Changes in Intestinal Microbiota Are Associated with Islet Function in a Mouse Model of Dietary Vitamin A Deficiency

Abstract

Aims: The underlying mechanisms involved in Vitamin A- (VA-) related changes in glucose metabolic disorders remain unclear. Recent evidence suggests that intestinal microbiota is closely linked to the metabolic syndrome. Here, we explored whether and how intestinal microbiota affects glucose homeostasis in VA-deficient diet-fed mice.

Methods: Six-week-old male C57BL/6 mice were randomly placed on either a VA-sufficient (VAS) or VA-deficient (VAD) diet for 10 weeks. Subsequently, a subclass of the VAD diet-fed mice was switched to a VA-deficient rescued (VADR) diet for an additional 8 weeks. The glucose metabolic phenotypes of the mice were assessed using glucose tolerance tests and immunohistochemistry staining. Changes in intestinal microbiota were assessed using 16S gene sequencing. The intestinal morphology, intestinal permeability, and inflammatory response activation signaling pathway were assessed using histological staining, western blots, quantitative-PCR, and enzyme-linked immunosorbent assays.

Results: VAD diet-fed mice displayed reduction of tissue VA levels, increased area under the curve (AUC) of glucose challenge, reduced glucose-stimulated insulin secretion, and loss of β cell mass. Redundancy analysis showed intestinal microbiota diversity was significantly associated with AUC of glucose challenge and β cell mass. Redundancy analysis showed intestinal microbiota diversity was significantly associated with AUC of glucose challenge and κB signaling pathway activation. Reintroduction of dietary VA to VAD diet-fed mice restored tissue VA levels, endocrine hormone profiles, and inflammatory response, which are similar to those observed following VAS-controlled changes in intestinal microbiota.

Conclusions: We found intestinal microbiota effect islet function via controlling intestinal inflammatory phenotype in VAD diet-fed mice. Intestinal microbiota influences could be considered as an additional mechanism for the effect of endocrine function in a VAD diet-driven mouse model.

Figure 1

VAD leads to reduction of tissue VA levels. (a) VA (retinol) levels in the serum, pancreas, intestines, and liver of mice from VAS, VAD, and VADR groups. (b) Representative photomicrographs of lipid droplets in the liver and islets using Oil red O staining from mice described for (a). (c) Heat map of PCR measurements of relative pancreatic/intestinal mRNA levels involved in VA metabolic signaling from mice described for (a). Magnification: 100x; scale bars, 20 μm. Error bars represent S.E. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 2

VAD leads to irregular islet morphology, loss of β cell mass, and impaired glycemic responses. (a) H & E staining of pancreatic islet and liver sections of mice from VAS, VAD, and VADR groups. Magnification, 40x; scale bars, 50 μm. (b, c) Blood glucose and insulin levels using the IPGTT test were analyzed of mice described for (a). The results of the IPGTT test were analyzed by AUC (i.e., AUC_{IPGTT-glucose} or AUC_{IPGTT-insulin}). (d) Representative immunofluorescence images of pancreatic islet sections of mice described for (a) stained with antibodies against insulin. Magnification: 20x; scale bars, 100 μm. (e) Mean islet area (μm²) of pancreas of mice described for (a). (f) β cell mass of mice described for (a). Error bars represent S.E. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

VAD causes reduced diversity/richness and alters cluster and composition of intestinal microbiota. (a) Alpha-diversity assay of intestinal microbiota of mice from VAS, VAD, and VADR groups. (b) Beta-diversity of intestinal microbiota of mice described for (a): unweighted UniFrac PCoA plotted against PC1 versus PC2 axes. (c) Cluster analysis for bacterial communities of mice described in (a). (c) Heat map of key OTUs of mice described for (a). (d, e) Bacterial composition at the phylum level of mice described for (a). (f) Akkermansia muciniphila composition at the phylum level of mice described for (a). Error bars represent S.E (n = 6/group). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

Correlation analysis of changes in intestinal microbiota and biochemical parameters of islet function. Redundancy analysis of the significant relationship between islet function and bacterial community of VAS, VAD, and VADR groups (n = 6/group).

Figure 5

VAD induces inflammatory responses in the intestine by activating the NF-κB signaling pathway. (a) Representative photomicrographs of intestinal sections of mice from VAS, VAD, and VADR groups using H & E staining. (b) Plasma LPS and TNF-α concentrations were analyzed of mice described for (a) by ELISA. (c, d) PCR measurements of relative intestinal inflammation markers (TNF-α, IFN-γ, IL-6, and IL-1β) and tight-junction integrity markers (ZO-1, occludin) of mice described for (a). (e) The protein levels in NF-κB signal pathways were analyzed of mice described for (a) using western blotting. (f) The protein levels of IKK were analyzed of mice described for (a) using ELISA. Error bars represent S.E (n = 6/group). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.