1 Section for Bacteriology, Pathology and Parasitology, National Veterinary Institute, Technical University of Denmark2 National Veterinary Institute, Technical University of Denmark3 Department of Microbiology, Technical University of Denmark4 Aarhus University
The impact of diet‐induced obesity on the dynamic and profile of gut microbiota
Obesity is increasing worldwide at an alarming rate which has reached epidemic proportions. Obesity and its co‐morbidities such as cardiovascular disease, type‐2 diabetes and cancer are of great economical burden to the countries affected. Efforts have been made to identify the causal factors that lead to obesity and maintenance of the obese state. Recently the gut microbiota has been implicated of being a contributing factor to obesity. Therefore the gut microbiota is a potential target for management of obesity and its co‐associated morbidities by changing the composition of the microbiota by pre‐ and probiotics diet supplements. In order to investigate the impact of diet and obesity on gut microbiota and vice versa in humans, there is a need for translational animal models that can contribute to the understanding of obesity and its related diseases. Pigs are often used in intervention studies, primarily due to the close resemblance of their anatomy and physiology to that of humans. The main focus of this Ph.D. thesis was to elucidate the changes in gut microbiota during the course of diet induced obesity in three porcine models and to relate their microbial profiles to a physiological trait, namely lean or obese phenotype. Furthermore, these porcine models of obesity were evaluated for their potential as animal models that may be used in diet‐induced obesity‐gut micriobiota related studies in the future. In this Ph.D. thesis, the faecal and intestinal microbiota was investigated in three different breeds of pigs that were used as models of obesity: the domestic Danish pig (Landrace x Yorkshire), Göttingen minipigs and Ossabaw minipigs. The L x Y pigs were divided into two groups; cloned pigs‐ and noncloned pigs used as the control group for the cloned pigs. The gut microbiota was primarily studied with molecular methods such as terminal restriction fragment length polymorphism (T‐RFLP), fluorescent in situ hybridization (FISH), next generation sequencing by Illumina and two quantitative real‐time PCR platforms namely the Rotor‐Gene Q instrument and high‐throughput microfluidics‐dynamic array. The faecal, colonic and ileal microbiota of lean and obese, cloned and non‐cloned pigs was investigated by T‐RFLP and the results showed that the gut microbiota was affected by high‐fat/high‐energy diet and obesity. A positive correlation was observed between body‐weight and percentage of body‐fat in obese cloned and in non‐cloned pigs. Based on gut microbial profile, the composition of the microbiota in cloned pigs did not have less inter‐individual variations among them as compared to the microbiota in non‐cloned pigs. The bacterial diversity was similar between cloned and non‐cloned pigs over time in both the lean and obese group. The colon microbiota of lean cloned pigs contained relatively more of bacteria belonging to the phylum Firmicutes and less bacteria belonging to the phylum Bacteroidetes than obese cloned pigs. In the obese group, body‐weight correlated positively with the relative abundance of Firmicutes and negatively with the abundance of Bacteroidetes; however the negative correlation with Bacteridetes was only observed in the cloned pigs. According to the next generation sequencing data (the V5 region of the 16S rRNA gene) the microbiota of cecum from obese Göttingen minipigs was characterised by significantly higher abundances of Spirochaetes, Tenericutes and Verrucomicrobia at phyla level than the lean Göttingen minipigs’ cecal microbiota. Generally, most of the differences in bacterial groups were observed in cecal microbiota. Based on qPCR, there was 7.6‐fold more Clostridium cluster XIV in cecal microbiota of obese Göttingen minipigs as compared to lean Göttingen. In lean Göttingen minipigs’ cecal microbiota, significantly higher abundances of the phyla Firmicutes and genera Clostridium, Akkermensia and Methanovibribacter was observed compared to obese Göttingen minipigs. Overall, most significant differences between lean and obese Göttingen minipigs cecal microbiota was observed in Gram‐negative bacteria both at phyla and genera taxonomic levels. The composition of the intestinal microbiota of lean and obese Ossabaw minipigs was investigated by sequencing as in the Göttingen study. The results revealed that the microbiota in colon and terminal ileum of obese Ossabaw minipigs had higher ratios of Firmicutes to Bacteroidetes than the lean Ossabaw minipigs. Furthermore, in obese Ossabaw minipigs, a significantly higher abundance of the genus Clostridium was observed in colon and terminal compared to lean Ossabaw minipigs. In lean Ossabaws the abundances of bacteria belonging to the genera Prevotella and Lactobacillus were significantly higher in both colon and terminal ileum than the obese Ossabaws. The high ratio of Firmicutes to Bacteroidetes has previously been connected to the obese phenotype in mice and humans. While low abundances of Lactobacillus in obesity has been reported to be connected to dysmetabolism in obese mice. In conclusion, the cloned pigs did not have reduced inter‐individual variation as compared to noncloned pigs in regard to their gut microbiota, in neither the obese nor the lean state. The gut‐microbial analysis of all the lean and obese domestic pigs suggests that high‐fat/high‐energy diet both in restricted amounts and ad libitum is associated with an increase in Firmicutes in the gut microbiota. Overall, high‐fat/high‐energy diet affects the gut microbiota differently than normal chow as observed in cloned/non‐cloned L x Y pigs, Ossabaw and Göttingen minipigs, respectively.