Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release

Abstract

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

Inhibition of the malate–aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion

“…Notably, we found that stimulation of αTC1-6 and INS-1 832/13 cells with glucose provoked increased levels of glutamate and glycerol 3-phosphate and decreased levels of aspartate, confirming previous findings in the INS-1 832/13 cell line [36]. These intermediates are all part of the mitochondrial shuttles.…”

“…Metabolite profiling was performed in αTC1-6 and INS-1 832/13 cells as previously described in detail [35,36]. In brief, α-TC1-6 and INS-1 832/13 cells were seeded in 12-and 24-well plates respectively and treated as described for hormone secretion.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…A similar time course can be seen in the respiratory response of clonal insulinoma cells and human islets to acutely elevated glucose, with an initial doubling of respiration followed by a further increase over the subsequent 50 min (112,184,491). Much of the enhanced proton current is directed through the ATP synthase, rather than the endogenous proton leak, judged by the extent of oligomycin inhibition (FIGURE 3D) (112,147,184,427).…”

“…A relatively low activity does not, of course, invalidate a pathway as long as it is sufficient to meet the ␤-cell's ill-defined NADPH requirements. Spegel et al (491) provided evidence for its significance in INS-1 832/13 cells: following the addition of high glucose, the NADPH/NADP ratio increased in the cells synchronously with the formation of ribose-5-phosphate. However, one difficulty with an obligatory role for the pentose phosphate pathway in facilitating insulin secretion is that alternative substrates, such as ␣-ketoisocaproate or pyruvate (in the case of INS-1-derived cells), are highly effective secretagogues while not providing substrate for the pathway.…”

“…However, after starvation in low glucose, the ␤-cell requires up to 60 min to achieve a new metabolic and bioenergetic steady state following a step elevation in glucose, with respiration (184), cytosolic ATP/ADP ratios (509), and metabolite levels (491), each showing different recovery kinetics. This greatly complicates interpretation of the resulting insulin release profile.…”