A great deal of evidence has shown that in mammals, the BW of IUGR neonate is significantly lower than that of normal neonate, and the syndrome would last for a long time to exert an adverse impact on their health [19]. In the present study, the FBW of IUGR-CON piglets declined by 20.95% compared with that of NBW-CON piglets, which is in agreement with previous studies [20, 21]. C. butyricum treatment significantly elevated the ADG and FBW of the IUGR-CB group compared with the IUGR-CON group. Similar to our study, many studies also indicated that C. butyricum supplementation could improve the growth performance of broilers, weanling pigs and Holstein heifers effectively [22–24].
As a central regulator of lipid homeostasis, the liver is responsible for the de novo synthesis, oxidation and export of FAs and, meanwhile, takes control of the biosynthesis and efflux of cholesterol [25, 26]. However, on account of the restricted condition of living, IUGR newborns would suffer from maldevelopment of the liver. The present study showed that IUGR severely damaged the hepatic morphological structure of infants, which is in line with a previous study [5]. Therefore, it may throw the lipid metabolism system into confusion. In the current study, increased TC content and decreased TBA levels were observed in the livers of the IUGR-CON piglets. BAs are the end products of cholesterol catabolism, and the conversion of cholesterol to BAs accounts for the daily turnover of a major fraction of cholesterol in mammals [27]. These results indicate that IUGR could lead to an excessive accumulation of cholesterol, which may result from the reduced transformation of BAs. Although no great difference in TG levels was found in either the liver or serum, a notable increase was found in the concentration of serum NEFAs in IUGR-CON piglets. Recently, more and more research has discovered that the accumulation of NEFAs is closely associated with a series of health problems, such as obesity, insulin resistance and vascular disease [28–30]. Thus, an elevated level of NEFAs is likely to have negative effects on the growth and development of these piglets. In addition, the activity of hepatic HL and the level of serum HDL-C decreased in IUGR-CON piglets. Herein, HL could play a critical role in hydrolysing triglycerides and promoting the uptake of HDL-C in circulating blood [31, 32]. HDL-C, considered “good cholesterol”, plays an important part in the pathway of reverse cholesterol transport (RCT), via which excess cholesterol can be transported from the periphery to the liver for clearance [33]. This evidence proved that IUGR could weaken the ability of clearing excess lipids of suckling piglets, which may lead to a potential risk of suffering from diseases related to lipid accumulation. On the contrary, the morphological structure of the liver was improved with effect in the IUGR-CB group. Besides that, the addition of C. butyricum lessened the deposition of excess lipids, such as TC and NEFAs of the piglets, and simultaneously increased the efflux of lipids through raising the level of hepatic TBA, HL and serum HDL-C of piglets. Similar effects of C. butyricum have been confirmed in HFD mice, which showed that C. butyricum intake could effectively improve the HFD-induced accumulation of lipid droplets in hepatocytes and decrease the content of hepatic TC and NEFAs in mice. Intriguingly, we also found that hepatic TG levels in the IUGR-CB group were elevated compared to the other two groups. We inferred that it was likely to be a kind of feedback regulation of a high level of cholesterol. Because cholesterol ester is less toxic than free cholesterol, the promotion of FA synthesis plays a role in cholesterol homeostasis with FAs being used as substrates for cholesterol esterification [34]. Hence, as shown above, supplemental C. butyricum could effectively regulate the disordered lipid metabolism of IUGR suckling piglets.
To further explore the molecular mechanism of lipid regulation of C. butyricum, we detected it in gene and protein levels from the aspects of FA and cholesterol metabolism. The uptake of circulating FAs by the liver is largely dependent on three major FA transporters located in the hepatocyte plasma membrane, which are known as fatty acid transport proteins (FATP), cluster of differentiation 36 (CD36) and caveolins [35, 36]. Following uptake, hydrophobic FAs cannot diffuse freely in the cytosol and must instead be shuttled between different organelles by fatty acid binding proteins (FABP) [35]. In this study, although no great difference was observed in the gene expression of FATP2, CD36 and FABP1 between the NBW-CON and IUGR-CON groups, the mRNA expression of CAV1 of IUGR-CON piglets was markedly decreased compared to the NBW-CON piglets. However, C. butyricum supplementation inversely increased the expression of CD36 and CAV1. Hereinto, CD36 has the function of facilitating the transport of long-chain FAs, and CAV1 plays a role in contributing to lipid trafficking and the formation of lipid droplets [36, 37]. Thus, it could be inferred that the elevated NEFA levels of IUGR-CON piglets in the serum might result from the weakened uptake of extracellular FAs by hepatocytes. However, C. butyricum treatment could increase lipid trafficking, consequently decreasing the NEFA content in the peripheral circulation and promoting its utilization and storage in the liver. The de novo biogenesis of FAs is mainly controlled by the sterol regulatory element-binding protein 1c (SREBP1c) and its downstream targets of ACC and FASN [25]. Thereinto, ACC is the first rate-limiting enzyme that converts acetyl-CoA to malonyl-CoA, and FASN is a key lipogenic enzyme that catalyses the terminal steps involved in synthesizing new FAs [38, 39]. Then, the newly synthesized FAs would be used for TG synthesis, and DGAT1 and DGAT2 catalyse its final step [40]. In the present study, IUGR had no great impact on the de novo synthesis of FAs, whereas it reduced its storage as a non-toxic form of triglycerides compared with the NBW-CON group. In contrast, C. butyricum treatment accelerated piglets’ biosynthesis of FAs and triglycerides. The reason for this phenomenon could be that IUGR impaired the uptake of FAs, which is considered the other important source of substrate to synthesize TG. Nevertheless, C. butyricum supplementation effectively improved it and simultaneously promoted the synthesis of FAs. Similar to our study, it was reported that C. butyricum supplementation could increase the expression of genes related to FA synthesis in chickens [8, 41]. Following uptake, these FAs are utilized by hepatocytes to generate adenosine triphosphate by means of oxidation. As the rate-limiting enzyme of FAO, carnitine palmitoyltransferase I (CPT1) catalyses the step of converting acyl-coenzyme As into acyl-carnitines, following which they can cross membranes to enter the mitochondria [42]. PPARα is a FA-activated nuclear receptor that plays a key role in the transcriptional regulation of genes involved in peroxisomal and mitochondrial FA β-oxidation, such as ACOX and LCAD [43–45]. The decreased expression of PPARα and LCAD in the IUGR-CON group in our study indicated that IUGR could weaken piglets’ ability of FAO during the suckling period. Considering the specificity of this time, the FAO dominates the generation of energy, so the growth of IUGR-CON piglets could be retarded because of the decreased FAO. However, C. butyricum treatment effectively improved the expression of FXR and PPARα and its target genes related to FAO. As a nuclear receptor, FXR is mainly expressed in the liver and intestine and has a comprehensive effect on lipid metabolism [46]. Similarly, Wang et al. (2020) found that the addition of C. butyricum could stimulate the peroxisomal FA β-oxidation probably through FXR–PPARα–ACOX pathway in hens [8]. Another study of human cells discovered that FXR activation induced the expression of PPARα and its downstream genes involved in FAO [47], which is in agreement with our study.
When it turns to cholesterol metabolism, LXRs work hand in hand with the sterol regulatory element-binding protein 2 (SREBP2) pathway to maintain cellular and systemic sterol levels [48]. On the one hand, LXRs could facilitate the elimination of excess cholesterol by stimulating biliary cholesterol excretion through the target genes of ABCG5 and ABCG8 [49]. On the other hand, SREBP2 could boost the biosynthesis of cholesterol by activating the transcription of the gene encoding of the rate limiting enzyme of HMGCR [50]. The increased gene expression of SREBF2 and the activity of HMGCR in the IUGR-CON group suggested that IUGR promoted the synthesis of cholesterol compared with the NBW-CON piglets, whereas the addition of C. butyricum down-regulated cholesterol synthesis by decreasing the expression of SREBF2 and up-regulated its efflux by elevating the expression of LXRα and its downstream ABCG8 gene. Herein, LXRα is one isoform of the LXR family that is highly expressed in metabolically active tissues, such as the liver and intestine.
Moreover, LXRα also plays a critical role in promoting RCT, through which excess cholesterol in peripheral tissues can be transferred to HDL and then transported to the liver for BA synthesis and excretion [48]. In this process, downstream genes of LXRα, such as ABCA1, ABCG1 and SR-BⅠ, work together to drive the assembly of HDL to initiate RCT [51–53]. Then, the excess cholesterol transported by HDL-C is used for the synthesis of BAs, which is critical for maintaining cholesterol homeostasis and preventing the accumulation of cholesterol in the liver [27]. BAs are synthesized by multi-step reactions catalysed in hepatocytes via two distinct routes: the “classical” (neutral) pathway and the “alternative” (acidic) pathway. The classic pathway is initiated by 7α-hydroxylation of cholesterol catalysed by the rate-limiting enzyme CYP7A1, followed by further transformations of the steroid nucleus and oxidative cleavage of the side chain involving CYP8B1 [54]. The alternative pathway is initiated by sterol 27-hydroxylase (CYP27A1). This reaction is followed by oxysterol 7α-hydroxylation, which is primarily mediated by CYP7B1 [55]. Finally, the synthesized BAs are secreted through the bile canalicular membrane by two ABC transporters (BSEP and MRP2) into the canalicular lumen [56]. Consistent with the variation in hepatic TBA levels, IUGR down-regulated the expression of CYP27A1 and CYP7B1 compared with the NBW-CON group. In the IUGR-CB group, the expression of CYP7A1, CYP27A1 and its downstream gene CYP7B1 was up-regulated markedly, and the expression of BSEP and MRP2 was increased accordingly. These results revealed that the reduced BAs of the IUGR-CON group were likely to result from the impaired acidic pathway and that C. butyricum treatment could effectively restore BA content to normal levels by promoting the classic and alternative pathways. A study focused on oxysterol 7α-hydroxylase, an important enzyme active in the acidic pathway for BA synthesis, confirmed the quantitative importance of the acidic pathway in early life in humans [57], which suggests that the acidic pathway might be the major route of BA synthesis in infants. Given the specific period of suckling, the malfunction of the alternative pathway would consequently lead to a mess in cholesterol metabolism. Moreover, Fu et al. (2001) discovered that increases in CYP27A1 activity could downregulate cholesterol synthesis through the SREBP pathway as well as enhance the efflux and elimination of cholesterol via LXR [58], which is highly in line with our findings above.
Plenty of evidence has confirmed that the composition of gut microbiota can have profound effects on the host [59, 60]. In our study, no great difference was found in the microbial α-diversity among these three groups except for a decreased Simpson index in the IUGR-CB group compared to the NBW-CON group. Combined with the changed bacterial community structure, it could be inferred that the addition of C. butyricum affected the homogeneity of the microbiota by modulating its composition. To further investigate the connection between the change in gut microbiota and the effect of lipid regulation in the IUGR-CB group, we analysed the differences among these groups both in phylum and genus levels. Although no difference was found in phylum level, piglets in the IUGR-CON group exhibited a significant increase in many opportunistic pathogens, such as Streptococcus, Enterococcus and Moraxella, which have the risk to cause an inflammatory response, and as a result, impair the normal function of the liver [61–63]. Intriguingly, C. butyricum treatment not only reduced the bacterium mentioned above but also decreased the abundance of Rothia and Acinetobacter, which were also considered opportunistic pathogens, and could have negative effects on the health of the host [64, 65]. Hence, C. butyricum’s modulation of gut microbiota might effectively protect against the invasion of pathogens caused by IUGR, which could be confirmed by the recovery of congestion in the liver portal vein and sinusoids.
Of note, among the changed microbes, Streptococcus and Enterococcus are both BSH-producing microbes whose common function is to generate the BSH enzymes used to deconjugate glycine- or taurine-conjugated BAs to form unconjugated BAs [66, 67]. As a result, the ileal BA profile of IUGR-CON piglets was altered compared with NBW-CON piglets, and it was characterized by decreased levels of conjugated BAs and elevated contents of unconjugated BAs. As we know, BAs can function as endogenous signalling molecules that bind to the BA receptors, such as FXR and LXRα, to regulate BA homeostasis in enterohepatic circulation and to modulate cholesterol and triglyceride metabolism [27, 68]. In the current study, C. butyricum treatment could elevate the proportion of conjugated BAs in the ileum, and their content of THCA, TCDCA and TCA were dramatically increased. THCA is a well-identified LXRα agonist, and TCA and TCDCA are well-recognized FXR agonists [16, 69]. Therefore, these signalling molecules might be transported to the liver via enterohepatic circulation and then play an important role in regulating lipid metabolism. To confirm this, we also analysed the BA profile in the liver. In line with the results of the ileum, C. butyricum supplementation increased the levels of LXRα agonist of THCA and the FXR agonist of TCDCA. Meanwhile, GUDCA, a kind of FXR antagonist [70], was also decreased in the IUGR-CB group compared with the IUGR-CON group. Hence, FXR and LXRα would be activated together to affect the lipid metabolism of the liver. Although the activation of FXR can repress the synthesis of BAs, it relies on the effect of small heterodimer partner (SHP). The importance of SHP in the feedback regulation of BA synthesis was demonstrated in SHP−/− mice, in which the repression of CYP7A1 was dismissed and the size of BA pool was enlarged [71]. In our study, despite the activation of FXR, no significant difference in SHP expression was observed in the IUGR-CB group compared to the IUGR-CON group. As a result, the repression of BA synthesis may not work out. Conversely, the synthesis of BAs could be up-regulated by the activation of LXRα. As a kind of hydrophilic BA used to treat hepatobiliary disorders, TUDCA can penetrate into the cell membrane and help transfer cholesterol from the cell membrane to HDL [72, 73]. A previous study showed that TUDCA treatment could effectively decrease serum and hepatic TC levels and increase the mRNA expression of CYP27A1 in a model of cholesterol gallstones [74]. Analogously, in our study, consistent with the decreased content of hepatic TUDCA in IUGR piglets, the concentration of serum HDL-C was decreased and the level of hepatic TC was increased in the IUGR-CON group compared with the NBW-CON group. However, C. butyricum supplementation significantly increased the hepatic content of TUDCA and, at the same time, promoted cholesterol efflux by increasing the level of serum HDL-C to drive the transport of cholesterol and up-regulating the expression of CYP27A1 to accelerate the synthesis of BAs.