulin resistance with demonstrated inhibitory effects on PI3K-Akt signaling [14
], may mediate neuro-cognitive deficits, brain insulin resistance, and neurodegeneration in the context of obesity, T2DM, and NASH. In this regard, we propose that ceramides generated from the periphery (i.e., liver or perhaps adipose tissue), cross the blood-brain barrier to mediate these adverse effects on brain structure and function. The lipid-soluble nature of ceramides makes it feasible for this class of lipids to regulate and alter brain function. This phenomenon could explain how obesity and T2DM pose increased risk of cognitive impairment and neurodegeneration in humans [77
]. In the present study, since it is now known which species of ceramides may be responsible for neurodegeneration in obesity and T2DM, and previous studies demonstrated that specific cell permeable synthetic ceramides impair insulin/IGF signaling and are cytotoxic in vitro
], we utilized synthetic bioactive (C2Cer) and inactive (C2DCer) ceramide molecules to test our hypothesis.
Corresponding to previous findings that ceramides promote insulin resistance [2
], we observed that following i.p. treatment of rat pups with synthetic C2Cer, as adolescents, the rats exhibited mild hyperglycemia and hyperlipidemia accompanied by increased serum ceramide levels. In addition, the livers showed evidence of on-going inflammation and injury with reduced lipid content but increased ceramide levels, and the brains exhibited normal histology, but had reduced lipid content and increased ceramide levels as well. The somewhat unexpected finding of reduced triglyceride levels in brain, liver and serum of C2-Ceramide treated versus control rats is consistent with results in another recent study in which it was suggested that ceramide treatments may mediate this effect by inhibiting adipogenesis [81
In most cells, lipids are mainly localized in membranes and have key roles in intracellular signaling [13
]. Lipid homeostasis is regulated in part by insulin signaling [13
Degradation of sphingolipids promotes ceramide generation, which can have adverse effect on intracellular signaling, cell survival, and inflammatory mediators [13
]. Correspondingly, we detected significant reductions in neuronal Hu expression in the temporal lobes of C2Cer-treated rats. This finding corresponds with the impairments in spatial learning and memory detected by Morris water maze testing. In the brain, myelin is the most abundant lipid, and myelin maintenance via oligodendroglial metabolism, is regulated by insulin and IGF signaling [83
]. Although we did not detect any reductions in MAG-1 immunoreactivity, conceivably, the impairments in insulin signaling effectuated by the C2Cer treatments, and as demonstrated in previous experiments [51
], led to dysregulated lipid metabolism and increased ceramide generation, which in turn, further impaired insulin/IGF signaling mechanisms, and at least in liver, also impaired cell survival.
Although there were no overt histopathological abnormalities detected in brain, the reductions in neutral lipid and triglyceride, and increased ceramide levels may reflect the early stage of neurodegeneration. Correspondingly in previous studies, it was demonstrated that relatively early abnormalities in AD include white matter atrophy and increased ceramide content in brain [26
]. Moreover, the impairments in cognitive and motor functions were also associated with reductions in Hu (reflecting neuronal injury or loss), and increased levels of phospho-tau and tau immunoreactivity in brains of C2Cer-treated rats. Therefore, neurobehavioral (functional), biochemical, and molecular abnormalities in brain may provide more sensitive indices of neurodegeneration, and precede many of the structural changes detected by histopathological examination. Even in humans with mild cognitive impairment, structural neurodegenerative lesions are often mild, focal, or absent [85
]. As ceramides and other toxic sphingolipids are generated by myelin breakdown or altered biosynthesis, virtually any pathophysiological process that leads to their accumulation would also impair CNS function. Therefore, we interpret the CNS functional impairments to be consequential to combined effects of neuronal loss/degeneration precipitated by C2Cer-mediated insulin/IGF resistance, and attendant locally increased ceramide generation. At this point, it is not possible to know the relative contributions of exogenous versus endogenous ceramides mediating brain insulin resistance and neurobehavioral deficits; nonetheless, what is clear is that the neurodegeneration process can be initiated by cytotoxic ceramides generated in the periphery, i.e. outside of the CNS. While ceramide levels are increased in sera, skeletal muscle, adipose tissue, and/or liver in peripheral insulin resistance diseases [13
], and in AD brains [90
], the levels cannot be accurately quantified for comparison with our experimental model due to heterogeneity of the expressed or accumulated ceramides and other sphingolipids and the lack of standardized methods for measuring such compounds.
The molecular and biochemical studies demonstrated that the early postnatal treatment with C2Cer impaired insulin and IGF signaling mechanisms in both livers and brains of the adolescent rats. The major impact was on the expression and/or phosphorylation state of the insulin receptor, IGF-1 receptor, IRS-1 or Akt. These findings are consistent with previous reports demonstrating that ceramides impair insulin signaling through the Akt pathway [14
], and also with our previous findings that bioactive synthetic ceramides (C2Cer or C6Cer) impair insulin/IGF signaling through inhibition of receptor and IRS expression and function.
The insulin/IGF-1-IRS-1-Akt signaling pathway mediates cell survival, energy metabolism, neuronal plasticity, and neurotransmitter function [53
]. Therefore, the observed C2Cer-mediated impairments of this pathway could account for the observed ongoing hepatocellular injury and cognitive-motor deficits. It is noteworthy that we detected increased levels of IRS-1 and/or insulin receptor protein vis-à-vis reduced relative levels of phosphorylated receptor and IRS-1. Conceivably, the increased protein levels reflect a compensatory protective response to the impairments in signaling that limited the degree and rate of cellular injury and death. The same argument could be made for the seemingly paradoxical increases in pS9-GSK-3β in C2Cer-exposed livers.
Although the magnitudes of these effects were variable, the aggregate effects of C2Cer treatment were to reduce insulin ± increase insulin receptor gene expression, and reduce IGF-1 receptor gene expression in liver and brain, inhibit signaling downstream through IRS-1 and Akt with increasing GSK-3β activity in liver, and constitutively impairing insulin signaling at the level of the receptor, IRS-1, or Akt in the brain. These results, together with the reduced levels of insulin gene expression in brain, are reminiscent of the findings of both insulin resistance and insulin deficiency in brains with AD [7
]. On the other hand, this model clearly does not replicate the abnormalities in AD, and instead seems more closely aligned with the effects of obesity and peripheral insulin resistance [43
]. However, in future studies, it will be of interest to examine the effects of aging in relation to ceramide-mediated neurodegeneration.
In conclusion, this study demonstrates that limited in vivo
exposure to bioactive toxic ceramides causes mild diabetes mellitus with hyperlipidemia, hepatocellular injury, deficits in cognitive and motor functions, and impairments in insulin/IGF signaling though IRS-1 and Akt. The importance of this work is that it demonstrates that peripherally generated ceramides, such as occurs in obesity, T2DM, alcoholic liver disease, and nitrosamine exposure [1
] can mediate cognitive impairment with deficits in brain insulin/IGF signaling that promote neurodegeneration. The results support our hypothesis that in peripheral insulin resistance disease states, cognitive impairment can be mediated via a liver/peripheral-brain axis of neurodegeneration due to increased ceramide production and trafficking across the blood-brain barrier. The consequential brain insulin resistance establishes a reverberating loop of neurodegeneration whereby inhibition of signaling through insulin and IGF receptors, IRS, and Akt, perturbs energy metabolism, lipid and cholinergic homeostasis, and neuronal plasticity. The findings suggest that individuals with peripheral insulin resistance diseases who are at risk for developing cognitive impairment and neurodegeneration may be identified by examining peripheral blood and possibly cerebrospinal fluid levels of ceramides and other toxic sphingolipids, and that preventive/treatment measures could include the use of agents that reduce or block the synthesis and accumulation of such compounds.