Gastrointestinal Autonomic Neuropathy Exacerbates Gut Microbiota Dysbiosis in Adult Patients With Type 2 Diabetes Mellitus

Abstract

Objective: The diabetic autonomic neuropathy is one of the most common complications in type 2 diabetes mellitus (T2DM), especially gastrointestinal autonomic neuropathy (GAN), which occurs in up to 75% of patients. The study aimed to investigate the gut microbiota composition, structure, and function in T2DM patients with GAN (T2DM_GAN) and set up a link between gut microbiota and clinical characteristics of patients.

Methods: DNA was extracted from fecal samples of three groups using the kit method: healthy volunteers (n = 19), the patients with T2DM (n = 76), and T2DM_GAN (n = 27). Sequencing of 16S ribosomal DNA was performed using the MiSeq platform.

Results: According to the clinical data, higher age, lower triglyceride, and lower body mass index were the main features of patients with T2DM_GAN. The gut microbiota analysis showed that Bacteroidetes, Firmicutes, and Proteobacteria constituted the three dominant phyla in healthy individuals. In addition, the gut microbiota structure and function of T2DM_GAN patients were clearly different from that of T2DM patients. T2DM patients were characterized by Fusobacteria, Fusobacteriia, Fusobacteriales, Fusobacteriaceae, Fusobacterium, Lachnoclostridium, and Fusobacterium_mortiferum. Those gut microbiota may be involved in carotenoid and flavonoid biosyntheses. Relatively, the Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Escherichia-Shigella, Megasphaera, Escherichia_coli, and Megasphaera_elsdenii were characteristic in the T2DM_GAN patients. Those may be involved in bacterial invasion of epithelial cells and pathogenic Escherichia coli infection.

Conclusions: GAN exacerbated gut microbiota dysbiosis in adult patients with T2DM. The findings indicated that phyla Fusobacteria and class Gammaproteobacteria were closely related to the occurrence of T2DM. Especially the latter may promote T2DM_GAN.

Keywords: diagnosis; gastrointestinal autonomic neuropathy; gastrointestinal symptoms; gut microbiota; type 2 diabetes mellitus.

Figure 1

The alpha diversity of the gut microbiota among the subjects. (A) Chao1 index, (B) observed_species, (C) ACE index, (D) PD_whole_tree, (E) Shannon, and (F) Simpson. One-way ANOVA (normal vs. T2DM vs. T2DM_GAN group), multiple comparisons are performed using FDR correction: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

T2DM_GAN induce significant changes in the gut microbiota. (A) Unconstrained PCoA (for principal coordinates PCo1 and PCo2) with unweighted UniFrac distance shows that gut microbiota of 119 subjects formed three distinct clusters, which separate along the third coordinate axis (p = 0.001, mrpp). Ellipses cover 80% of the data for each group. (B) Distribution map of relative abundance of each group at the top 5 phyla.

Figure 3

Gastrointestinal autonomic neuropathy exacerbates gut microbiota dysbiosis in adult patients with type 2 diabetes mellitus. Kruskal–Wallis test (normal vs. T2DM vs. T2DM_GAN group), multiple comparisons are performed using FDR correction: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

The biomarkers characteristic of the gut microbiota among the subjects. (A) Histogram of LDA scores: the height of the column represents the size of the LDA score, and the higher this value, the more significant the impact on the final classification (LDA > 3.5). (B) Cladogram showing differentially abundant taxonomic clades with an LDA score >3.5.

Figure 5

Association between clinical information and microbiota composition at the previously depicted differential bacteria. Notes: *p < 0.05, **p < 0.01, ***p < 0.001. BMI, body mass index; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; DBIL, direct bilirubin; UBIL, unconjugated bilirubin; T_CHOL, total cholesterol; HDL_C, high-density lipoprotein cholesterol; LDL_C, low-density lipoprotein cholesterol; FBG, fasting blood glucose; HbA1c, glycosylated hemoglobin; BUN, blood urea nitrogen; WBC, white blood cells; RBC, red blood cells.

Figure 6

The significantly and differentially pathway between T2DM and T2DM_GAN patients. The left X-axis represents different groups, the Y-axis represents the average relative abundance of a species in different groups, and the right represents the confidence interval and p-value.

Figure 7

Association between differential KEGG pathways and microbiota composition at the previously depicted differential bacteria. Notes: *p < 0.05, **p < 0.01, ***p < 0.001.