Cell-free supernatant of Levilactobacillus brevis (RAMULAB51) from coconut inflorescence sap (Neera) enhances glucose uptake and PPAR-γ in 3T3-L1 adipocytes and inhibits α-glucosidase and α-amylase

Abstract

Introduction: Lactic acid bacteria are prized for their probiotic benefits and gut health improvements. This study assessed five LAB isolates from Neera, with RAMULAB51 (Levilactobacillus brevis, GenBank ON171686.1) standing out for its high hydrophobicity, auto-aggregation, antimicrobial activity, and enzyme inhibition. It evaluated RAMULAB51's potential in probiotics and diabetes management, focusing on its effects on digestive enzyme inhibition, glucose uptake, and adipocyte function.

Methods: Isolates were characterized by Gram staining, catalase reaction, growth at 37°C, and tolerance to phenol, pH, and gastrointestinal conditions. Molecular identification using 16S rRNA sequencing. Evaluations included hydrophobicity, auto-aggregation, HT-29 cell line adhesion, antimicrobial activity, and antibiotic susceptibility. Enzyme inhibition was measured for α-glucosidase and α-amylase using cell-free supernatant, cell extract, and intact cells. Adipogenesis was assessed through Oil-Red O staining, gene expression analysis (PPAR-γ, C/EBPα, Adiponectin, Glut-4, FAS), and glucose uptake assays on 3T3-L1 cells.

Results: All isolates showed good tolerance to pH (≤9.15 CFU/ml), phenol (≤9.90 CFU/ml), hydrophobicity (≤78.14%), and auto-aggregation (≤92.23%). RAMULAB51 demonstrated the highest tolerance, hydrophobicity, and auto-aggregation. It strongly co-aggregated with Micrococcus luteus and Bacillus subtilis, showing significant antimicrobial activity with a 24 mm inhibition zone against Micrococcus luteus. All isolates were sensitive to Ampicillin, Azithromycin, Streptomycin, and Tetracycline, but resistant to Methicillin and Vancomycin. RAMULAB51 demonstrated the highest enzyme inhibition: α-glucosidase (68.45% CFS, 60.18% CE, 42.15% IC) and α-amylase (80.74% CFS, 61.23% CE, 35.12% IC). By inhibiting these digestive enzymes, RAMULAB51 reduces the conversion of carbohydrates into glucose, thereby decreasing blood glucose levels. This reduction in circulating glucose subsequently influences adipocyte function, as evidenced by the enhanced glucose uptake (1000 µg/mL) and upregulation of PPAR-γ, C/EBPα, Adiponectin, and Glut-4, alongside the downregulation of FAS.

Conclusion: The study highlights RAMULAB51's potential for improving glucose and lipid metabolism. Further, in vivo research is needed to explore its full therapeutic benefits. These findings confirm RAMULAB51's significant probiotic potential and its promise for diabetes management, warranting further clinical investigation.

Keywords: 3T3-L1 adipocytes; Neera; PPAR-γ activation; Type 2 Diabetes Mellitus; probiotics; α-amylase; α-glucosidase.

Figure 1

Comparison of phylogenetic trees for strains isolated from Neera (Ingroup) and reference outgroup, using maximum likelihood bootstrap 1,000 analysis of 16S rRNA sequences.

Figure 2

The survival rates of isolates at acidic pH 2 and various bile salt concentrations were assessed by incubating the strains for 2 and 4 h at 37°C with (A) 0.3 and 1% bile salt concentrations. Additionally, survival rates in (B) Gastric and intestinal juices were measured at 1, 3, 5, and 8 h, (C) Autoaggregation, and (D) Coaggregation of isolates with M ± SD compared using Duncan’s MRT and significant differences denoted by different superscripts (#, a–e) (p < 0.05).

Figure 3

Enzymes inhibition by the isolates against α-glucosidase (A) and α-amylase (B). Data are articulated as M ± SD. Duncan’s MRT indicates significant differences among means within the same column, with different letters (a–c) representing statistically distinct groups (p ≤ 0.05).

Figure 4

The effect of CFS from Levilactobacillus brevis RAMULAB51 on cell viability at various concentrations. The data points represent M ± SD of cell viability percentages. The sigmoidal curve fit shows a dose-dependent reduction in cell viability was observed with increasing concentrations of CFS. The IC50 value, where cell viability is reduced to 50%, is approximately 1,336.17 μg/mL, indicated by the vertical dashed line on the graph.

Figure 5

Graph showing the relative expression levels of PPAR-γ, C/EBPα, FAS, Adiponectin, and Glut-4 in 3T3-L1 adipocytes treated with varying concentrations of CFS on days 5, 8, and 12. Values existing, as the M ± SD, normalized to β-actin levels. Duncan’s MRT was employed to identify significant differences. Statistical significance is denoted as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6

Glucose uptake in 3T3-L1 adipocytes subjected to various concentrations of CFS (500 μg/mL and 1,000 μg/mL) and insulin (100 nM) across different time points (15, 30, and 240 min). Data existing, as the M ± SD, normalized to β-actin levels. Duncan’s MRT was employed to identify significant differences. Statistical significance is denoted as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.