Pancreatic β-cells play a key role in the glucose homeostasis, secreting insulin in response to blood nutrient fluctuations. This is due to a complex relationship between metabolism and insulin secretion which comprises both triggering (ATP and Ca²⁺ dependent [1]) and amplifying (mitochondrial metabolite dependent [2]) pathways of insulin secretion. This study has attempted to develop a mathematical model of insulin release from pancreatic β-cells with particular regard to the role of glutamate. The model’s input is made up of three nutrient components: glucose, alanine [3] and glutamine [4] while the ouput of the model is represented by insulin secretion. A systems biology approach was employed to integrate biological experimental work with mathematical tools to gain novel insights about the role played by glutamate in insulin secretion. A simplified kinetic model of the glucose-stimulated insulin secretion in pancreatic β-cells which takes into account glycolysis, Krebs cycle, alanine uptake and glutamine-glutamate metabolism was built [5]. Experimental work was carried out on a functional clonal insulin-secreting cell line (BRIN-BD11) to validate the model. Cell viability and death assays were performed following incubation in various concentrations of nutrients. The utilization and production of the major components of the mathematical model include: glucose, alanine, glutamine, pyruvate, lactate, glutamate, ATP/ADP ratio and insulin. All the determinations described above were performed following BRIN-BD11 incubation in the presence of 10 mM of alanine and various concentrations of glucose (5, 16.7, 30 mM) and glutamine (0, 1, 2 mM). A quantitatively accurate description of the oscillating behaviour of pyruvate and others metabolites of the Krebs cycle and ATP was obtained. ATP, ADP, pyruvate and glucose-6-phosphate concentrations were associated with coordinated oscillations. Differences in the oscillating features were found when values obtained by experimental procedures were used since desensitization to glucose induced by 24hr incubation in high glucose (30mM) resulted in reduced oscillations of the metabolites and lower insulin secretion. Cell viability was not altered by incubation in various nutrient combinations (maximum difference 12.1%). Insulin secretion was biphasic in the presence of glucose and alanine (AUC value: 126 ± 12 and 117 ± 14 µg/mg protein during the 1^st and 2^nd phase respectively) but was not affected by acute (1-20 min) glutamine stimulation, indicating that only a small amount of glutamine-derived glutamate is converted into α-ketoglutarate in glucose stimulatory conditions. It is likely that changes in glutamate and α-ketoglutarate production represent a more important coupling pathway when low concentrations (below 5mM) of extracellular glucose are present.