Associations between hepatic miRNA expression,liver triacylglycerols and gut microbiota during metabolic adaptation to high-fat diet in mice

Aims/hypothesis

Despite the current pandemic of metabolic diseases, our understanding of the diverse nature of the development of metabolic alterations in people who eat a high-fat diet (HFD) is still poor. We recently demonstrated a cardio-metabolic adaptation in mice fed an HFD, which was characterised by a specific gut and periodontal microbiota profile. Since the severity of hepatic disease is characterised by specific microRNA (miRNA) signatures and the gut microbiota is a key driver of both hepatic disease and miRNA expression, we analysed the expression of three hepatic miRNA and studied their correlation with hepatic triacylglycerol content and gut microbiota.

Methods

Two cohorts of C57BL/6 4-week-old wild-type (WT) male mice (n?=?62 and n?=?96) were fed an HFD for 3 months to provide a model of metabolic adaptation. Additionally 8-week-old C57BL/6 mice, either WT or of different genotypes, with diverse gut microbiota (ob/ob, Nod1, Cd14 knockout [Cd14KO] and Nod2) or without gut microbiota (axenic mice) were fed a normal chow diet. Following which, glycaemic index, body weight, blood glucose levels and hepatic triacylglycerol levels were measured. Gut (caecum) microbiota taxa were analysed by pyrosequencing. To analyse hepatic miRNA expression, real-time PCR was performed on total extracted miRNA samples. Data were analysed using two-way ANOVA followed by the Dunnett’s post hoc test, or by the unpaired Student’s t test. A cluster analysis and multivariate analyses were also performed.

Results

Our results demonstrated that the expression of miR-181a, miR-666 and miR-21 in primary murine hepatocytes is controlled by lipopolysaccharide in a dose-dependent manner. Of the gut microbiota, Firmicutes were positively correlated and Proteobacteria and Bacteroides acidifaciens were negatively correlated with liver triacylglycerol levels. Furthermore, the relative abundance of Firmicutes was negatively correlated with hepatic expression of miR-666 and miR-21. In contrast, the relative abundance of B. acidifaciens was positively correlated with miR-21.

Conclusions/interpretation

We propose the involvement of hepatic miRNA, liver triacylglycerols and gut microbiota as a new triad that underlies the molecular mechanisms by which gut microbiota governs hepatic pathophysiology during metabolic adaptation to HFD.

     

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.The human intestinal tract harbors the most abundant, and among the most diverse, microbial community of all body sites (Ley et al. 2008; Costello et al. 2009). As in most mammals, the gut microbiome is dominated by four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (Ley et al. 2008), which represent more than 1000 different molecular species or phylotypes (Dethlefsen et al. 2008; Claesson et al. 2009). Remarkably, this phylotype composition can be specific and stable for each individual. Repeated sampling of the same individuals indicates that samples from the same subject are more similar than samples from different subjects (Costello et al. 2009; Turnbaugh et al. 2009), and in a 2-yr interval an individual conserves over 60% of phylotypes of the gut microbiome (Manichanh et al. 2008).The gut is considered the primary site for cross-talk between the host immune system and microorganisms, in part because of the size and complexity of its microbiota and the presence of specialized lymphoid structures in the mucosa (Guarner et al. 2006). This close relationship is important for maintaining an adequate homeostasis between the individual and the external environment (Backhed et al. 2005; Guarner et al. 2006). Imbalances of the intestinal microbial composition, named dysbiosis, may disturb homeostasis, and therefore lead to a dysfunction or disease state. For instance, specific changes of this microbial ecosystem were recently associated with two of the major inflammatory bowel diseases (IBD) (Ott et al. 2004; Manichanh et al. 2006; Frank et al. 2007; Dicksved et al. 2008). A large reduction of microbial diversity was found in patients with Crohn''s disease (Manichanh et al. 2006; Dicksved et al. 2008), and a selective reduction of Faecalibacterium prausnitzii, a member of the Firmicutes phylum, was reported in patients with ulcerative colitis (Sokol et al. 2009). Remarkably, in both inflammatory bowel diseases, most bacteria that decrease in abundance relative to healthy controls are producers of butyrate, which has strong anti-inflammatory effects (Nancey et al. 2002; Hamer et al. 2009). Therefore, although the mechanisms underlying these disorders are yet unclear, it is now well accepted that intestinal microorganisms play a key role in the initiation and maintenance of IBD (Round and Mazmanian 2009).Experimental manipulation has great potential to go beyond observational studies and allow us to decode the physiological roles of the gut bacterial community, and also define new therapeutic strategies based on altering this microbiome. In principle, the stability of the gut microbiome could be disrupted by the use of prebiotics, probiotics, and antibiotics. Intake of prebiotics (i.e., specific nondigestible food ingredients) is expected to stimulate the growth and/or bacterial activity in the gut. So far, however, no prebiotic has been shown to have a persistent effect in modifying the gut microbial composition. Similarly, intake of probiotics (i.e., live microorganisms) confers only transient effects on digestive physiology, and long-term persistent alteration of the indigenous gut microbial composition remains controversial. Attempts to manipulate the composition of the intestinal microbiome by fecal bacteriotherapy have now become the focus of an extensive body of clinical case reports with promising results (Borody et al. 2003; You et al. 2008; Khoruts et al. 2009; Shanahan 2009). For instance, it has recently been shown that fecal transplantation from a healthy donor restored both gut microbiota composition and function in a human patient that suffered from recurrent Clostridium difficile–associated diarrhea (Khoruts et al. 2009). Finally, in contrast to prebiotic and probiotic intake, antibiotics have been shown to produce drastic short- and long-term alterations of the human indigenous microbiota. In these studies, microbial compositions were examined using DNA fingerprint techniques (Lofmark et al. 2006; Jernberg et al. 2007), microarrays (Palmer et al. 2007), and, more recently, by taking advantage of DNA pyrosequencing (Dethlefsen et al. 2008; Antonopoulos et al. 2009). All of the above studies indicated that after antibiotic intake there is a drastic disruption of the intestinal microbiota, resulting in a long-term decrease of its overall diversity.The above observations clearly anticipate that experimental manipulation of the gut bacterial community should be feasible to some extent, for example, in the well-established transplantation of exogenous microbiota into germ-free animals. The result of this procedure is a stable colonization by the transplanted community that keeps most of its original diversity (Rawls et al. 2006; Alpert et al. 2008). Therefore, although host factors probably have a major effect in broadly shaping the intestinal microbial ecosystem, long-term alterations of an indigenous consortium might also be induced, especially at the phylotype level. Such changes can now be uncovered due to the rapid development of genomic approaches and computational methods, which permit more detailed comparisons of the compositions of microbial ecosystems. In the present study, we used recently developed tools for deep sequencing and phylogenetic clustering to examine the degree to which the gut ecosystem could be intentionally manipulated. Using rats as a model system, we compared the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. We tested the hypothesis that antibiotics, by reducing bacterial load, would promote establishment of the transferred microbiota, this outcome would have important implications for clinical practice in situations where the goal is to colonize the gut with a new microbiota. The results were surprising, and indicated that the indigenous gut microbial composition could be reshaped to an extent not anticipated in previous studies.     

Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of lynch syndrome patients

Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair‐deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene‐specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3′ end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2‐specific PCR primers and MLPA probes, designed on PSVs, in the 3′ duplicated region is unreliable, and present an RNA‐based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV‐based mutation detection methods. Hum Mutat 31:578–587, 2010. © 2010 Wiley‐Liss, Inc.     

Serrated Flippers and Directional Asymmetry in the Appendicular Skeleton of the Commerson's Dolphin (Cephalorhynchus commersonii)

Presence of saw‐toothed structures (serrations) on the leading edge of the flippers in the Commerson's dolphin and their relation with directional asymmetry in the appendicular skeleton were investigated in individuals from the Tierra del Fuego population, Argentina. Serrations were more frequent in the left flipper than in the right (P < 0.001) and in males than in females (P < 0.001). Serration length was significantly longer in the left flipper than in the right (P = 0.023), in males than in females (P = 0.004), and in older individuals than young (P < 0.001). The length of the radius (P = 0.028) and the length (P = 0.004), width (P < 0.001) and weight (P = 0.006) of the scapula showed significant directional asymmetry favoring the right side, whereas the length (P < 0.001) and width (P < 0.001) of the second digit favored the left side. The asymmetry appears to be innate in the species but is likely to be enhanced by differential mechanical stress between flippers as a result of lateralized behavior. We propose that the left flipper would be more flexible and preferably used in sensory or tactile activities that involve the serrations, whereas the right flipper would be more responsible for actions requiring a larger muscular exercise, possibly related to the maintenance of stability during swimming. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.