Date of Defense
20-3-2025 9:00 AM
Location
F3-129
Document Type
Dissertation Defense
Degree Name
Doctor of Philosophy in Cellular and Molecular Biology
College
COS
Department
Biology
First Advisor
Dr. Khalid Muhammad
Keywords
Camel Milk, Diabetes, Bioactive proteins, BRET, Insulin Receptor, GLP-1R.
Abstract
Camel milk (CM), renowned for its exceptional nutritional and medicinal properties, has been a crucial resource in arid regions for centuries. Its notable antidiabetic effects, supported by extensive research through in vitro, in vivo, and clinical studies, have gained significant scientific attention. However, the underlying cellular and molecular mechanisms remain largely unclear. Our previous research demonstrated that camel milk whey hydrolysates (CWHs) induce insulin receptor (IR) activation and downstream signaling, promoting glucose uptake in vitro using HEK293 and HepG2 cells. Building on this foundation, the current study focused on synthesizing nine of the most potent peptides (designated P1-9) derived from CM whey hydrolysates, examining their bioactivity both in vitro and in silico. This investigation assessed the pharmacological effects of these synthetic peptides on IR and GLP1R (Glucagon-like peptide-1) using in vitro models, including human embryonic kidney (HEK293), human hepatocarcinoma (HepG2), and INS1 832/13 cell lines. The functional activities of the peptides were evaluated through phosphorylation of IR, AKT, and ERK 1/2. Bioluminescence resonance energy transfer (BRET)--based analysis revealed differential effects of synthetic CM-derived peptides on IR and GLP1R activity in HEK293 cells. For IR, single peptide treatment—except for P4 and P5—partially induced IR activation compared to Insulin in HEK293 cells. In combined treatment with insulin, the peptides demonstrated three distinct profiles: P1, P3, P4, P5 being non-effective; P6, P7, P8 significantly enhancing insulin-mediated IR activation; and P2 antagonizing insulin’s effect. On GLP1R, peptides partially induced GLP1R activation through Gs, Gq, and β arrestin signaling, in combination treatment, BRET signals showed significant increases, especially with P1-P4 showing high efficacy, P5-P7 with lower efficacy, and P8-P9 with moderately high efficiency. These findings were consistent with AKT and ERK1/2 phosphorylation observed in INS-1 832/13 cells. Furthermore, in IR, all peptides slightly promoted IR and AKT phosphorylation when administered alone, whereas combination with insulin significantly increased AKT phosphorylation, especially with P6 and P7. In glucose uptake assays using HepG2 cells, all peptides enhanced glucose uptake, with P6, P7, and P8 demonstrating the highest potency. Interestingly, in cAMP assays, except for P1 and P2, all other peptides exhibited antagonistic effects when combined with GLP1. Molecular docking studies on insulin-bound IR and GLP1R complexes revealed binding sites with high Dscore values. Specifically, on IR, peptides P1 and P4 displayed the highest binding affinity at site 2, while on GLP1R, peptides P1, P2, and P3 showed the highest binding affinity at site 1 among the other tested sites. Overall, the results confirm the bioactivity of synthetic CM-derived peptides on IR and GLP1R, as well as their downstream signaling pathways. In conclusion, this study provides a comprehensive validation of bioactive peptides generated from camel milk protein hydrolysis, further supporting the therapeutic potential of CM peptides in modulating key molecular pathways involved in glucose metabolism. These synthetic CM-derived peptides offer promising prospects for future development of antidiabetic therapies and the creation of novel CM-derived drugs with potential applications in managing diabetes.
MOLECULAR MECHANISMS AND ROLE OF CAMEL MILK DERIVED BIOACTIVE PEPTIDES TOWARDS INSULIN AND GLP-1 RECEPTOR ACTIVATION
F3-129
Camel milk (CM), renowned for its exceptional nutritional and medicinal properties, has been a crucial resource in arid regions for centuries. Its notable antidiabetic effects, supported by extensive research through in vitro, in vivo, and clinical studies, have gained significant scientific attention. However, the underlying cellular and molecular mechanisms remain largely unclear. Our previous research demonstrated that camel milk whey hydrolysates (CWHs) induce insulin receptor (IR) activation and downstream signaling, promoting glucose uptake in vitro using HEK293 and HepG2 cells. Building on this foundation, the current study focused on synthesizing nine of the most potent peptides (designated P1-9) derived from CM whey hydrolysates, examining their bioactivity both in vitro and in silico. This investigation assessed the pharmacological effects of these synthetic peptides on IR and GLP1R (Glucagon-like peptide-1) using in vitro models, including human embryonic kidney (HEK293), human hepatocarcinoma (HepG2), and INS1 832/13 cell lines. The functional activities of the peptides were evaluated through phosphorylation of IR, AKT, and ERK 1/2. Bioluminescence resonance energy transfer (BRET)--based analysis revealed differential effects of synthetic CM-derived peptides on IR and GLP1R activity in HEK293 cells. For IR, single peptide treatment—except for P4 and P5—partially induced IR activation compared to Insulin in HEK293 cells. In combined treatment with insulin, the peptides demonstrated three distinct profiles: P1, P3, P4, P5 being non-effective; P6, P7, P8 significantly enhancing insulin-mediated IR activation; and P2 antagonizing insulin’s effect. On GLP1R, peptides partially induced GLP1R activation through Gs, Gq, and β arrestin signaling, in combination treatment, BRET signals showed significant increases, especially with P1-P4 showing high efficacy, P5-P7 with lower efficacy, and P8-P9 with moderately high efficiency. These findings were consistent with AKT and ERK1/2 phosphorylation observed in INS-1 832/13 cells. Furthermore, in IR, all peptides slightly promoted IR and AKT phosphorylation when administered alone, whereas combination with insulin significantly increased AKT phosphorylation, especially with P6 and P7. In glucose uptake assays using HepG2 cells, all peptides enhanced glucose uptake, with P6, P7, and P8 demonstrating the highest potency. Interestingly, in cAMP assays, except for P1 and P2, all other peptides exhibited antagonistic effects when combined with GLP1. Molecular docking studies on insulin-bound IR and GLP1R complexes revealed binding sites with high Dscore values. Specifically, on IR, peptides P1 and P4 displayed the highest binding affinity at site 2, while on GLP1R, peptides P1, P2, and P3 showed the highest binding affinity at site 1 among the other tested sites. Overall, the results confirm the bioactivity of synthetic CM-derived peptides on IR and GLP1R, as well as their downstream signaling pathways. In conclusion, this study provides a comprehensive validation of bioactive peptides generated from camel milk protein hydrolysis, further supporting the therapeutic potential of CM peptides in modulating key molecular pathways involved in glucose metabolism. These synthetic CM-derived peptides offer promising prospects for future development of antidiabetic therapies and the creation of novel CM-derived drugs with potential applications in managing diabetes.
