Exploring the potential of pheophorbide A, a chlorophyll-derived compound in modulating GLUT for maintaining glucose homeostasis

Abstract

Introduction: Pheophorbide A, a chlorophyll-breakdown product, is primarily investigated for its anti-oxidant and anti-inflammatory activity. Recent reports on pheophorbide A have shown its potential in lowering blood glucose levels, thus leading to the exploration of its use in diabetes management. Literature has also shown its effect on enhanced insulin secretion, whereas its mechanism on glucose stimulated insulin secretion (GSIS) in pancreatic β cells remains unexplored.

Methods: In-silico and in-vitro investigations were used to explore the effect of pheophorbide A on class I glucose transporters (GLUTs). In-silico studies include - Molecular docking studies and stability assessment using GROMACS. In-vitro studies include - MTT assay, Glucose uptake assay, Live-cell imaging and tracking of GLUTs in presence of Pheophorbide A compared to control.

Results: Molecular docking studies revealed better binding affinity of pheophorbide A with GLUT4 (-11.2 Kcal/mol) and GLUT1 (-10.7 Kcal/mol) when compared with metformin (-5.0 Kcal/mol and -4.9 Kcal/mol, respectively). Glucose levels are largely regulated by GLUTs where GLUT1 is one of the transporters that is ubiquitously present in human β cells. Thus, we confirmed the stability of the complex, that is, pheophorbide A-GLUT1 using GROMACS for 100 ns. We further assessed its effect on a pancreatic β cell line (INS-1) for its viability using an MTT assay. Pheophorbide A (0.1-1 µM) showed a dose-dependent response on cell viability and was comparable to standard metformin. To assess how pheophorbide A mechanistically acts on GLUT1 in pancreatic β cell, we transfected INS-1 cells with GLUT1-enhanced green fluorescent protein and checked how the treatment of pheophorbide A (0.50 µM) modulates GLUT1 trafficking using live-cell imaging. We observed a significant increase in GLUT1 density when treated with pheophorbide A (0.442 ± 0.01 µm-2) at 20 mM glucose concentration when compared to GLUT1 control (0.234 ± 0.01 µm-2) and metformin (0.296 ± 0.02 µm-2). The average speed and distance travelled by GLUT1 puncta were observed to decrease when treated with pheophorbide A. The present study also demonstrated the potential of pheophorbide A to enhance glucose uptake in β cells.

Conclusion: The current study's findings were validated by in-silico and cellular analyses, suggesting that pheophorbide A may regulate GLUT1 and might be regarded as a potential lead for boosting the GSIS pathway, thus maintaining glucose homeostasis.

Keywords: GLUT1 trafficking; cell viability; live cell imaging; molecular docking; molecular dynamics simulation; pheophorbide A.

Figure 1

(A–C) Physicochemical analysis of the selected compounds; (A, B) ProTox ii–generated radar plot for pheophorbide A and Metformin; (C) heatmap for Lipinski and SwissADME (Lipinski criteria are mentioned in the properties column, remaining accounts as the maximum and minimum bars represented in the illustration).

Figure 2

(A–C) Molecular docking showing the 2D image of amino acids involved in the binding site and a 3D image of the hydrogen bond interaction of GLUT1 with the ligands; (A) GLUT1 and glucose; (B) GLUT1 and Metformin; (C) GLUT1 and pheophorbide A.

Figure 3

(A–E) Molecular dynamics simulation of GLUT1 with pheophorbide A using GROMACS; (A) Radius of gyration (Rg) value up to 100 ns; (B) root-mean-square deviation of the backbone atom of the complex; (C) root-mean-square fluctuation of c-alpha atoms of the complex; (D) inter-hydrogen bond of the complex; (E) intra-hydrogen bond of the complex.

Figure 4

(A, B) Effects of pheophorbide A and Metformin on INS-1 cell viability by using MTT assay; (A) dose-dependent effect of pheophorbide A and Metformin on the cell viability of INS-1 cells. Data represent the mean ± SE, (n = 9; N = 3). Statistical analysis using two-way ANOVA by Dunnett’s multiple comparisons test indicates significant differences (****p< 0.0001). (B) Representative bright-field microscopy images illustrating the influence of pheophorbide A at concentrations of 0.5 µM and 1.0 µM on INS-1 cells. (C) Representative bright-field microscopy images of INS-1 cells under non-treatment conditions. For all images, scale bar is 1 µm.

Figure 5

(A–C) Effect of pheophorbide A and Metformin on density of GLUT1 at the plasma membrane and uptake of glucose under 3 mM and 20 mM glucose concentration in INS-1 cells; (A) GLUT1 density at the plasma membrane, images were taken at 100× using TIRF-M; (B) bar graph representing density of GLUT1 with respect to pheophorbide A and Metformin treatment (n = 14; N = 3); (C) bar graph representing glucose uptake by INS-1 cells in treated (pheophorbide A/Metformin) and untreated conditions, cultured at 3 mM and 20 mM glucose media (n = 9; N = 2). Statistical analysis using two-way ANOVA by Dunnett’s multiple comparisons test indicates significant differences (****p< 0.0001; *p 0.0488). For all images, scale bar is 1 µm.

Figure 6

(A–H) Effect of pheophorbide A and Metformin on GLUT1 trafficking. Cells were imaged for up to 1 min at 1 s time interval using TIRF-M; the generated tracks were then analyzed; (A, B) display of INS-1 transfected cells and their corresponding tracks generated using the TrackMate function in ImageJ; (C) quantification of the total distance traveled by GLUT1 puncta in 1 min was calculated (n = 15 tracks, N = 5 cells); (D) calculation of the average speed of GLUT1 puncta observed at 1 s intervals up to 1 min (n = 15 tracks, N = 5 cells); (E–G) depiction of trends observed in GLUT1 under untreated and treated conditions with Metformin/pheophorbide A for single puncta using tracks generated by ImageJ; (F) measurement of the distance traveled by a single puncta in untreated and treated conditions (Metformin/pheophorbide A) up to 40 s. Statistical analysis using one-way ANOVA by Dunnett’s multiple comparisons test indicates significant differences (****p< 0.0001; *p 0.0478). For all images, the scale bar is 1 µm. (F) During the tracking process, the high mobility of the control blue dot and metformin black dot made accurate tracking challenging, hence, is not represented after 20 s.