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. 2016 Nov 15:7:498.
doi: 10.3389/fphys.2016.00498. eCollection 2016.

Vitamin D Signaling through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models

Danmei Su  1 , Yuanyang Nie  1 , Airu Zhu  1 , Zishuo Chen  1 , Pengfei Wu  1 , Li Zhang  1 , Mei Luo  2 , Qun Sun  1 , Linbi Cai  1 , Yuchen Lai  1 , Zhixiong Xiao  1 , Zhongping Duan  3 , Sujun Zheng  3 , Guihui Wu  4 , Richard Hu  5 , Hidekazu Tsukamoto  6 , Aurelia Lugea  7 , Zhenqui Liu  7 , Stephen J Pandol  7 , Yuan-Ping Han  8
Affiliations

Vitamin D Signaling through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models

Danmei Su et al. Front Physiol. .

Abstract

Metabolic syndrome (MetS), characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD), is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR) is highly expressed in the ileum of the small intestine, which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD) is necessary but not sufficient, while additional vitamin D deficiency (VDD) as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD), the Paneth cell-specific alpha-defensins including α-defensin 5 (DEFA5), MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD), Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR) knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with resolving metabolic disorders and fatty liver in the HFD+VDD mice. An in vitro analysis showed that DEFA5 peptide could directly suppress Helicobacter hepaticus. Thus, the results of this study reveal critical roles of a vitamin D/VDR axis in optimal expression of defensins and tight junction genes in support of intestinal integrity and eubiosis to suppress NAFLD and metabolic disorders.

Keywords: Akkermansia muciniphila; defensins; gut microbiota; helicobacter; metabolic syndrome; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); vitamin D.

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Figures

Figure 1
Figure 1
High fat feeding is not sufficient to induce robust hepatic steatosis and metabolic disorders, but additional vitamin D deficiency is needed. Balb/C mice were fed for 18 weeks in the following 4 conditions (n = 10 per group): (1) control chow, AIN93 with standard VD3 supplement at 1000 IU/kg, (2) vitamin D deficient AIN93 chow, VDD, (3) high fat diet (60% calorie from fat) with standard VD3 supplement, HFD, (4) vitamin D deficient high fat diet, HFD + VDD. (A) Representative images of liver tissues, H&E staining. NAS scores are indicated. (B) Representative images of liver tissues, Masson's Trichrome staining. (C) Intraperitoneal glucose tolerance test (IPGTT). (D) Homeostatic model assessment (HOMA-IR). (E) Plasma total cholesterol, low-density lipoprotein (LDL), plasma lipase, and plasma insulin levels in fast. Data represent two independent experiments (n = 6–10 mice/group for each measurement). Error bars represent the SEM of samples within a group. *p ≤ 0.05, **p ≤ 0.01 (Student's t-test). The arrows indicate the impact of vitamin D supplement to ameliorate the metabolic disorders. #indicates the impact of vitamin D.
Figure 2
Figure 2
Systemic and local inflammation is aggravated by dietary vitamin D deficiency. Balb/C mice were fed with four conditions for 18 weeks as described in Figure 1. (A) Plasma TNF-α levels were measured by an ELISA kit. (B–D) In the visceral fat tissue, the mRNA levels of TNF-α, PAI-1, and MCP-1 were measured by RT-qPCR analysis. (E,F) In the pancreas, the mRNA levels of MMP13 and TNF-α were determined by RT-qPCR analysis. (G) Plasma, alanine aminotransferase (ALT) levels. (H) Lymphocyte infiltration in the liver was determined by CD3 staining. Data represent two independent experiments (n = 6–10 mice/group for each measurement). Error bars represent the SEM of samples within a group. *p ≤ 0.05, **p ≤ 0.01 in comparison with the control.
Figure 3
Figure 3
Vitamin D maintains intestinal homeostasis. The animal feeding conditions are described as in Figure 1. (A) Plasma LPS levels were measured by a Limulus Amebocyte Lysate kit. (B) Gut permeability was determined by fluorescein isothiocyanate (FITC)-dextran presented in plasma after oral gavage administration. (C) Expression of ZO-1, occluding, and claudin 2 in ileum region was determined by RT-qPCR analysis. (D) Periodic Acid/Schiff (PAS) staining. Green box highlights the mucosa and goblet cells in the control. (E) Expression of VDR in the ileum, skin, pancreas, and liver by the mice was determined by RT-qPCR analysis. Data represent two independent experiments (n = 6–10 mice/group for each measurement). Error bars represent the SEM of samples within a group. *p ≤ 0.05, **p ≤ 0.01 (Student's t-test). The arrows indicate the impact of dietary vitamin D on intestinal integrity and plasma endotoxin. #indicates the impact of vitamin D.
Figure 4
Figure 4
Vitamin D signaling maintains the steady expression of Paneth cell defensins and their converting enzyme, MMP7. (A) Expression of defensins in the ileum by the mice under the four feeding conditions was determined by RT-qPCR analysis. (B) Immunohistochemical staining of the ileal region for DEFA1, and immunofluorescent staining for MMP7 in the crypts of Lieberkühn. Data (n = 4–10 mice/group for each measurement) are presented as mean ± standard error. Comparison was conducted by t-test between the experimental groups with the control, and *P < 0.05, **P < 0.01. #indicates the impact of vitamin D.
Figure 5
Figure 5
Initial dysbiosis in the ileum induced by high fat feeding is exacerbated by additional vitamin D deficiency. (A) The microbiota in the ileal lumen of the mice by the four feeding conditions were determined by 16S ribosomal (rRNA) gene sequencing analysis (n = 6), and presented as hierarchical heat-map with bacterial taxa (n = 3). (B) Principle component analysis (PCA). (C) The relative abundances of Helicobacter hepaticus and Akkermansia muciniphila were determined by qPCR analysis by species-specific primers. Data represent two independent experiments (n = 6 for each measurement). Error bars represent the SEM of samples within a group. *p ≤ 0.05, **p ≤ 0.01 (Student's t-test). The arrows indicate the impact of vitamin D supplement to ameliorate the dysbiosis. #indicates the impact of vitamin D.
Figure 6
Figure 6
Oral administration of human alpha-defensin 5 rebalances gut microbiota and resolves hepatic steatosis. (A) Experimental design. Balb/C mice were fed with HFD+VDD for 18 w, followed by administrated synthetic human DEFA5 (10 μg /dose or mock with saline) by oral gavage for 4 times within 25 d, and the mice were terminated in additional 10 days (n = 6 for each group). (B) Relative abundance of A. muciniphila presented in the feces during the treatments was quantitated by 16S rDNA qPCR analysis. Changing folds over the mock were plotted. (C) Relative abundance of H. hepaticus in the lumen of ileum after the treatment was quantitated by 16S rDNA qPCR analysis. (D) Relative abundance of A. muciniphila in the lumen of ileum after the treatment was quantitated by 16S rDNA qPCR analysis. (E) The plasma levels of endotoxin after the treatment were determined by Limulus Amebocyte Lysate test. (F) The plasma levels of TNF-α was determined by ELISA analysis. (G) Fasting plasma glucose during the treatments. (H) Representative images of the liver sections, H&E staining. (I) The mRNA levels of IL-1β in the liver after the treatment were determined by RT-qPCR analysis. The data (n = 6 for each measurement) are presented as mean ± standard error. Comparison was conducted by t-test between the experimental groups with the control, and *P < 0.05, **P < 0.01.
Figure 7
Figure 7
VDR KO mice exhibit ileal dysbiosis, impaired mucosa, and hepatic steatosis. VDR KO and WT littermates in the genetic background (B6.129S4) fed with fortified phosphate and calcium chow for 20w were examined. (A) Representative images of liver tissues, H&E and Masson's trichrome staining. (B) The mRNA levels of DEFA5, MMP7, occludin, and mucin 2 (MUC2) in the ileum of WT, heterozygote, and homozygous VDR KO were determined by RT-qPCR analysis. (C) H. hepaticus and A. muciniphila in the ileum lumen were determined by 16S rDNA qPCR analysis and presented as relative fold of changes. (D) Immunohistochemical staining of MMP7 and DEFA1 in the ileum by WT and VDR KO mice. The data (n = 4–6 for each measurement) are presented as mean ± standard error. Comparison was conducted by t-test between the experimental groups with the control, and *P < 0.05, **P < 0.01.
Figure 8
Figure 8
A working model showing how vitamin D and its signaling in maintain the homeostasis of gut microbiota, and how lacking of vitamin D may exacerbate the high-fat-diet initiated fatty liver and metabolic syndromes. (A) Sufficient VD and its signaling in maintaining the integrity of small intestine. Gut VDR/Paneth-cells/defensins axis may configure the enterotype of microbiota in eubiosis. Moreover, VD signal up regulates the key components of tight junctions and mucin. (B) The primary dysbiosis initiated by HFD is worsened by hypovitaminosis D into second-phase dysbiosis that promotes systemic and hepatic inflammation, leading to insulin resistance, fatty liver and metabolic syndromes, and even transition of simple steatosis and into NASH.
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