NAFLD includes a wide spectrum of liver diseases, starting with the accumulation of lipid molecules in hepatocytes and evolving into NASH state that can degenerate to liver cirrhosis and/or hepatocellular carcinoma5,6. Despite the main feature is liver dysfunction, the detrimental impact of lipid accumulation can affect the whole metabolic state also predisposing to cardiovascular and neurological diseases36. In this view, the aim of our study was to investigate the effects of the altered lipid metabolism induced by NASH in the thalamus. In particular, we monitored and quantified over time the putative indicator of inflammation Taurine, and the levels of Glutamate, tNAA, tCho, tCr to study the impact of metabolic fluctuation on brain energetic status, structure and function.
In the global population, NAFLD prevalence is estimated around 25% although its systemic impact on body metabolism can expand this valuation to a wide population37. Thus, recently, it has been coined a new definition of this pathological state known as MAFLD2. In this context, a high-fat diet is considered the main cause of a range of systemic dysfunctions that include a gain of weight and fasting glucose, abnormal fasting insulin levels, raised lipid biosynthesis in the liver, elevated levels of circulating fatty acids and glucose intolerance38.
The occurrence of these events was studied in depth in an animal model of MAFLD resembling the human features of disease development39, confirming that chronic intake of a diet enriched in fats and carbohydrates contributes to induce inflammation and oxidative damage primarily to the hepatic microcirculation and then, at systemic level40,41. This suggests a more generalized endothelial dysfunction, also involving blood-brain barrier that, once damaged, permits the infiltration of circulating inflammatory cells into the brain42.
In accordance with this latter hypothesis, recent studies have shown that ceramides and other toxic lipids, generated by the liver during NASH, are able to mediate adverse effects in the brain, due to their ability to cross the blood brain barrier and, consequently, to cause neuroinflammation, oxidative stress, metabolic impairment and neurotransmitter transmission deficit43,44.
The occurrence of steatosis, lobular inflammation and hepatocellular ballooning, characterizing WD SW-induced NASH in our experiments, further support the hypothesis of its possible impact on the brain, as shown by the strong correlation between the reduction of brain volume and MAFLD progression, overtime.
At thalamic level, the detrimental role of inflammation and oxidative stress, subsequent to high fat diet consumption, has been highlighted by the onset of microgliosis and astrocytosis, contributing to neuronal damage8 and that can progress in apoptotic death after the alteration of oxidative phosphorylation and mitochondrial dysfunction14.In addition, similarly to other brain area, such as prefrontal cortex, hippocampus, amygdala and mammillary bodies, the alteration of metabolic activity in the thalamus also cause a functional impairment, particularly cognitive deficits characterized by memory and learning disorders15.
Spectroscopic analysis, carried out in thalamus of high-fed diet mice, showed a time-dependent increase in the concentration of taurine, considered a hypothetical marker of inflammation, up to twentieth week. This gradual increase was accompanied, until the end of the experiment, by an enhancement in total choline which, normally, represents the sum of the levels of glycerophosphorylcholine and phosphorylcholine, both precursors of phosphatidylcholine and sphingomyelin45,46,31.
Taken together, these results indicate that the solubilization of glycerophosphorylcholine and phosphorylcholine, probably due to oxidative/inflammatory insults affecting membranes, could be responsible not only for neuronal demyelination, but also for the alteration of plasma membrane permeability and polarization, and for the dysfunction of neurotransmitter vesicular release,47,48,49,29. Furthermore, the same structural membrane alteration of astrocytes and microglia could affect their function, too50.
At the end of the experiment, in thalamus of WD SW mice, increased NAA and glutamate levels were also highlighted, although they were not associated with any changes in glutamine levels (data not shown). This suggests that, in the presence of increased glucose tolerance and increased levels of circulating fatty acids, as typically observed at that time point in DIAMOND mice28, cerebral tissue activates an alternative mechanism to the use of glucose, capable to equally satisfy its energy needs. Indeed, although glucose has always been recognized as the primary source of brain energy, growing evidence shows that other metabolites, such as glutamate and acetate, are used as energy sources, mainly by astrocytes, both in physiological and pathological conditions51. In this perspective, the increase in astrocytic glutamate could represent the substrate needed to an anaplerotic reaction aimed to ensure the right homeostasis of Krebs cycle and to the production of necessary lactate for neuronal survival.
The use of glutamate as a mitochondrial substrate52,53,54 in turn, could justify its vesicular depletion at synaptic level. Consequently, the lack of glutamate release, compared to the unchanged levels of glutamine measured over time, could explain the cognitive deficits characterizing NASH55.
In conditions of impaired metabolism, the brain can also use free fatty acids to produce energy56. Thus, it is plausible that astrocytes further compensate the energy deficit due to decreased glucose levels through fatty acid β-oxidation.
The main source of free fatty acids crossing the blood brain barrier may come from long-chain fatty acid/albumin complexes and, to a lesser extent, from circulating lipoproteins56. Once inside the astrocytes, the conversion into acetyl-CoA, operated by the acyl-CoA synthetase, allows its translocation into the mitochondrial matrix for β-oxidation and for the production of ketone bodies, such as acetoacetate, beta-hydroxybutyrate and acetone that results from their spontaneous decomposition57. Ketone bodies are synthesized starting from two acetyl-CoA molecules also at the peripheral level, mainly by the liver, especially in conditions of decreased glucose bioavailability. Subsequently, they are transported to the extrahepatic tissues, where they are used, after conversion into acetyl-CoA and introduction into the cycle of tricarboxylic acids, for energy production58.
Therefore, the ketone bodies produced by astrocytes or coming from the bloodstream in conditions of more marked metabolic alterations migrate within neurons, where they are converted into acetyl-CoA and used in the Krebs cycle. On the other hand, acetyl-CoA excess is converted into NAA and stored inside neuronal mitochondria for satisfying a possible sudden increase in energy needs59.
In our experiments, the increased amount of NAA, found in thalamus of mice fed a high-fat diet, were also associated with a raise of creatine/phosphocreatine levels, indicating also the formation of phosphate reservoirs needed for ATP synthesis.
Overall, the production of NAA and creatine/phosphocreatine appears necessary to constantly ensure correct mitochondrial functionality and, consequently, the energy needed for brain functions potentially compromised by the inflammatory insult triggered at the peripheral level by NASH. On the other hand, the tight correlation between reduced brain volume and NAFLD development, revealed by our experiments, further supports the hypothesis of an increased risk of functional deficits of specific brain areas.
Therefore, since the thalamus represents a key element in the integration of neuronal impulses within the network including prefrontal cortex, hippocampus, amygdala and mammillary bodies, a constant energy supply must be always maintained60.
In this scenario, the use of newly synthesized glutamate as an energy source, rather than as a neurotransmitter reserve, could represent a key element for the compensation of the energetic deficit to prevent neuronal damage, but at the same time, the triggering cause of the learning and memory deficits that are often found in NASH affected patients15.
Finally, our results also suggest the need for pharmacological interventions aimed to counteract inflammatory degeneration of MAFLD which, despite being a very widespread phenomenon with detrimental consequences at CNS level, is still not treated with a specific therapy.