Six-week-old C57BL/6 wild-type male mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China). The mice were housed (five per cage) in a conventional animal room and subjected to a 12:12 h light/dark cycle in low-stress conditions with free access to food and water ad libitum. The mice of the control group were fed a normal diet (ND) containing 10% kcal fat (cat. no. D12450J; Research Diet, New Brunswick, NJ, United States) and regular tap water. The western diet group mice were fed with a liquid high-fat: high-fructose diet (HFHFD) containing 40% kcal fat and 20% kcal fructose (made up with cultured media) (cat. no. D09100308L; Research Diet, New Brunswick, NJ, United States) The food composition and calories were shown in Figure 1A and supplemental material. Additionally, the mice were exposed to IH during their sleeping hours or intermittent room air (IA) as control conditions for 15 weeks, and started treatment with the diet concurrently.

The IH chamber was monitored with an automated gas controller and designed for mimicking different levels of hypoxia exposure by regulating the ratio of the gaseous mixture. As described in our previous work, the IH paradigm was consisted of repeated cycles of 20.9% O₂/8% O2 FiO₂ (30 episodes per hour) with 20 s at the nadir FiO₂ during the 12-h light phase (Liu et al., 2020) (Figure 1B). The mice were divided into four groups: ND + IA, ND + IH, HFHFD + IA, and HFHFD + IH. All the animal procedures were approved by the Animal Experimental Ethical Inspection of the Laboratory Animal Centre of Wenzhou Medical University prior to the start of the study.

The body weight and food consumption of animals were monitored weekly. In the 15th week, the mice were anesthetized with ketamine/xylazine (100/15 mg/kg, i.p.) and sacrificed. Blood was collected from the vena cava by cardiac puncture prior to exsanguination, centrifuged to separate serum, and then stored at −80°C. Epididymal fat pads and liver were harvested and weighed. Portions of the liver, intestine, and adipose tissue were frozen in liquid nitrogen or fixed in 10% neutral buffered formalin for further analysis.

The day before sacrificing, the mice were fasted overnight and underwent a glucose tolerance test (GTT) and insulin tolerance test (ITT). Glucose solution (2 g/kg) and insulin (0.75 U/kg) were administered by intraperitoneal injection. Tail blood was collected at 0, 15, 30, 60 and 120 min after glucose administration, and glucose concentrations were measured using a glucometer and test strips (ACCU-CHEK Performa; Roche, United States). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined by using standard kits purchased from Cusabio (Wuhan, China). Hepatic, adipose or serum lipids including triglyceride (TG), free fatty acid (FFA) and cholesterol, were determined by commercial lipids assay kits (abcam, Cambridge, MA, United States) according to the manufacturer’s protocols. Four-hydroxynonenal (4-HNE) level in the liver was measured by colorimetric assay using an ELISA kit (Cusabio, China). Formalin-fixed mouse liver and epididymal white adipose tissues were embedded in paraffin and sliced at 4.5 μm. Hematoxylin and eosin (H&E) staining was used for tissue morphology assessment. Pathology was scored in a blinded manner. Scoring ranges were as follows: Degree of steatosis (0–3), lobular inflammation (0–3), hepatocyte ballooning (0–2). In addition, the NAFLD activity score (NAS) was scored according to the non-alcoholic steatohepatitis (NASH) clinical research network scoring system (Kleiner et al., 2005). Hepatic lipids were visualized by Oil Red O (ORO) stain (Servicebio, Wuhan, China). Image Pro plus 6.0 was used for ORO staining quantification. Neutrophil infiltration in the liver was assessed by a naphthol AS-D chloroacetate (CAE) kit (Sigma-Aldrich, United States) according to the manufacturer’s directions. Apoptosis was detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; EMD Millipore, Billerica, MA). CAE and TUNEL positive cells were counted by Image J software (NIH, United States) and expressed as the ratio of positive cells per 1,000 hepatocytes.

Protein was extracted from frozen liver and epididymal white adipose tissues (eWAT). Nuclear protein was prepared from mouse livers as described previously (Alam et al., 2017). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA). Western blotting was performed as described previously (Liu et al., 2020). Primary antibodies (1:500–1:1,000) against Phospho-HSL, ATGL, PPAR-γ, CPT-1a, Nrf2, BAX, BCL-2, Adiponectin, Cleaved-Caspase3, pro-Caspase3 and CYP7A1 were from Cell Signaling Technology (MA, United States). β-Actin and Lamin B antibody were the reference of total protein and nuclear protein, respectively, and purchased from Abcam (Cambridge, MA, United States). Densitometric analysis was performed using UN-SAN-IT Gel (Silk Scientific, Orem, UT) software.

Total mRNA was extracted from fresh liver tissue, eWAT and ileum by TRIzol (Invitrogen) reagent according to the manufacturer’s protocol. Total RNA was used for reverse transcription with the cDNA cycle kit (Invitrogen). Primers for the experiment are listed in Table 1. Real-time PCR was performed on the ABI 7300 fast real-time PCR system (Applied Biosystems) using SYBR green PCR Master Mixture (Applied Biosystems). The 2^−△△CT method was used to determine fold differences between the target genes and an endogenous reference (GAPDH).

Quantitative analysis of hepatic bile acids was measured following published methods with modifications (García-Cañaveras et al., 2012; Xie et al., 2013; Xie et al., 2015). Mouse liver homogenate samples or standard solutions were mixed with acetonitrile (1:3, v/v) containing internal standards, then the extractions were centrifuged and the supernatants were combined and dried. The dried residues were resuspended in acetonitrile/methanol (95:5, v/v) and centrifuged at 13,500 g at 4°C for 20 min for further solid-phase extraction. The supernatant was transferred to a 96-well plate for LC-MS analysis. A Waters ACQUITY ultra performance LC (UPLC) system equipped with a binary solvent delivery manager and a sample manager (Waters, Milford, MA) was used throughout the study. UPLC-MS raw data obtained with negative mode were analyzed using TargetLynx applications manager version 4.1 (Waters Corp., Milford, MA) to obtain calibration equations and the quantitative concentration of each bile acid in the samples. Total hepatic bile acids were calculated from the sum of individual bile acid species and normalized to tissue weight.

Results were reported as means ± SEM (N = 4–6) and analyzed using the GraphPad Prism version 6 (GraphPad Software Inc., San Diego, CA, United States). Two-way analysis of variance (ANOVA) with Bonferroni post hoc test or one-way ANOVA with Tukey’s post hoc test or Student’s t-test where it was appropriate. A p-value <0.05 was considered to be a significant difference.

During the period of animal modeling, mice body weight and food consumption were monitored weekly. There was no significant difference in food intake observed in all the groups (Figure 2B). Mice of HFHFD showed significant weight gain from the third week by comparison to their control groups, and IH treatment significantly decreased the mice body weight in both ND and HFHFD groups at the 15th week (Figure 2A). HFHFD increased liver and eWAT weight as expressed by the ratio to body weight. IH decreased liver to body weight ratios in the HFHFD feeding mice, however, the parameter for eWAT was not significantly altered by IH exposure (Figures 2C, D). To assess mice glucose metabolism, we determined GTT and ITT at the end of the experimentation period. HFHFD-fed mice showed a decreased glucose tolerance as represented by elevated peak blood glucose levels at the 15th min time point compared to that of ND mice (Figure 2E). Additionally, HFHFD decreased insulin sensitivity as determined by the ITT measurement (Figure 2F). However, no differences in GTT or ITT were found between HFHFD + IA and HFHFD + IH groups.

General hepatic morphology was visualized by representative microphotographs of H&E staining. Figure 3B showed that normal histology was observed in the ND group, and IH caused no overt pathological change in the presence of ND. Compared with mice in the ND group, significant pathological findings such as marked steatosis, ballooning hepatocytes, lobular inflammation, and NAS were observed in the HFHFD group. IH exposure alleviated these morphological changes and decreased NAS score in the HFHFD feeding mice. Similarly, elevated transaminases including ALT and AST in HFHFD mice were significantly decreased by IH (Figure 3C). Taken together, these data suggested that IH reduced HFHFD-associated liver injury.

Liver sections were stained for ORO to evaluate lipids storage (Figure 4A). HFHFD exacerbated steatosis was alleviated by IH treatment. Quantitative lipids content showed a similar trend to staining. FFA, TG, and cholesterol levels in both liver and serum were increased by HFHFD, while IH significantly decreased these targets (Figure 4B). To investigate the mechanisms of reduced steatosis, we measured hepatic mRNA as well as protein levels of typical enzymes or transcriptional factors involved in lipid metabolism in the group of HFHFD + IA and HFHFD + IH (Figures 4C, D). HSL and ATGL are the key enzymes that catalyze lipolysis (Grabner et al., 2021). Cd36 and Fatp5, are known as the major players that mediate fatty acids uptake (Pohl et al., 2004; Hao et al., 2020). CPT-1α is the rate-limiting enzyme for fatty acid β-oxidation (Schlaepfer and Joshi, 2020). Fasn, Chrebp, Srebp-1c and Scd-1 are the important transcription factors associated with lipogenesis (Yu et al., 2018). PPAR-γ can be activated by Chrebp and participates in lipid synthesis (Witte et al., 2015). Our data showed that IH enhanced the protein expression of p-HSL, AGTL and CPT-1α in the HFHFD group while decreasing PPAR-γ level as determined by western blot. For gene expression levels, IH significantly decreased mRNA expression levels of Fatp5, Chrebp, Srebp-1c and Scd-1 in HFHFD mice, whereas Cd36 and Fasn were not altered by IH (Figures 4C, D). We furtherly measured oxidative stress in the liver. 4-HNE, the main byproduct of lipid peroxidation, was elevated in HFHFD compared to their control mice. IH exposure caused no obvious change in the 4-HNE level of ND mice but reduced it in the mice of HFHFD (Figure 4E). Nrf2, known as the famous antioxidant, was also determined in our work. IH caused no change in total Nrf2 expression in HFHFD liver, but significantly increased nuclear Nrf2 expression level (Figure 4G). Moreover, the well-known Nrf2 downstream target genes, SOD and CAT, were upregulated in the interaction of IH + HFHFD mice (Figure 4F). These data proved that IH exposure protected liver from HFHFD via manipulating lipid metabolism and suppressing hepatic oxidative stress.

The finding of decreased serum FFA levels in HFHFD + IH mice suggested that lipid metabolism in adipose tissue was regulated by IH. Therefore, we determined the general pathological feature of eWAT by H&E staining. HFHFD increased lipid storage in adipose tissue and that was enhanced by IH exposure, which was proved by adipocyte size evaluation (Figure 5A) as well as the quantitative TG level in eWAT (Figure 5B). The major enzymes of fat mobilization in adipose tissue were also measured. IH caused a significant reduction of AGTL protein expression but not p-HSL. And the factors involved in lipogenesis including PPAR-γ, Fasn and Chrebp, were increased by IH in the HFHFD mice, as measured by western blot and rt-qPCR (Figures 5C, D). However, CPT-1α, which served as the critical enzyme for FFA oxidation, was not affected by IH (Figure 5D).

To evaluate liver inflammation, CAE staining and genes involved in inflammatory modulation were performed. Visible CAE-positive staining was observed in the HFHFD + IA group, which was also reflected in cell counting. IH exposure significantly increased neutrophil recruitments in the absence of HFHFD, while it showed a robust reduction in neutrophil infiltration in the mice of HFHFD (Figure 6A). CXCL1 is the main chemokine that controls neutrophil recruitment and contributes to inflammation. A decreased mRNA expression level of CXCL1 was shown in the HFHFD + IH compared to HFHFD + IA mice. MCP-1 is the key chemokine that regulates the migration and infiltration of monocytes/macrophages. In the HFHFD + IH mice, the expression of MCP-1 was not statistically different from that of HFHFD + IA mice. The pro-inflammatory cytokines including IL-6 and TNFα but not IL-1β were inhibited by IH exposure in mice treated with HFHFD. In addition, IH reduced other inflammatory mediator gene levels such as BNIP3 in the presence of HFHFD, but no significant change in the HIF-1a expression was observed in these groups (Figure 6B). Furthermore, we measured adipokines expression in the eWAT for inflammation assessment. The relative protein level of adiponectin was not affected by IH in the HFHFD mice (Figure 6C). And the mRNA levels of pro-inflammatory adipokines TNFα, IL-6 and ANGPTL2, were not significantly different between HFHFD + IA and HFHFD + IH (Figure 6D).

For histologic analysis of apoptosis, representative photomicrographs of the TUNEL staining in the liver and positive cell counting were performed in Figure 7A. HFHFD increased TUNEL-positive stained cell counts of hepatocytes and non-parenchymal cells compared to ND. IH caused a severer degree of TUNEL staining in the ND mice but moderated this phenomenon in the HFHFD (Figure 7A). For assessment of cell death pathway, Caspase3, BAX and BCL-2 protein levels were analyzed by western blot. IH decreased caspase3 cleavage and BAX expression in the liver of HFHFD mice, while did no significant change in pro-caspase3 and BCL-2 protein levels. The ratio of cleaved-to pro-caspase3 and the ratio of BAX to BCL-2 were downregulated by IH exposure in the HFHFD (Figure 7B), indicating that IH decreased apoptosis caused by HFHFD.

Bile acid serves critical physiological functions in regulating whole-body metabolism especially lipid homeostasis in the liver. The heatmap of liver bile acid profiles showed that IH exposure with HFHFD decreased some of the differential bile acids compared to that of HFHFD + IA mice (Figure 8A), but the total bile acids content from the sum of individual bile acid species in HFHFD + IH was not statistically different from that in the HFHFD + IA liver (Figure 8B). Nuclear FXR is identified as a ligand-activated transcription factor and biological sensor for metabolic regulation. FXR directly activates SHP and FXR/SHP axis plays a role in modulating lipid homeostasis (Watanabe et al., 2004; Kim et al., 2017). However, neither FXR nor SHP mRNA expression was different between HFHFD + IA and HFHFD + IH mice (Figure 8E). Bile acids such as TCA, TCDCA, TDCA, DCA, CDCA, and CA, act as the potent agonists of FXR, whereas TUDCA, THDCA, UDCA, TMCAs, and MCAs function as FXR antagonists (Tu et al., 2000; Wang et al., 2008; Feng et al., 2011; Kumari et al., 2020). Administration of IH altered the composition of bile acids pool in HFHFD, as shown by decreased concentration of TCA, CA, TaMCA, wMCA, bMCA and UDCA (Figure 8B). As a result, IH rose the percentage of FXR agonistic bile acids in total bile acids content from 23.2% to 31.4%. The ratio of FXR agonistic to FXR antagonistic bile acids species in HFHFD + IH performed approximately 1.5-fold of that in HFHFD + IA (Figure 8C), suggesting that IH yielded bile acids pool prone to be, in sum, more FXR agonistic. To determine the basis for the altered bile acids profile, the protein expression of CYP7A1, responsible for bile acid synthesis, and mRNA of ileal ASBT which is critical for bile acids absorption, were detected by western blot and qPCR, respectively. In the HFHFD mice, IH showed a statistically increased CYP7A1 expression in the liver and decreased ASBT gene level in the ileum. (Figure 9). These data implied us bile acids metabolism regulation was involved in the interaction of IH and HFHFD.