Fuel stimulation of insulin secretion from pancreatic beta-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, as for instance aspartate, glutamate or glutamine. The goal of the present study was to evaluate the role of such factors when insulin release (IR) is stimulated by glucose or amino acids (AA) using NMR-technology and respirometry in superfused -HC9 cells. Exposing -HC9 cells to high glucose or an AA mixture plus glutamine (Q) caused a marked stimulation of IR associated with increased oxygen consumption, cAMP release and phosphorylation potential. However, levels of [MgATP] and [MgADP] changed only slowly and little while the rate of insulin release increased fast and very markedly. High glucose or AA+Q increased intracellular pH. Intracellular Na⁺ levels were greatly elevated by AAM. However, glutamine decreased -cell Na levels. Stimulation of -cells by glucose in the presence of AA+Q was associated with rising cellular concentrations of glutamate, glutamine and strikingly lower aspartate levels. Methionine sulfoximine, an inhibitor of glutamine synthetase, blocked the glucose enhancement of AM+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 IR but illustrate that determining the identity and relative importance of all participating coupling factors and second messengers remains a challenge largely unmet.