Effect of Akkermansia muciniphila on Glp and Insulin Secretion

1. Introduction

Diabetes mellitus is a chronic metabolic disease demarcated by elevated blood glucose levels due to impaired insulin secretion, insulin action, at times both. Persistent hyperglycemia often leads to serious complications that affect both small and large blood vessels. Diabetes is a complication that is globally rising, with the greatest increase observed in low- and middle-income countries, especially as urbanization and lifestyle changes accelerate (Yadav et al., 2014). The International Diabetes Federation estimates that the number of adults affected by diabetes may rise to 589 million globally. About 11.1% of the population aged 20–79 is living with diabetes, and over 40% are still undiagnosed. Projections indicate that if current trends continue, the number of people with diabetes could reach 853 million by 2050. Rapid urbanization and shifts in dietary patterns have contributed to a particularly high and growing prevalence in these regions. These figures underscore the urgent need for effective prevention, early detection, and management strategies to address the increasing global burden of diabetes.

A critical angle of diabetes is the dysbiosis in the existence of insulin which is a peptide hormone produced by pancreatic β-cells that plays a central role in maintaining glucose homeostasis by promoting glucose uptake into skeletal muscle and adipose tissue, stimulating glycogen synthesis in the liver, and suppressing hepatic glucose production (Rahman et al., 2021). Beyond its effects on carbohydrate metabolism, insulin also facilitates lipid storage, inhibits lipolysis, and enhances protein synthesis, thereby supporting overall metabolic health. Disruption of insulin secretion-whether due to autoimmune destruction of β-cells in type 1 diabetes or insulin resistance in peripheral tissues as seen in type 2 diabetes, is fundamental to the pathogenesis of diabetes and its associated complications (Nakrani et al., 2023).

As the global prevalence of diabetes continues to rise, largely driven by increasing rates of obesity, sedentary lifestyles, and poor dietary habits, effective management of insulin secretion and sensitivity becomes imperative for a healthy life. Therefore, the development of therapies that enhance insulin action remains a major focus of research.

Yet another key player in diabetes dysbiosis is the gut hormone glucagon-like peptide-1 (GLP-1), which is secreted by intestinal L-cells. GLP-1 is a critical regulator of glucose homeostasis and appetite (Zheng et al., 2024). It stimulates glucose-dependent insulin secretion, suppresses glucagon release, and delays gastric emptying, making it a cornerstone in managing type 2 diabetes and obesity. GLP-1 secretion is modulated by nutrient sensing in L-cells, particularly through protein-rich foods, unsaturated fatty acids, and soluble fiber, which enhance its release. Furthermore, GLP-1 receptor agonists (GLP-1 RAs) have demonstrated significant clinical benefits, including sustained weight loss (10–20% body weight reduction) and cardiovascular risk reduction through mechanisms such as improved endothelial function, reduced inflammation, and attenuation of atherosclerotic plaque progression. Recent studies highlight their potential neuroprotective effects, with a 10–20% lower risk of Alzheimer’s disease and dementia, likely due to anti-inflammatory actions and modulation of brain reward pathways.

The gut is revered as the second brain. Scientific studies have established the significant influence of the gut microbiome on insulin secretion and sensitivity. Certain gut bacteria produce metabolites such as short-chain fatty acids (SCFAs) like butyrate and acetate, which have improved insulin sensitivity and promote insulin secretion, partly by modulating inflammatory pathways implicated in insulin resistance. This insinuates that targeting the gut microbiota with probiotics or postbiotic metabolites may offer promising, natural strategies for improving insulin function and managing diabetes and related metabolic disorders.

Akkermansia muciniphila, a keystone gut microbe, enhances host health through unique mucin degradation (Pellegrino et al., 2023) and metabolic activities. Its distinctive capability to break down intestinal mucin glycoproteins-via glycosyl hydrolases, sulfatases, and sialidases-generates short-chain fatty acids (SCFAs) like acetate and propionate (Li et al., 2021). These SCFAs serve as energy sources for colonocytes, promote regulatory T-cell differentiation, and suppress inflammation by inhibiting histone deacetylases (HDACs) (Li et al., 2023, Pellegrino et al., 2023).

In this regard, we endeavored to conduct clinical studies to evaluate whether the bacterial cell extracts of the probiotic Akkermansia muciniphila can stimulate insulin secretion in pancreatic beta-cells (using the INS-1 cell line) and GLP-1 secretion in human L-cells (using the NCI-H716 cell line). Both cell lines are well-established models for evaluating GLP-1 and insulin secretion, respectively, and for screening compounds that modulate these hormones. By surveying the effects of VH (Vidya Herbs) A. muciniphila extracts on hormone secretion in these cell lines, the feasibility of exploring their potential as metabolic interventions for improving glucose regulation and supporting diabetes and weight management increases exponentially.

2. Materials and Methods:

2.1. Bacterial Cell Preparation, Protein Quantification:

VH Akkermansia muciniphila cells were cultured under anaerobic conditions to obtain sufficient biomass at Vidya Herbs sterile facility. Bacterial cells were lysed via ultra-strength sonication, applying high-intensity sound waves to disrupt cell walls. The lysate was centrifuged into pellet insoluble debris, and the soluble protein-rich supernatant was collected. Protein quantification was performed using a bicinchoninic acid (BCA) assay.

2.1.1. Insulin Study

• Treatment Formulation

A dose-response experiment was designed with protein concentrations ranging from 0 μg/mL (negative control) to 500 μg/mL of total protein. INS-1 -cells were treated with these formulated doses, and glucose-stimulated insulin secretion (GSIS) was measured via ELISA at 0, 30, and 60 minutes to assess time-dependent effects. This protocol ensured precise evaluation of VH A. muciniphila-derived proteins on insulin secretion dynamics.

• Cell Line Prep, Culture Conditions and Insulin Measurement

INS-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified incubator with 5% CO₂. Cells were seeded into 12-well plates and allowed to reach confluency prior to treatment, with all assays performed in biological triplicates. The two controls included were a negative control consisting of untreated cells (0 µg/mL extract) and a positive control with cells exposed to 10 mM glucose, a known stimulator of insulin secretion in pancreatic beta-cells. Following treatment, cell culture supernatants were collected at 0, 30, and 60 minutes, and insulin concentrations were quantified using a commercially available enzyme-linked immunosorbent assay (ELISA). All insulin measurements were performed in triplicate to ensure accuracy and reproducibility of results.

2.1.2. GLP-1 Study:

• Treatment Formulation

A dose-response experiment was designed with protein concentrations ranging from 0 μg/mL (negative control) to 500 μg/mL of total protein. The treatment regimen involved exposing the NCI-H716 cells to these concentrations and measured GLP-1 secretion at three time points: 0, 30, and 60 minutes.

• Cell Line Prep, Culture Conditions and GLP-1 Measurement

Human L-cell lines (NCI-H716) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained under standard conditions (37°C, 5% CO2). Cells were seeded into 12-well plates at a density that allowed them to reach confluency before the treatment. Two controls were included in the experiment. A negative control: cells without any treatment (0 µg/mL extract) and a positive control: cells treated with 10 mM glutamine, known to stimulate GLP-1 secretion. After the treatment, cell culture supernatants were collected at 0, 30 and 60 min and analyzed for GLP-1 secretion using a commercially available enzyme-linked immunosorbent assay (ELISA). GLP-1 concentrations were measured in triplicates.

2.3. Data Analysis:

Data was analyzed using standard statistical software, including Excel and GraphPad Prism and expressed as means ± standard deviation (SD) and standard error of the mean (SEM). Statistical significance between groups was evaluated using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons.

3. Results

Insulin concentrations were seen to improve with the treatment. The data in Table 1 and Table 2 shows the treatment of INS-1 beta cells with increasing concentrations of VH A. muciniphila cell extracts that affect insulin secretion over time, compared to negative (untreated) and positive (glucose-stimulated) controls. At baseline (0 min), insulin levels are low and similar across all groups, indicating no immediate effect from the extract (Figure 1). After 30 and 60 minutes of treatment, there is a clear, dose-dependent increase in insulin secretion. Higher concentrations of VH A. muciniphila extract result in significantly greater insulin release. This is visually summarized in the (Figure 1) heatmap (panel a), where the color shifts from red (low) to green (high) as both dose and time increase. After 30 and 60 minutes, insulin secretion increases in a dose-dependent manner with higher concentrations of VH A. muciniphila extract with an optimum at the extract dose of 250 µg/mL. The positive control (10 mM glucose) produces a much larger increase in insulin secretion (15.9 mU/L at 30 min, 27.7 mU/L at 60 min), consistent with the robust glucose-stimulated response expected in INS-1 cells. When focusing only on the treated groups (Table 2, Figure 2), the data reinforces that even at the lowest tested dose (31.2 µg/mL), insulin secretion is significantly higher than the negative control after 30 and 60 minutes. Overall, the results indicate that VH A. muciniphila cell extracts stimulate insulin secretion in a dose-dependent manner, though to a lesser extent than direct glucose stimulation, supporting their potential to modulate beta-cell function.

In the GLP study, all groups show low GLP-1 levels (2–5 pM), indicating similar starting points before treatment (Table 3). The negative control remains low (2.76 pM). All VH Akkermansia muciniphila extract-treated groups show a marked, dose-dependent increase in GLP-1 secretionFigure 3a (Heatmap) shows a clear transition from low (red) to high (green) GLP-1 levels with increasing extract dose and time. While the Figure 1b-d (Bar Graphs) illustrates the dose- and time-dependent increase in GLP-1 secretion, with the highest levels at 60 minutes and 500 µg/mL. Figure 4 illustrates the percent change in GLP-1 secretion from NCI-H716 cells after treatment with various doses of VH Akkermansia muciniphila cell extract, compared to a negative control and a positive control (glutamine), measured at 0, 30, and 60 minutes. The heatmap shows a clear progression from red, low GLP-1 secretion at baseline, to yellow and green, higher secretion, as both the extract dose and incubation time increase. Error bars (SD) are relatively small, indicating consistent results across replicates. VH Akkermansia muciniphila cell extract significantly increases GLP-1 secretion from NCI-H716 cells in a dose- and time-dependent manner, with effects that approach those of the positive control (glutamine) at higher doses and longer incubation. This supports the potential of VH Akkermansia muciniphila as a beneficial modulator of gut hormone secretion and metabolic health.