Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

Zhanxiang Wang,Debbie C Thurmond,Eunjin Oh

doi:10.1074/jbc.m610553200

Zhanxiang Wang, Debbie C Thurmond + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m610553200

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Abstract

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

Highlights

We and others have presented evidence to suggest that the key to actin remodeling in the islet ␤-cell may lie in the glucosespecific activation of the small Rho family GTPase protein Cdc42 [1,2,3,4,5]
Secretion in ␤-Cells—We and others have demonstrated that impairment of Cdc42 activation or cycling leads to inhibition of glucose-stimulated insulin secretion from pancreatic ␤-cells [1,2,3, 23]
Data are shown as the ratio of insulin released after stimulation relative to basal level from three independent experiments; *, p Ͻ 0.05 versus basal

Summary

Introduction

We and others have presented evidence to suggest that the key to actin remodeling in the islet ␤-cell may lie in the glucosespecific activation of the small Rho family GTPase protein Cdc42 [1,2,3,4,5]. Rac1 activation was dependent upon Cdc42, MIN6 cells were transfected with control or Cdc42 siRNA oligonucleotides (using siCon or siA as in Fig. 1), and whole cell lysates were prepared for assessment of Rac1 activation. As a result of the Pak1 depletion, glucose-induced Rac1 activation was abolished in MIN6 cells (Fig. 9, A and B), similar to the effect observed with depletion of Cdc42.

Results

Conclusion