Glucose-induced Cytosolic pH Changes in β-Cells and Insulin Secretion Are Not Causally Related: STUDIES IN ISLETS LACKING THE NA+/H+ EXCHANGER NHE1

Myriam Nenquin,Patrick Stiernet,Jean-Christophe Jonas,Jean-Claude Henquin,Pierre Moulin

doi:10.1074/jbc.m702862200

Abstract

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.

Highlights

Normal glucose homeostasis requires precise regulation of insulin secretion, a complex process that pancreatic ␤-cells achieve through changes in their metabolism [1,2,3,4]
Except for two studies reporting no effect of glucose on pHc in mouse ␤-cells [11] or an acidification in rat islets [12], there is a large consensus that high glucose increases pHc in mouse islets [13,14,15,16,17], rat islets [18, 19] and insulin-secreting cell lines [20, 21]
We recently showed that NHE1 is strongly expressed in rat islets and is present in mouse islets [35]

Summary

Introduction

Normal glucose homeostasis requires precise regulation of insulin secretion, a complex process that pancreatic ␤-cells achieve through changes in their metabolism [1,2,3,4]. Studies in which changes in ␤-cell pHc were imposed by manipulation of pH and ionic composition of the extracellular medium or by exposure to weak bases or acids have led to the contradictory proposals that alkalinization augments [14, 22] or inhibits [23] insulin secretion, and that acidification increases it (24 –26). Glucose-induced priming of insulin secretion (i.e. augmentation of the response to a second stimulation), which is thought to be mediated by the amplifying pathway [29], has been linked to ␤-cell acidification [26, 30].These proposals are at odds with the fact that glucose increases islet pHc and with the correlative evidence that only those fuels which increase ␤-cell pHc amplify insulin secretion [31].

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