Fuel stimulation of insulin secretion from pancreatic β-cells is thought to be mediated by metabolic coupling factors that are generated by energized mitochondria, including protons, adenine nucleotides, and perhaps certain amino acids (AA), as for instance aspartate, glutamate, or glutamine (Q). The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or AA, alone or combined, using ³¹P, ²³Na and ¹H NMR technology, respirometry, and biochemical analysis to study the metabolic events that occur in continuously superfused mouse β-HC9 cells contained in agarose beads and enhanced by the phosphodiesterase inhibitor IBMX. Exposing β-HC9 cells to high glucose or 3.5 mM of a physiological mixture of 18 AA (AAM) plus 2 mM glutamine caused a marked stimulation of insulin secretion associated with increased oxygen consumption, cAMP release, and phosphorylation potential as evidenced by higher phosphocreatine and lower P_i peak areas of ³¹P NMR spectra. Diazoxide blocked stimulation of IR completely, suggesting involvement of ATP-dependent potassium (K_ATP) channels in this process. However, levels of MgATP and MgADP concentrations, which regulate channel activity, changed only slowly and little, whereas the rate of insulin release increased fast and very markedly. The involvement of other candidate coupling factors was therefore considered. High glucose or AAM + Q increased pH_i. The availability of temporal pH profiles allowed the precise computation of the phosphate potential (ATP/P_i × ADP) in fuel-stimulated IR. Intracellular Na⁺ levels were greatly elevated by AAM + Q. However, glutamine alone or together with 2-amino-2-norbornanecarboxylic acid (which activates glutamate dehydrogenase) decreased β-cell Na levels. Stimulation of β-cells by glucose in the presence of AAM + Q (0.5 mM) was associated with rising cellular concentrations of glutamate and glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AMM + Q-induced IR and associated changes in glutamine and aspartate but did not prevent the accumulation of glutamate. The results of this study demonstrate again that an increased phosphate potential and a functional K_ATP channel are essential for metabolic coupling during fuel-stimulated insulin release but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet.