Bioenergetic Analysis of Single Pancreatic β-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects

Abstract

Impaired activation of mitochondrial energy metabolism by glucose has been demonstrated in type 2 diabetic β-cells. The cause of this dysfunction is unknown. The aim of this study was to identify segments of energy metabolism with normal or with altered function in human type 2 diabetes mellitus. The mitochondrial membrane potential (ΔψM), and its response to glucose, is the main driver of mitochondrial ATP synthesis and is hence a central mediator of glucose-induced insulin secretion, but its quantitative determination in β-cells from human donors has not been attempted, due to limitations in assay technology. Here, novel fluorescence microscopic assays are exploited to quantify ΔψM and its response to glucose and other secretagogues in β-cells of dispersed pancreatic islet cells from 4 normal and 3 type 2 diabetic organ donors. Mitochondrial volume densities and the magnitude of ΔψM in low glucose were not consistently altered in diabetic β-cells. However, ΔψM was consistently less responsive to elevation of glucose concentration, whereas the decreased response was not observed with metabolizable secretagogue mixtures that feed directly into the tricarboxylic acid cycle. Single-cell analysis of the heterogeneous responses to metabolizable secretagogues indicated no dysfunction in relaying ΔψM hyperpolarization to plasma membrane potential depolarization in diabetic β-cells. ΔψM of diabetic β-cells was distinctly responsive to acute inhibition of ATP synthesis during glucose stimulation. It is concluded that the mechanistic deficit in glucose-induced insulin secretion and mitochondrial hyperpolarization of diabetic human β-cells is located upstream of the tricarboxylic acid cycle and manifests in dampening the control of ΔψM by glucose metabolism.

The Pancreatic β-Cell: A Bioenergetic Perspective

“…However, Tanaka et al (506) utilized the targeted ATP reporter GO-ATeam and found that while 10 mM glutamine had no effect on mitochondrial or cytosolic ATP levels of islets in the presence of 2.8 mM glucose, the combination of glutamine and leucine caused a rapid increase in ATP in both compartments of a magnitude comparable to that seen with 25 mM glucose. Similarly, Gerencser (173) demonstrated that the combination of glutamine and BCH hyperpolarized ⌬ m in human islets.…”

“…rescence of a cationic probe such as TMRM (173,183), or the more slowly equilibrating rhodamine 123 (R123) (135,286,304). This technique is complicated by the plasma membrane potential, ⌬ p , which controls the gradient of the probe between the incubation medium and the cytosol.…”

“…173, 174, 211) is more quantifiable, but the probes respond slowly and are affected equally by changes in ⌬ m and ⌬ p . Changes in ⌬ p , which are particularly relevant in the ␤-cell context, can be allowed for by including a fluorescent anion together with TMRM (173,174,378). These approaches have been recently reviewed (61,380).…”

“…Formerly, these have been either qualitative ("mitochondria hyperpolarize or depolarize") or semi-quantitative (with estimates of the extent of the change in ⌬ m in transitioning from low to high glucose, Ref. 183); however, a novel technique employing simultaneous cationic and anionic probes has allowed quantitative determination (173,174).…”

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

“…While rare mitochondrial defects can cause diabetes [15], it is possible that more subtle derangements in cellular energy metabolism contribute more generally to β-cell impairment in diabetes. In support of this idea, the dampened response of ΔψM to glucose in T2D human β-cells may reflect a subtle bioenergetic supply-demand dysfunction [16]. …”