In Silico and In Vivo Investigation of the Anti-Hyperglycemic Effects of Caffeic Acid

Abstract

Hyperglycemia, characterized by elevated blood glucose levels, is a major risk factor for diabetes mellitus and its complications. While conventional therapies are effective, they are often associated with side effects and high costs, necessitating alternative strategies. This study evaluates the potential of caffeic acid (CA), a phenolic compound with reported antihyperglycemic properties, using both in silico and in vivo approaches. Molecular docking simulations revealed that CA demonstrates a strong binding affinity to protein tyrosine phosphatase 1B (PTP1B), a critical enzyme in glucose metabolism, with superior interaction profiles compared to the reference drug, ertiprotafib. In the in vivo studies, a Drosophila melanogaster model was used to investigate the effects of CA under hyperglycemic conditions induced by a high-sugar diet. Treatment with CA, particularly at a concentration of 500 μM, significantly reduced hemolymph glucose levels and improved several physiological and behavioral parameters, including survival rates, body size, body weight, and larval movement. Furthermore, gene expression analysis demonstrated that CA modulates key metabolic and stress-related pathways, enhancing glucose homeostasis and reducing metabolic stress. These findings highlight the dual utility of in silico and in vivo methodologies in elucidating the antihyperglycemic potential of CA. The results support the development of CA as a cost-effective and ethically viable therapeutic candidate with implications for diabetes management in resource-limited settings.

Figure 1

Experimental design used in this study. Five groups of 3rd instar larvae of D. melanogaster larvae were used to assess the effects of a HSD with or without CA treatment at varying concentrations (31.25, 125, and 500 μM). One group, reared without any treatment, served as the untreated control. Created with BioRender.com.

Figure 2

Grid box was positioned around the binding site of the PTP1B receptor (1C83) (A) and highlighting the redocked ligand (pink) and the native ligand (blue) (B), with an RMSD value of 0.001 Å.

Figure 3

Docking view of CA in the binding site of PTP1B (PDB ID: 1C83). The stereo view of the docked complex between CA and 1C83 (A) and the interactions between CA and 1C83 presented in a 2D format (B). The diagram was created using Discovery Studio, with hydrogen bonds represented by green dashed lines along with their distances in Å, RMSD 0.159.

Figure 4

Molecular dynamics results radius of gyration (A), RMSD (B), and RMSF (C) of PTP1B.

Figure 5

Measurement of hemolymph glucose level of Drosophila melanogaster after HSD treatment with or without CA. Comparison of larval hemolymph glucose levels between the normal control and HSD (A) and the comparison of glucose levels between HSD-fed larvae and those treated with varying concentrations of CA (B). Statistical significance is indicated by asterisks (**p < 0.01, ****p < 0.0001), with “ns” denoting no significant difference.

Figure 6

Improvement of Drosophila melanogaster survival at various developmental stages after HSD treatment in the presence or absence of CA. Comparison of larval-to-pupal survival between flies maintained on normal food and those on HSD (A), comparison of larval-to-pupal survival between flies maintained on HSD with and without CA treatment (B), comparison of pupal-to-adult fly survival between flies maintained on normal food and those on HSD (C), and comparison of pupal-to-adult fly survival between flies maintained on HSD with and without CA treatment (D). Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), with “ns” denoting no significant difference.

Figure 7

Measurement of larval length and width after HSD treatment in the presence or absence of CA. Comparison of larval length between normal control and HSD (A), comparison of larval length between HSD-fed larvae in the presence or absence of CA treatment (B), comparison of larval width between normal control and HSD (C), and comparison of larval width between HSD-fed larvae in the presence or absence of CA treatment (D). Statistical significance is indicated by asterisks (**p < 0.01, ****p < 0.0001), with “ns” denoting no significant difference.

Figure 8

Measurement of body weight and larval crawling after HSD treatment in the presence or absence of CA. Comparison of larval body weight between normal control and HSD (A), comparison of larval body weight between HSD-fed larvae in the presence or absence of CA treatment (B), comparison of larval crawling between normal control and HSD (C), and comparison of larval crawling between HSD-fed larvae in the presence or absence of CA treatment (D). Statistical significance is indicated by asterisks (*p < 0.05, ***p < 0.001, ****p < 0.0001), with “ns” denoting no significant difference.

Figure 9

Measurement of srl (A), pepck (B), and totA (C) expressions following HSD treatment with or without CA. Treatment with CA at 500 μM significantly upregulated srl (A) and pepck (B) expression compared to the HSD control, while totA (C) expression was significantly reduced. Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01), with “ns” denoting no significant difference.

Figure 10

Illustration on the induction of hyperglycemia and CA treatment in Drosophila melanogaster. Larvae were exposed to a 30% sucrose solution to induce hyperglycemia, with phenotypic outcomes following CA treatment shown in (A). The correlation between phenotypic and molecular results after treatment is presented in (B). Created in BioRender. Nainu, F. (2025) https://BioRender.com/zbumoqj.