| Following the development of Human Genome Project, researches focus on another huge "organ" in human beings or animals, gastrointestinal microflora, have become more and more important. Recent researches show that some gut microbes such as Bacteroides, Firmicutes, as well as methanogens may closely associated with host’s energy homeostasis and adipose metabolism. In current study,16S rRNA clone libraries of fecal methanogens of Erhulian and Landrace pig (different body fat mass) was constructed first. Results showed that there was a difference of composition of methanognes between the two breeds, which suggested that the different diversity of methanogens in hindgut of individuals with different body fat mass. Therefore, gut methanogens might have an effect on host’s energy and lipid metabolism. Secondly, SD rat was selected as animal model, the variation of the diversity and amount of intestinal methanogens, obesity associated parameters in blood serum, daily gain, the weight of epididymal fat pad, the expression of lipid metabolism associated genes was investigated through the addition of a methanogne inhibitor, BCM. Results showed that compared with control, treatment showed more body fat mass and a trend to obesity. The possible pathway about the effect of methanogens on host’s energy and lipid metabolism of SD rat was also summarized.1 Library of 16S rRAN gene of methanogens from fecal sample of erhualian and landrace pigTwo methanogen 16S rRNA gene clone libraries were constructed from fecal sample of Erhualian and Landrace pig. Sequence analysis showed that the 381 Erhulian clones were divided into 14 phylotypes with 176 sequences of 11 phylotypes affiliated with the Methanobrevibacter sp. (46.2% of clones) and 111 sequences of 1 phylotype affiliated with Methanobrevibacter smithii(29.1% of clones). The 384 Landrace clones were divided into 43 phylotypes with 134 sequences of 2 phylotypes affiliated with Methanobrevibacter smithii (34.9% of clones),62 sequences of 5 phylotypes affiliated with Methanobrevibacter sp. (16.1% of clones), and 29 sequences of 6 phylotypes affiliated with Methanosphaera stadtmanae (7.6% of clones) which did not find in the library of Erhualian. The percentage of unidentified clones in the two library was 24.7% and 41.4% respectively. Results showed that the diversity of methanogen 16S rRNA gene in Landrace was more than that in Erhulian. Erhulian was famous for it’s high fat composition, while Landrace is well known for it’s high lean meat content. Therefore, with results above, we might conclude that there was an interaction between the energy and lipid metabolism of host and the diversity of intestinal methanogens although there might be other influencing factors such as inheritance and environment.2 The construction of SD rat model with inhibited intestinal methanogens with BCMIn the present study, we successfully constructed a rat model with inhibition of gut methanogens by intragastric administration of BCM. There was no disadvantageous effect of BCM on other intestinal bacteria in vitro. Results showed that treating with BCM (0.012g/mol) for 10 days, the amount of methanogens in fecal samples was markedly reduced compared to control with Real-time PCR. After treating with BCM for 30 days, the copy number of mcrA gene of methanogens in feces was decresed about 10 times compared to control, and the quantity of methanogens was found stable from 30th to 60th day. According to the result, experimental period for BCM treating on rat could be determined as 30 days. Results for in vitro fermentation of fecal sample showed that, compared with control, the same concentration of BCM could significantly reduce the methane production. After feeding BCM for 24h, the methane production was lowest, while from 24h to 72h, the methane level was slightly recovered but not clearly. DGGE results showed that there was no obvious variation on the diversity of bacteria in the fermentation at different time point (Oh,4h,8h,12h,48,60h, and 72h), which suggested that the addition of BCM concentrated at 0.012g/ml had no effect on the normal growth of bacteria.3 Intragastric administration of bcm influences the diversity and quantity of gut methanogens of SD ratPCR-DGGE was used to investigate the variation of diversity and quantity of gut methanogens of SD rat treated with BCM by intragastric administration, stopped with BCM treating or stopped with BCM treating followed by inoculation of fecal fermentation from healthy rat. DGGE results showed that after treating with BCM for 10 days, some bands initially disappeared from the profile. After treating with BCM for 30 days, a large number of bands further disappeared from the profile of treatment. These disappeared bands were indentified close to Methanobrevibacter gottschalkii, only a small part of them were classified into Methanobrevibacter smithii and Methanobrevibacter sp.. Real-time PCR results showed that treating with BCM for 30 days, the quantity of fecal methanogens of treatment was clearly decrease compared to control. Compared with the profile of those rats continued with BCM treating, after the recolonization of gut methanogens, a few bands exhibited gradually from the DGGE profile. Sequencing results showed that these recovered bands were indentified as the same bands disappeared from the beginning. qPCR results showed that rats stopped with BCM treating or inoculated with fecal fermentation from healthy rat markedly got more quantity of fecal methanogens compared to those continued with BCM treating. Results above might suggest that after the intragastric administration of BCM, large number of gut methanognes were inhibited in SD rat, and most of the inhibited methanoges were classified to Methanobrevibacter gottschalkii.4 Inhibition of gastrointestinal methanogens influences serum biochemical parameters, daily gain, and weight of epididymal fat pad of SD ratThe variation of several obesity associated biochemical parameters of blood serum: high density lipoprotein (HDL), low density lipoprotein (LDL), glucose, cholesterol, and triglyceride as well as daily gain and the weight of epididymal fat pad was investigated by intragastric administration of BCM on SD rat. Results showed that after treating with BCM for 30 days, the level of serum LDL, cholesterol, the weight of epididymal fat pad, and daily gain of treatment was significantly higher than that of control (P<0.01), while stopped with BCM treating (St) or stopped with intragastric administration and inoculated with fecal fermentation from healthy rat (In), the level of serum HDL, LDL, cholesterol, the weight of epididymal fat pad, and daily gain was markedly lower than that of rat with continued BCM treating (P<0.05). Nevertheless, no significant difference observed between group St and In (P>0.05). Results above showed that after the inhibition of gut methanogens, the body fat of SD rats was clearly increased. Meanwhile, these rats showed potential obese characteristics, while these disadvantages were improved by the recolonlization of intestinal methanogens.5 The inhibition of gut methanogens influences the expression of lipid metabolism associated genes of SD ratThe variation of expression of lipid metabolism associated genes in epididymal fat pad, liver, and colon of SD rat with BCM treating or recolonlization of gut methanogens was detected by Real-time PCR. The detected genes included peroxisome proliferator-activated receptor y (PPARy),fasting-induced adipose factor (Fiaf),lipoprotein lipase (LPL), Leptin, protein phosphatase 2 A (PP2A),sterol regulatory element-binding protein-lc (SREBP-lc),carbohydrate response element-binding protein (ChREBP), adiponectin (Adi), and fatty acid synthase (FASN). Results showed that compared with control, after treating with BCM for 30 days, the expression of PPARy, Fiaf, LPL, PP2A, SREBP, ChREBP, and FASN in all of the tree tissues and Adi in epididymal fat pad was significantly up-regulated (P<0.05 or P<0.01), while the expression of Leptin was found no change (P>0.05). In the second experimental period, compared with rats continued with BCM treating, with the recolonization of gut methanogens, the expression of PPARy, Fiaf, LPL, SREBP, ChREBP, and FASN in all of the three tissues was markedly down-regulated (P<0.05), and the expression of Adi in epididymal fat pad was clearly up-regulated (P<0.05). The expression of PP2A gene was only found significantly up-regulated in liver (P<0.05). The expression of Leptin was only found significantly up-regulated in the epdidymal fat pad of rat inoculated with fecal fermentation from healthy rat (In), while the expression of Leptin was markedly up-regulated in liver and colon of group stopped with BCM treating (St) or In (P<0.05). No expression of Adi was detected in liver and colon. Results above suggested that after the inhibition of intestinal methanogens, in all of the three tissues of SD rat, the expressions of most lipid metabolism associated genes were significantly up-regulated, while with the recolonization of gut methanognes, the expression of the genes showed contrary variation, which illustrated that rat with inhibited methanogens can absorb more lipid that stimulated the deposition of body fat.6 Inhibition of gut methanogens influences the level of metabolites in colonic digesta of SD ratThe variation of metabolites in colonic digesta between control and SD rat with BCM treating, and among rat stopped with BCM treating (St), stopped with BCM treating and inoculated with fecal fermentation from healthy rat (In), and continued with BCM treating (Co) was detected through’H-NMR. The PCA score profile showed that all of the samples could be totally distinguished. The loading profile showed that the level of a large number of metabolites in colonic digesta was changed after treating with BCM. Compared to control, the level of lactate, valine, isoleucine, propionate, acetate and some unknown sugars and amino acids was decreased, while the level of butyrate and succinate was elevated. At the end of the second experiment period, compared to Co, the level of valine of colonic digesta of St and In was decreased, while the level of propionate, butyrate, isoleucine, acetate and some unidentified sugars and amino acids was increased. Results above might suggest that after the inhibition of intestinal methanogens, there was an variation on the level of volatile fatty acid, amino acid, as well as sugar. The interaction of these materials finally impact on the energy and lipid metabolism of SD rat. |
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