Partial MCT1 invalidation protects against diet-induced non-alcoholic fatty liver disease and the associated brain dysfunction

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Introduction
Non-alcoholic fatty liver disease (NAFLD) is a metabolic syndrome, affecting approximately 25% of the population and >80% of morbidly obese people.Over the last decade, NAFLD has become known as a multisystem disease, affecting extrahepatic organs, with its clinical burden not only confined to liverrelated morbidity and mortality. 1Indeed, the most common liverrelated complication of individuals with NAFLD with advanced liver disease (F3/F4) that has progressed to decompensation over a long follow-up period, is hepatic encephalopathy. 2everal studies have reported the negative effects of unhealthy diet and obesity on cerebral function and cognition. 3It is now clear that not only advanced forms of chronic liver disease, but even precirrhotic stages of NAFLD can be linked to impaired cognitive performance 4 and cerebral function 5 (independently of cardiometabolic disorders), altered behaviour, 6 and low total cerebral volume. 4These combine to compromise brain health. 7,8However, more precise alterations of cerebral physiology and the mechanisms behind them are yet to be determined.
To function, the brain depends on continuous delivery of oxygen and energy substrates.The cerebral vasculature is well suited for this purpose, and additional mechanisms have evolved to closely regulate blood flow, matching oxygen supply with demand. 9Therefore, any changes in these mechanisms or the structure of the cerebrovascular system can have detrimental effects on brain tissue oxygenation and overall physiology. 10Several studies have reinforced this view by highlighting an association of cerebral hypoxia with neurodegenerative conditions, either predisposing the brain for developing neurodegeneration or occurring as the disease progresses. 11ecently, it has been observed that hypoxia not only directly induces neuronal damage, but also initiates cerebral inflammation through microglial and astrocytic responses. 12Toxic inflammatory mediators produced by glial cells under hypoxic conditions are key in the development of brain inflammation, which exacerbates neuronal injury, synaptic remodelling, and neurodegeneration. 13Growing evidence implicates low-grade chronic systemic inflammation and consequently brain inflammation in the pathogenesis of diet-induced obesity and cerebral dysfunction. 14However, brain tissue oxygenation and the role of NAFLD in any detected alterations have yet to be investigated directly.
The monocarboxylate transporter-1 (MCT1, or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues, playing an important role in energy homoeostasis. 15As previously shown, genetically modified mice haploinsufficient for Mct1( +/− ) developed normally but exhibited a unique phenotype on a high-fat diet (HFD), characterised by resistance to hepatic steatosis and associated metabolic alterations, with some of the possible protective mechanisms described. 16,17n this study, we tested the hypothesis that NAFLD and associated systemic abnormalities lead to the development of an encephalopathy by altering cerebral physiology and behaviour.We also investigated whether a global MCT1 downregulation, which prevents NAFLD development, may offer cerebral protection.Using an animal model of diet-induced NAFLD, and the Mct1 haploinsufficient mouse line described above, we obtained data from a battery of in vivo and ex vivo studies, which suggest that NAFLD in the presence of obesity is associated with low-grade brain tissue hypoxia and inflammation, as well as cerebrovascular, metabolic, and behavioural changes, indicating early stages of an obesogenic diet-induced encephalopathy.Mct1 haploinsufficient mice, despite fat accumulation in adipose tissue, were protected, indicating the potential for MCT1 as a novel preventive or therapeutic target.

Materials and methods
All experiments were performed in accordance with the Swiss animal welfare laws approved by the Committee on Animal Experimentation for the Canton of Vaud, Switzerland (VD 3401.c) and the ARRIVE guidelines. 18Further technical details on all experimental tests used are available in the Supplementary material.
At the end of the 16 weeks, blood (plasma) via cardiac puncture, brain, and liver tissues were collected under terminal anaesthesia for further analysis.

Body weight and composition measurements
Body composition was measured in all mice using EchoMRI (LLC, Houston, TX, USA), at week 0 and 16 of their feeding regime.At the end of the feeding duration, % fat mass and lean mass were compared between groups.Body weight was obtained weekly using a digital balance, and total food intake per mouse per cage was calculated at the end of the feeding period for animals on the HFD HF/HG diet.

Open field
Open field (OF) to assess anxiety-like behaviour was performed using an arena divided into virtual quadrants.Each mouse was placed in the centre and allowed to explore for 15 min.Average speed and total distance travelled were measured to assess locomotion, whereas anxiety was evaluated according to the time spent exploring the centre zone.

Forced swim test
Following OF and 1 resting day, depressive-like behaviour was assessed using the forced swim test (FST).Mice were individually placed in a glass cylinder and allowed to swim for 4 min.Duration of immobility (floating) was recorded.

Brain tissue oxygen measurements
Optical fluorescence Under isoflurane anaesthesia, tissue partial pressure of oxygen (PO 2 ) was monitored in the somatosensory (forelimb) region of the cortex (S1FL 0.5 mm below the cortical surface) by optical fluorescence technology (OxyliteTM, Oxford Optronics, Oxford, UK).Following a 15-min recovery period, parenchymal PO 2 sampling was started until a stable reading was achieved.

Blood gas manipulations
After baseline PO 2 was recorded, systemic hypercapnia was induced by 10% CO 2 inhalation (in 21% O 2 with the gas balance made of nitrogen) for 5 min.

Multispectral optoacoustic tomography
In a different cohort of mice, brain tissue oxygenation was measured under isoflurane anaesthesia with the inVision 128 small animal multispectral optoacoustic tomography (MSOT) system (iThera Medical GmbH, Munich, Germany).
MSOT images (Fig. 1D) were reconstructed as described in the Supplementary material.Regions of interest (ROIs) were drawn over the left and right cortex on deoxygenated haemoglobin (Hb)/oxygenated haemoglobin (HbO 2 ) maps using ImageJ (NIH, USA).ROIs for each region were averaged.Tissue oxygenation as a measure of tissue oxygen saturation (SO 2 ) of the ROIs was calculated by the following: Cerebral blood volume (CBV) was calculated by the following:
Respiratory capacities were expressed as oxygen consumption per wet mass of tissue (pmol s −1 mg −1 ) or per protein mass (pmol s −1 lg −1 ) and corrected for residual oxygen consumption (ROX).

Statistical analysis
Oxygen measurements via optical fluorescence were digitised using a Power 1401 interface (CED, Cambridge, UK) and processed using Spike 2 software (CED, Cambridge, UK).Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, California, USA).Data are expressed as mean ± SEM.Differences were ascertained by 1way or 2-way ANOVA followed by Tukey's multiplecomparisons post hoc test, the Mann-Whitney U test, or the Kruskal-Wallis test followed by Dunn's multiple-comparisons post hoc test where appropriate.Differences with a p value of <0.05 were considered significant.

Animal model characterisation
Mct1 +/+ but not Mct1 +/− animals on HFD HF/HG experience a NAFLD-associated anxiety-and depressionrelated behaviour At the end of the feeding regime, OF and FST were performed.Average speed and distance of exploration in OF were not different between groups (Fig. 3A and B) indicating preserved locomotion irrespective of diet and body composition.However, obese Mct1 +/+ HFD HF/HG animals with NAFLD spent significantly less time exploring the centre zone (n = 10, 41 ± was not different from MCT +/− NC controls (n = 7, 72 ± 11 s, p = 0.7; Fig. 3C and D).
Mct1 +/+ HFD HF/HG mice also displayed an elevation in some cytokines (interferon gamma [IFN-c] and IL-10 [Fig.S4]; no other cytokine alterations detected [Supplementary material]).to a known vasodilatory stimulus, hypercapnic acidosis was induced by changing the inspired gas mixture to include 10% CO 2 , which led to a significant increase in parenchymal PO 2 from baseline in all groups (p <0.05; Fig. 1A and C).

Brain oxygenation is compromised in obese Mct1
To exclude the impact of an acute inflammatory response following craniotomy, non-invasive MSOT was used to measure brain tissue oxygenation.In Mct1 +/+ HFD HF/HG mice, CBV (an indicator of blood/ oxygen supply to the brain and vascular density) was significantly decreased (1 ± 0.2 a.u., p = 0.04) compared with Mct1 +/+ NC controls (2 ± 0.1 a.u.; Fig. 1F), but cerebral blood flow (CBF), measured from arterial spin labelling magnetic resonance imaging (MRI), was unaltered (Fig. S5).The reduction in CBV was also associated with a reduction in vascular density (Fig. S6A and B) and a decrease in vessel diameter (Fig. S6C).
Obese Mct1 +/+ animals with NAFLD present higher mitochondrial respiratory capacities but not improved metabolic outcome, whereas Mct1 +/− animals remain unaffected To investigate further the lower brain oxygenation observed in obese Mct1 +/+ animals with NAFLD, oxygen consumption by mitochondrial respiration was measured ex vivo in homogenised cortical tissue (constant mass) using highresolution respirometry.
Despite the increase in cortical respiratory capacities of Mct1 +/+ HFD HF/HG mice, no positive changes were observed in cerebral metabolite ratios measured by ex vivo nuclear magnetic resonance (NMR) spectroscopy (Table S2) or in protein content of OXPHOS complex subunits and proteins involved in mitochondrial remodelling (Fig. S7).Differences in respiratory capacities were eliminated when measurements were normalised to lg of protein/mg of brain tissue used (Fig. 6B).

Discussion
Individuals with NAFLD have been reported to experience cognitive impairment, 8 with the aetiology and precise cerebral dysfunction yet to be thoroughly characterised.In our study, HFD HF/HG caused obesity, hormonal and metabolic alterations, and NAFLD in Mct1 +/+ animals, which resulted in specific brain clinico-pathological features.These features included anxiety-and depression-related behaviour, also reported in obese people 21 and those with NAFLD, which were even worse in the presence of insulin resistance 22 (as seen in Mct1 +/+ HFD HF/HG mice).Additionally, low-grade brain tissue hypoxia, likely caused by the low-grade brain inflammation and decreased CBV, was accompanied by microglial and astrocytic morphological and metabolic alterations.Unlike Mct1 +/+ mice, animals with a lower global expression of MCT1 (Mct1 +/− ) did not develop NAFLD or associated hormonal and metabolic alterations when fed with HFD HF/HG, despite the substantial fat accumulation in adipose tissue.Such phenotype allowed us to investigate the liver-brain interactions during HFD HF/HG and obesity, with data indicating that liver protection and associated induced systemic alterations, effectively prevent behavioural and cerebral dysfunction, emphasising the role of the liver-brain axis in NAFLD-induced encephalopathy, that up until now was mainly attributed to obesity.
Adiposity is associated with increased secretion of inflammatory and fibrotic mediators, which can reach the liver and contribute to chronic low-grade inflammation, 23 while prompting brain inflammation, with astrocytes and microglia playing a pivotal role. 24Indeed, obese Mct1 +/+ animals with NAFLD presented peripheral and cerebral upregulation of certain pro-and anti-inflammatory cytokines, as well as remarkable alterations in cortical microglia (also seen in individuals with steatohepatitis 25 ) and astrocytes, all of which were strikingly prevented in Mct1 +/− HFD HF/HG animals without NAFLD.
The morphological alterations of cortical microglia and astrocytes, as well as the moderate cytokine elevation, suggest that brain inflammation is mild with cells just undergoing a lowgrade chronic response.IL-10, an anti-inflammatory cytokine, redirects active astrocytes to produce mediators, which attenuate microglial response. 26IFN-c stimulates microglia and astrocytes to produce pro-inflammatory cytokines and chemokines. 27However, the net effect of IFN-c signalling in the central nervous system (CNS) is similar to that in the periphery, also anti-inflammatory.Therefore, it is not clear whether the cellular response in Mct1 +/+ HFD HF/HG mice is a pro-or antiinflammatory reaction.Nonetheless, our results show that brain inflammation in diet-induced NAFLD extends to the cortex (beyond the hippocampus and hypothalamus as seen during obesity 28 ), increasing the risk of cerebral damage as it persists.Brain inflammation can also occur when peripheral cytokines communicate with the brain across the intact blood-brain barrier (BBB), by activating the endothelium, which in turn signals to perivascular macrophages inducing further microglial response.
MCT1 is widely expressed in the brain, 29 and its role in promoting microglial response and associated proinflammatory effects by enhancing glycolysis has been shown. 30In other conditions, such as ischaemia, 31 MCT1 expression is upregulated in responsive astrocytes.Therefore, it is possible that the partial invalidation of MCT1 (without compensation from other MCTs; Fig. S8) provides protection by preventing the above response processes.The involvement of the vagus nerve sensory afferents, direct active transport of cytokines across the BBB, compromised vascular permeability and alterations in the gut microbiome in developing brain inflammation, 32 and the possible protective role of MCT1 downregulation in these has not been investigated here but cannot be excluded as contributing factors.
Tissue inflammation and hypoxia share an interdependent relationship in the periphery and CNS during various pathologies. 33NAFLD is characterised by a pro-inflammatory and pro-coagulant state that promotes processes that induce cerebrovascular damage and, consequently, contributes to clinical and subclinical cerebrovascular pathologies. 34  tissue oxygenation, which was only observed in Mct1 +/+ HFD HF/HG mice, that also displayed lower cortical CBV.
In these mice, CBF, which is the product of volume and velocity of blood flowing through the brain, was not significantly decreased.This could be as a result of the early stages of the disease and the compensatory action of high blood pressure (Fig. S9) observed in Mct1 +/+ HFD HF/HG mice.Elevated transmitted cerebral blood velocity (as blood volume was decreased and CBF was constant) results in potentially harmful pulsatile energy to be transferred to the microvasculature leading to damage, contributing to the decreased CBV.Additionally, the observed decrease in vascular density and vessel diameter in the cortex of Mct1 +/+ HFD HF/HG mice could further compromise blood/oxygen supply, contributing to the reported low-grade hypoxia.These vascular alterations may occur owing to volume changes associated with the reported glial responses, either as a compensatory mechanism to preserve the extracellular space volume and avoid oedema or as a consequence of compression.As these vascular alterations were also associated with an increase in average distance between 2 vessels in close proximity (Fig. S6D), the impact of increased oxygen diffusion distance and therefore limited oxygen diffusion range within the tissue, contributing to the observed brain hypoxia, cannot be excluded.Despite this decrease in vessel diameter, cerebrovascular reactivity was preserved, suggesting that remaining vessels are still functional, indicating a potential for restoring brain oxygenation by cerebrovascular dilation, as long as vascular density does not decline dramatically.The additional impact of an altered vascular tone cannot be excluded.The preserved cerebrovascular reactivity also indicates an overall preserved pericyte viability (lack of death in rigor) and function, as they normally respond to CO 2 by inducing capillary dilation, contributing to the increase in CBF and brain oxygenation. 35lthough Mct1 +/− HFD HF/HG animals also have reduced (but not significant) vascular density, they have a normal vessel diameter.In parallel, in vivo electrophysiology recordings (Fig. S10A) indicate a higher neuronal firing rate in these animals.These results together suggest a compensatory mechanism, via the neurovascular coupling, by which the increase in firing activity results in vasodilation during active brain states but returns to normal diameter when CNS depression is achieved by pentobarbital overdose 36 during fixation.Therefore, despite the small decrease in density, CBV and oxygenation are maintained at normal levels in Mct1 +/− HFD HF/HG animals.
Our results also suggest that the observed alterations in glial cells contribute further to (and possibly become exacerbated by) the decrease in brain tissue oxygenation observed in obese Mct1 +/+ animals with NAFLD.Higher respiratory capacity (i.e.oxygen consumption) was recorded in the cortex of these mice, which was eliminated when the measurements were normalised to micrograms of protein per milligram of brain tissue used.As the protein content of OXPHOS complex subunits and proteins involved in mitochondrial remodelling were unchanged, it is likely that only functional changes are responsible for the higher respiratory capacities when normalised to tissue mass.Neurons are unlikely to contribute to the oxygen consumption changes as in vivo extracellular single-unit recordings indicated no differences in cortical neuronal activity between obese Mct1 +/+ animals with NAFLD and Mct1 +/+ NC controls (Fig. S10A and B).However, as neuronal excitability can be slightly altered in Mct1 +/− mice by the diet, it cannot be excluded that this aspect contributes to modifying brain responses to peripheral signals.
Mct1 +/− HFD HF/HG mice without NAFLD showed protection against all cerebral alterations.These mice did not experience brain inflammation and glial alterations, explaining the normal respiratory capacities and oxygen consumption.As Mct1 +/− HFD HF/HG mice develop fat accumulation but not NAFLD, metabolic syndrome, or systemic inflammation, it is suggested that the protection against cerebral alterations is partly caused by the absence of liver disease, emphasising its importance in studies focusing on obesity.Furthermore, because these mice presented less fat accumulation in the adipose tissue compared with Mct1 +/+ HFD HF/HG animals, and because of the absence of metabolic syndrome, it implies that no substantial damage has occurred yet to trigger the damaging inflammatory and cerebrovascular alterations seen in Mct1 +/+ mice, which exhibit higher % fat mass.
MCT1 is found in several other tissues including muscles, adipose tissue, heart, and intestine, all of which are involved in energy homoeostasis regulations 37 and are altered during obesity and NAFLD.These organs can communicate with the brain, and therefore, their contributing role the reported protective phenotype cannot be ruled out when using mice with global haploinsufficiency.Additionally, the partial invalidation of MCT1 has an impact on the immune system in the periphery (considering its role in immune cell function 38 ), and it might participate in the beneficial effects observed by reducing peripheral inflammation and consequently preserving brain function.Similarly, other glial cells in the CNS, such as tanycytes, which have been shown to express MCT1 39 and be implicated in the control of systemic metabolism by acting as glucose 40 and lipid 41 sensors, are also likely to participate in the phenotype of resistance to diet-induced NAFLD.Conditional knockdown models in future studies will allow answers to these questions.Further limitations include the lack of precise mechanistic pathway responsible for the protective phenotype.The impact of other pathological factors, such as ammonia, and the reversibility of NAFLD and induced encephalopathy by partial blockage of MCT1 will need to be investigated further.
In conclusion, this study provides evidence indicating a key role of NAFLD in inducing low-grade brain tissue hypoxia and inflammation, as well as cerebrovascular, glial, metabolic, and behavioural alterations.Such effects are expected to persist chronically or even worsen with disease progression, leading to the early stages of a NAFLD-induced brain dysfunction, while increasing the risk of neurodegenerative conditions, such as Alzheimer's disease, that share the above pathophysiological mechanisms. 42Mct1 haploinsufficient mice, despite fat accumulation in adipose tissue, were protected from NAFLD and the above detrimental cerebral alterations, emphasising the importance of the liver-brain axis in developing cognitive decline observed in obese individuals.Finally, this protective phenotype indicates the potential of MCT1 as a novel therapeutic target for preventing and/or treating NAFLD and the associated multifactorial encephalopathy.

Fig. 1 .
Fig. 1.Cerebral oxygenation and cerebrovascular reactivity in Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG.(A) Grouped data traces of PO 2 measurements and quantification of (B) basal PO 2 and (C) cerebrovascular reactivity to hypercapnia (10% inspired CO 2 ) recorded via optical fluorescence in the somatosensory cortex of Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG.(D) Representative coronal maps of Hb and HbO 2 in vivo with MSOT.Quantification of (E) brain tissue oxygen saturation and (F) CBV in the somatosensory cortex of Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG derived from Hb and HbO 2 measurements.A p value of <0.05 indicates significance level, 1-way ANOVA followed by Tukey's multiple-comparisons post hoc test and the Kruskal-Wallis test followed by Dunn's multiple-comparisons post hoc test.CBV, cerebral blood volume; Hb, deoxygenated haemoglobin; HbO 2 , oxygenated haemoglobin; HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; MCT1, monocarboxylate transporter-1; MSOT, multispectral optoacoustic tomography; NC, normal chow; PO 2 , partial pressure of oxygen.(This figure appears in color on the web.)

Fig. 2 .
Fig. 2. Characterisation of body composition and liver histology of mice fed with NC or HFD HF/HG.(A) Percentage of fat mass and (B) lean mass assessed in Mct1 +/+ and Mct1 +/− mice by EchoMRI at 16 weeks of either NC or HFD HF/HG diet.(C) Total HFD intake over a 16 weeks period per mouse per cage for Mct1 +/+ and Mct1 +/− mice.(D) Quantitative histology analysis indicating percentage of FPA in liver slices of Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG diet.(E) Representative examples of FPA in liver slices of Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG diet.A p value of <0.05 indicates significance level, the Mann-Whitney U test and 1-way ANOVA followed by Tukey's multiple-comparisons post hoc test and the Kruskal-Wallis test followed by Dunn's multiple-comparison post hoc test.FPA, fat proportionate area; HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; MCT1, monocarboxylate transporter-1; NC, normal chow.(This figure appears in color on the web.)

Fig. 3 .
Fig. 3. Assessment of anxiety-and depression-related behaviour in Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG.Measurement of mean (A) speed, (B) distance, and (C) time spent by Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG exploring the centre zone of the arena, assessed by the OF test.(D) Representative heatmaps indicating the time spent by Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG exploring each zone during the OF test.(E) Measurement of time spent floating by Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG during the FST.A p value of <0.05 indicates significance level, the Kruskal-Wallis test followed by Dunn's multiple-comparisons post hoc test and 1-way ANOVA followed by Tukey's multiple-comparisons post hoc test.FST, forced swim test; HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; MCT1, monocarboxylate transporter-1; NC, normal chow; OF, open field.(This figure appears in color on the web.)

Fig. 5 .
Fig. 5. Determination of cortical astrocytic response in Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG.(A) Representative confocal z-stack projections and (B) quantification of percentage area fraction of GFAP positive astrocytes in the cortex of Mct1 +/+ and Mct1 +/− mice fed with NC or HFD HF/HG.p value of <0.05 indicates significance level, one-way ANOVA followed by Tukey's multiple comparisons post hoc test.(This figure appears in color on the web.)