Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells

Abstract

Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic β-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contribute to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of β-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of β-cells to glucose.

Control of pancreatic β-cell bioenergetics

“…Notably, however, the loss of bioenergetic glucose responsiveness in islets from individuals with T2D can be restored by glucokinase activators, and impaired GSIS is consequently rescued [58] . This effect underscores the importance of glucokinase for GSIS, but also leaves open the possibility that this enzyme is the endpoint of the recently discovered positive feedback loop that is effected by a factor that emerges downstream from mitochondrial ATP synthesis [39] . In this model, bioenergetic glucose sensitivity would be lost in T2D because of failing feedback.…”

“…2A). The glucose effect on ATP supply is comparably strong because most of the respiratory rise is coupled to phosphorylation [25,36] , whilst the proportion of basal respiration used to make ATP is exceptionally low [28,33,[37][38][39] . Glucose increases ATP-synthesis-coupled respiration more than it raises respiration linked to mitochondrial proton leak, which means it stimulates both the rate and coupling efficiency of oxidative phosphorylation [26,36] .…”

“…Attenuated bioenergetic glucose sensitivity is mirrored by dampened GSIS [26] , which suggests a role for mitochondrial proton leak in the regulation of glucose-sensing. A recent metabolic control analysis has indeed demonstrated that proton leak exerts significant control over both the magnitude and homeostasis of the mitochondrial membrane potential in insulinoma cells incubated at low glucose levels [39] . Low mitochondrial ATP synthesis efficiency thus appears to ensure that the membrane potential as well as the mitochondrial and cytosolic ATP/ADP ratios are kept relatively low when insulin secretion is not required.…”

“…At stimulatory glucose levels, homeostatic control of the mitochondrial membrane potential is almost exclusively exerted by glucose oxidation, a process that is regulated itself by factors that arise downstream from ATP synthesis [39] . These factors have not yet been identified, but positive feedback reinforces bioenergetic responsiveness to glucose and supports the notion that glucose-sensing by beta cells is not accounted for by glucokinase alone (cf.…”

“…Modular-kinetic and metabolic control analyses have provided quantitative information on oxidative phosphorylation in beta cells [39] and in isolated beta cell mitochondria [52] . Extending such analyses to cellular energy metabolism surrounding the cytosolic ATP/ADP ratio might offer insight in some unresolved issues in beta cell biology.…”