GOS Ameliorates Obesity In DIO Rats
During the administration, the average daily food intake of SD rats was recorded, and the results showed that GOS had no appetite inhibition (Fig. 1a and Table S2). After GOS and orlistat treatment, the weight gain trends were slower than that of the Model group, among which GOS-H had the best effect (Fig. 1b-c). These results indicated that GOS can decelerate the trend of weight gain without affecting appetite in DIO rats. After weighing the fat mass (fat pad) that was anatomically obtained, the fat/ body ratio of the rat was calculated. The results showed that GOS significantly reduced the fat content and the fat/body ratio caused by HFD (Fig. 1d-e and Table S3).
GOS Improves Serum and Liver Lipid Metabolism In DIO Rats
The effects of GOS on serum and liver lipid metabolism levels (TC, TG, LDL-C and HDL-C) were determined. Compared with the Model group, GOS significantly increased serum HDL-C level and decreased TC, TG and LDL-C levels (Fig. 2a-d and Table S4), although the GOS-H was slightly inferior to the orlistat. Similarly the levels of TC, TG and LDL-C in liver were obviously lower, while the levels of HDL-C were markedly increased after GOS treatment (Fig. 2e-h and Table S5), indicating that GOS can improve dyslipidemia induced by HFD in rats.
GOS PromotesFecal LipidExcretion
The contents of TC and TG in the feces of DIO rats were examined, the results are presented in Fig. 2i-j and Table S6. The TC and TG levels after orlistat treatment were obviously higher than those of the Model group, due to the fact that orlistat is a lipase inhibitor, which can inhibit the absorption of fat in the body. The contents of TC and TG in GOS group were significantly higher than Model group. The results showed that GOS can improve obesity symptoms by promoting the excretion of TC and TG.
GOS Improves Serum Glucose Level In DIO Rats
Blood glucose is the main source of normal energy supply in the body. Glucose tolerance is the body's ability to regulate blood glucose concentration. As shown in Fig. 2k, serum glucose levels were measured. Blood glucose were decreased after GOS treatment, but only GOS-H showed significant difference,which indicates that GOS had a certain hypoglycemic effect. In order to observe whether the glucose tolerance of rats was abnormal after the administration of GOS, a glucose tolerance test was conducted to observe the body's sensitivity to glucose level. The results are shown in Fig. 2l-m, indicated that GOS-H reduced glucose tolerance in obese rats.
GOS Reduces Lipogenesis In the Liver and Inhibits Adipocyte Hypertrophy
The DIO rats were dissected, the liver was removed, photographed and the overall morphology was observed, as shown in Fig. 3a. The liver of the Model group was hypertrophy, the surface was not smooth and the color was white. In contrast, the livers in the Control group were bright red in color, small in size, soft in texture and with tissue elasticity. Interestingly, the livers of GOS groups were bright red in color, soft in texture, and smooth in leaf. In addition, liver tissues of DIO rats were sectioned and stained, as shown in Fig. 3b. The hepatocytes in the Control group were orderly and uniform in size, there was no inflammatory cell infiltration. However, liver cells in Model group were deformed and had uneven size and more fat drop accumulation. After GOS administration, the fat droplets were significantly reduced.
HE staining was performed on epididymis (Fig. 3c), perirenal (Fig. 3d) and subcutaneous adipose tissue (Fig. 3e). The adipocytes in the Control group had normal structure, compact arrangement, uniform size, large number and small cell volume, while the adipocytes in the Model group were not uniform in size. After GOS administration, the size of fat cells significantly decreased and the hypertrophy were improved. All data indicated that GOS can relieve fatty liver, inhibit the growth and accumulation of fat cells in WAT, so as to play an anti-obesity role.
DGE In Liver and Adipose Tissue
To explore the mechanism of GOS weight loss and lipid-lowering, genes differentially expressed in the liver and epididymal fat were identified. The differentially expressed genes were mapped by differential gene analysis as shown in Fig. 4a and Fig. 5a, respectively. The significantly up-regulated and down-regulated genes in liver and epididymal fat were counted. Compared with Model, GOS up-regulated 300 genes and down-regulated 266 genes in liver. Similarly, GOS up-regulated 120 genes and down-regulated 428 genes in adipose tissue. The above results showed that feeding on different diets (normal diet and high-fat diet), and the administration of GOS can regulate gene transcription levels in DIO rats. The results of the Gene Ontology (GO) annotation analysis in liver and epididymal fat are shown in Fig. 4b and Fig. 5b, respectively. In the liver, the impacted biological processes mainly involved cell process, cell development process, biological regulation, metabolic process and multicellular tissue process. The cell composition was mainly associated with cell membrane and organelle. The molecular function mainly covered binding function and catalytic function (Fig. 4b). Furthermore, the results of GO annotation analysis in the epididymal fat were the same as those in the liver (Fig. 5b).
In order to further understand the connections between genes in organisms, pathway analysis was conducted, the cooperative effects of different genes were deeply explored to complete certain functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis in liver and epididymal fat are shown in Fig. 4c and Fig. 5c, respectively. It can be known that the KEGG pathway classification in liver and epididymal fat mainly involved energy metabolism, bile acid metabolism, amino acid metabolism, lipid metabolism, sterol and steroid metabolism. In addition, enriched bubble map analysis was performed on the KEGG pathway analysis of differential genes in liver and epididymal fat, and the results are presented in Fig. 4d and Fig. 5d, respectively. The results suggested that the differential genes in liver were concentrated on metabolism-related pathways, including ether-lipid metabolism, lipid metabolism, mitogen-activated protein kinase (MAPK) signaling pathway, metabolism of cytochrome P450 to heterologous organisms, biliary secretion, and tyrosine metabolism (Fig. 4d). These results indicated that GOS was associated with lipid metabolism pathways in the liver. Therefore, the lipid metabolism-related proteins in the liver were selected for lipid lowering mechanism study.
In addition, the results in epididymal fat suggested that the differential genes were mainly related to thermogenesis, including MAPK, peroxisome proliferators-activated receptors (PPAR), phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway, cholesterol metabolism, fatty acid metabolism, and arachidonic acid metabolism (Fig. 5d). These results indicated that GOS was associated with the browning and lipid metabolism of white fat in epididymal fat. Therefore, proteins related to the browning and lipid metabolism of white fat were selected for anti-obesity mechanism study.
GOS Promotes Epididymal Fat Browning
Based on the results of gene sequencing, genes related to lipid metabolism and energy metabolism were verified (Fig. 6a), including browning marker genes and thermogenic genes, namely, UCP1, peroxisome proliferative proliferator-activated receptor γ (PPARγ), peroxlsome proliferator-activated receptor-γ coactlvator-1α (PGC1α), zinc lipoprotein 16 (PR domain-containing 16, PRDM16) and activating transcription factor2 (ATF2). The results indicated that protein levels of UCP1, PPARγ, PGC1α and PRDM16 were significantly increased after GOS treatment (Fig. 6b-e). Protein levels of ATF2 in GOS-H and GOS-M group was markedly higher than those in the Model group (Fig. 6f). According to these results, we can preliminarily infer that GOS may promote the browning of epididymal fat cells in DIO rats.
GOS Promotes Brown Fat Formation
BAT loses weight by increasing the amount of energy consumed by nontremor heat production. In order to investigate the changes of BAT in DIO rats after GOS intervention, we detected the expression of thermogenic protein in BAT (Fig. 7a). The results were consistent with the expression of these proteins in epididymal fat. Compared with HFD alone, the protein expression levels of UCP1, PRDM16 and PPARγ in the brown fat of DIO rats treated with GOS were obviously increased (Fig. 7b,c,e). In addition, the high and medium dose GOS significantly increased PGC1α protein expression (Fig. 7d). On the premise of suppressing weight gain and promoting the increase of brown fat cells, the results indicated that GOS can promote the formation of BAT and thermogenesis.
GOS Regulates Cholesterol Catabolism In Liver andEpididymal Fat
Cholesterol metabolic-related proteins, cholesterol 7α-hydroxylase (CYP7A1), low density lipoprotein receptor (LDLR), liver X receptor α (LXRα), peroxisome proliferative proliferator-activated receptor α (PPARα), sterol-responsive element binding protein-2 (SREBP2) in liver, and CYP7A1, LDLR, PPARα, LXRα in epididymal fat were detected, as shown in Fig. 8a and Fig. 9a, respectively. The results suggested that the protein expression levels of CYP7A1 and PPARα in the liver of DIO rats treated with GOS were significantly increased compared with that given only HFD (Fig. 8b,e).
Compared with the Model group, the expression level of LDLR and LXRα protein were significantly increased after GOS-H and GOS-M treatment (Fig. 8c,d). GOS, especially the medium and low dose, reversed the upregulated expression of SREBP2 induced by HFD (Fig. 8f). Moreover, the results in epididymal fat were consistent with the expression of these protein in liver. Protein expression levels of CYP7A1, LDLR, LXRα, PPARα in epididymal fat were markedly higher in DIO rats treated with GOS than in those using HFD intervention (Fig. 9b-e). These results suggested that GOS can promote the decomposition of cholesterol in liver and epididymal fat by regulating the expression of proteins involved in cholesterol catabolism to relieve obesity and its related metabolic diseases.