4‐PBA inhibits hypoxia‐induced lipolysis in rat adipose tissue and lipid accumulation in the liver through regulating ER stress

Abstract High‐altitude hypoxia may disturb the metabolic modulation and function of both adipose tissue and liver. The endoplasmic reticulum (ER) is a crucial organelle in lipid metabolism and ER stress is closely correlated with lipid metabolism dysfunction. The aim of this study is to elucidate whether the inhibition of ER stress could alleviate hypoxia‐induced white adipose tissue (WAT) lipolysis and liver lipid accumulation‐mediated hepatic injury. A rat model of high‐altitude hypoxia (5500 m) was established using hypobaric chamber. The response of ER stress and lipolysis‐related pathways were analyzed in WAT under hypoxia exposure with or without 4‐phenylbutyric acid (PBA) treatment. Liver lipid accumulation, liver injury, and apoptosis were evaluated. Hypoxia evoked significant ER stress in WAT, evidenced by increased GRP78, CHOP, and phosphorylation of IRE1α, PERK. Moreover, Lipolysis in perirenal WAT significantly increased under hypoxia, accompanied with increased phosphorylation of hormone‐sensitive lipase (HSL) and perilipin. Treatment with 4‐PBA, inhibitor of ER stress, effectively attenuated hypoxia‐induced lipolysis via cAMP‐PKA‐HSL/perilipin pathway. In addition, 4‐PBA treatment significantly inhibited the increase in fatty acid transporters (CD36, FABP1, FABP4) and ameliorated liver FFA accumulation. 4‐PBA treatment significantly attenuated liver injury and apoptosis, which is likely resulting from decreased liver lipid accumulation. Our results highlight the importance of ER stress in hypoxia‐induced WAT lipolysis and liver lipid accumulation.

was proved to accelerate lipolysis and suppress lipogenesis of WAT (Xiong et al., 2014). Under normal conditions, the lipid metabolism is a dynamic equilibrium process between different organs. However, under hypoxia environment, the activation of lipolysis promotes excessive free fatty acids (FFA) release, which is taken up by the liver, contributing to ectopic lipid accumulation and pathogenesis of liver (Lefere et al., 2016). Adipose tissue dysfunction could lead to increased delivery of FFA and glycerol to the liver which drives hepatic gluconeogenesis and facilitates the accumulation of lipids and insulin signaling inhibiting lipid intermediates (Bosy-Westphal et al., 2019). Herein, hypoxia caused lipid metabolism disorder of WAT may further influence liver function, leading to the maladaptation to high-altitude environment and increasing the incidence of acute mountain sickness (AMS).
The endoplasmic reticulum (ER) is an organelle that functions to synthesize, fold, and transport proteins. It is also the site of triglyceride synthesis and nascent lipid droplet formation (Nettebrock & Bohnert, 2019). The sensing, metabolizing, and signaling mechanisms for lipid metabolism exist within or on the ER membrane domain (Balla et al., 2020). Dysregulation of ER homeostasis led to accumulation of misfolded proteins in the ER lumen and evoke ER stress (Henne, 2019). To reduce ER stress, the unfolded protein response (UPR) signal pathways are activated. Recently, accumulated evidence suggested that ER homeostasis and UPR activation play an important homeostatic role in lipid metabolism (Basseri & Austin, 2012;Mohan et al., 2019). As reported by Deng et al., ER stress could induce lipolysis by activating cAMP/PKA and ERK1/2 pathways (Deng et al., 2012).
Previous study also found that burned patients displayed significant ER stress within adipose tissue and ER stress could augment lipolysis in cultured human adipocytes (Bogdanovic et al., 2015).
The disulfide bond formation during protein synthesis is independent of oxygen, however, the post-translational protein folding and isomerization process is oxygen-dependent (Koritzinsky et al., 2013). Herein, hypoxia exposure could induce extensive protein modification in the ER and result in the accumulation of misfolded/unfolded proteins, which activate UPR and evoke ER stress (Chipurupalli et al., 2019;Maekawa & Inagi, 2017). We decided to test the hypothesis that ER stress may modulate hypoxia-induced WAT metabolic derangement and liver dysfunction based on the following evidence: (1) ER is one of the major sites of lipid metabolism.
(2) lipid metabolism and function are sensitive to oxygen concentration. (3) Hypoxia could induce ER stress due to the accumulation of misfolded proteins (Xu et al., 2015;Yang et al., 2014). (4) ER stress is closely correlated with lipid metabolism dysfunction (Mohan et al., 2019). (5) lipid metabolism in WAT plays a critical role in the progression of liver dysfunction (Dong et al., 2020).
To address this issue, we investigated the effects of ER stress in hypoxia-induced lipolysis using chemical chaperone 4-PBA, antagonist of ER stress. The main objective of this study was to clarify the role of ER stress which regulates WAT lipolysis and liver lipid accumulation under continuous high-altitude hypoxia exposure. An understanding of the interplay between tissues and these proposed mechanisms may provide novel therapeutic strategies for the treatment of the whole-body metabolism dysfunction at high altitude.

| Animals care
Adult male Sprague-Dawley rats (280-330 g) were purchased from Weitong Lihua Laboratory Animal Limited Company. The rats were housed at room temperature (22°C-25°C) and in a 12-12 h lightdark cycle with free access to food and water and adapted to the condition above for 1 week before experiment. All experiments were conducted in strict accordance with the laboratory animal care guidelines published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). All protocols concerning animal use were approved by the Institutional Animal Care and

| Hypoxic challenge
Hypoxia group rats were placed in a hypobaric chamber (Guizhou Fenglei Air Ordnance Co., Ltd.) and subjected to hypoxia mimicking an altitude of 5500 m for 10 days. The chamber was opened daily for 30 min to clean and replenish food and water and room temperature was kept at 20°C-22°C. We monitored the body weights of rats every day. 4-PBA (P21005) was commercially purchased (Sigma-Aldrich). Rats were randomly divided into four groups: (1) Control group, (2) Hypoxia group, (3) Control + 4-PBA (30 mg/kg /day), and (4) Hypoxia + 4-PBA (30 mg/kg/day). The dose of 4-PBA was set based on previous reports (Luo et al., 2015;You et al., 2019;Zeng et al., 2017). All the rats were sacrificed by decapitation and serum was obtained by centrifugation and stored at −80°C. The perirenal fat pads were collected and weighed immediately, frozen in liquid nitrogen, and stored at −80°C.

| Histology staining
WAT and liver tissue were fixed in 4% paraformaldehyde overnight, followed by embedment in paraffin and longitudinal slicing, with 4μm-thick sections obtained for hematoxylin-eosin (HE) staining. The stained slides were examined by microscopy for histomorphological analyses. A commercial terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit (Roche) was employed to assess the degree of hepatic cell apoptosis. Histological alterations were assessed in randomly selected histological fields at ×400 magnification and apoptosis index (AI) was calculated.

| Western blotting and densitometry analyses
Homogenized rat WAT was lysed in 200 μl RIPA lysis buffer (Beyotime, P0013B) with 1% phenylmethyl sulfurylfluoride and 4% complete protease inhibitor cocktail mix (Roche). Extracts were centrifuged at 14,000 g for 15 min at 4°C. Eighty micrograms of total protein was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transferring blotting to nitrocellulose membrane (Millipore Corp., Billerica). Membranes were then blocked with 5% non-fat-dried milk in PBS for 1 h with gentle shaking. Membranes were incubated first with primary antibodies (dilution: 1:1000) overnight at 4°C, in 1% BSA in PBS overnight at 4°C with shaking. The following primary antibodies were purchased from Abcam. Then, membranes were washed and incubated with secondary antibodies for 2 h at room temperature. Finally, the samples were visualized by enhanced chemiluminescence using Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation). After scanning, band density was analyzed using Image J 1.33 software (National Institutes of Health).

| Reverse-transcription PCR and quantitative real-time PCR
Total RNA was prepared from frozen liver tissues with TRIZOL (Invitrogen) reagent and the cDNA was synthesized using TransScript TM First-Strand cDNA Synthesis Super-Mix (TransGen Biotech, AT301). The program was run on a S1000 Thermal Cycler.
Quantitative real-time PCR was performed using the SYBR®Premix Ex TaqTMkit (Takara, RR420A) and analyzed in a step-one plus RT-PCR system (life science, Applied Biosystems). The primer sequences are listed in Table 1.
These assays were performed according to manufacturer's instructions. Serum levels of triglyceride (TG), total cholesterol (TC), highdensity lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by an automatic biochemical analyzer (Chemray 240, Rayto Life and Analytical Sciences).
Serum alanine (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) microplate test kits were obtained from Nanjing Jiancheng Bioengineering Institute. These assays were performed as previously described . Briefly, ALT, AST, and ALP activities were evaluated at 37°C for 15 min by assessing for a decrease in absorbance at a wavelength of 510 nm, with Chemi Lab ALT, AST, and ALP assay kits, respectively.

| Statistical analysis
The data are presented as mean ± standard error (SE). For Western blot, protein levels were normalized to β-actin. Statistical significance is determined by one-way Analysis of variance (ANOVA) or nonparametric for more than three groups. p-Value < .05 was considered statistically significant (SPSS 18.0 software).

| Hypoxia exposure induces endoplasmic reticulum stress in WAT
To investigate the role of ER stress in WAT under hypoxia treatment, we first examined the expression of ER stress markers, namely GRP78 and CHOP (Figure 1a). Under ER stress conditions, increased GRP78 is dissociated from unfolded proteins and activates ER stress receptors triggering the UPR. As shown in   To investigate the effect of inhibition of ER stress on WAT lipolysis under hypoxia, we first evaluated the body weight and wet weight of perirenal fat in hypoxia rats with or without 4-PBA treatment. 4-PBA significantly attenuated the reduction of body weight and wet weight of perirenal fat after 10 days exposure to hypoxia (Figure 2a,b). In addition, inhibition of ER stress via 4-PBA was associated with a significant reduction of lipolysis, evidenced by a significant reduction in serum glycerol and FFA levels (Figure 2c,d).

| ER stress inhibition ameliorate hypoxiainduced WAT lipolysis via cAMP/PKA pathway
Endoplasmic reticulum stress has been suggested to trigger lipolysis in adipocytes. The lipolysis process is closely correlated with the production of cAMP and activation of cAMP-dependent protein kinase A (PKA). In our study, hypoxia challenge significantly increased pPKA production (Figure 3a,b), which phosphorylates HSL and perilipin (Miyoshi et al., 2006;Sztalryd et al., 2003). The p-

| PBA treatment ameliorated hypoxia-induced liver lipid transport and accumulation
Under continuous hypoxia exposure, increased delivery of free fatty acids (FFA) caused by enhanced lipolysis in WAT may contribute to the lipid accumulation in the liver. As shown in Figure 4a, levels of FFA content significantly increased in hypoxia group rat liver, which was attenuated by 4-PBA treatment. Lipid uptake in the liver was regulated by many transporters, including cluster of differentiation (CD36), fatty acid binding protein 1(FABP1), and FABP4. mRNA levels of CD36, FABP1, and FABP4 that regulate the entry of fatty acids into hepatocyte, are generally upregulated to cope with increased circulation FFAs (Figure 4b-d).

| PBA treatment ameliorated hypoxia-induced live hepatic injury and apoptosis
Hypoxia-induced liver lipid accumulation may further trigger the pathogenesis of liver injury, serum levels of liver enzyme were tested to confirm our speculation. As shown in Figure 5a-c, the hypoxia group rat exhibited a marked increase in the levels of AST, ALT, F I G U R E 1 Hypoxia exposure induces endoplasmic reticulum stress in the WAT. The expression levels of ER stress-related genes in WAT are shown. (a) GRP78, CHOP, p-PERK, PERK, p-IRE1α, and IRE1α protein expression levels; (b) Relative GRP78 protein expression levels; (c) relative CHOP protein expression levels; (d) p-PERK/PERK ratio; (e) p-IRE1α/IRE1α ratio. Data are shown as the mean ± SE of at least two independent western blots, *p < .05, **p < .01, and ***p < .001 (control group vs. hypoxia group, n = 6/group). # p < .05, ## p < .01 (hypoxia group vs. hypoxia + 4-PBA group, n = 6/group) and ALP (p<.05), indicating potential liver injury. However, the hypoxia + 4-PBA group significantly decreased the levels of AST and ALT (p<.05) when compared with hypoxia group, indicating that 4-PBA inhibits hypoxia-induced hepatocellular injury.
The apoptosis status of rat liver exposed to hypoxia was evaluated with a TUNEL assay. As shown in Figure 5d,e, the percentage of apoptotic cells was significantly increased in hypoxia group as compared with control group, which was effectively attenuated by 4-PBA treatment.

| DISCUSS ION
Lipid metabolism in white adipose tissue played an essential role in maintaining energy homeostasis at high-altitude area. In this study, WAT ER stress-mediated lipolysis is enhanced in a rat model of highaltitude hypoxia. Moreover, we found that increased FFA release results in liver lipid accumulation and liver dysfunction, which was attenuated by the inhibition of ER stress using 4-PBA.
As ER membrane are located with a variety of lipid metabolismrelated enzymes and ER is the major site of lipid metabolism, ER is involved in the control of metabolic homeostasis via regulating lipid metabolism. Under normal conditions, ER in the adipocyte functions to meet the demands of protein synthesis and secretion, triglyceride synthesis, nascent lipid droplet formation, and nutrient sensing.
However, ER function is overwhelmed and the UPR is activated under stressful conditions (Menikdiwela et al., 2019;Sikkeland et al., 2019). Therefore, perturbations in ER homeostasis exerts a vital pathogenic mechanism in multi metabolic disorders of adipose tissue (Khan & Wang, 2014;Suzuki et al., 2017). Adverse stimuli like hypoxia may pose challenges to adipocyte and induce ER stress. In the present study, continuous hypoxia exposure evoked ER stress in adipose tissue, evidenced by increased GRP78, CHOP, p-PERK, and p-IRE1α expression in rat WAT. Our finding is in accordance with previous studies showing that hypoxia exposure induce ER stress in 3 T3-F442A and 3 T3-L1 adipocytes (Mihai & Schroder, 2015). UPR pathways were activated to ameliorate the overload of unfolded proteins under ER stress, which in turn influence lipid metabolism . Data are shown as the mean ± SE, *p < .05, **p < .01, and ***p < .001(control group vs. hypoxia group, n = 6/group). #p < .05, ##p < .01 (hypoxia group vs. hypoxia + 4-PBA group, n = 6/group) (Song et al., 2016). The activation of ER stress in adipose tissue may further induce lipolysis and elevated circulating FFAs (Song et al., 2017).
To confirm the potential role of the ER stress and UPR in the modulation of the lipolysis, we treated rat with 4-PBA, an ER stress inhibitor. 4-PBA treatment led to significant reduction in lipolysis, which blocked the phosphorylation of HSL and perilipin. As the results from upstream regulation, 4-PBA treatment then effectively reduced glycerol and FFA release from adipose tissue, suggesting that ER stress-mediated lipolysis mainly by regulating cAMP-PKA/ HSL under hypoxia. Similar to our study, enhanced lipolysis and ER stress occurred in the visceral WAT and inhibition of ER stress F I G U R E 5 4-PBA ameliorates hypoxia-induced hepatic injury and apoptosis. (a) Serum levels of AST in rats exposed to hypoxic (n = 6) or normoxic (n = 6) conditions. (b) Serum levels of ALT; (c) serum levels of ALP; (d)apoptosis index of four group rats; (e) representative images of TUNEL-stained sections of liver (magnification, 400×). Data are shown as mean ± SE, *p < .05, **p < .01, (control group vs. hypoxia group). #p < .05, ##p < .01 (hypoxia group vs. hypoxia + 4-PBA group) F I G U R E 6 Protective mechanisms of 4-PBA inhibit hypoxia-induced lipolysis in WAT and lipid accumulation in the liver through regulating ER stress. Treatment with 4-PBA, inhibitor of ER stress, effectively attenuated hypoxia-induced lipolysis via cAMP-PKA-HSL/perilipin pathway. In addition, 4-PBA treatment significantly attenuated liver injury and apoptosis, which is likely resulting from decreased liver lipid accumulation via inhibiting FFA transport.
alleviated lipolysis in a rat model of chronic kidney disease (Zhu et al., 2014). In addition, curcumin was reported to suppress the ER stress-mediated lipolysis via cAMP/PKA/HSL pathway (Wang et al., 2016). Deng et al., also reported that ER stress involved lipolysis through up-regulation of GRP78 and activation of phosphorylation status of PERK and eIF2α in rat adipocytes (Deng et al., 2012).
Since the liver is the largest metabolic organ and regulates various physiological and metabolic processes, it also performs a key role in high-altitude adaptation . Adipose dysfunction is closely associated with metabolism-related liver diseases, an understanding of the interplay between tissues and these proposed mechanisms is still necessary (Da Silva Rosa et al., 2020).
Accumulating data are pointing out the pathophysiological role of ectopic fat accumulation in different organs, including the liver injury and apoptosis, is likely resulting from decreased liver lipid accumulation via inhibiting FFA transport. Lines of evidence proved that excess FFA may modify the biology and function of hepatocyte and play an essential role in the pathogenesis liver dysfunction (Pereira et al., 2021). A high serum level of saturated FFAs is associated with hepatocyte lipo-apoptosis (Takahara et al., 2017). In line with our fundings, Hubel, E., et al. found that repetitive Amiodarone treatment led to ER stress and aggravated lipolysis in adipose tissue while inducing a lipotoxic hepatic lipid environment and hepatic injury (Hubel et al., 2021).
In conclusion, enhanced ER stress-mediated WAT lipolysis was observed in a rat model of high-altitude hypoxia, which contributes to hepatic dysfunction and apoptosis through excess release of FFA. Our findings highlight the vital role of 4-PBA in WAT lipolysis and liver dysfunction via regulating ER stress, which may provide novel insights into systemic metabolic disturbances in high-altitude area.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.