L-Carnitine Is Involved in Hyperbaric Oxygen-Mediated Therapeutic Effects in High Fat Diet-Induced Lipid Metabolism Dysfunction

Lipid metabolism dysfunction and obesity are serious health issues to human beings. The current study investigated the effects of hyperbaric oxygen (HBO) against high fat diet (HFD)-induced lipid metabolism dysfunction and the roles of L-carnitine. C57/B6 mice were fed with HFD or normal chew diet, with or without HBO treatment. Histopathological methods were used to assess the adipose tissues, serum free fatty acid (FFA) levels were assessed with enzymatic methods, and the endogenous circulation and skeletal muscle L-carnitine levels were assessed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, western blotting was used to assess the expression levels of PPARα, CPT1b, pHSL/HSL, and UCP1. HFD treatment increased body/adipose tissue weight, serum FFA levels, circulation L-carnitines and decreased skeletal muscle L-carnitine levels, while HBO treatment alleviated such changes. Moreover, HFD treatment increased fatty acid deposition in adipose tissues and decreased the expression of HSL, while HBO treatment alleviated such changes. Additionally, HFD treatment decreased the expression levels of PPARα and increased those of CPT1b in skeletal muscle, while HBO treatment effectively reverted such changes as well. In brown adipose tissues, HFD increased the expression of UCP1 and the phosphorylation of HSL, which was abolished by HBO treatment as well. In summary, HBO treatment may alleviate HFD-induced fatty acid metabolism dysfunction in C57/B6 mice, which seems to be associated with circulation and skeletal muscle L-carnitine levels and PPARα expression.


Introduction
Obesity is a global health issue, affecting both developed and developing countries. Due to the increasing consumption of higher energy diets as well as lower energy expenditure, the prevalence of obesity is increasing remarkably throughout the world [1]. According to the world health organization (WHO), the global prevalence of obesity almost doubled from 1980 to 2008, and the prevalence of general obesity in China increased by about 90% [2], which is now attracting attention.

Experimental Design and General Parameters
The timeline design of the experiments was reported in Figure 1A. After 0, 10, or 14 weeks of treatments, the body weights were reported in Figure 1B. 10 or 14 weeks of HFD treatment resulted in significantly higher body weight of the animals than those received normal diet, indicating successful establishment of HFD-induced obesity model. On the other hand, HBO co-treatment for 4 weeks effectively abolished such changes, suggesting protective effects of HBO. The epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) weights were reported in Figure 1C,D,E, respectively. The EWAT and IWAT weights significantly changed in a similar pattern as the body weight, while no significant changes were observed in the BAT weight.
Molecules 2020, 25, x 3 of 20 provided evidence for applying HBO treatment as a potential therapy for fatty acid metabolism dysfunction and obesity.

Experimental Design and General Parameters
The timeline design of the experiments was reported in Figure 1A. After 0, 10, or 14 weeks of treatments, the body weights were reported in Figure 1B. 10 or 14 weeks of HFD treatment resulted in significantly higher body weight of the animals than those received normal diet, indicating successful establishment of HFD-induced obesity model. On the other hand, HBO co-treatment for 4 weeks effectively abolished such changes, suggesting protective effects of HBO. The epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) weights were reported in Figure 1 C, D and E, respectively. The EWAT and IWAT weights significantly changed in a similar pattern as the body weight, while no significant changes were observed in the BAT weight. Figure 1. Experimental design, whole body, epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) weight. C57/B6 mice were kept in 21-25 °C environment with 12 h light/dark cycle. Food and water were provided ad labium. Animals received normal chew diet, or HFD (15% lard, 3% soybean oil, 5% egg yolk, 18% sugar, and 59% chew diet) for a total of 14 weeks. HBO treatment (5 min pressure rise stage, 60 min stabilization stage with 2.0 atmospheres absolute and 100% oxygen, 5 min depression stage) were applied during the last 4 weeks Figure 1. Experimental design, whole body, epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) weight. C57/B6 mice were kept in 21-25 • C environment with 12 h light/dark cycle. Food and water were provided ad labium. Animals received normal chew diet, or HFD (15% lard, 3% soybean oil, 5% egg yolk, 18% sugar, and 59% chew diet) for a total of 14 weeks. HBO treatment (5 min pressure rise stage, 60 min stabilization stage with 2.0 atmospheres absolute and 100% oxygen, 5 min depression stage) were applied during the last 4 weeks of diet treatment. At the end of treatments, whole body weight was measured, then animals were sacrificed, EWAT, IWAT, BAT, and skeletal muscle were weighed and collected. Three to five animals were included in each group. Error bars represent standard derivation.

Hematoxylin and Eosin Staining for EWAT
Representative hematoxylin and eosin staining pictures for EWAT were shown in Figure 2A-D. Quantification for the average adipocyte size was reported in Figure 2E. It was revealed that the adipocyte size significantly increased in the samples from HFD-treated animals, while HBO treatment may effectively alleviate such changes. of diet treatment. At the end of treatments, whole body weight was measured, then animals were sacrificed, EWAT, IWAT, BAT, and skeletal muscle were weighed and collected. Three to five animals were included in each group. Error bars represent standard derivation. (A): The whole experimental design. (B): The body weights at 0-, 10-and 14-week. (C): EWAT weight at the end of study. (D): IWAT weight at the end of study. (E): BAT weight at the end of study. *: statistically different from control group animals (p < 0.05).

Hematoxylin and Eosin Staining for EWAT
Representative hematoxylin and eosin staining pictures for EWAT were shown in Figure 2A-D. Quantification for the average adipocyte size was reported in Figure 2E. It was revealed that the adipocyte size significantly increased in the samples from HFD-treated animals, while HBO treatment may effectively alleviate such changes.

Hematoxyin and Eosin Staining for BAT
Representative hematoxylin and eosin staining pictures for BAT were shown in Figure 3A-D. Quantification for the average size of fat droplets was reported in Figure 3E. It was revealed that the size of fat droplets significantly increased in the samples from HFD-treated animals, while HBO treatment may effectively alleviate such changes.

Immunohistochemistry for HSL in EWAT
Representative immunohistochemistry pictures for HSL in EWAT were shown in Figure 4A-D. Quantification for the positively stained area percentages were reported in Figure 4E. The results indicated that the expression levels of HSL were significantly decreased in samples from HFD-treated animals, while HBO treatment effectively alleviated such changes. Decreased expression of the rate limiting lipolysis enzyme HSL indicated that HFD decreased the capacity of lipolysis in EWAT, which might be contributing to the observed larger adipocyte size. Representative immunohistochemistry pictures for HSL in EWAT were shown in Figure 4A-D. Quantification for the positively stained area percentages were reported in Figure 4E. The results indicated that the expression levels of HSL were significantly decreased in samples from HFD-treated animals, while HBO treatment effectively alleviated such changes. Decreased expression of the rate limiting lipolysis enzyme HSL indicated that HFD decreased the capacity of lipolysis in EWAT, which might be contributing to the observed larger adipocyte size.

Immunohistochemistry for HSL in BAT
Representative immunohistochemistry pictures for HSL in BAT were shown in Figure 5A-D. Quantification for the positively stained area percentages were reported in Figure 5E. The results indicated that the expression levels of HSL were significantly decreased in samples from HFD-treated animals, while HBO treatment effectively alleviated such changes.

Immunohistochemistry for HSL in BAT
Representative immunohistochemistry pictures for HSL in BAT were shown in Figure 5A-D. Quantification for the positively stained area percentages were reported in Figure 5E. The results indicated that the expression levels of HSL were significantly decreased in samples from HFD-treated animals, while HBO treatment effectively alleviated such changes.

Serum FFA Levels
Serum FFA levels were reported in Figure 6. Following HFD treatment, the serum FFA levels remarkably increased, while HBO treatment effectively abolished such changes. Notably, HBO solo treatment also led to increased serum FFA levels.

Serum FFA Levels
Serum FFA levels were reported in Figure 6. Following HFD treatment, the serum FFA levels remarkably increased, while HBO treatment effectively abolished such changes. Notably, HBO solo treatment also led to increased serum FFA levels. Figure 6. Serum free fatty acid (FFA) levels. Serum FFA levels were measured with a commercially available kit (BC0595, Solarbio, China). Three to five samples from independent animals were included in the tests. Error bars represent standard derivation. *: statistically different from control group animals (p < 0.05). #: statistically different from HFD group animals (p < 0.05).

Endogenous L-Carnitine Levels in Serum and Skeletal Muscle
LC-MS/MS results of serum and skeletal muscle L-carnitine levels were reported in Figure 7 Interestingly, differential patterns were observed: L-carnitine levels remarkably increased in the serum of HFD treated animals and HBO treatment abolished such changes ( Figure 7A). On the other hand, no statistical differences were observed in the L-carnitine levels in the skeletal muscle of HFD treated animals, while HBO treatment significantly increased the levels of L-carnitine ( Figure 7B). The data suggested that HFD changed the distribution of L-carnitine in the system, increasing circulatory levels, but not the skeletal muscle levels while HBO treatment returned circulatory levels of L-carnitine to normal, but dramatically increased skeletal muscle levels of L-carnitine.

Endogenous L-Carnitine Levels in Serum and Skeletal Muscle
LC-MS/MS results of serum and skeletal muscle L-carnitine levels were reported in Figure 7 Interestingly, differential patterns were observed: L-carnitine levels remarkably increased in the serum of HFD treated animals and HBO treatment abolished such changes ( Figure 7A). On the other hand, no statistical differences were observed in the L-carnitine levels in the skeletal muscle of HFD treated animals, while HBO treatment significantly increased the levels of L-carnitine ( Figure 7B). The data suggested that HFD changed the distribution of L-carnitine in the system, increasing circulatory levels, but not the skeletal muscle levels while HBO treatment returned circulatory levels of L-carnitine to normal, but dramatically increased skeletal muscle levels of L-carnitine.

Western Blotting for pHSL/HSL and UCP1 in Brown Adipose Tissues
Western blotting results revealed that HFD treatment effectively enhanced the phosphorylation of HSL in brown adipose tissues, while HBO co-treatment returned the phosphorylation of HSL to normal level ( Figure 8A). Similarly, UCP1 expression was remarkably enhanced in the brown adipose tissues from HFD treated animals, while HBO co-treatment abolished such changes ( Figure 8B).
Molecules 2020, 25, x 11 of 20 Figure 8. Western blotting for pHSL/HSL and UCP1 in brown adipose tissues. Brown adipose tissues from C57/B6 mice were homogenized in RIPA buffer with 1:100 PMSF and 1:100 phosphatase inhibitor cocktails, centrifuged at 14,000 g for 10 min, and the resulting supernatants were subjected to western blotting analysis for pHSL/HSL and UCP1. Images were acquired from a Vilber Lourmet gel imaging system and analyzed with ImageJ. Three samples from independent animals were included per group. Error bars represent standard derivation. (A): Representative blot images and quantifications of pHSL/HSL. (B): Representative blot images and quantifications of UCP1. *: statistically different from control group animals (p < 0.05). #: statistically different from HFD group animals (p < 0.05).

Western Blotting for CPT1b and PPARα in Skeletal Muscle
Western blotting results indicated that HFD treatment significantly enhanced the expression of CPT1b relative to control, while HBO co-treatment brought the expression levels back to normal levels ( Figure 9A). The enhanced expression of CPT1b following HFD treatment is likely compensation. Meanwhile, HFD treatment remarkably decreased the expression levels of PPARα, while HBO co-treatment reverted it back to normal ( Figure 9B), suggesting that HFD treatment interrupted with beta oxidation, while HBO may exert its protective effects via protection of PPARα signaling. Interestingly, HBO treatment only seemed to decrease the expression levels of PPARα as well.

Western Blotting for CPT1b and PPARα in Skeletal Muscle
Western blotting results indicated that HFD treatment significantly enhanced the expression of CPT1b relative to control, while HBO co-treatment brought the expression levels back to normal levels ( Figure 9A). The enhanced expression of CPT1b following HFD treatment is likely compensation. Meanwhile, HFD treatment remarkably decreased the expression levels of PPARα, while HBO co-treatment reverted it back to normal ( Figure 9B), suggesting that HFD treatment interrupted with beta oxidation, while HBO may exert its protective effects via protection of PPARα signaling. Interestingly, HBO treatment only seemed to decrease the expression levels of PPARα as well.

Discussion
Obesity and associated fatty acid metabolism dysfunction are major health issues across the world. Many efforts had been made towards effective methods for symptom alleviation and complication prevention, but current methods (pharmaceutical agents and physical therapies) are associated with adverse effects as well as limited efficacy. The mechanism of obesity is also being extensively investigated, with many candidates identified as potential therapeutic targets, among them, L-carnitine is a promising one. In the current study, the effects of HBO treatment against HFDinduced fatty acid metabolism dysfunction were investigated, HBO's effect on endogenous levels of L-carnitine was investigated as the potential mechanism of action.

HBO and Fatty Acid Metabolism
HBO therapy has been utilized for multiple health conditions such as carbon monoxide poisoning, neurological damage, and radiation damage [26]. There is only limited evidence for its use in diabetes treatment [27], and it has not been approved to be used in obesity or fatty acid metabolism dysfunction. However, since HBO provides high level of oxygen, it is highly likely to facilitate fatty acid metabolism thus may serve as a potential therapy for such disorders. In previous studies, it had been reported that HBO treatment may improve metabolic capacity of the skeletal muscle [28] and decrease mouse body weight [29], suggesting that HBO may be beneficial in obesity and fatty acid metabolism dysfunction. Notably, these previous studies used either genetically modified animal model or chemically induced animal models, while the current study used diet-induced animal model, providing better relevancy. On the other hand, due to the potentially increased oxidative

Discussion
Obesity and associated fatty acid metabolism dysfunction are major health issues across the world. Many efforts had been made towards effective methods for symptom alleviation and complication prevention, but current methods (pharmaceutical agents and physical therapies) are associated with adverse effects as well as limited efficacy. The mechanism of obesity is also being extensively investigated, with many candidates identified as potential therapeutic targets, among them, L-carnitine is a promising one. In the current study, the effects of HBO treatment against HFD-induced fatty acid metabolism dysfunction were investigated, HBO's effect on endogenous levels of L-carnitine was investigated as the potential mechanism of action.

HBO and Fatty Acid Metabolism
HBO therapy has been utilized for multiple health conditions such as carbon monoxide poisoning, neurological damage, and radiation damage [26]. There is only limited evidence for its use in diabetes treatment [27], and it has not been approved to be used in obesity or fatty acid metabolism dysfunction. However, since HBO provides high level of oxygen, it is highly likely to facilitate fatty acid metabolism thus may serve as a potential therapy for such disorders. In previous studies, it had been reported that HBO treatment may improve metabolic capacity of the skeletal muscle [28] and decrease mouse body weight [29], suggesting that HBO may be beneficial in obesity and fatty acid metabolism dysfunction. Notably, these previous studies used either genetically modified animal model or chemically induced animal models, while the current study used diet-induced animal model, providing better relevancy. On the other hand, due to the potentially increased oxidative stress burden, higher level of HBO treatment (2.5 ATA) was also reported including liver damage [29], pulmonary edema, and maybe inflammation [30], while lower level of HBO treatment (2 ATA) did not induce such damages [31]. This phenomenon is mainly due to the nature of oxidative stress, in which a balance needs to be established, ensuring the necessary oxidation reactions in the organism to be performed effectively, while avoiding too many free radicals to be generated. In the current study, decreased body weight, less fat deposition and normal phosphorylation state of HSL (the major fatty acid mobilizing enzyme) in adipose tissues were observed in HFD-fed animals treated with HBO, which is consistent with the previous report [27], suggesting the potential of HBO to be used for obesity management. Regarding the potential adverse effects of HBO, HBO treatment without HFD indeed resulted in somewhat elevated serum FFA and brown adipose tissue weight, as well as increased fatty acid deposit in both white and brown adipose tissue. While none of these changes were statistically significant, it did indicate that hyperoxia may result in deleterious effects, especially in the organ systems not challenged with HFD. Interestingly, these endpoints all improved when animals received HFD, suggesting differential effects of HBO treatment depending on the diet.

Roles of L-Carnitine in HBO Mediated Protective Effects Against HFD-Induced Fatty Acid Metabolism Dysfunction
L-carnitine and its metabolites were known to exert protective effects in multiple organisms, such as the cardiovascular system, skeletal muscle, liver, and adipose tissue [32][33][34][35], probably due to their critical roles in fatty acid beta oxidation [36]. Focusing on fatty acid metabolism, L-carnitine supplementation was reported to decrease body weight in HFD-fed mice [37] and alleviate the pathological changes in adipose tissues [34]. However, few studies reported the relationship between HBO treatment and endogenous L-carnitine levels. HBO treatment was reported to improve fatty acid beta oxidation, which was reported to be mediated through enhanced glucose and lipid metabolism [28], which is closely associated with L-carnitine [38]. On the other hand, endogenous L-carnitine levels in circulation and in skeletal muscles were reported to be associated with fatty acid metabolism dysfunction: increased circulation L-carnitine levels were reported to be associated with metabolic syndrome [39], while lower skeletal muscle L-carnitine levels were reported to contribute to insulin resistance and metabolic syndrome as well [40]. In the current study, endogenous L-carnitine levels in both serum and skeletal muscle were measured. The results indicated that HFD induced higher circulation L-carnitine level, which is consistent with previous reports [39]. Slightly higher skeletal muscle L-carnitine levels were also observed, probably due to compensation. HBO treatment decreased circulating L-carnitine levels while increased skeletal muscle L-carnitine levels, suggesting that L-carnitine is involved in HBO-mediated protection against fatty acid metabolism dysfunction. It seems like HBO facilitates beta oxidation in skeletal muscle, just as reported by Takemura et al. [41]. Our study made one step further focusing on the effects of HBO on circulating and skeletal L-carnitine contents. HBO treatment seemed to redistribute endogenous L-carnitine, translocating L-carnitine from circulation to skeletal muscle. While further investigation is still needed, this is a good indicator that L-carnitine is critical for HBO treatment in obesity/fatty acid metabolism dysfunction.

L-Carnitine and PPARα/CPT1b
PPARα is a classical key regulator of fatty acid metabolism, which regulates several key enzymes in fatty acid beta oxidation, such as ACOX1, FABPs and CPT1 [25,42,43]. Although the strongest expression of PPARα was observed in the liver, it is also expressed in the heart and skeletal muscles, and regulates fatty acid metabolism in these striated muscle tissue as well [44][45][46]. In skeletal muscle, it has been observed that PPARα, not PPARδ mediates responses to PPAR agonists [47]. PPARα and L-carnitine are both critical participants in fatty acid beta oxidation, and can often interact with each other: PPARα agonists may decrease serum L-carnitine levels [48], while L-carnitine supplementation may enhance PPARα expression [49]. Cooperative interactions between L-carnitine and PPARα were also reported [50]. In the current study, PPARα expression was significantly depressed by HFD challenge, which is consistent with a previous report [51]. HBO treatment effectively restored the expression levels of PPARα in the skeletal muscle, suggesting that HBO's protective effects may at least partially be mediated through the restoration of PPARα signaling. Notably, the expression levels of PPARα in animals treated with only HBO also decreased, although the average value is still higher than those with HFD. This result is consistent with observed serum FFA level changes and adipose tissue pathological changes, suggesting that HBO treatment should be cautiously applied on metabolically normal individuals.
Among the PPARα downstream molecules, CPT1 is the rate-limiting enzyme for the carnitine palmitoyltransferase system, connecting fatty acid acyl groups to carnitine. Interruption of CPT1 is frequently associated with HFD induced metabolic dysfunction and diabetes [52,53]. Furthermore, the function of CPT1 is directly linked to the endogenous levels of L-carnitine, thus it is highly likely to be affected when L-carnitine levels change [54]. In the current study, CPT1b (skeletal muscle isoform) expression levels remarkably elevated in HFD-treated animals, while HBO treatment returned it to normal levels. This result is consistent with Leduc-Gaudet et al. [55], in which HFD increased CPT1b expression levels in skeletal muscles. Another study reported no statistical difference in CPT1b expression levels following HFD, but fenofibrate, a PPARα agonist significantly decreased its expression [56]. In the current study, HBO treatment both enhanced PPARα expression and decreased CPT1b expression. One possible explanation is that other molecules modulates CPT1b under HFD challenge. When HBO treatment recovered PPARα signaling, such changes were reverted. Another possibility is that the actual activity of PPARα/CPT1b is different from their expression levels. Assays of PPARα and CPT1b activities under HFD and/or HBO treatment have been planned. Our results suggest that HBO may enhance PPARα expression in skeletal muscle in a pattern similar to PPARα agonists, which is likely part of its mechanism of action in protecting against HFD induced fatty acid metabolism dysfunction and obesity.

Limitations of the Current Study and Future Directions
The current study revealed the protective effects of HBO treatment against HFD-induced fatty acid metabolism dysfunction/obesity, as well as the roles of L-carnitine and associated PPAR signaling. The proposed mode of action of HBO and L-carnitine was reported in Figure 10. However, interventions such as extraneous L-carnitine supplementation and PPARα agonist/antagonist treatments were absent due to the limited capacity of animal housing and handling. These experiments are guaranteed in the future studies.
Molecules 2020, 25, x 14 of 20 effectively restored the expression levels of PPARα in the skeletal muscle, suggesting that HBO's protective effects may at least partially be mediated through the restoration of PPARα signaling. Notably, the expression levels of PPARα in animals treated with only HBO also decreased, although the average value is still higher than those with HFD. This result is consistent with observed serum FFA level changes and adipose tissue pathological changes, suggesting that HBO treatment should be cautiously applied on metabolically normal individuals. Among the PPARα downstream molecules, CPT1 is the rate-limiting enzyme for the carnitine palmitoyltransferase system, connecting fatty acid acyl groups to carnitine. Interruption of CPT1 is frequently associated with HFD induced metabolic dysfunction and diabetes [52,53]. Furthermore, the function of CPT1 is directly linked to the endogenous levels of L-carnitine, thus it is highly likely to be affected when L-carnitine levels change [54]. In the current study, CPT1b (skeletal muscle isoform) expression levels remarkably elevated in HFD-treated animals, while HBO treatment returned it to normal levels. This result is consistent with Leduc-Gaudet et al. [55], in which HFD increased CPT1b expression levels in skeletal muscles. Another study reported no statistical difference in CPT1b expression levels following HFD, but fenofibrate, a PPARα agonist significantly decreased its expression [56]. In the current study, HBO treatment both enhanced PPARα expression and decreased CPT1b expression. One possible explanation is that other molecules modulates CPT1b under HFD challenge. When HBO treatment recovered PPARα signaling, such changes were reverted. Another possibility is that the actual activity of PPARα/CPT1b is different from their expression levels. Assays of PPARα and CPT1b activities under HFD and/or HBO treatment have been planned. Our results suggest that HBO may enhance PPARα expression in skeletal muscle in a pattern similar to PPARα agonists, which is likely part of its mechanism of action in protecting against HFD induced fatty acid metabolism dysfunction and obesity.

Limitations of the Current Study and Future Directions
The current study revealed the protective effects of HBO treatment against HFD-induced fatty acid metabolism dysfunction/obesity, as well as the roles of L-carnitine and associated PPAR signaling. The proposed mode of action of HBO and L-carnitine was reported in Figure 10. However, interventions such as extraneous L-carnitine supplementation and PPARα agonist/antagonist treatments were absent due to the limited capacity of animal housing and handling. These experiments are guaranteed in the future studies.

Animal Housing, Treatment and Sample Collection
C57/B6 mice were purchased from Qingdao Institute of Drug Control. Upon arrival, animals were kept in 21-25 • C environment with 12-h light/dark cycle. Food and water were provided Ad libitum. After one-week adaptation, animals were randomly assigned into control, HFD, HBO treatment, and HFD + HBO treatment groups. Six animals were included for each group. Control and HBO treatment animals received normal chew diet, while HFD and HFD + HBO treatment animals received 40% HFD (15% Lard, 3% soybean oil, 5% egg yolk, 18% sugar, and 59% chow diet) for ten weeks. Then, HBO treatment and HFD + HBO treatment animals were subjected to hyperbaric treatment (5 min pressure rise stage, 60 min stabilization stage with 2.0 atmospheres absolute and 100% oxygen, 5 min depression stage) [57] for four more weeks, in which the diet treatments continued. Please refer to Figure 1A for the timeline of the study. The 12-h cumulative food intake was measured after the first HBO treatment session, in which HFD had elevated food intake as expected, but HBO had no significant effects (Supplementary material 2). At the end of the 14-week study, animals were sacrificed under anesthesia (33 mg/kg pentobarbital, intraperitoneal injection). Serum, skeletal muscle, EWAT, IWAT and BAT tissues were collected and stored either in −80 • C freezer or fixed in 4% paraformaldehyde for later use. All the procedures used in this study have been approved by the Qingdao University Animal Care and Use Committee in keeping with the National Institutes of Health guidelines (Approve code: 20190603).

Histological Methods
After fixing in 4% formaldehyde for 24 h, the EWAT and BAT tissues were histologically processed and embedded in paraffin as described in [58] with minor modifications. The paraffin blocks were then sectioned on a microtome (Leica RM2016, Leica, Germany) at 6-micron thickness. The sections were dried in oven at 37 • C overnight, and then subjected to hematoxylin and eosin staining or immunohistochemistry following manufacturer's instructions. The primary antibody dilution for the HSL antibody was 1:50. ImageJ (NIH, US) was used to semi-quantify the fraction of positively stained area. Three independent experiments were performed per group.

Serum FFA Level Measurement
The serum FFA levels were determined with a serum FFA measurement kit (BC0595, Solarbio, CN) following manufacturer's instructions. A standard curve was established along with actual samples. After data acquisition with a plate reader (M5, MD-SpectraMax, Molecular Devices, San Jose, CA, USA) at 550 nm absorption, the serum concentration of FFA was calculated according to the standard curve.

Western Blotting
Western blotting for PPARα and CPT1b expression levels in skeletal muscles as well as for UCP1, pHSL and HSL in brown adipose tissues were performed as described in [59] with minor modifications. Briefly, samples were homogenized in RIPA buffer with 1:100 phenylmethylsulfonyl fluoride and 1:100 phosphatase inhibitor cocktail (Epizyme, China, GRF102) added and centrifuged at 14,000× g for 10 min. The resulting supernatants were subjected to BCA assay for protein concentrations. Equal amounts of protein were mixed with sampling buffer and denatured at 95 • C for 5 min, and then subjected to SDS-PAGE electrophoresis. After transferring proteins to PVDF membrane and blocked with non-fat milk, primary antibodies (1:1000 for PPARα, CPT1b, UCP1, pHSL, and HSL) and 1:5000 for GAPDH or β-actin) were used to probe for the target proteins, and the bands were visualized by ECL system with a Fusion Solo S gel imaging system (Vilber Lourmat, Collégien, France). ImageJ (NIH, US) was used to semi-quantify the band densities. Three independent experiments were performed per target.

Statistics
Statistical analysis was performed with SPSS 17.0. The body weights at 0 week and 10 weeks were analyzed with one way analysis of variance (ANOVA), and all the other data were analyzed with two by two factorial design ANOVA. Results were considered statistically significant when p < 0.05.

Conclusions
In the current study, HBO treatment may alleviate HFD-induced fatty acid metabolism dysfunction/obesity in C57/B6 mice, which seems to be associated with corrected circulation and skeletal muscle L-carnitine levels and PPARα expression levels. HBO treatment is a promising physical therapy for management of metabolic syndrome and obesity.