Feeding a HFD is the most common method for animal models of obesity, thus HFD continues to be an indispensable method for discovering mechanisms of metabolic syndromes . Similarly, untargeted metabolomics, an effective method to measure metabolites, plays an important role in understanding the physiological functions of metabolites and the potential causes of metabolic disorders .
Feeding rabbits with a HFD will result in damage to the normal function of PAT, destruction of the balance between lipid formation and degradation, and over accumulation of lipids in PAT. The LC-MS/MS metabolite analyses showed that the PAT lipid cycle in rabbits fed a HFD was disturbed, resulting in significant changes in the levels of phospholipids, fatty acids, steroid hormones, and L-methionine (Fig. 4C). Similarly, feeding mice with a HFD caused a metabolic imbalance that resulted in metabolic disorders such as IR and nonalcoholic steatohepatitis .
The symptoms of obesity closely resemble the spectrum of metabolic changes in PAT, including phospholipids, fatty acids, steroid hormones, and amino acids, among which phospholipids and lysophosphatides are the most abundant metabolites. Phospholipids are the main components of plasma membranes including phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs), which are precursors of lysophosphatidylethanolamine (LPEs) and lysophosphatidylcholines (LysoPCs/LPCs), respectively . In our study, PCs and PEs were the most frequently found phospholipids (most of them down-regulated in HFD rabbits), whereas levels of all LPCs were reduced. Concerning the 14 PCs and 8 PEs in PAT, some PCs, such as PC (18:4e/20:5) and PC (17:2/22:6), increased in HFD rabbits, which was similar to a previous study in humans where some PCs to be significantly higher in the obese group than that in the control group . The level of PE (18:2/18:2) was lower in mice fed a HFD than mice fed a normal diet , consistent with our results. Further, the level of LPCs (LPC 15:0, LPC 19:0) decreased in rabbits fed a HFD for 4 weeks, which was partially in agreement with a decrease in plasma in human Obesity and T2D , and low-abundance of LPCs in serum of hyperlipidemic mice fed a HFD . And PE is methylated to PC . Changes in phospholipid levels can inhibit calcium ion transport and affect the transfer of phospholipids between the endoplasmic reticulum (ER) and mitochondria, inducing ER stress and mitochondrial dysfunction, which will decrease fatty acid oxidation and acetyl CoA levels . In addition, an imbalance in the PC/PE ratio will affect the mitochondria-associated ER membranes, leading to an excessive accumulation of sphingomyelin (SM) in the ER, inducing the activation of PKC, inhibiting the activity of AKT, and disrupting energy supply and metabolic homeostasis . SM is produced by a group transfer in phosphatidylcholine combined with the associated skeleton, which is closely related to sphingomyelin synthase (SMS). A significant increase in SM may reduce reverse cholesterol transport, increasing the risk of atherosclerosis lesions and other metabolic diseases . Further, as an important signal molecule, reduced levels of LPC combined with some cell-specific G-coupled protein receptors can cause an increase in insulin secretion through glucose stimulation, which will damage β cell function and lead to insulin resistance, stimulate the production of adipocytes, and aggravate the risk of obesity and other diseases [43, 44]. These are the factors that may cause insulin resistance. The decrease of LPCs in rabbits from this study may be related to an increase in insulin resistance, thus LPCs could be considered as potential biomarkers for metabolic diseases caused by obesity due to HFD. Our results here indicated that SM levels were significantly up-regulated in the HFD compared to the SND rabbit groups, in agreement with reports of obesity and insulin sensitivity in obese adult humans  and a study on the plasma metabolic fingerprints of atherosclerosis rabbits. These results suggest that changes of phospholipids levels may reduce insulin sensitivity, lead to insulin resistance, and increase the risk of atherosclerosis.
Arachidonic acid (ARA) and adrenic acid are omega-6 polyunsaturated fatty acids. In the current study, levels of ARA were higher in the HFD than in the SND rabbit group, in agreement with a significant increase in serum ARA levels in rats fed a HFD . According to our identification results, ARA was the main metabolite of the arachidonic acid metabolic, ovarian steroidogenesis, biosynthesis of unsaturated fatty acids, and ferroptosis metabolism pathways. However, a ‘one-to-many’ type of relationship was pointed out between metabolic pathways that have been annotated and identified compounds. ARA matched 18 associated metabolic pathways, which showed that at least ARA was comparatively important for PAT. The most important metabolic pathway for ARA in this study was platelet activation (P < 0.01). Under normal circumstances the release of fatty acids in PAT is strictly controlled to meet energy requirements. Conversely, metabolic disorders cause excessive release of fatty acids relative to tissue requirements. It is widely accepted that disturbances in fatty acid metabolism lead to increased inflammatory signaling, which are central factors in IR . ARA promotes the production of several prostaglandins, which are associated with lipopolysaccharide (LPS) induced inflammation . It is hypothesized that the increase of ARA is associated with PAT metabolic disorders and may induce inflammation to further produce insulin resistance. Previous studies have shown that the concentration of body fat and adipocytokines in the (HFD + ARA) group was significantly increased after six weeks of induction . Further, excessive levels of ARA can cause oxidative stress and activate pro-inflammatory signals that induce endoplasmic reticulum (ER) stress leading to IR [50, 51]. Hence, ARA can be used as an indicator of PAT metabolic disorders in obese patients consuming HFD. Endogenous adrenal acid is produced by ARA, and it is mainly oxidized in the peroxisome. Consistent with results here, plasma levels of adrenal acid were found to be up-regulated in an adipose hepatitis model, and primarily caused by instability of peroxidase β-oxidation . Docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) are long-chain omega-3 polyunsaturated fatty acids (PUFAs). The levels of DPA and DHA were significantly elevated in PAT from HFD-fed rabbits compared to SND-fed rabbits. The higher levels of DPA and DHA in the HFD rabbit group disagreed with significantly lower levels of DPA and DHA in 12-wk old rats fed a HFD relative to rats in the control group. Further, a strong positive association existed between the reduced levels of these two metabolites and the insulin sensitivity index . A possible explanation is that a higher concentration of n-3 PUFAs inhibits the release of free fatty acids from PAT , which in turn inhibits the inflammatory signaling pathway and decreases the risk of IR, thus playing a protective role. In addition, DHA and DPA have strong anti-inflammatory effects and can activate peroxisome, thus increasing insulin sensitivity [54, 55]. The levels of DPA and DHA in the HFD rabbit group were significantly increased, which may indicate a protective effect. Therefore, rabbits in the HFD group may have IR and other metabolic syndromes, but they may also produce metabolites such as DHA and DPA to protect them against adverse factors. However, no free fatty acids were detected in plasma/serum of HFD rabbits, thus additional research is needed.
Steroid hormones such as testosterone, 2-hydroxyestradiol, and epitestosterone were also found in this study. These hormones play vital roles in the production and metabolic function of adipose tissue via hormone receptors. Levels of testosterone, 2-hydroxyestradiol, and epitestosterone were significantly lower in PAT from the HFD than the SND rabbit group. The hormone 2-hydroxyestradiol has strong inhibitory effects on NADPH during lipid peroxidation in rat microsomes. In addition, lipid peroxides depend on specific ions at the initial stage, and are strongly inhibited by oxygen absorption , which may inhibit the PAT lipid metabolic pathway and cause lipid accumulation. Changes in testosterone were the result of rats fed HFD to indced IR , strongly suggesting that changes in testosterone levels are due to rabbits fed HFD induced insulin resistance. Furthermore, a study showed that testosterone gradually increased in visceral fat rather than in subcutaneous adipose tissue in human females . This result agreed with the significant increase adipocytes cells and density of PAT in HFD rabbits relative to SND rabbits. Thus, changes in 2-hydroxyestradiol and testosterone levels in this study may have led to lipid accumulation by increasing adipocytes cells and density of PAT and inhibiting the absorption of oxygen by lipid metabolism. However, the specific mechanism of steroid hormones in PAT needs further study.
L-methionine produces methionine, and it functions not only as an essential amino acid but also as a physiological effector . We found higher levels of L-methionine in HFD than in SND rabbits. The methionine cycle provides methyl units for various reactions including methylation in lipids. The S-adenosine methionine (SAM) is used as a major methyl donor molecule and it is synthesized from the essential amino acid methionine . Choline, produced by phosphatidylcholine, and the subsequent substances produced by choline oxidation, such as betaine, can not only help to adjust cell volume, but also act as methyl donors in the homocysteine-methionine (HM) cycle, transporting excess fatty acids to corresponding organelles for metabolism . Methionine supplementation increases homocysteine (Hcy) concentration and is associated with vitamin B6. Therefore, in our study, one possible explanation is the excessive accumulation of fatty acids in PAT of rabbits fed with a HFD, and the increase of L-methionine level, thus further increasing the level of Hcy, and finally disturbing the HM cycle. However, previous studies have been reported that high circulating Hcy concentrations are related to elevated risk of atherosclerosis, steatohepatitis and lipid metabolic disturbances [62, 63]. And changes in HM cycle after feeding lean Iberian sows with a HFD were associated with obesity-related diseases and T2D , indicating that the higher levels of L-methionine may be related to atherosclerotic diseases and T2D by affecting the HM cycle.