To explore the hypobaric hypoxia influence on lipid metabolism, we first built a hypobaric hypoxia animal model using Sprague-Dawley male rats, following previous studies. Serum alanine aminotransferase (ALT) and blood glucose (BG) were not significantly changed after exposure to hypobaric hypoxia (Fig. 1C and D). However, serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were significantly affected by hypobaric hypoxia treatment. Interestingly, cholesterol and TG had the highest levels (p < 0.05) after exposure for 3 days, and were restored to normal levels on day 30 (Fig. 1E and F). At the same time, HDL showed an opposite trend and decreased on day 3, after which it increased gradually to normal levels on day 30 (Fig. 1G). The LDL levels also increased, with the largest effects being evident on day 3 (p < 0.05) (Fig. 1H), which is consistent with a previous study [16].

Effects of the Hypobaric Hypoxia treatment on rat biochemical parameters. A Schematic diagram of the hypobaric hypoxia rat and cell model. B-H, different biochemical parameters after hypobaric hypoxia and Fenofibrate treatment. There is an observable increase in CHO, TG and LDL after hypobaric hypoxia treatment. At the same time, the parameters weight, GLU and HDL significantly decrease. ALT: Serum alanine aminotransferase, GLU: blood glucose, TG: serum triglycerides, CHO: total cholesterol, LDL: low-density lipoprotein, HDL: high-density lipoprotein. H: Hypobaric hypoxia treatment, H + F: Hypobaric hypoxia and Fenofibrate treatment. Student’s t-test, paired tail, * < 0.05

It has been well established that PPARA and ANGPTL4 are important regulators of lipid metabolism [17, 18]. To assess whether the PPARA-ANGPTL4 pathway also regulates lipid metabolism caused by hypobaric hypoxia, we applied Fenofibrate, a PPARA specific agonist, to evaluate changes in biochemical parameters and lipid profiles (Fig. 1A). Our results showed that the PPARA agonist relieved the effects caused by hypobaric hypoxia on lipid metabolism (Fig. 1 B-H). Importantly, lipid profile results demonstrated that hypobaric hypoxia plays an important role in lipid metabolism and that different lipid components change dynamically at different stages of hypobaric hypoxia. The PPARA-ANGPTL4 pathway may be central regulator of lipid metabolism.

To test whether PPARA and ANGTL4 are involved in regulating lipid metabolism during hypobaric hypoxia, we built a hypobaric hypoxia model using the liver cell line HL-7702. We evaluated the expression levels of both PPARA and ANGTL4 at 4 h, 8 h, and 24 h. The qRT-PCR results demonstrated ANGPTL4 and PPARA are significantly up- and down-regulated after hypobaric hypoxia treatment, respectively (Fig. 2A and B). These observations are consistent with previous studies [18, 19] and support the idea that the PPARA-ANGPTL4 pathway plays a role on lipid metabolism. We further investigated the detailed mechanism behind lipid metabolism associated with hypobaric hypoxia by scanning differential metabolic genes and pathways in across the entire genome. Considering the importance of the liver in lipid metabolism, including its association with lipid biosynthesis, lipoprotein secretion, and cholesterol transport, we exposed a normal human liver cell line (HL-7702) to hypobaric hypoxia for 24 hours. This allowed us to identify the key genes regulating lipid metabolism using microarrays (Fig. 1A). We detected a total of 49, 395 transcripts in our microarray data and an almost perfect correlation between the different replicates (Fig. 2A). Microarray analysis identified differentially expressed genes with log2FC in expression levels (p < 0.05). We combined the replicated data and identified 528 and 765 genes with significantly down-regulation and up-regulation, respectively, in the hypobaric hypoxia treated samples (Fig. 2B and Table S1). Furthermore, we obtained a larger number of genes related to lipid metabolism, including CPT1, ANGPTL4, and LPL. Notably, we found PPARA expression levels are significantly decreased after hypobaric hypoxia treatment (p < 0.01), while ANGPTL4 shows a different trend (Fig. 2C). These results indicate PPARA and ANGPTL4 may be key regulators of metabolism caused by hypobaric hypoxia.

Differentially expressed genes after Hypobaric Hypoxia treatment in HL-7702. A and B Relative mRNA and protein expression levels of PPARA and ANGPTL4 after hypobaric hypoxia treatment. PPARA was down-regulated after hypobaric hypoxia treatment and inhibited mostly at 24 h. ANGPTL was up-regulated after hypobaric hypoxia treatment and expressed at the highest level at 8 h. C Heatmap of differentially expressed genes after hypobaric hypoxia treatment. We obtained 528 and 765 significantly down- and up-regulated genes, respectively, in the hypobaric hypoxia treated samples. The heatmap results demonstrated our data has high reproducibility. The red frame represents the genes up-regulated by ANGPTL4

The Hypobaric hypoxia-induced decrease in PPARA expression levels attenuates the inhibition of ANGPTL4 [20]. Accordingly, we aimed to investigate which novel pathways and genes may be involved in the lipid metabolism mediated by ANGPTL4. Hence, we analyzed differentially expressed genes using an ingenuity pathway analysis (IPA) and obtained numerous enriched pathways (Fig. 3A and Table S2), including cancer, cellular growth and proliferation (Fig. 3A and B), PPAR Regulation of Inflammatory Signaling, LXR/RXR Activation and β-adrenergic signaling. These enriched pathways were further observed to show ANGPTL4 is involved in lipid metabolism and might be, as previous studies proposed, an important cancer regulator [21,22,23]. Notably, we also observed a high number of novel pathways are seemingly associated with lipid metabolism. For example, interferon signaling, ErbB2-ErbB3 signaling, PPARα/RXRα activation, fatty acid synthesis, and NF-κB activation are shown in Fig. 3B. Importantly, we also identified an immune response pathway that is mediated by HMGB1 and associated with inflammation, cell differentiation and tumor cell migration. We hypothesize that a crosstalk between the PPARA-ANGPTL4 pathway and cancer related pathways occurs during the regulation of lipid metabolism. The enriched pathways obtained from the differentially expressed genes provide detailed information on the genes likely participating in lipid metabolism and advance our understanding of this process as caused by hypobaric hypoxia.

Ingenuity pathway analysis (IPA) and enriched pathways for differentially expressed genes after Hypobaric Hypoxia treatment. A Differentially, expressed genes due to hypobaric hypoxia. Enriched pathways from the IPA results include cancer, immunity and lipid metabolism. B Regulation network obtained from the IPA results. C Detail of HMGB1 related pathway in the IPA results. Several immune response-related genes, such IL-1R, MAPK and ERK, are involved in the regulation of hypobaric hypoxia

To evaluate the role of PPARA-ANGPTL4 in the regulation of lipid metabolism, we first validated the expression levels of hypobaric hypoxia liver tissues by qRT-PCR. Our results are consistent with those observed in HL-7702 cell line sequencing data (Fig. 2A), with significant changes occurring on days 3 and 15 after hypobaric hypoxia treatment (Fig. 4A). We then tested the effects of PPARA and ANGPTL4 on key lipid components using fenofibrate, which is a PPARA agonist that activates PPARA and inhibits ANGPTL4 expression [24, 25]. The qRT-PCR results demonstrated ANGPTL4 is mostly down-regulated on day 3 post-fenofibrate treatment, and restored to normal levels on day 15 (Fig. 4A). In addition, PPARA mRNA levels did not significantly changed compared to the hypobaric hypoxia group without fenofibrate treatment (Fig. 4A). Immunohistochemistry staining on rat liver tissues also confirmed hypobaric hypoxia treatment significantly increased the expression of ANGPTL4, and that subsequent treatment with fenofibrate inhibited ANGPTL4 by activating PPARA (Fig. 4B).

ANGPTL4 and PPARA are key regulators of lipid metabolism following Hypobaric Hypoxia. A The ANGPTL4 and PPARA relative mRNA levels in hypobaric hypoxia rats. ANGPTL4 was up-regulated to the highest level at day 3, while PPARA was down-regulated to the lowest level at day 3. This trend was abated by Fenofibrate, which is consistent with results obtained in HL-7702 cells. Student’s t-test, paired tail, * < 0.05. B Immunohistochemistry of ANGPTL4 in mouse liver tissues. ANGPTL4 was up-regulated due to hypobaric hypoxia treatment and inhibited by Fenofibrate. C Electron microscopic biopsy of rat livers in different groups. Hypobaric hypoxia treatment increases the amount of lipid droplets, while fenofibrate treatment significantly attenuates these changes due to the inhibition of ANGPTL4. D Statistical results for the lipid droplets. Hypobaric hypoxia treated rat livers contain a higher number of lipid droplets. This number decreases following fenofibrate treatment

We further investigated the influence of ANGPTL4 inhibition on lipid metabolism. Rats treated with fenofibrate showed a reduced body weight, TG, TC, HDL and LDL compared with the hypobaric hypoxia treated group (Fig. 1). Electron microscopic biopsy on rat livers showed an increase in the amount of lipid droplets in the hypobaric hypoxia treated group. Treatment with fenofibrate significantly attenuated these changes due to the inhibition of ANGPTL4 (Fig. 4C and D). These results support the idea that PPARA-ANGPTL4 are differentially regulated by hypobaric hypoxia and mediate lipid metabolism.

We then tested whether PPARA-ANGPTL4 regulate downstream target genes mediating lipid metabolism by overexpressing ANGPTL4 in HL-7702 cells and measuring its influence on target genes. The qRT-PCR analysis revealed a 500-fold increase in mRNA levels compared with controls (Fig. 5A). ANGPTL4 target genes were significantly affected due to the expression of ANGPTL4 (Fig. 5B and S2). In particular, the expression of ACACA, LIPE and LPL, which are key regulators of lipid metabolism, was significantly downregulated after overexpressing ANGPTL4 (Fig. 5B). Furthermore, we also evaluated the influence of ANGPTL4 overexpression on lipid metabolism. The oil red O staining assays confirmed that ANGPTL4 overexpression regulates lipid metabolism by increasing the number of lipid droplets in treated cells compared to controls (Fig. 5C). We performed IPA based on qRT-PCR results after overexpressing ANGPTL4 and built a pathway revealing ANGPTL4 is central regulator of lipid metabolism (Fig. 5D). The overexpression of ANGPTL4 affected the key regulator of IL1 and the TGF-β pathways (Fig. 5D).

Overexpression of ANGPTL4 has an equal effect on the regulation of pathway genes and lipid metabolism caused by Hypobaric Hypoxia. A The overexpression of ANGPTL4 on mRNA levels. B Lipid metabolism-related genes relative mRNA levels after the expression of ANGPTL4, showing the up-regulation of ACACA, LIPE and LPL. Student’s t-test, paired tail, * < 0.05. C Oil red O staining assays validated the overexpression of ANGPTL4 regulates lipid metabolism by increasing the number of lipid droplets in treated cells compared to controls. D IPA predicted pathways based on qRT-PCR results. The predicted pathways are enriched in lipid metabolism, similar to the observations following hypobaric hypoxia treatment

The above results demonstrate ANGPTL4 and PPARA respond to hypobaric hypoxia and play an important role in the regulation of lipid metabolism.