Aims/Hypothesis Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. (Seahorse XF24 Extracellular Flux Analyzer) glucose utilization (radiometric) lactate release (enzymatic colorimetric) ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels respiration and insulin secretion were examined in human islets. Results Glucose increased insulin release glucose utilization raised ATP production and respiratory rates in both lines and pyruvate increased insulin secretion Eriocitrin and respiration. EndoC-βH1 cells exhibited higher insulin secretion while plasma membrane depolarization was attenuated and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines but responses were weaker in EndoC-βH1 cells similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells. Conclusions/Interpretation Functions associated with early stimulus-secretion coupling with the exception of plasma membrane potential and Ca2+ oscillations were similar in the two cell lines; insulin secretion respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable models with the caveat of replicating key findings in isolated islets Eriocitrin EndoC-βH1 cells have the advantage of carrying the human Eriocitrin genome allowing studies of human genetic variants epigenetics and regulatory RNA molecules. Introduction Defective insulin secretion by pancreatic beta cells underlies type 2 diabetes mellitus (T2D) a disease that increases globally and soon is estimated to Eriocitrin affect >500 million people . Despite decades of research neither the regulation of insulin secretion nor the mechanism underlying the disease is completely understood. Stimulus-secretion STAT2 coupling in the beta cell links a rise in postprandial blood glucose levels to insulin release. Glucose is transported into the beta cell and metabolized to yield pyruvate which in turn is further metabolized to raise ATP-levels . This increase in the ATP/ADP-ratio closes ATP-dependent K+-channels (K+ATP-channels) in the plasma membrane . Closure of K+-channels depolarizes the cell membrane causing an opening of voltage-gated Ca2+-channels and release of insulin . This pathway known as the triggering pathway is complemented by an amplifying pathway . Numerous studies have been devoted to elucidate the nature of the latter enigmatic pathway . Stimulus-secretion coupling has primarily been studied in insulinoma cell lines and rodent isolated islets. These studies imply differences between species as well as between clonal and primary cells. In recent times human islets have been made available to research but their number is limited. In addition to beta cells islets also contain significant numbers of α- δ- PP ε-cells and blood vessel endothelial cells [6 7 limiting the use of islets as a specific beta cell model. Moreover rodent and human beta cells and islets show differences in the expression of key enzymes in glucose metabolism in the insulin gene (two genes in rodents while one gene in humans)  glucose transporters  and islet structure . Attempts have been made to develop human beta cell lines; however these lines show low levels of insulin production slow growth rate or limited phenotypic and functional stability [11 12 Recently a stable human beta cell line EndoC-βH1 was derived using targeted oncogenesis in human fetal pancreatic tissue . EndoC-βH1 cells produce and secrete insulin in response to glucose are stable in culture and express beta cell-specific markers such as PDX1 and MAFA. Transplantation of EndoC-βH1 cells reinstated normoglycemia in STZ-induced diabetic mice . In the present study we attempted to provide a comprehensive characterization of stimulus-secretion coupling in the EndoC-βH1 beta cell line by comparing glucose metabolism in this cell line and in the clonal rat cell line INS-1 832/13 [14 15.