Gut microbiota, intestinal permeability and inflammation in females and males in a murine model of diet induced-obesity.

Background and aim: Nowadays, the rapidly growing prevalence of obesity is regarded as one of the most serious healthcare issues of our society. The main burden of obesity lies in its pathophysiologic relationship with a variety of metabolic and non-metabolic diseases. Currently, lowgrade systemic inflammation is considered a relevant link between obesity and its associated disorders. The mechanisms linking obesity to inflammation might depend partly on alterations of gut microbiota and enteric barrier. The aim of this study was to evaluate changes in gut microbiota, enteric permeability and inflammation in a mouse model of diet-induced obesity. Methods: Male and female C57BL/6 mice (4-5-weeks old) were fed with standard diet (SD, 10% of energy from fat) or high fat diet (HFD, 60% of energy from fat) for 8 weeks, to elicit obesity. Animals were weighed weekly and growth curves were determined. Fasting metabolic parameters (glucose, tryglicerides, cholesterol) were measured in blood samples. The microbiota of colonic fecal pellets was analyzed by 16rRNA amplicon sequencing. The integrity of intestinal mucosal barrier was evaluated both functionally (plasma level assessment of low molecular weight (4 kDa) fluorescein isothiocyanate dextran following its oral administration) and by assessing the expression of tight junctions (claudin-1, claudin-4 and occludin by western blotting; zonulin-1 [ZO-1] by ELISA). Neuronal (HuC/D) and glial (S100beta and glial fibrillary acidic protein [GFAP]) markers in longitudinal muscle-myenteric plexus preparations were examined by immunofluorescence staining. The following parameters were evaluated also: expression of IL-1β in the ileum and TGFβ1 in serum (ELISA); SIRT1 and SOD1 protein expression in the ileum (western blotting); malondialdehyde (MDA) levels in ileal tissue (colorimetric assay). Results: In male mice, the HFD-induced weight gain occurred earlier and was much greater than females. All HFD mice developed hyperglycemia, hypercholesterolemia, but only HFD female mice developed hypertriglyceridemia. HFD altered the composition of gut microbial community in both males and females. The taxonomic analysis of phylum distribution in HFD mice showed a significant increase in Firmicutes along with a decrease in Bacteroidetes, as compared to SD. This finding was confirmed by an increase in Firmicutes:Bacteroidetes ratio, which was more remarkable in HFD females than HFD males, as compared to their respective SD groups. Intestinal permeability increased in all HFD mice, as compared to SD, with a significant increment in females. Accordingly, HFD mice displayed a decreased expression of claudin-1, claudin-4, occludin and ZO-1. Both females and males showed a significant decrease in the density of HuC/D+ myenteric neurons along with a remarkable increase in S100β+ and GFAP+ glial cells. In HFD females, but not in males, IL-1β and TGFβ1 expression displayed a trend to increase, as compared to SD animals. Moreover, in both genders fed with HFD there was a decrease in the antioxidant factors SIRT1 and SOD1, while an increase in MDA ileal levels was observed only in HFD females. Discussion and conclusion: HFD-induced obesity resulted in metabolic alterations, which were associated with an increase in intestinal permeability, an alteration of enteric barrier, and changes in gut microbiota, particularly in females. This gender showed also an increase in inflammatory cytokines. These findings suggest that HFD promotes changes in gut microbiota and impairments of enteric barrier, which might allow an enhanced passage of luminal contents (i.e. xenobiotics and/or bacterial materials) into bowel tissues, thus contributing to a chronic low-grade activation of the immune/inflammatory systems, an imbalance between oxidative stress and endogenous antioxidant pathways, a reactive enteric gliosis, and a loss of myenteric neurons.