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Corresponding authors. Addresses: The Roger Williams Institute of Hepatology London, Foundation for Liver Research, London, UK. Tel.: +44 207 255 9852.
Department of Biomedical Sciences, University of Lausanne, Lausanne, SwitzerlandThe Roger Williams Institute of Hepatology London, Foundation for Liver Research, London, UKFaculty of Life Sciences and Medicine, King’s College London, London, UK
The Roger Williams Institute of Hepatology London, Foundation for Liver Research, London, UKFaculty of Life Sciences and Medicine, King’s College London, London, UK
Institute for Biomedical Engineering, University of Zurich and ETH Zurich, Zurich, SwitzerlandNeuroscience Centre Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland
Department of Biomedical Sciences, University of Lausanne, Lausanne, SwitzerlandInstitute of Sports Science, University of Lausanne, Lausanne, Switzerland
The Roger Williams Institute of Hepatology London, Foundation for Liver Research, London, UKFaculty of Life Sciences and Medicine, King’s College London, London, UK
The Roger Williams Institute of Hepatology London, Foundation for Liver Research, London, UKFaculty of Life Sciences and Medicine, King’s College London, London, UK
Diet-induced NAFLD and associated systemic alterations result in behavioural changes and low-grade brain tissue hypoxia.
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Brain hypoxia is likely linked to the induced low-grade brain inflammation, as well as cerebrovascular, glial, and metabolic alterations.
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Mct1 haploinsufficient mice are protected from NAFLD and detrimental cerebral alterations.
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MCT1 is a potential novel therapeutic target for preventing and/or treating NAFLD and the associated multifactorial encephalopathy.
Background & Aims
Non-alcoholic fatty liver disease (NAFLD) has been associated with mild cerebral dysfunction and cognitive decline, although the exact pathophysiological mechanism remains ambiguous. Using a diet-induced model of NAFLD and monocarboxylate transporter-1 (Mct1+/−) haploinsufficient mice, which resist high-fat diet-induced hepatic steatosis, we investigated the hypothesis that NAFLD leads to an encephalopathy by altering cognition, behaviour, and cerebral physiology. We also proposed that global MCT1 downregulation offers cerebral protection.
Methods
Behavioural tests were performed in mice following 16 weeks of control diet (normal chow) or high-fat diet with high fructose/glucose in water. Tissue oxygenation, cerebrovascular reactivity, and cerebral blood volume were monitored under anaesthesia by multispectral optoacoustic tomography and optical fluorescence. Cortical mitochondrial oxygen consumption and respiratory capacities were measured using ex vivo high-resolution respirometry. Microglial and astrocytic changes were evaluated by immunofluorescence and 3D reconstructions. Body composition was assessed using EchoMRI, and liver steatosis was confirmed by histology.
Results
NAFLD concomitant with obesity is associated with anxiety- and depression-related behaviour. Low-grade brain tissue hypoxia was observed, likely attributed to the low-grade brain inflammation and decreased cerebral blood volume. It is also accompanied by microglial and astrocytic morphological and metabolic alterations (higher oxygen consumption), suggesting the early stages of an obesogenic diet-induced encephalopathy. Mct1 haploinsufficient mice, despite fat accumulation in adipose tissue, were protected from NAFLD and associated cerebral alterations.
Conclusions
This study provides evidence of compromised brain health in obesity and NAFLD, emphasising the importance of the liver–brain axis. The protective effect of Mct1 haploinsufficiency points to this protein as a novel therapeutic target for preventing and/or treating NAFLD and the associated brain dysfunction.
Impact and implications
This study is focused on unravelling the pathophysiological mechanism by which cerebral dysfunction and cognitive decline occurs during NAFLD and exploring the potential of monocarboxylate transporter-1 (MCT1) as a novel preventive or therapeutic target. Our findings point to NAFLD as a serious health risk and its adverse impact on the brain as a potential global health system and economic burden. These results highlight the utility of Mct1 transgenic mice as a model for NAFLD and associated brain dysfunction and call for systematic screening by physicians for early signs of psychological symptoms, and an awareness by individuals at risk of these potential neurological effects. This study is expected to bring attention to the need for early diagnosis and treatment of NAFLD, while having a direct impact on policies worldwide regarding the health risk associated with NAFLD, and its prevention and treatment.
We thank Sandforth and colleagues for their interest in our study.1,2 In their letter to the editor, Sandforth et al., refer to “body weight data missing in the report”. However, they do not indicate what would be the added value of reporting such data. Body weight was determined weekly, but these data were not presented as considered not of interest in the present context. More relevant to the focus of the article are the differences in fat mass. We would like to kindly point to Fig. 2A-B in our original paper, which depicts % fat mass and lean mass assessed by EchoMRI at the end of the 16-week feeding protocol, the time point at which all experiments were performed.
We read with great interest the spectacular work of Hadjihambi and colleagues, published in August 2022 online ahead of print in the Journal.1 The authors aimed to establish the role of non-alcoholic fatty liver disease (NAFLD) in the development of brain dysfunction. To establish this role, the authors used a mouse model that they generated more than 10 years ago,2 monocarboxylate transporter-1 haploinsufficient (Mct1+/−) mice. MCT1 or SLC16A1 is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues, including the liver, brain and adipose tissue, playing an important role in energy homeostasis in health and disease, including obesity, type 2 diabetes and cancer.
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 liver-related morbidity and mortality.
Indeed, the most common liver-related 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.
It 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
However, 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.
Therefore, any changes in these mechanisms or the structure of the cerebrovascular system can have detrimental effects on brain tissue oxygenation and overall physiology.
Several 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.
Recently, it has been observed that hypoxia not only directly induces neuronal damage, but also initiates cerebral inflammation through microglial and astrocytic responses.
Toxic 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.
Growing evidence implicates low-grade chronic systemic inflammation and consequently brain inflammation in the pathogenesis of diet-induced obesity and cerebral dysfunction.
However, 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.
As 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.
In 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.
were bred in the animal facility of the Biomedical Sciences department at the University of Lausanne, Switzerland, to produce male Mct1+/− and Mct1+/+ littermate controls. Animals were group-housed in individually ventilated cages, enriched with rails and cardboard tubes, in a room of 20–22 °C, relative moisture 50–60%, and 12-h light–dark cycle (light 7 a.m. to 7 p.m.). At 8 weeks old, mice were given ad libitum access to either a standard diet of normal chow (NC; 3242.PX.F12, Granovit, Kaiseraugst, Switzerland) and water or a HFD (Cat no. TD.93075.PWD; adjusted calories diet [55% fat], Envigo, Harlan Teklad, Indianapolis, Indiana, USA) with fructose and glucose in their water (HFD HF/HG; 23.1 g/L d-fructose [10021753, Axonlab, Baden, Switzerland] + 18.9 g/L d-glucose [A3666,1000, PanReac AppliChem, Darmstadt, Germany]) for 16 weeks.
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.
Behavioural experiments
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 (PO2) 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 PO2 sampling was started until a stable reading was achieved.
Blood gas manipulations
After baseline PO2 was recorded, systemic hypercapnia was induced by 10% CO2 inhalation (in 21% O2 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 (HbO2) maps using ImageJ (NIH, USA). ROIs for each region were averaged. Tissue oxygenation as a measure of tissue oxygen saturation (SO2) of the ROIs was calculated by the following:
Fig. 1Cerebral oxygenation and cerebrovascular reactivity in Mct1+/+ and Mct1+/− mice fed with NC or HFD HF/HG.
(A) Grouped data traces of PO2 measurements and quantification of (B) basal PO2 and (C) cerebrovascular reactivity to hypercapnia (10% inspired CO2) 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 HbO2in 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 HbO2 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; HbO2, 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; PO2, partial pressure of oxygen. (This figure appears in color on the web.)
Cerebral blood volume (CBV) was calculated by the following:
High-resolution respirometry
Brain homogenates (somatosensory cortex) were prepared in prechilled MiRO5 buffer and transferred into calibrated Oxygraph-2k (O2k, Oroboros Instruments, Innsbruck, Austria) 2-ml chambers, where oxygen concentration (μM) and oxygen flux per tissue mass (pmol O2 s−1 mg−1) were recorded. The established substrate, uncoupler, inhibitor titration (SUIT) protocol SUIT-008 (https://www.mitofit.org/index.php/SUIT-008) was used (Fig. S1).
Respiratory capacities were expressed as oxygen consumption per wet mass of tissue (pmol s−1 mg−1) or per protein mass (pmol s−1 μg−1) and corrected for residual oxygen consumption (ROX).
Immunofluorescence staining
Mice were terminally anaesthetised with sodium pentobarbital (150 mg/kg i.p.) and transcardially perfused with ice-cold 0.9% sodium chloride solution followed by 4% paraformaldehyde (PFA) at 4 °C. Brain and liver were removed, post-fixed in PFA overnight, and kept in PBS until processed. Liver slices were stained for H&E (Supplementary material). Surface of lipid droplets was measured, and fat proportionate area (FPA; % of total tissue surface) was calculated. Immunofluorescence labelling of microglia and astrocytes was performed on 60 μm brain slices. Sections were incubated overnight at 4 °C with primary antibodies (ionised calcium binding adaptor molecule 1 [Iba1], 1:1,000, 019-19741, FUJIFILM Wako, Neuss, Germany; glial fibrillary acidic protein [GFAP], 1:500, Z0334, Dako, Santa Carla, California) and incubated with secondary antibodies (2 h at room temperature; Alexa Fluor Plus 555, Donkey anti-Rabbit, 1:1000, A32794, Thermo Fisher, Basel, Switzerland). Nuclei were labelled by incubation with Hoechst 33342 (1:10,000; ab228551; Abcam, Cambridge, UK) for 10 min at room temperature. Sections were imaged using a Zeiss LSM 700 confocal microscope (Jena, Germany) at 20× and 60× magnification. Semi-automated analysis was performed using ImageJ on 20× magnification images evaluating cell density and % area fraction of Iba1/GFAP positive cells. Confocal stacks acquired with 60× magnification were reconstructed in 3D using Imaris software (Bitplane, Schlieren, Switzerland) based on Iba1 intensity, and volume of individual cells was assessed.
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 1-way or 2-way ANOVA followed by Tukey's multiple-comparisons 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.
Sample sizes were calculated by Gpower 3 v3.1.9.2 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) using a ‘means: ANOVA (2 groups)’ test, with a desired power of 90% and a significance level of 5%. The effect size was calculated from brain oxygen measurements performed in a preliminary study (n = 3). Based on this calculation, n = 6–10 was used. The same calculations were performed for other experiments, when possible, to determine appropriate n numbers.
Results
Animal model characterisation
Following 16 weeks of HFD HF/HG administration, a significant increase in % fat mass was observed in Mct1+/+ (n = 25, 36 ± 1%, p <0.0001) and Mct1+/− (n = 24, 25 ± 2%, p <0.0001) animals, compared with that in their analogous NC controls (Mct1+/+ NC, n = 19, 12 ± 1%; Mct1+/− NC, n = 15, 11 ± 1%), with no changes in lean mass and total food intake between the groups (Fig. 2A–C). However, % fat mass in Mct1+/- HFD HF/HG animals was lower compared with Mct1+/+ HFD HF/HG animals (p <0.0001).
Fig. 2Characterisation 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.)
Liver histology confirmed the presence of hepatic steatosis (Fig. 2D–E) only in Mct1+/+ HFD HF/HG mice, which was accompanied by an increase in total brain lipids (Table S1), plasma cytokines (Fig. S2D–H), obesity-related endocrine abnormalities (increase in leptin and insulin and decrease in glucagon-like peptide-1 [GLP-1]) compared with Mct1+/+ NC controls (Fig. S2A–C), recapitulating features observed in obese individuals.
systemic inflammation, and hormonal abnormalities (Fig. 2D and E and Fig. S2), despite substantial adiposity.
Mct1+/+ but not Mct1+/− animals on HFD HF/HG experience a NAFLD-associated anxiety- and depression-related 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 ± 5 s, p = 0.005) compared with Mct1+/+ NC controls (n = 10, 93 ± 12 s; Fig. 3C and D). Mct1+/− HFD HF/HG mice without NAFLD did not reveal the same anxiety-like behaviour, as they spent significantly more time exploring the centre zone (n = 8, 90 ± 15 s, p = 0.01) compared with Mct1+/+ HFD HF/HG mice, which was not different from MCT+/− NC controls (n = 7, 72 ± 11 s, p = 0.7; Fig. 3C and D).
Fig. 3Assessment 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.)
In the FST, obese Mct1+/+ HFD HF/HG animals with NAFLD exhibited a significantly longer floating duration (n = 10, 84 ± 7 s, p = 0.01) compared with Mct1+/+ diet controls (n = 12, 51 ± 6 s; Fig. 3E), indicating a depression-related behaviour. Mct1+/- HFD HF/HG animals without NAFLD showed protection against depression development despite high fat mass, as seen by lower floating duration (n = 7, 6 ± 3 s, p <0.0001), compared with Mct1+/+ HFD HF/HG mice, which was not significantly different from Mct1+/− NC controls (n = 11, 37 ± 9 s, p = 0.05; Fig. 3E). These results are not attributable to changes in swimming abilities, which were not different between groups (Fig. 4C). No learning and memory deficits were detected in these mice (Fig. S3), in agreement with other animal studies.
(A) Representative confocal z-stack projections and (B) 3D reconstruction of Iba1-positive microglial cells in the cortex of Mct1+/+ and Mct1+/− mice fed with NC or HFD HF/HG. Quantification of (C) density, (D) percentage area fraction, and (E) volume of Iba1-positive microglia in the cortex of Mct1+/+ and Mct1+/− mice fed with NC or HFD HF/HG. 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. HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; Iba1, ionised calcium binding adaptor molecule 1; MCT1, monocarboxylate transporter-1; NC, normal chow. (This figure appears in color on the web.)
In the cortex of Mct1+/+ HFD HF/HG mice, responsive astrocytes were detected by an increase in % area fraction of GFAP-positive cells (n = 6, 46 ± 6%, p <0.0001) compared with Mct1+/+ NC controls (Fig. 5; n = 6, 10 ± 1%, p <0.0001). Like microglia, % area fraction of astrocytes was lower in Mct1+/- HFD HF/HG animals (n = 6, 8 ± 3%, p <0.0001) compared with Mct1+/+ HFD HF/HG mice and not different from Mct1+/- NC controls (Fig. 5; n = 6, 7 ± 0.9%, p = 0.9).
Fig. 5Determination 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.)
Mct1+/+ HFD HF/HG mice also displayed an elevation in some cytokines (interferon gamma [IFN-γ] and IL-10 [Fig. S4]; no other cytokine alterations detected [Supplementary material]).
Brain oxygenation is compromised in obese Mct1+/+ mice with NAFLD, but not in Mct1+/− animals
Using an optic fibre oxygen sensor positioned above the somatosensory cortex, a lower PO2 was measured in obese Mct1+/+ HFD HF/HG mice with NAFLD (n = 10, 18 ± 2 mmHg, p = 0.02) compared with Mct1+/+ NC controls (n = 8, 25 ± 2 mmHg; Fig. 1B). In contrast, Mct1+/− HFD HF/HG mice presented a similar PO2 (n = 8, 30 ± 2 mmHg, p = 0.9) to Mct1+/- NC controls (n = 8, 30 ± 2 mmHg), which was significantly higher than in Mct1+/+ HFD HF/HG animals (p = 0.0001, Fig. 1B). To evaluate the ability of cerebral vessels to respond to a known vasodilatory stimulus, hypercapnic acidosis was induced by changing the inspired gas mixture to include 10% CO2, which led to a significant increase in parenchymal PO2 from baseline in all groups (p <0.05; Fig. 1A and C).
To exclude the impact of an acute inflammatory response following craniotomy, non-invasive MSOT was used to measure brain tissue oxygenation. Mct1+/+ HFD HF/HG mice with NAFLD had a significantly lower SO2 (n = 3, 6 ROIs/animal, 35 ± 4%, p = 0.047) compared with Mct1+/+ NC controls (n = 5, 6 ROIs/animal, 51 ± 1%; Fig. 1E). Mct1+/- HFD HF/HG mice had normal levels of SO2 (n = 7, 6 ROIs/animal, 50 ± 1%, p = 0.06) compared with Mct1+/- NC controls (n = 6, 6 ROIs/animal, 54 ± 1%), which were higher than those of Mct1+/+ HFD HF/HG animals (p = 0.006; Fig. 1E).
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). Mct1+/- HFD HF/HG mice had normal CBV (3 ± 0.1 a.u.), similar to Mct1+/− NC controls (2 ± 0.1 a.u., p = 0.5), which was higher than that of Mct1+/+ HFD HF/HG animals (p = 0.0001; Fig. 1F). Vascular density and vessel diameter were not significantly different between Mct1+/- HFD HF/HG and Mct1+/− NC animals (Fig. S6A–C).
Fig. 6Determination of cortical mitochondrial respiratory capacities by high-resolution respirometry in Mct1+/+ and Mct1+/− mice fed with NC or HFD HF/HG.
(A) Tissue mass and (B) protein mass specific oxygen fluxes in somatosensory cortical tissue samples from Mct1+/+ and Mct1+/− mice fed with NC or HFD HF/HG: OXPHOS capacity (NP and NSP) and E capacity (NSE). A p value of <0.05 indicates significance level, 2-way ANOVA followed by Tukey's multiple-comparisons post hoc test. E, electron transfer; HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; MCT1, monocarboxylate transporter-1; NC, normal chow; NP, NADH pathway OXPHOS capacity; NS, NADH and succinate-linked pathway; OXPHOS, oxidative phosphorylation; P, OXPHOS.
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 high-resolution respirometry.
For tissue mass-specific fluxes (Fig. 6A), only Mct1+/+ HFD HF/HG mice showed a significantly higher NADH pathway oxidative phosphorylation (OXPHOS) (P) capacity (NP) but no differences in OXPHOS (NSP) respiratory capacities compared with NC controls. Upon stepwise titration of the uncoupler carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), there was a significantly higher combined NADH and succinate-linked pathway (NS) electron transfer (E) capacity in Mct1+/+ HFD HF/HG mice compared with controls and Mct1+/− HFD HF/HG mice, which were not different from Mct1+/− NC controls.
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 μg of protein/mg of brain tissue used (Fig. 6B).
Discussion
Individuals with NAFLD have been reported to experience cognitive impairment,
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
Is fatty liver associated with depression? A meta-analysis and systematic review on the prevalence, risk factors, and outcomes of depression and non-alcoholic fatty liver disease.
(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,
Indeed, 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
) 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 low-grade chronic response. IL-10, an anti-inflammatory cytokine, redirects active astrocytes to produce mediators, which attenuate microglial response.
However, the net effect of IFN-γ 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 anti-inflammatory 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
), 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.
Expression of monocarboxylate transporter mRNAs in mouse brain: support for a distinct role of lactate as an energy substrate for the neonatal vs. adult brain.
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,
NAFLD 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.
Using 2 lines of evidence (optical fluorescence and MSOT), we demonstrated that NAFLD is associated with reduced cortical 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 CO2 by inducing capillary dilation, contributing to the increase in CBF and brain oxygenation.
Although 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
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
and are altered during obesity and NAFLD. These organs can communicate with the brain, and therefore, their contributing role in 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
), 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
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.
Mct1 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.
Abbreviations
BBB, blood–brain barrier; CBF, cerebral blood flow; CBV, cerebral blood volume; CNS, central nervous system; E, electron transfer; FCCP, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; FPA, fat proportionate area; FST, forced swim test; GFAP, glial fibrillary acidic protein; GLP-1, glucagon-like peptide-1; Hb, deoxygenated haemoglobin; HbO2, oxygenated haemoglobin; HFD, high-fat diet; HFD HF/HG, HFD with high fructose/glucose in water; Iba1, ionised calcium binding adaptor molecule 1; IFN-γ, interferon gamma; MCT1, monocarboxylate transporter-1; MRI, magnetic resonance imaging; MSOT, multispectral optoacoustic tomography; N, NADH-linked pathway; NAFLD, non-alcoholic fatty liver disease; NC, normal chow; NMR, nuclear magnetic resonance; NP, NADH pathway OXPHOS capacity; NS, NADH and succinate-linked pathway; O2k, Oxygraph-2k; OF, open field; OXPHOS, oxidative phosphorylation; P, OXPHOS; PFA, paraformaldehyde; PO2, partial pressure of oxygen; ROI, region of interest; ROX, residual oxygen consumption; S1FL, somatosensory (forelimb) region of the cortex; SO2, tissue oxygen saturation; SUIT, substrate, uncoupler, inhibitor titration.
Financial support
This study was supported by the internal funding of UNIL allocated to LP and the allocated project funding for AH from The Roger Williams Institute of Hepatology, Foundation for Liver Research. CD was supported by the Swiss National Science Foundation under grant agreement nº 194964.
Authors’ contributions
Study concept and design: AH, LP. Acquisition of data: AH, CK, JK, ALG, PSH, SL. Analysis and interpretation of data: AH, JK, CD, IJC, KM, RCP. Drafting of the manuscript: AH. Critical revision of the manuscript for important intellectual content: AH, LP, RJ. Statistical analysis: AH. Obtained funding: AH, LP. Administrative, technical, or material support: CK, CD, MM, RJ, AK, US, RCP. Study supervision: AH, LP.
Data availability statement
The data that support the findings of this study are available from the corresponding authors upon reasonable request.
Conflicts of interest
RJ has research collaborations with Takeda and Yaqrit and consults for Yaqrit. RJ is the founder of Yaqrit Limited, which is developing UCL inventions for treatment of individuals with cirrhosis. RJ is an inventor of ornithine phenylacetate, which was licensed by UCL to Mallinckrodt. He is also the inventor of Yaq-001, DIALIVE, and Yaq-005, the patents for which have been licensed by his University into a UCL spinout company, Yaqrit Ltd. All other authors report no conflicts of interest.
Please refer to the accompanying ICMJE disclosure forms for further details.
Acknowledgements
The authors are grateful to the colleagues from UNIL, Dr Nadège Zanou, Dr Sylviane Lagarrigue, Cendrine Repond, Dr Marie-Laure Possovre, Dr Charlotte Jollé, and Cathy Gouelle, for their help with animal husbandry and knowledge exchange, as well as technical advice and stimulating discussions about the project. The authors would also like to acknowledge Mark Augath and Dr Ruiqing Ni for their technical support with MRI and multispectral optoacoustic tomography performed at ETH, as well as Dr Alexandre Sarre and Dr Corrine Berthonneche for their services of blood pressure measurements performed at UNIL. We are also very grateful to Prof. Alexander Gourine, who provided the equipment for the optical fluorescence oxygen measurements, as well as Dr Antonio Riva for his advice on cytokine selection. We thank the Wohl Cellular Imaging Centre at King’s College London for help with confocal microscopy. Finally, we are thankful to Prof. Bengt Kayser and Prof. Nicolas Place (Institute of Sports Science and Department of Biomedical Sciences, University of Lausanne, Lausanne 1005, Switzerland) for their provision of the O2k machine and reagents used in the Oroboros experiments, as well as Stijn den Daas and Dr Svetlana Mastitskaya for their efforts on the ImageJ plug in and antibody selection.
Supplementary data
The following are the supplementary data to this article:
Is fatty liver associated with depression? A meta-analysis and systematic review on the prevalence, risk factors, and outcomes of depression and non-alcoholic fatty liver disease.
Expression of monocarboxylate transporter mRNAs in mouse brain: support for a distinct role of lactate as an energy substrate for the neonatal vs. adult brain.
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