Flaxseed Polysaccharide Altering Colonic Gene Expression of Lipid Metabolism and Energy Metabolism in Obese Rat

Obesity is one of the most serious public health challenges. Recently, we found that axseed polysaccharide (FP) had an anti-obesity effect through promoting lipid metabolism, inducing satiety and regulating gut microbiota, but how FP promote lipid metabolism through altering the colonic epithelial cells remains to be elucidated. In this study, a transcriptome study was performed to investigate the effect of FP altering the gene expression of colonic epithelial cells in an obese rat model. Results The transcriptome analysis showed that 3,785 genes were differentially expressed after FP intervention in colonic epithelial cells, including 374 down-regulated and 3,411 up-regulated genes. Through KEGG analysis, we found out three classical pathways related to lipid metabolism and energy metabolism, including PPAR signaling pathway, nitrogen metabolism and oxidative phosphorylation (OXPHOS). Moreover, qRT-PCR results showed consistent expression trends of differential genes with transcriptome analysis. The anti-obesity effect of FP may be achieved by regulating the expression of lipid metabolism- and energy metabolism-related proteins acting on the PPAR (peroxisome proliferator-activated receptor) signaling pathway, nitrogen metabolism and OXPHOS pathway in vivo. M, Montgomery MK, Reehorst CM, Nightingale R, Ng I, Anderton H, et al. Deletion of intestinal Hdac3 remodels the lipidome of enterocytes and protects mice from diet-induced obesity.

Introduction transcriptional responses to fermented bers, which regulates genes involved in energy metabolism, in particularly lipid catabolism [7]. What's more, intestinal microbiota can affect both sides of the energy balance equation, as a factor affecting the harvest of energy from dietary ingredients, and as a factor affecting host genes that regulate energy consumption and storage, for example, they can diminish mitochondrial fatty acid oxidation, suppress intestinal epithelial production of Angptl4 to facilitate lipid storage in adipose tissues, show greater inhibition of Cpt1 and so on [8].
In the previous research, we had found axseed polysaccharides (FP) had an anti-obesity effect via promoting lipid metabolism, inducing satiety and regulating the intestinal ora [9][10]. However, as a polysaccharide, FP can not directly enter the body to play a role, so we speculate that it can promote weight loss through the interaction with intestinal ora, change the gene expression of intestinal epithelial tissue, and induce the metabolism of fat and energy in vivo. However, it is not clear which intestinal epithelial tissue gene expression is changed by axseed polysaccharide. In the present study, we aimed to nd out the signi cantly different genes affected by FP involved in lipid and energy metabolism in colonic epithelial cells, further investigating the mechanism of FP in weight loss.

Materials And Methods
Animals, diets, and sample preparation 18 male Sprague-Dawley (SPF grade), 4 weeks old, were bought from Guangdong Medical Laboratory Animal Center (Guangzhou, China.). Group Con (n = 6) was fed with a standard diet (D12450B), others were given high-fat diet (D12492) to establish the obesity model, which were nally divided into two groups, Group HFD and Group FPD (6 rats per group). After that, Group Con and Group HFD were fed with the control diet, AIN-93M (3.77 kcal/g), while Group FPD was fed with a 10% FP diet, replaced 10% corn starch (in AIN-93M) with equivalent FP (3.37 kcal/g). The rats were weighed weekly. The details of the diets in this research are as labeled in Table 1.

Statistical Analysis
Numerical data were presented as means ± standard deviation (SD). The difference between means was analyzed using SPSS 25.0 software (IBM Corporation, Armonk, New York). Two-tailed Student's t-tests were conducted to compare the data in different groups. Besides, P < 0.05 (*) or P < 0.01 (**) was considered as statistically signi cant.

Results
Gene expression pro le of rat colonic epithelial cells As showed in our previous research, the body weight of rats in Group FPD was signi cantly lower than that of group HFD, and nally became very similar to those in Group Con at the end of the trial, which indicated that FP induced body weight loss [9]. Approximately 52.16 million raw reads were obtained from three groups. A huge proportion (95.14%) of high-quality clean reads was generated after adapter trimming and ltering low-quality reads. The percentages of clean reads having a base quality greater or equal than Q30 were above 95.14% with 48.37-50.25% of GC content, indicating that the data produced by sequencing are of high quality. On average, 96.04% of clean reads were mapped to the reference genome, among which 90.06% were aligned to unique locations.
As shown in Fig. 1a, 80.45% of the genes were in common among all three groups. Besides, the gene composition between Group Con and Group HFD were somewhat similar, while Group FPD was obviously different compared with them ( Fig. 1b). This result suggested that FP intervention would change the gene composition of colonic epithelial tissues.

Identi cation of Differentially Expressed Genes
The volcano plot showed the variation of mRNA expression between Group HFD and Group Con (Fig. 2a).
In total, 28 DEGs, including 14 upregulated and 14 downregulated, were identi ed. Besides, 3,785 DEGs were identi ed between Group HFD and Group FPD (Fig. 2b), including 374 downregulated and 3411 upregulated DEGs (P < 0.05). Compared with Group HFD, there were a lot of DEGs in Group FPD, but few in Group Con. Therefore, our subsequent analysis would focus on Group HFD and Group FPD.

KEGG Pathway Analysis of Differentially Expressed Genes
The DEGs were mapped in the KEGG pathway database. Compared with Group HFD and Group FPD, the annotated genes were classi ed into six categories. Most of them were enriched in signal transduction. Besides, 15 DEGs were enriched in the pathway of Energy metabolism and 26 DEGs in PPAR signaling pathway (Fig. 3).

KEGG Pathway Enrichment Analysis of Differentially Expressed Genes
It has been reported that weight loss involves a variety of mechanisms, such as inhibiting energy intake, stimulating energy expenditure and so on. Thus, KEGG pathway enrichment analysis of 15 DEGs in 'Energy metabolism' between Group HFD and Group FPD was performed. As a result, 13 metabolic pathways with signi cantly differential expressions (p < 0.05) were identi ed between Group HFD and Group FPD, of which 10 pathways showed the most signi cant difference (p < 0.01). Figure 4 shows the top 13 ranked KEGG pathway of DEGs. Nitrogen metabolism occupied the strongest enrichment degree as it possessed the highest Rich factor, followed by monobactam biosynthesis. Nitrogen metabolism had the most DEGs, and the then is OXPHOS. The DEGs in the two pathways were listed in Table 2, and all of them were upregulated. Especially, there were 9 genes of carbonic anhydrase enriched in nitrogen metabolism. The DEGs enriched in PPAR signaling pathway were further analyzed. Based on the correlation of gene expression, the correlation coe cient between gene and gene is obtained by Spearman algorithm. To systemically analyze the functions of these DEGs, we mapped them to PPI data and obtained an PPI network (Fig. 5). The network showed the association between 23 DEGs ( Table 2). The result suggested that the upregulation of Pparg, Lpl, Fabp1 and Acsl1 might play an important role in colonic epithelial tissues.

Veri cation of differentially expressed genes
In order to verify the results of transcriptome sequencing, 17 important DEGs were chosen for quantitative RT-PCR. The results of qRT-PCR are shown in Fig. 6. The expression trends were consistent with those obtained by RNA-seq, suggesting that the RNA-seq datas reliably re ected the gene expression alterations.

Discussion
This experiment investigated the gene expression of colonic epithelial cells affected by FP intervention in an obese rat model. After FP intervention, 3,785 DEGs were found in the obese rats, 15 of which were enriched in energy metabolism. Among them, 10 DEGs were enriched in nitrogen metabolism, including 9 genes of carbonic anhydrase and Cps1. Four DEGs were enriched in OXPHOS, including Cox4i2, Cox6b2, Atp6v1g2 and Ndufa4l2. Besides, 23 DEGs in PPAR signaling pathway showed strong connection.
As showed in our previous research, obese rats in Group FPD showed the reduction of body weight loss as well as body fat [9]. It is well-known that obesity is always related to an imbalance between the accumulation and consumption of lipids and energy. Thus, since FP intervention had been proved to lose weight, we speculated that it could improve lipid metabolism and energy metabolism. Our present results were consistent with our previous report, and more importantly, we found that FP intervention did improve the lipid metabolism, mainly by regulation of the PPAR signaling pathway in colonic epithielial tissues.

FP intervention promoted lipid metabolism of the colonic epithelial tissue
Most of the dietary lipids are long-chain TGs, which are hydrolyzed in the intestine to produce FAs and monoglycerides [11]. Then, they will enter enterocytes. As the members of the lipid-binding protein superfamily, FABPs bind to saturated and unsaturated LCFA and other hydrophobic ligands such as monoacylglycerols, mediating FA transportation to different cell organelles [12]. Thus, FP intervention upregulated the expression of Fabp1-Fabp5, heightening the transshipment of FA. After entering enterocytes, FFAs and glycerol arrive at the crossroads of several pathways; they can be metabolized within mitochondria, or be re-esteri ed to TGs, packaged into chylomicrons (CMs), and secreted from enterocytes to the lymph [13], which results in elevated TG levels in the circulation. CMs are the main contributors to the serum lipid level, and the decreased clearance or increased production of them can lead to hypertriglyceridemia [14]. High intake of dietary fat increases postprandial plasma TG levels signi cantly, and the regulation of lipid metabolism in intestinal epithelial cells could affect postprandial hyperlipidemia [15]. Most dietary fatty acids are processed as CM-TG, which can be later metabolized at the tissue level by LPL, releasing FA for tissue uptake [16]. Thus, FP intervention upregulated Lpl, inhibiting the release of CMs and the rise of plasma TG levels.
As a key enzyme in glycerol metabolism, GK catalyzes the conversion of glycerol to glycerol 3-phosphate, which is the main substrate in TG synthesis [17]. As a pore-forming transmembrane protein, the aquaglyceroporin AQP7 facilitates the transport of glycerol across cell membranes [18]. AQP7 involves in rapid uid movement through the villus epithelium in the small and large intestine [19]. Therefore, FP intervention downregulated Gk to inhibit the synthesis of TG, and upregulated Aqp7 to facilitate the transport of glycerol that would be metabolized in other tissues.
It is also very important to accelerate the oxidation of FAs, which in turn helps inhibit their reesteri cation. In the present study, the upregulation of Pparg and Rxrg was found. Several prior studies have demonstrated that administering mice PPAR agonists can induce the expression of lipid oxidation genes and β -oxidation in intestinal cells, and inhibit enterocytic lipid accumulation and post-prandial lipidemia [20]. RXR is a heterodimeric partner of PPARs, and LCFA may increase transcriptional activity of PPARs by acting as a physiological ligand of RXR [21]. Researchers found that the activation of PPARγ could control pathways connected with FA metabolism and mitochondrial function [13]. Thus, FP intervention enhanced the activation of PPARγ and RXR, altering DEGs of colonic epithelial tissue to perform biological responses and transcription of genes related to lipids.
Most cellular FA metabolic pathways need to be preferentially activated by thioesteri cation, and acyl-CoA synthetase catalyzes the formation of active FA [22]. ACSLs catalyze the conversion of free longchain fatty acids into their acyl-coenzyme A (CoA) forms [23]. The transport of long-chain acyl-CoA esters into the mitochondria matrix is mediated by the CPT system, among which CPT1 is responsible for converting acyl-CoAs into acylcarnitine to shuttle across the mitochondria membranes [24]. Thus, FP intervention upregulated Acsl1, Acsl4, Acsl6 and Cpt1c, which promoted the conversion of FA to acyl-CoA and the start of mitochondrial fatty acid β-oxidation. Besides, SCFAs and MCFAs permeate the inner mitochondrial membrane in the non-esteri ed form, which are activated into their CoA-derivatives in the mitochondrial matrix [25]. Afterwards, these FAs participate in β-oxidation and the TCA cycle. The nal products include CO 2 , NADH and FADH 2 .
In addition to accelerate FA oxidation, FP intervention also heightened cholesterol catabolism. As the key enzyme in the bile acid formation pathway, Cyp8b1 was found to be inhibited expressed in high fat diet mice, which inhibited the catabolism of cholesterol into bile acids, causing the elevated cholesterol [26]. Researchers found the elevation of PLTP activity results in rapid disposal of cholesterol from body through increased conversion into bile acids and subsequent excretion [27]. Thus, FP intervention upregulated Cyp8b1 and Pltp to accelerate cholesterol catabolism. Besides, FP intervention also played an important role in the prevention of in ammation. Two or more members of the perilipin family of lipid droplet surface proteins decorate lipid droplets in chordates [28]. As metabolically dynamic cellular organelles, lipid droplets are specialized in storing free fatty acids, the excess of which have toxic effects and can lead to in ammation [29]. Researchers found that Plin1 de ciency secreted pro-in ammatory lipid metabolites, exacerbating adipose tissue in ammation [29]. The colonic epithelial cells responded to elevated FAs by increasing expression of Perilipin under FP intervention, inhibiting in ammation. Adiponectin is encoded by the Adipoq gene, controls the metabolism of glucose and FA and has a general anti-in ammatory effect [30]. Adiponectin protects against murine colitis and maintains intestinal homeostasis through by modulating adaptive immunity and interactions with its receptor AdipoR1 [31]. Angptl4 protected against acute colonic in ammation and that its absence worsened the severity of in ammation [32]. As the critical enzyme in FA metabolism, SCD is responsible for the conversion of saturated FAs to unsaturated FAs, and oleic acid is one of the major products [33]. Researchers found that gut-speci c SCD-1 activity is necessary to control intestinal epithelial in ammation and synthetize oleate, and that dietary oleic acid provides protection against intestinal in ammation in vivo [34]. Thus, FP intervention upregulated Plin1, Plin4, Adipoq, Angptl4 and Scd to regulate and attenuate in ammation.
In conclusion, FP intervention modulated PPAR signaling pathways to suppress triglyceride accumulation mainly by stimulating fatty acid oxidation, as well as reduce in ammation.

FP intervention promoted energy metabolism of the colonic epithelial tissue
We found plenty of CAs were upregulated. As isozymes, CAs catalyze the carboxylation of water to form carbonic acid, which spontaneously decomposes into protons and bicarbonate (H 2 O + CO 2 ↔ H 2 CO 3 ↔ HCO 3 − + H + ) [35]. The CAs we found includes several isoforms: cytosolic (CA I, CA II, CA III, CA XII), membrane-bound (CA IV, CA XII and CA XIV), mitochondrial (CA V) and catalytically inactive (CA VIII) [36][37][38]. The luminal mucus from the colon and stomach of guinea pigs, mice and humans showed a large amount of CA activity, by which the hydration rate of CO 2 was increased by 1000-2000 times, about 1 / 10 of the rate found in red cells [39]. Thus, as showed in Fig. 7, FP intervention up-regulated CAs and accelerated the consumption of CO 2 , reducing the accumulation of it in cells and enhancing the TCA cycle. This would further increased energy consumption.
We also found the upregulation of Ca5b and Cps1. Localized in mitochondria, Cps1 catalyzes the condensation of metabolic ammonia and HCO 3 − to carbamyl phosphate [40]. Researchers suggested CA V in enterocytes provided bicarbonate ions to CPS1 and facilitated the conversion of ammonia to citrulline, the rst two steps of the urea cycle, which played an important role in detoxi cation and disposal of ammonia produced in the gut [41]. Approximately 12-18 g of protein reaches the human colon daily, and the main pathway of amino acid fermentation is deamination, leading to the production of ammonia, most of which is absorbed and metabolized into urea by the liver and excreted in urine [42]. Thus, FP intervention reduced the accumulation of bicarbonate ions in mitochondria, enhancing the initiation of the urea cycle and ammonium removal.
Generated from the TCA cycle and β-oxidation, NADH and FADH 2 provide electrons to the ETC [43]. The electrons are transferred to O 2 at complex IV to produce H 2 O, and the pumped protons produce an electrochemical gradient on the inner membrane of mitochondria, which is the driving force for complex V to produce ATP [44]. As a component of the ETC complex I subunit, NDUFA4L2 ne-tunes complex I activity, and mediates mitochondrial activation of OXPHOS [45]. COX6B2 facilitates the assembly of complex IV to support mitochondrial respiration and OXPHOS-induced generation of ATP [46]. We also found the upregulation of ATP6V1G2, which encoded subunit G2 of vacuolar ATPase (V-ATPase), transporting protons from the cytoplasm into the lysosome and maintaining lysosomal acidi cation [47].
V-ATPases are highly conserved proton pumps, and V1 is the peripheral membrane subcomplex of them, which contains the sites of ATP hydrolysis [48]. Thus, FP intervention upregulated Ndufa4l2 and Cox6b2 to heighten OXPHOS, accelerating the generation of ATP and stimulating energy consumption. Besides, FP intervention also upregulated Atp6v1g2, hydrolyzing ATP to provide energy for H + transport.
As the TCA progress and oxygen consumption in the ETC increase, however, the oxygen concentration in the colonic epithelial cells decreases. As the major consumers of oxygen in the cells, mitochondria are severely affected by decreased oxygen availability, and thus OXPHOS is adapted to hypoxia by remodeling ETC as well as the activity of the TCA cycle [49]. The COX4 subunit optimizes respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2, and COX4i2 facilitates a decrease in cytochrome c oxidase a nity to oxygen [50]. Both NDUFA4L2 and COX4I2 can reduce the capacity for mitochondrial oxygen consumption and act to limit mitochondrial ROS production during hypoxia, by reducing the activity of complex I and cytochrome c oxidase respectively [51]. Researchers found the deterioration of respiratory capacity due to differences at the level of respiratory chain complexes in obese individuals, among which, particularly, electron transport at the level of complexes I and IV was most affected [52]. Therefore, FP intervention upregulated Ndufa4l2 and Cox4i2, preventing mitochondria from hypoxia, which kept the energy expenditure steady and healthy.
As we have mentioned above, the Adipoq gene of colonic epithelial tissues that encode adiponectin was upregulated. In the previous researches, we had found FP intervention signi cantly upregulated adiponectin probably via the gut-brain axis. There is growing evidence that the metabolic bene ts of bariatric surgery are the result of complex gastrointestinal regulation, which produces signals that "educate" the brain to adapt to the new gut environment, and these signals somehow lower the defense levels of body fat [53]. Besides, the gut-brain axis also plays a key role in the control of energy balance [54]. Therefore, FP intervention might improve lipid metabolism and energy metabolism via the gut-brain axis to achieve some of the impressive effects of bariatric surgery with medications.

Conclusion
In summary, this study further sheds light on the mechanisms involved in the reported effects of FP intake. FP intervention had directly or indirectly altered the gene expression in epithelial cells, which further changed the host's lipid metabolism and energy metabolism, nally resulting in weight loss. By altering gene expression of the PPAR signaling pathway in the colonic epithelial cells, FP intervention accelerated FA catabolism to reduce accumulation. By promoting energy metabolism, FP intervention facilitated the transformation of FA oxidation products to ATP. As shown here, this study offers convincing evidence that FP may lose weight by regulating some potential key genes, which are involved in lipid metabolism and energy metabolism. Proving the role of FP in the prevention of obesity and obesity-related chronic diseases, these novel ndings may provide a powerful and novel mean to clarify FP as an e cient functional food candidate in therapy of obesity.   DEGs between different treatment groups. The horizontal axis indicates expression changes (log) of the genes in different treatment groups while the vertical axis shows the differences of gene expression. The discrepancy was more signi cant with smaller p-values and bigger −log10 (adjusted p-value). Splashes were for different genes, among which grey dots were genes with no signi cant discrepancy, red dots were genes signi cantly up-regulated and blue dots were signi cantly down-regulated genes.

Abbreviations
Abbreviations: DEGs, differentially expressed genes; Group Con indicates rats fed D12450B during the obesity model building and AIN-93M thereafter. Group HFD indicates rats fed D12492 during model building and AIN-93M thereafter. Group FPD indicates rats fed D12492 during model building and an FP containing diet thereafter.
Page 20/25    Illustration of potential regulatory mechanism involved in mitochondrial energy metabolism of FP intervention in the colonic epithelial cell. FP intervention upregulated ACS and CPT-1, promoting the transformation of FA to acyl-CoAs and the transfer into mitochondria for β-oxidation and TCA cycle.
Then, CO2, NADH and FADH2 were generated. FA intervention upregulated the relative proteins of complexes I and IV, promoting oxidative phosphorylation and generated H2O. CA V catalyzed H2O and CO2 into HCO3-, and then co-catalyzed with CPS1 for the transformation of NH3 and HCO3-to Citrulline. Therefore, the anti-obesity effect of FP intervention functioned through promoting the catabolism of FA to facilitate lipid metabolism and energy metabolism. Note that rectangles in green indicate upregulation of proteins in group FPD compared with group HFD, while that in grey indicates no signi cantly alteration.
Group HFD indicates rats fed D12492 during model building and AIN-93M thereafter. Group FPD indicates rats fed D12492 during model building and an FP containing diet thereafter.

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