Small G proteins in islet beta-cell function

Abstract

Glucose-stimulated insulin secretion from the islet beta-cell involves a sequence of metabolic events and an interplay between a wide range of signaling pathways leading to the generation of second messengers (e.g., cyclic nucleotides, adenine and guanine nucleotides, soluble lipid messengers) and mobilization of calcium ions. Consequent to the generation of necessary signals, the insulin-laden secretory granules are transported from distal sites to the plasma membrane for fusion and release of their cargo into the circulation. The secretory granule transport underlies precise changes in cytoskeletal architecture involving a well-coordinated cross-talk between various signaling proteins, including small molecular mass GTP-binding proteins (G proteins) and their respective effector proteins. The purpose of this article is to provide an overview of current understanding of the identity of small G proteins (e.g., Cdc42, Rac1, and ARF-6) and their corresponding regulatory factors (e.g., GDP/GTP-exchange factors, GDP-dissociation inhibitors) in the pancreatic beta-cell. Plausible mechanisms underlying regulation of these signaling proteins by insulin secretagogues are also discussed. In addition to their positive modulatory roles, certain small G proteins also contribute to the metabolic dysfunction and demise of the islet beta-cell seen in in vitro and in vivo models of impaired insulin secretion and diabetes. Emerging evidence also suggests significant insulin secretory abnormalities in small G protein knockout animals, further emphasizing vital roles for these proteins in normal health and function of the islet beta-cell. Potential significance of these experimental observations from multiple laboratories and possible avenues for future research in this area of islet research are highlighted.

Figure 1

Schematic representation of the biosynthesis of farnesyl and geranyl pyrophosphates. HMG-CoA is synthesized from acetyl-CoA and acetoacetyl-CoA; this step is catalyzed by HMG-CoA-synthetase. HMG-CoA-reductase catalyzes the conversion of HMG-CoA to MVA, which is the precursor for cholesterol biosynthesis. MVA also serves as the precursor molecule for the biosynthesis of farnesyl pyrophosphate (farnesyl-pp) and geranylgeranyl pyrophosphates (geranylgeranyl-pp). These MVA derivatives, in turn, are incorporated into candidate substrate proteins via the prenylation reaction catalyzed by FTase and GGTase. Due to the paucity of specific inhibitors for FTase/GGTase, initial studies used LOVA to decipher the roles for protein prenylation in insulin secretion. Follow-up studies using more specific inhibitors of these enzymes further confirmed the novel roles of these lipid modification steps in insulin secretion (25,47).

Figure 2

Modulatory roles of various classes of small G proteins and their regulatory proteins/factors in insulin secretion. Small G proteins, such as Rac1, Cdc42, and ARF-6 (and potentially Rho) regulate cytoskeletal remodeling and vesicular fusion in the islet. Rab3A, Rab27A, and Rap1 are implicated docking and priming of secretory vesicles at the exocytotic sites. Glucose-mediated activation of some of these signaling proteins is under the fine control of various regulatory proteins including GDIs (e.g., Rho GDI and Cav-1) and GEFs (Tiam1, ARNO, and Epac2).

Figure 3

Guanine nucleotide regulatory proteins/factors involved in the activation-deactivation cycle of G proteins. The small G proteins (e.g., Cdc42 and Rac1) in their GDP-bound (inactive) conformation remain associated with their respective GDIs. The principal role of GDIs is to prevent dissociation of GDP from the corresponding G protein. After the receipt of the appropriate signal, the G protein/GDI complex dissociates, thereby facilitating GTP/GDP exchange mediated by various guanine nucleotide exchange factors (GEFs). The GTP-bound, functionally active G protein, in turn, regulates its effector proteins and downstream signaling steps leading to cellular (de)activation. GTP bound to these G proteins is hydrolyzed by the GTPase activity intrinsic to the candidate G protein, to GDP yielding the inactive conformation of the G protein. Under specific conditions, the GTPase activity can be stimulated by additional regulatory factors, such as the GAP.

Figure 4

A model for potential cross-talk between ARF-6, Cdc42, and Rac1 leading to GSIS. Glucose metabolism leads to activation of Cdc42 and ARF-6, which, in turn, culminates in the activation of lipid-metabolizing enzymes within the β-cell. The lipid second messengers (e.g., PIP₂ and PA) generated from PLase activation promote dissociation of Rac1 from the Rac1/GDI complex, paving the way for Rac1 activation (GTP-Rac1) mediated by Tiam1, a GEF specific for Rac1. Activation of Rac1 promotes cytoskeletal remodeling, which facilitates the trafficking of insulin-laden secretory granules to the plasma membrane for their fusion and exocytotic secretion of insulin. It should be noted that whereas previous studies have provided evidence to suggest that glucose-mediated activation of Cdc42 is upstream to Rac1 activation, the time course for ARF-6 activation by glucose remains to be determined in cognate cellular preparations.

Figure 5

A model for glucose-mediated activation of farnesylated and geranylgeranylated proteins leading to GSIS. Identification of ERK and PAK1 as target proteins. Glucose metabolism leads to the activation of islet endogenous FTases and GGTases culminating in the activation of farnesylated and geranylated proteins, respectively. Such conclusions were reached by pharmacological and molecular biological approaches. It appears that activation of a yet to be identified farnesylated protein(s) is necessary for glucose-mediated activation of ERK and subsequent effects on insulin gene transcription and insulin secretion. On the other hand, glucose-mediated activation of Cdc42 and Rac1 leads to regulation of PAK1 activity. Potential phosphoprotein substrates for PAK1 have not been identified in the β-cell up until now, but could include Rho-GDI. The activation of ERK and PAK (and other effector proteins) facilitate reorganization of the actin cytoskeletal architecture leading to translocation and fusion of insulin granules with the plasma membrane and release of insulin. An active involvement of IQGAPs and gelsolins in these signaling pathways in the organization of the cytoskeletal architecture is also proposed.

Figure 6

Positive modulatory roles for Rac1 (and Rap1?) in the activation of NOX and generation of ROS and subsequent oxidative stress in β-cells under the duress of various stimuli. Based on the current knowledge, it is evident that NOX plays a significant role(s) in the generation of ROS and associated increase in the oxidative stress in various in vitro and in vivo models of glucolipotoxicity and diabetes. Such conditions promote the activation of Rac1 (and Rap1) in the islet β-cell. After activation of Rac1, the cytosolic core complex of NOX (i.e., p67^phox/p47^phox/p40^phox/ Rac1-GTP) translocates to the membrane to associate with the gp91^phox, p22^phox, and Rap1 for the holoenzyme assembly and functional activation of NOX.