Enterococcus faecalis YM0831 suppresses sucrose-induced hyperglycemia in a silkworm model and in humans

Abstract

Hyperglycemia caused by excessive intake of sucrose leads to lifestyle-related diseases such as diabetes. Administration of a lactic acid bacterial strain to mice suppresses sucrose-induced hyperglycemia, but evidence for a similar effect in humans is lacking. Here we show that Enterococcus faecalis YM0831, identified using an in vivo screening system with silkworms, suppressed sucrose-induced hyperglycemia in humans. E. faecalis YM0831 also suppressed glucose-induced hyperglycemia in silkworms. E. faecalis YM0831 inhibited glucose uptake by the human intestinal epithelial cell line Caco-2. A transposon insertion mutant of E. faecalis YM0831, which showed decreased inhibitory activity against glucose uptake by Caco-2 cells, also exhibited decreased inhibitory activity against both sucrose-induced and glucose-induced hyperglycemia in silkworms. In human clinical trials, oral ingestion of E. faecalis YM0831 suppressed the increase in blood glucose in a sucrose tolerance test. These findings suggest that E. faecalis YM0831 inhibits intestinal glucose transport and suppresses sucrose-induced hyperglycemia in humans.

Keywords: Animal disease models; Experimental models of disease; Metabolic disorders; Preclinical research; Translational research.

Fig. 1

Inhibitory effect of the E. faecalis YM0831 against an increase in hemolymph glucose levels in silkworms induced by intake of sucrose or glucose. a Electron microscope image of E. faecalis YM0831 is shown. Scale bar indicates 1 µm. b Silkworms were fed a diet containing 10% (w/w) sucrose with or without E. faecalis YM0831 (6.3%, 12.5%, 25% [w/w] in the diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 14/group). c Silkworms were fed a diet containing 10% (w/w) sucrose or glucose with or without E. faecalis YM0831 (25% [w/w] in diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 7/group). d Silkworms were fed a diet containing 10% (w/w) sucrose with or without E. faecalis YM0831 (YM0831, 12.5% [w/w] in diet) or autoclaved E. faecalis YM0831 (autoclaved YM0831, 12.5% [w/w] in the diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 14/group). e Silkworms were fed a diet containing 10% (w/w) glucose with or without E. faecalis YM0831 (YM0831, 25% [w/w] in diet) or autoclaved YM0831 (autoclaved YM0831, 25% [w/w] in the diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 6–7/group). Data represent mean ± SEM. Statistically significant differences between groups were evaluated using Student’s t-test. **P < 0.01. ***P < 0.001. NS: P > 0.05

Fig. 2

Inhibitory effect of E. faecalis YM0831 on glucose transport in the silkworm intestinal tract using isolated intestine and glucose uptake by Caco-2 cells. a Glucose solution with or without added E. faecalis YM0831 (250 mg wet weight/ml) were enclosed in isolated silkworm intestinal tract. The intestinal samples were incubated in PBS at 27 °C. Glucose levels outside of the intestine were determined. b Glucose solution with or without added E. faecalis YM0831 (YM0831, 250 mg wet weight/ml), or glucose solution with added autoclaved E. faecalis YM0831 (autoclaved YM0831, 250 mg wet weight/ml) were enclosed in isolated silkworm intestinal tract. Intestinal samples were incubated in PBS at 27 °C for 10 min. Glucose levels outside of the intestine were determined. n = 3–4/group. c E. faecalis YM0831 (62.5 mg wet weight cells/ml) was added in the uptake system of 2-NBDG in Caco-2 cells and fluorescence uptake by the Caco-2 cells was measured over time. d Various numbers of E. faecalis YM0831 were added in the uptake system of 2-NBDG in Caco-2 cells and fluorescence uptake by the Caco-2 cells was measured. n = 3/group. e E. faecalis YM0831 (62.5 mg wet weight cells/ml) or autoclaved E. faecalis YM0831 (autoclaved YM0831, 62.5 mg wet weight cells/ml) was added in the uptake system of 2-NBDG in Caco-2 cells and fluorescence uptake by the Caco-2 cells was measured. n = 3/group. f Viability of Caco-2 cells following the addition or absence of E. faecalis YM0831 (62.5 mg wet weight cells/ml) or addition of 20% ethanol solution was measured. n = 3/group. Data represent mean ± SEM. Statistically significant differences between control and groups in the presence of samples were evaluated using Student’s t-test. **P < 0.01, ***P < 0.001, NS: P > 0.05

Fig. 3

Characterization of a transposon mutant with attenuated inhibitory activity of E. faecalis YM0831 against glucose uptake by Caco-2 cells. a Experimental scheme of the screening to obtain a transposon mutant with attenuated inhibitory activity of E. faecalis YM0831 against glucose uptake by Caco-2 cells. b Decrease in inhibitory activity of E. faecalis YM0831 transposon mutant (Tp10-72) against glucose uptake by Caco-2 cells. E. faecalis YM0831DR (parent, 62.5 mg wet weight cells/ml) or Tp10-72 (Tp10-72, 62.5 mg wet weight cells/ml) was added in the uptake system of 2-NBDG in Caco-2 cells and fluorescence uptake by the Caco-2 cells was measured. n = 3–6/group. c Decrease in inhibitory activity of Tp10-72 against sucrose-induced hyperglycemia in silkworms. Silkworms were fed a diet containing 10% (w/w) sucrose with or without E. faecalis parent (YM0831DR, 12.5% [w/w] in diet) or Tp10-72 (Tp10-72, 12.5% [w/w] in the diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 12–14/group). d Decrease in inhibitory activity of Tp10-72 against glucose-induced hyperglycemia in silkworms. Silkworms were fed a diet containing 10% (w/w) glucose with or without E. faecalis YM0831DR (parent, 12.5% [w/w] in diet) or Tp10-72 (Tp10-72, 12.5% [w/w] in the diet) for 1 h. Glucose levels in the silkworm hemolymph were measured (n = 7/group). e Inserted region of transposon Tn916 in Tp10-72 genome determined by whole genome sequencing analysis. f Functions of ManX, ManY, and ManZ coded by the man operon. g Decreases in mRNA amounts of genes in the man operon in TP10-72 revealed by RT-PCR analysis. h Complementation of decreased inhibitory activity of TP10-72 on glucose uptake by Caco-2 cells. E. faecalis YM0831DR/pND50 (parent/vector, 70 mg wet weight cells/ml), Tp10-72/pND50 (Tp10-72/vector, 70 mg wet weight cells/ml), or Tp10-72/pMan operon (Tp10-72/pMan operon, 70 mg wet weight cells/ml) were added in the uptake system of 2-NBDG in Caco-2 cells and fluorescence uptake by the Caco-2 cells was measured. n = 3–15/group. Data represent mean ± SEM. Statistically significant differences between groups were evaluated using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Comparative genome analysis between the E. faecalis YM0831 and high-risk strains. a The phylogenic tree of the E. faecalis YM0831 and strains analyzed for sequencing (ST) and clonal complex (CC) are shown. HiRECC high-risk enterococcal clonal complexes. b Comparative gene analysis regarding the presence of cytolysin and Esp contained in the Enterococcus faecalis pathogenic island (37) in the YM0831 with the high-risk strain, MMH 594

Fig. 5

Inhibition of blood glucose increases by ingestion of E. faecalis YM0831 in a sucrose tolerance test in humans. Three sucrose tolerance tests were performed in each of the 14 healthy human subjects; a non-ingestion control, ingestion of bacterial cell suspension, and ingestion of heat-treated bacterial cell suspension. Subjects took the sample solution (YM0831 bacterial cells: 4 × 10¹⁰ cells/50 ml) suspended in saline 15 min before sucrose loading. Subsequently, the subjects drank 150 ml of 50% (w/v) sucrose solution. Blood glucose levels of the subjects were determined at 0, 15, 30, 45, 60, 90, and 120 min after sucrose challenge. Blood was collected from the fingertip and the blood sugar level was measured using a simple blood glucose meter. a Experimental schedule is shown. b Blood glucose levels in E. faecalis YM0831 cell suspension ingestion group (YM0831), autoclaved cell suspension ingestion group (autoclaved YM0831), and non-ingestion group (control) for each subject after sucrose loading are shown. *P < 0.017 after Bonferroni correction (paired Student’s t-test). c Sucrose tolerance tests in E. faecalis YM0831 cell suspension ingestion group (YM0831), autoclaved cell suspension ingestion group (Autoclaved YM0831), and non-ingestion group (control) are shown. *P < 0.017 after Bonferroni correction (paired Student’s t-test). Data represent mean ± SEM

Fig. 6

Inhibition by ingestion of a yogurt produced by E. faecalis YM0831 against blood glucose increases in a sucrose tolerance test in humans. Sucrose tolerance tests were conducted on 10 healthy human subjects by the crossover method with or without yogurt ingestion. The subjects consumed the yogurt sample (200 ml, YM0831 bacterial cells: 4 × 10¹⁰ cells/200 ml yogurt) 10 min before sucrose loading. Subsequently, the subjects drank 150 ml of 50% (w/v) sucrose solution. Blood glucose levels of the subjects were determined at 0, 15, 30, 45, 60, 90, and 120 min after sucrose challenge. Blood was collected from the fingertip and the blood sugar level was measured by using a simple blood glucose meter. a Experimental schedule is shown. b Blood glucose levels in the yogurt-ingestion group (yogurt [YM0831]) and non-ingestion group (control) for each subject for each time after sucrose loading are shown. *P < 0.05 (paired Student’s t-test). c Sucrose tolerance tests in the yogurt-ingestion group (yogurt [YM0831]) and non-ingestion group (control) are shown. *P < 0.05 (paired Student’s t-test). Data represent mean ± SEM