Upon glucose stimulation, metabolic pathways of pancreatic beta-cells promptly adapt metabolite levels inducing insulin secretion fine-tuned by mitochondrial glutamate dehydrogenase (GDH). Although well described in vitro, these responses cannot yet be captured in vivo due to the intrinsic nature of the islets scattered throughout the pancreas. Tested first in vitro, glutamate precursor glutamine enhanced glucose-stimulated insulin secretion without eliciting oxidative catabolism, as opposed to glucose. Then, to be as close as possible to the in vivo state, we collected the pancreas of mouse models in fasted versus fed states and at the peak of a glucose tolerance test, immediately followed by snap freezing before in situ analysis of metabolic pathways. On the same series of pancreatic cryosections, islets were identified by dithizone beta-cell staining for metabolic analyses combining spatial in situ redox enzymatic assay with targeted metabolomics using time-of-flight secondary ion MS high-resolution imaging. Direct measurements in cryopreserved pancreatic sections of control and beta-cell specific GDH knockout mice showed tight coupling between glycolysis and mitochondrial pathways favored by low lactate dehydrogenase activity and strong succinate dehydrogenase velocity. In response to regular feeding, intra-islet glutamate and glutamine levels were elevated, an effect dependent on beta-cell GDH. Acute in vivo glucose stimulation increased both alanine and glutamate intra-islet levels. Lack of beta-cell GDH abrogated the rise in glutamate and reduced insulin secretion without impacting alanine levels. Overall, the hallmark of in vivo beta-cell stimulation was a strong mitochondrial activity and GDH-dependent elevation of glutamate required for the full development of insulin secretion.