Evaluation of Betanin on Key Enzymes Related to Obesity, Diabetes, Insulin Signaling Pathway, and Metabolic Disorders: In Vitro, Cellular, and In Silico Study

Background/Objectives: Betanin (Bet), a natural compound, exhibits potent antioxidant and metabolic regulatory properties, yet its effect on cellular glucose utilization remains unclear. This study investigated, for the first time, the impact of Bet on glucose consumption and the activation of key carbohydrate–catabolic pathways in human erythrocytes. Methods: In vitro assays were performed to evaluate enzyme inhibition and activation. Human erythrocytes were incubated with Bet to assess glucose consumption. Enzyme activities were measured spectrophotometrically, and molecular docking was used to analyze binding interactions. Results: Our results demonstrate that Bet inhibits digestive enzymes in a dose-dependent manner, with maximal inhibition at 90 µg/mL for pancreatic lipase and 70 µg/mL for α-amylase, showing IC50 values of 48.8 and 31.9 µg/mL, respectively, supported by strong binding affinities of −9.3 and −8.9 Kcal/mol. These interactions are stronger than those of orlistat (−6.9 Kcal/mol) and acarbose (−7.7 Kcal/mol). Bet also induced the activity of AMPK with an IC50 of 1.83 µg/mL and a BE of −7.90 Kcal/mol, compared to the specific AMPK activator A-769662, which had an IC50 of 1.29 µg/mL and a binding energy of −10.0 Kcal/mol. Consequently, Bet stimulated key glycolytic enzymes, reaching maximal activation (~62%) at 1.4 µg/mL for hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD), and at 1.6 µg/mL for pyruvate kinase (PK), supported by binding energies of −7.2, −7.5, and −9.0 Kcal/mol and AC50 values of 0.87, 0.98, and 0.91 µg/mL, respectively. Moreover, Bet enhanced key Krebs cycle enzymes (IDH, SDH, MDH, LDH) in a dose-dependent manner, with AC50 values of 0.76, 0.80, 0.72, and 0.52 µg/mL and strong binding energies (−7.8, −7.8, and −8.4 Kcal/mol), reaching maximal activation near 1.4 µg/mL. Bet also increased glucose consumption by human erythrocytes. Conclusions: Bet enhances glucose utilization by inhibiting digestive enzymes and activating intracellular metabolic pathways, suggest potential metabolic regulatory effects.