Mafld Causes Alterations in Thalamus Energy Metabolism and Brain Structure


 Background

Metabolic associated fatty liver disease (MAFLD), commonly known as non-alcoholic fatty liver disease, represents a continuum of events characterized by excessive hepatic fat accumulation which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and in some severe cases hepatocellular carcinoma. MAFLD might be considered as a multisystem disease that affects not only the liver but involves wider implications, relating to several organs and systems, the brain included. The present study aims to investigate changes associated with MAFLD-induced alteration of thalamic metabolism in vivo.
Methods

DIAMOND mice were fed a chow diet and tap water (NC NW) or fat Western Diet (WD SW) for up to 28 weeks. At the baseline and weeks 4, 8, 20, 28 the thalamic neurochemical profile and total cerebral brain volume were evaluated longitudinally in both diet groups using 1H-MRS. To confirm the disease progression, at each time point, a subgroup of animals was sacrificed, the livers excised and placed in formalin. Liver histology was assessed and reviewed by an expert liver pathologist.
Results

MAFLD development significantly increases the thalamic levels of total N-acetylaspartate, total creatine, total choline, and taurine. Furthermore, in the WD SW group a reduction in total cerebral brain volume has been observed (p < 0.05 vs NC NW).
Conclusion

Our results suggest that thalamic energy metabolism is affected by MAFLD progression. This metabolic imbalance, that is quantifiable by 1H-MRS in vivo, might cause structural damage to brain cells and dysfunctions of neurotransmitter release.


Introduction
Metabolic (dysfunction)-associated fatty liver disease (MAFLD) 1,2 formerly known as non-alcoholic fatty liver disease (NAFLD) 3 is a heterogeneous condition of fatty liver disease which might be in uenced by multiple factors including age, gender, hormonal status, ethnicity, diet, alcohol intake, smoking, genetic predisposition, the microbiota and metabolic status 4 . The spectrum of the disease extends from steatosis to hepatocellular carcinoma (HCC) 5 and though hepatic steatosis is highly prevalent, in ammation occurs only in a minority. Moreover, liver-related complications (i.e., cirrhosis or cancer) are likely in patients with steatohepatitis 6 , but the progression is not inevitable or consequential. Indeed, cirrhosis is not a fundamental stage for HCC development 7 . This heterogeneity also underlines the possible impact of MAFLD on several organs and systems, included the brain. In fact, nervous dysfunctions 8 , brain lesions, changes in perfusion and brain activity 9 , brain aging, increased risk of ischemic and hemorrhagic stroke 10,11 are some of the consequences of the wide spectrum of extrahepatic alterations induced by MAFLD.
In particular, oxidative stress, in the disease progression, leads to alterations in mitochondrial function and structure with a consequent reduction in neuronal metabolism 12,13,14 . In turn, the alteration of metabolic activity in speci c brain areas (i.e., thalamus, hippocampus, pre-frontal cortex) could cause cognitive de cits 15 because various metabolites, such as N-acetylaspartate, creatine, choline, glutamate and taurine 16,17,18,19 are involved in energy metabolism and in the maintenance of brain functions 20 .
Changes in the cerebral levels of those metabolites, following a fatty diet, have been reported in preclinical studies 21 and evidence exists that the consumption of a high caloric diet also leads to high concentrations of in ammatory cytokines in the brain, resulting in microgliosis, astrocytosis and neuronal damage 22,8 . Moreover, patients suffering from steatohepatitis have a reduced brain volume 23 and are at higher risk to suffer from neurological diseases, which are, most probably, related to the volume reductions as well 24 .
Altogether, these discoveries provide a rationale to further evaluate the role of MAFLD in brain damage, through the identi cation, visualization and quanti cation of brain biochemical markers and neurotransmitters, and the alteration of the brain volume that, overall, could re ect physiologic or pathologic conditions 25 . In this perspective, advances in neuroimaging provide unique opportunities to evaluate brain structure, biochemistry and function 26 . In particular, the proton magnetic resonance spectroscopy ( 1 H-MRS) represents a non-invasive method useful to study brain metabolism, longitudinally and in a non-invasive manner 27 .
Therefore, the aim of our work was to analyze the total brain volume in a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) 28 and to assess and quantify the main metabolites present in brain tissue, including N-acetylaspartate (NAA), the predominant MRS signal in the healthy neurons 16 ; total creatine (tCr), involved in cellular energy metabolism; total choline (tCho), also involved in the synthesis and the breakdown of cellular membranes 29 ; the neuroin ammation modulator taurine (Tau) 30,31 and the excitatory neurotransmitter glutamate (Glu) 32 . As the concentration of brain metabolites often re ects the state of its metabolic activity and energetic status, the 1 H-MRS analysis of the metabolic uctuation might be predictive of the potential MAFLD implications at brain level.

Materials And Methods
Animals. Male DIAMOND (diet-induced animal model of non-alcoholic fatty liver disease) mice were purchased from Sanyal Biotechnology (Virginia Beach, VA, USA) kept under standard laboratory conditions in a speci c-pathogen-free animal facility and maintained at 22 ± 2°C with alternating 12 h light-dark cycle. All the experimental procedures were performed according to protocols approved by the Animal Care of University Magna Graecia of Catanzaro. The experimental procedures were carried out in compliance with the ARRIVE guidelines. All experiments were performed in accordance with the European Commission guidelines (Directive 2010/63/EU) for the animals used for scienti c purposes.
Study design. Mice of 8-12 weeks of age and weight of 20.54 ± 0.53 grams were divided into two groups and fed ad libitum a normal chow diet (NC, Harlan TD.2019) and tap water (NW) or a high fat/high carbohydrate diet (Western diet, WD, Harlan TD.88137) with a high fructose-glucose water solution (SW, 23.1 g/L d-fructose + 18.9 g/L d-glucose) for up to 28 weeks. The choice of the animals and the diet used to develop steatosis and steatohepatitis have been based on previously published studies 6,28 . One day before starting diet regimen, baseline body weight and MRS were assessed. On weeks 4, 8, 20 and 28, thalamic neurochemical pro le was evaluated in the two diet cohorts using 1 H-MRS. Animals body weight was assessed weekly. The day of the sacri ce, animals were exposed to inhaled iso urane prior to being euthanized. Euthanasia was performed by cervical dislocation. The entire liver was removed from the abdominal cavity and weighed. The liver was sectioned in a sagittal plane and placed in containers of 10% formalin, for later histologic processing and analysis. 1 H-MRS data analysis was performed using TARQUIN 4.3.10, a new accurate and robust modeling algorithm 33 which allows to quantify the concentrations of metabolites within the voxel.
Volumetric Analysis. Volumetric analysis was performed using OsiriX imaging software (v. 12.0.2, Pixmeo SARL, Switzerland). Total cerebral brain volumes (TCBVs) were estimated using T1_FLASH_3D images in coronal view with a thickness of 112.78 µm per slice, obtained with a T1_FLASH_3D_iso sequence (TE: 8 ms, TR: 50 ms, averages: 1, dummy scans: 20, image size: 133x133x80, eld of view: 15x15x10 mm 3 ). By the "Draw tool" the ROIs were traced on the MRI sections. To improve the viewing of cerebral margins, a Default WL / WW, "French Clut" and a linear opacity table were used. Total brain's area was de ned every 3 slices, starting from the olfactory bulbs up to the last part of the cerebellum. Subsequently, "Generate Missing ROIs" function was used to outline total brain area for all slices. Then, "Compute ROI Volume" feature was used to merge the ROIs of the entire brain and estimate its volume.
Histological analysis. Liver histology was assessed from para n-embedded tissue sections stained with hematoxylin and eosin. Histology was reviewed using the NASH-Clinical Research Network (CRN) criteria and fatty liver inhibition of progression (FLIP) algorithm by a liver pathologist.
Ballooning hepatocytes was graded as 0 (none), 1 (when few hepatocytes presented a rounded shaped, reticulated, and pale cytoplasm, but with normal dimensions), and 2 (when there is a cluster of prominent ballooning hepatocytes).
Statistical analysis. Data were analyzed with GraphPad PRISM 9.1.2 (GraphPad Software, Inc., La Jolla, CA, USA). All results were expressed as mean ± S.E.M. (Standard error of the mean). Normality was tested using Shapiro-Wilk normality test. Normally distributed data were analyzed by one way ANOVA followed by Tukey's test, while data without normal distribution were analyzed using Kruskal-Wallis analysis of variance and subsequent Dunn's tests. The Unpaired Two-tailed Student's t test was used for comparison of data derived from two groups. Value with p < 0.05 were considered statistically signi cant. Correlation analysis was assessed using Spearman's correlation coe cient, using NAS as pathology marker.

Results
Mice fed a high fat diet and sugar water develop NAFLD. Animals fed a WD SW developed obesity compared to CD NW-fed mice (Fig. 1A). The weight gain was accompanied by an increase in liver weight at all time points (Fig. 1B).
Mice on a high fat diet with ad libitum sugar water administration developed steatohepatitis (Fig. 1C,D), which was characterized by steatosis, lobular in ammation, and hepatocellular ballooning (Fig. 1D,E).
The NAFLD activity score (NAS) increased by week 8 and remained higher than NC NW mice by week 28 (Fig. 1E). Speci cally, histology of the liver showed an extensive development of steatosis by week 8 in WD SW-fed mice (Fig. 1D,E). At 8 weeks mice had a mean steatosis grade of 2.5 ± 0.3 ( Fig. 1E) with 60 ± 11% cells with steatosis (not shown). This remained nearly constant after 20 weeks, with a mean steatosis grade of 2.7 ± 0.1 (Fig. 1E), and 68.75 ± 4.7% cells with steatosis (not shown). At week 28 weeks mice had a mean steatosis grade of 2.9 ± 0.1 (Fig. 1E) and 81 ± 3.4% cells with steatosis (not shown). Following initiation of WD SW diet, steatohepatitis developed in almost all mice and at week 20, all mice had NASH with a prominent in ammation (Fig. 1C,D,E), whereas at week 28, 8 out of 10 mice developed steatohepatitis (Fig. 1C). Stage 1 brosis was present by week 20 after initiation of the WD SW diet (Fig. 1E). In contrast, none of the animals on chow diet developed NAFLD (Fig. 1C).
Volumetric Analysis of control and high-fat diet-fed animals. Volumetric analysis showed the same total cerebral brain volumes (TCBVs) in both animal cohorts at 0 weeks.
Smaller total cerebral brain volumes were associated with Steatosis and Steatohepatitis. Smaller TCBVs were strongly correlated with high NAS (r = -0.9120; p < 0.001, Fig. 4A) already after 8 weeks (steatosis). A strong correlation between TCBV and NAFLD was also observed during the progression of the disease either after 20 weeks (r = -0.9415; p < 0.001, Fig. 4B) either after 28 weeks (r = -0.9498; p < 0.001, Fig. 4C) 1 H-MRS of control and high-fat diet-fed animals. 1 H-MRS spectra of the mouse thalamus (Fig. 5A) for the NC NW and WD SW animals underlined the presence of the main brain metabolites between 0.7-4 PPM (Fig. 5B). The quanti cation of 1 H-MRS spectra (Fig. 5B) showed similar concentrations at the baseline in both cohorts (Fig. 6).
Comparisons at speci c times showed that the concentrations of tNAA (p < 0.05) and tCr (p < 0.01) were signi cantly higher in the WD SW group than in the NC NW control group at 20 weeks, as well as the concentrations of tNAA (p < 0.05), tCr (p < 0.01), tCho (p < 0.05) and Glu (p < 0.05) were signi cantly increased in the WD SW than NC NW cohort at 28 weeks (Fig. 6).

Discussion
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 carcinoma 5,6 . 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 diseases 36 . 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 quanti ed over time the putative indicator of in ammation Taurine, and the levels of Glutamate, tNAA, tCho, tCr to study the impact of metabolic uctuation 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 population 37 . Thus, recently, it has been coined a new de nition of this pathological state known as MAFLD 2 . 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 intolerance 38 .
The occurrence of these events was studied in depth in an animal model of MAFLD resembling the human features of disease development 39 , con rming that chronic intake of a diet enriched in fats and carbohydrates contributes to induce in ammation and oxidative damage primarily to the hepatic microcirculation and then, at systemic level 40,41 . This suggests a more generalized endothelial dysfunction, also involving blood-brain barrier that, once damaged, permits the in ltration of circulating in ammatory cells into the brain 42 .
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 neuroin ammation, oxidative stress, metabolic impairment and neurotransmitter transmission de cit 43,44 .
The occurrence of steatosis, lobular in ammation and hepatocellular ballooning, characterizing WD SWinduced 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 in ammation and oxidative stress, subsequent to high fat diet consumption, has been highlighted by the onset of microgliosis and astrocytosis, contributing to neuronal damage 8 and that can progress in apoptotic death after the alteration of oxidative phosphorylation and mitochondrial dysfunction 14 .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 de cits characterized by memory and learning disorders 15 .
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 in ammation, 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 sphingomyelin 45,46,31 .
Taken together, these results indicate that the solubilization of glycerophosphorylcholine and phosphorylcholine, probably due to oxidative/in ammatory 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, too 50 .
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 mice 28 , 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 conditions 51 . 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 substrate 52,53,54 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 de cits characterizing NASH 55 .
In conditions of impaired metabolism, the brain can also use free fatty acids to produce energy 56 . Thus, it is plausible that astrocytes further compensate the energy de cit 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 lipoproteins 56 . 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 decomposition 57 .
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 production 58 .
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 needs 59 .
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 in ammatory 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 de cits of speci c 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 maintained 60 .
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 de cit to prevent neuronal damage, but at the same time, the triggering cause of the learning and memory de cits that are often found in NASH affected patients 15 .
Finally, our results also suggest the need for pharmacological interventions aimed to counteract in ammatory degeneration of MAFLD which, despite being a very widespread phenomenon with detrimental consequences at CNS level, is still not treated with a speci c therapy. Histology score for steatosis, hepatocyte ballooning, lobular in ammation, brosis and NAFLD Activity Total cerebral brain volume for the NC NW (red) and WD SW ( Relationship between TCBV (Total cerebral brain volume, cm3) and NAS (NAFLD Activity Score, 0-8) by