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-esterified 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 significantly, 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 fluid 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 re-esterification. 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 thioesterification, and acyl-CoA synthetase catalyzes the formation of active FA [22]. ACSLs catalyze the conversion of free long-chain 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-esterified 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 final products include CO2, NADH and FADH2.
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 inflammation. 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 inflammation [29]. Researchers found that Plin1 deficiency secreted pro-inflammatory lipid metabolites, exacerbating adipose tissue inflammation [29]. The colonic epithelial cells responded to elevated FAs by increasing expression of Perilipin under FP intervention, inhibiting inflammation. Adiponectin is encoded by the Adipoq gene, controls the metabolism of glucose and FA and has a general anti-inflammatory 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 inflammation and that its absence worsened the severity of inflammation [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-specific SCD-1 activity is necessary to control intestinal epithelial inflammation and synthetize oleate, and that dietary oleic acid provides protection against intestinal inflammation in vivo [34]. Thus, FP intervention upregulated Plin1, Plin4, Adipoq, Angptl4 and Scd to regulate and attenuate inflammation.
In conclusion, FP intervention modulated PPAR signaling pathways to suppress triglyceride accumulation mainly by stimulating fatty acid oxidation, as well as reduce inflammation.
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 (H2O + CO2 ↔ H2CO3 ↔ HCO3− + 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) [36–38]. 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 CO2 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 CO2, 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 HCO3− to carbamyl phosphate [40]. Researchers suggested CA V in enterocytes provided bicarbonate ions to CPS1 and facilitated the conversion of ammonia to citrulline, the first two steps of the urea cycle, which played an important role in detoxification 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 FADH2 provide electrons to the ETC [43]. The electrons are transferred to O2 at complex IV to produce H2O, 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 fine-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 acidification [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 affinity 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 significantly upregulated adiponectin probably via the gut-brain axis. There is growing evidence that the metabolic benefits 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.