Symbiotic probiotic communities with multiple targets successfully combat obesity in high-fat-diet-fed mice

Abstract

Probiotics hold great potential for treating metabolic diseases such as obesity. Given the complex and multifactorial nature of these diseases, research on probiotic combination with multiple targets has become popular. Here, we choose four obesity-related targets to perform high-throughput screening, including pancreatic lipase activity, bile salt hydrolase activity, glucagon-like peptide-1 secretion and adipocyte differentiation. Then, we obtained 649 multi-strain combinations with the requirement that each must cover all these targets in principle. After in vitro co-culture and in vivo co-colonization experiments, only four (<0.7%) combinations were selected as symbiotic probiotic communities (SPCs). Next, genome-scale metabolic model analysis revealed that these SPCs showed lower metabolic resource overlap and higher metabolic interaction potential involving amino acid metabolism (Ammonium, L-Lysine, etc.) and energy metabolism (Phosphate, etc.). Further animal experiments demonstrated that all SPCs exhibited a good safety profile and excellent effects in improving obesity and associated glucose metabolism disruptions and depression-like behaviors in high-fat-diet-fed mice. This anti-obesity improvement was achieved through reduced cholesterol level, fat accumulation and inhibited adipocyte differentiation. Taken together, our study provides a new perspective for designing multi-strain combinations, which may facilitate greater therapeutic effect on obesity and other complex diseases in the future.

Keywords: Obesity; genome-scale metabolic model (GSMM); multi-strain combination; probiotics; species coexistence; symbiotic probiotic community (SPC).

Figure 1.

In vitro high-throughput screening of probiotics with anti-obesity targets. (a) Schematic diagram showing the experimental design and screening process. (b) Phylogenetic tree depicting 28 probiotics with different targets. PL, pancreatic lipase activity; GLP-1, GLP-1 secretion; differentiation, differentiation; BSH, BSH activity. Asterisks represent probiotics with two targets. The reference strains were highlighted in red.

Figure 2.

Random muti-strain combination possibly worsens the anti-obesity outcomes. (a) Flowchart of experiments. (b) Six probiotic groups with single or multiple strains. (c) The percentage of initial weight gain were measured once a week. (d) Bodyweight gain at the end point. (e) Unilateral gonadal white adipose tissue (WAT) weight at the end point. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Symbiotic probiotic community (SPC) with multiple targets selected through in vitro and in vivo screening. (a) Co-cultures of 649 multi-strain combinations were grown overnight and detected the OD600. Red-circled data represent group G6, as mentioned in figure 2B. (b) Flowchart depicting the secondary assessment of the 63 combinations selected from (a). (c) Results of the secondary assessment. Red and blue circles indicate positive and negative results, respectively. White circles represent combinations that were not subjected to detection. (d-e) five combinations selected from (c) and all the strains were administered using oral gavage to detect survival days in gut. (d) Electropherogram of fecal DNA PCR showing survival at day 3, day 4, and day 5, respectively. (e) Quantification of survival time in (d); bacterial ID is indicated in parentheses.

Figure 4.

GSMM analysis – coexistence and potential microbial metabolic interactions. (a) Schematic diagram of GSMM analysis model. (b-c) the concept of metabolic resource overlap (MRO, B) and metabolic interaction potential (MIP, C). (d) Results analyzed via GSMM model under restriction of MIP ≥6 and MRO <0.065. Three of four SPCs obtained through in vitro experiments were included in the twenty-one gsmm-predicted SPCs. (e) Alluvial diagram showing potential microbial metabolic interactions in the four SPCs (SPC A, SPC B, SPC C and SPC D).

Figure 5.

SPCs exhibited excellent anti-obesity effects in HFD mice. (a) Flowchart of experiments. (b) The percentage of initial weight gain. (c) Bodyweight gain at the end point. (d) Mice were gavaged with glucose. Serum glucose content was measured at 30, 60, 90, and 120 minutes after injection. (e) Area under the curve (AUC) for glucose. (f) Serum GLP-1 content. (g) Cumulative food intake. h) Serum total cholesterol (TC) content. (i) WAT weight. (j-k) H&E staining of adipocytes (k) and the adipocyte area (j) in the WAT. (l) Schematic of the behavioral abnormalities test in the open field box. (m) The total travel distance of mice during the OFT. (n) The time of mice spent in the center zone during the OFT. ND, normal diet. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Symbiotic probiotic community with multiple targets successfully combats obesity in high-fat-diet-fed mice.