Potential roles of PP2A-Rac1 signaling axis in pancreatic β-cell dysfunction under metabolic stress: Progress and Promise

Abstract

Recent estimates by the International Diabetes Federation suggest that the incidence of diabetes soared to an all-time high of 463 million in 2019, and the federation predicts that by 2045 the number of individuals afflicted with this disease will increase to 700 million. Therefore, efforts to understand the pathophysiology of diabetes are critical for moving toward the development of novel therapeutic strategies for this disease. Several contributors (oxidative stress, endoplasmic reticulum stress and others) have been proposed for the onset of metabolic dysfunction and demise of the islet β-cell leading to the pathogenesis of diabetes. Existing experimental evidence revealed sustained activation of PP2A and Rac1 in pancreatic β-cells exposed to metabolic stress (diabetogenic) conditions. Evidence in a variety of cell types implicates modulatory roles for specific signaling proteins (α4, SET, nm23-H1, Pak1) in the functional regulation of PP2A and Rac1. In this Commentary, I overviewed potential cross-talk between PP2A and Rac1 signaling modules in the onset of metabolic dysregulation of the islet β-cell leading to impaired glucose-stimulated insulin secretion (GSIS), loss of β-cell mass and the onset of diabetes. Potential knowledge gaps and future directions in this fertile area of islet biology are also highlighted. It is hoped that this Commentary will provide a basis for future studies toward a better understanding of roles of PP2A-Rac1 signaling module in pancreatic β-cell dysfunction, and identification of therapeutic targets for the treatment of islet β-cell dysfunction in diabetes.

Graphical abstract

Potential cross talk between PP2A and Rac1 signaling modules leading to dysfunction of the islet β-cell under metabolic stress conditions.

1. Introduction

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is mediated largely via the generation of soluble second messengers, including cAMP, ATP, and hydrolytic products of phospholipases (lyso-phospholipids and inositol phosphates; refs. ^1–3). In addition to ATP, published evidence affirms the contributory roles for GTP in physiological insulin secretion (4). Although putative molecular and cellular mechanisms underlying the modulatory role(s) of GTP in islet β-cell function remain elusive, available evidence indicates that they might involve activation of one (or more) GTP binding proteins (referred to as G proteins, hereinafter; ^4–6). Two major groups of G proteins, namely the heterotrimeric (G_i, G_s, G_z) and small G proteins (Arf6, Cdc42, Rap1 and Rac1) have been identified in β-cells. A wide variety of regulatory factors/proteins have also been identified in the islet β-cell that govern regulation of G proteins, which cycle between their inactive (GDP-bound) and active (GTP-bound) conformations (4–6). Published evidence from numerous laboratories provided compelling evidence implicating roles of G proteins (and their regulatory factors/proteins) in islet function including GSIS. The reader is referred to recent reviews for advances in this field of islet biology (4–6). In addition to G protein-mediated regulatory control of islet functions, earlier studies have demonstrated localization of several protein kinases in normal rat islets as well as clonal β-cells; these include Ca²⁺-, Ca²⁺/calmodulin-, cAMP-, and phospholipid-dependent protein kinases (7–9). The phosphorylation status of proteins is tightly regulated by the balance of the activities of protein kinases and protein phosphatases. Even though numerous earlier studies were focused on the identification and characterization of protein kinases in islets, relatively less is understood about the localization and regulation of protein phosphatases (e.g., protein phosphatase 2A; PP2A) in pancreatic β-cells. Available evidence on identification, characterization and regulatory functions of protein phosphatases in the pancreatic islet β-cell are reviewed in (10).

In the context of islet dysfunction and the pathology of T2DM, recent estimates by the International Diabetes Federation suggest that the incidence of diabetes soared to an all-time high of 463 million in 2019 compared to 382 million in 2013 and 371 million in 2012. The federation predicts that by 2045 the number of individuals afflicted with this disease will increase to 700 million. According to the Centers for Disease Control, 34.2 million Americans have diabetes, in addition to more than 88 million Americans with prediabetes. It is estimated that the absolute global economic burden will escalate from U.S. $1.3 trillion in 2015 to $2.5 trillion in 2030, which represents a staggering increase in costs as a share of global GDP from 1.8% in 2015 to 2.2% in 2030. Therefore, efforts to understand the pathophysiology of diabetes are critical for moving toward the development of novel therapeutic strategies for this disease.

Several mechanisms/models have been put forth in recent years for the onset of metabolic dysfunction and demise of the islet β-cell leading to the pathogenesis of diabetes (11–15). It is widely accepted that the onset of type 2 diabetes (T2DM) may, in part, be due to an intricate interplay between the genetic expression of the disease and a host of environmental factors including increased metabolic (e.g., oxidative and endoplasmic reticulum) stress under the duress of chronic exposure to elevated glucose (glucotoxicity), saturated fatty acids (lipotoxicity), glucose plus saturated fatty acids (glucolipotoxicity), pro-inflammatory cytokines and biologically active sphingolipids (ceramide; CER; ^11–16). In the context of the current Commentary, published evidence, in in vitro and in vivo models of metabolic stress and diabetes, implicates sustained activation of specific signaling pathways, including G proteins (e.g., Rac1) and protein phosphatases (e.g., PP2A) as potential contributors to the onset of pancreatic β-cell dysmetabolism and demise under the duress of metabolic stress and diabetes (17–20).

The overall objective of this Commentary, therefore, is to propose a Working Model that explores potential crosstalk between the PP2A-Rac1 signaling modules as regulators of islet β-cell dysfunction and demise in diabetes. For brevity, this Commentary is divided into four sections. In the first, I will overview known roles of Rac1 signaling pathway in islet function and dysfunction. The second section will highlight regulatory roles of PP2A signaling axis in islet function and dysregulation. The third section will focus on potential crosstalk between Rac1 and PP2A pathways in islet β-cell dysfunction, with special emphasis on key regulatory proteins that appear to bridge the two signaling modules in the cascade of events leading to β-cell dysfunction and demise in diabetes. The last section will identify potential knowledge gaps in the field and avenues for future research.

2. Rac1 in islet function and dysfunction

Didsbury and coworkers first isolated two cDNAs encoding proteins with ∼92% homology, which were designated as Rac1 and Rac2. They suggested that Rac proteins uniquely differed in their tissue distribution and biological functions, including secretory functions (21). A recent (April 2020) PubMed search for Rac1 yielded more than 8,500 publications, to substantiate the proposal that it is involved in the regulation of a variety of cellular functions, including cytoskeletal organization and remodeling, superoxide generation and secretion. The reader is referred to recent reviews on Rac1 for its roles in the regulation of cellular function (17,22,23).

2.1. Regulatory mechanisms underlying glucose-induced, Rac1-mediated insulin secretion

Pharmacological and molecular biological investigations from multiple laboratories have suggested key roles for Rac1 in GSIS from clonal β-cells and rodent islets. These studies have demonstrated that Rac1 is activated by physiological concentrations of glucose within 15–20 min of exposure, leading to its translocation from the cytosolic fraction to the membrane fraction for regulation of various effector proteins, which are requisite for insulin secretion to occur (4–6,17). Follow-up investigations have further documented that glucose-induced activation of Rac1 is necessary for cytoskeletal remodeling for optimal translocation and docking of insulin granules with the plasma membrane for exocytotic secretion of insulin (4–6). Taken together, the findings reviewed above provide support to the proposal that Rac1 plays positive modulatory roles in islet function including GSIS.

As depicted in Figure 1, Rac1 functions in pancreatic β-cells are modulated by a variety of mechanisms. These include regulation by: (i) regulatory factors such as guanine nucleotide exchange factors (GEFs) and GDP-dissociation inhibitors (GDIs); (ii) post-translational modifications; and (iii) Rac1 interaction partners. Several lines of evidence in multiple cell types, including the islet β-cell, support the formulation that subcellular distribution (soluble vs. membranous) of Rac1 also dictates its roles in the regulation of cell function. For brevity, regulation of Rac1 function via these mechanisms in discussed briefly below.

Figure 1: Potential mechanisms that underlie functional (in)activation of Rac1 in pancreatic β-cells.

Published evidence suggests that Rac1 is regulated by a variety of proteins/factors, including GEFs, GDIs. In addition, using proteomics approaches, we have identified novel interaction partners for Rac1 in pancreatic β-cells under basal and hyperglycemic conditions. Interestingly, association of some of these proteins/factors with Rac1 was significantly increased under high glucose-treatment conditions. Furthermore, Rac1 has been shown to undergo a variety of post-translational modifications, including prenylation, palmitoylation, phosphorylation, ubiquitination and SUMOylation. As discussed in the text, prenylation is the most studied for Rac1 in the islet β-cell. Potential regulation of its function by other post-translational modification steps need to be assessed in the context of its role in the pathogenesis of islet function under metabolic stress. Lastly, Rac1, which is primarily cytosolic and translocates to the plasma membrane fraction, following cellular activation for optimal regulation of its effectors. We have recently demonstrated that Rac1 associates with the nuclear fraction (in an active, but unprenylated configuration) under conditions of metabolic stress. We proposed that it might regulate apoptotic factors in the nuclear fraction to elicit regulatory effects leading to cell demise (see text for additional details).

Regulation of Rac1 functions by GDIs and GEFs:

Several studies have described the expression and regulation of GDIs in the pancreatic β-cell function (4–6). These regulatory proteins are known to: (i) prevent dissociation of GDP from candidate G proteins; (ii) inhibit the intrinsic GTPase-activating protein-catalyzed hydrolysis of GTP; (iii) sequester back specific G proteins from their membranous sites, thereby inhibiting their interaction with their respective effector proteins; and (iv) regulate spatial determination in the actin cytoskeletal control. In this context, recent studies have identified at least three GDIs (GDIα, GDIβ and GDIγ) in clonal β-cells, normal rodent islets and human islets (6). GDIα has been implicated in the functional regulation of Rac1 and Cdc42 in the cascade of events leading to GSIS (4–6). Studies from our laboratory have also suggested roles of GDIβ in islet function (6). In addition to GDIs, expression of a variety of GEFs was also reported in the islet β-cell. These factors promote exchange of GDP for GTP on G proteins. At least two GEFS, namely Tiam1 and Vav2 have been shown to regulate glucose-mediated Rac1 functions in clonal β-cells and rodent islets. Specific inhibitors of Tiam1 (NSC23766) and Vav2 (Ehop-016) have been shown to attenuate glucose-induced Rac1 activation and insulin secretion (6,15). It should be noted that the functional activation of GDIs and GEFs is under the regulatory control of post-translational modifications, such as phosphorylation-dephosphorylation, which, in turn, appear to regulate their biological functions. The reader is referred to recent reviews on regulatory control of insulin secretion by various GDIs and GEFs (4–6).

Regulation of Rac1 functions by post-translational modifications:

Most of small G proteins and the γ-subunits of trimeric G proteins undergo post-translational modification at their unique C-terminal sequence (the CAAX motif). The first modification involves attachment of either 15-carbon (farnesylation) or a 20-carbon (geranylgeranylation) derivative of mevalonic acid at the C-terminal cysteine via a thioester linkage. These steps are catalyzed by farnesyltransferase (FTase) and geranylgeranyl-transferases (GGTases), respectively. Ras and nuclear lamins are examples of farnesylated proteins. Cdc42 and Rac1 undergo geranylgeranylation. Following prenylation, the three terminal amino acids after the prenylated cysteine are cleaved by a protease, resulting in the exposure of the carboxylate anion of the prenylated cysteine residue. This site is then subjected to carboxylmethylation (CML) by a prenylcysteine methyltransferase, which neutralizes the carboxylate anion, thus making the candidate G proteins more hydrophobic resulting in their translocation to the membranous sites for interaction with their effector proteins (4,6,15). Using a variety of experimental approaches, we have demonstrated that prenylation and CML of Rac1 are necessary for GSIS to occur (4,6,15). In addition, Rac1 has been shown to undergo other modifications including palmitoylation, phosphorylation, adenylation, ubiquitination and SUMOylating (22). Potential roles of these modification steps in the overall function of the islet β-cell, including glucose-induced Rac1 activation and insulin secretion remain an understudied area of investigation.

Regulation of Rac1 functions by virtue of its subcellular localization:

Rac1 is localized predominantly in the cytosolic fraction as a complex with GDI in its GDP-bound, inactive configuration. Cell stimulation by appropriate signals leads to its dissociation from the GDI followed by GEF-mediated exchange of GTP for GDP (activation), and translocation to the membrane fraction (4,6). In general, membrane association of Rac1 is a hallmark signaling step for propagation of cellular events mediated by active Rac1. Growth factors have been shown to promote nuclear translocation of Rac1 in other cell types (22). The cytoplasmic-nuclear shuttling of Rac1 appears to be mediated via nuclear export sequence (NES) and nuclear localization sequence (NLS) motifs (6,22). Despite recent advances in this field in other cell types, these aspects of Rac1 biology have not been explored in the islet to date. However, we recently reported that Rac1 associates with the nuclear compartment in clonal β-cells, normal rat islets and human islets following exposure to hyperglycemic conditions. Using non-prenylatable, constitutively active mutants of Rac1 we have demonstrated that prenylation of Rac1 is not necessary for nuclear translocation of Rac1 (24). Additional studies are needed to further understand potential roles and significance of this signaling step in high glucose-induced metabolic dysfunction of the islet β-cell.

Regulation of Rac1 functions by its interacting partners:

In addition to the above regulatory mechanisms, functional activation of G proteins has been shown to be under the precise control of interacting proteins/factors, which might associate with Rac1 under specific experimental conditions (4–6, 23). Using proteomics approaches we have recently reported novel interacting partners for Rac1 in pancreatic β-cells under basal as well as hyperglycemic conditions (25; see below for additional details). These include Rho GTPases, cytoskeletal proteins, GEFs, and nuclear proteins etc. Potential roles of these proteins in the regulation of Rac1, specifically in the regulatory control of Rac1-PP2A signaling module, are highlighted in the following sections.

2.2. Roles of Rac1 in islet β-cell dysfunction: Lessons learnt from cell culture and animal models

Several lines of evidence support the formulation that functional inactivation of Rac1 leads to impaired insulin secretion. For example, Li and coworkers have demonstrated that overexpression of a dominant negative Rac1 mutant (N17Rac1) markedly attenuated glucose- and forskolin-, but not KCl-induced insulin secretion in INS-1 cells (26). N17Rac1 also induced significant morphological changes including the disappearance of F-actin. It was concluded that Rac1 activation may be necessary for the recruitment of secretory granules through actin cytoskeletal reorganization and remodeling (26). Along these lines, we reported that siRNA-mediated knockdown of Rac1 in INS 832/13 cells significantly impaired GSIS, without affecting basal secretion, suggesting positive modulatory roles for Rac1 in insulin secretion (17).

Additional support for a regulatory role for Rac1 in insulin secretion emerged from the Rac1 knockout animal models (27). For example, Rac1-null [βRac1^−/−] mice exhibited impaired glucose tolerance and hypoinsulinemia. Glucose-, but not KCl-induced insulin secretion, was markedly attenuated in islets from the Rac1 null mice. The β-cell mass or islet density remained unaltered in these mice. Based on these findings, it was concluded that Rac1 plays a key regulatory role in insulin secretion primarily through regulating cytoskeletal reorganization (27). In another related study, Greiner et al demonstrated that Rac1 null mice exhibited marked alterations in islet morphogenesis. The β-cell spreading and migration were significantly reduced in this model. Cell to cell contact of D-cadherin was also increased in Rac1-null mice. Actin remodeling and cell spreading induced by betacellulin was also not demonstrable in the transgenic islets. This is the first study to suggest a role for Rac1 in islet morphogenesis (28). Using β-cell-specific inducible cilia knockout animal models, Volta and coworkers have demonstrated novel roles for endosomal Ephrin type A (Epha)-processing in β-cell cilia facilitated glucose homeostasis. Notably, these studies have implicated Rac1 and Tiam1 (a GEF for Rac1) in these signaling steps (29).

More recent studies have defined roles of Rac1 signaling axis in the onset of islet β-cell dysregulation and demise under conditions of chronic exposure to metabolic stress (glucolipotoxicity, exposure to proinflammatory cytokines and sphingolipids). Data from these investigations indicated paradoxical (sustained) activation of Rac1, which appears to play critical regulatory roles in the induction of islet dysfunction by accelerating oxidative and stress kinase pathways (4,6,17). Key findings from these investigations were also confirmed in islets from T2DM human donors and animal models of T2DM. These findings are highlighted in recent reviews (4,6,17,30). Lastly, compatible with our in vitro observations suggesting key regulatory roles for Rac1 in cytokine-induced metabolic dysregulation of the islet β-cell (4,6,15), we reported prevention of the onset of spontaneous diabetes in the NOD mouse model following treatment with a Rac1 inhibitor (31). A significant reduction in Rac1 activation was also noted in islets derived from the Rac1 inhibitor-treated mice. Based on these observations we proposed that Rac1 signaling pathway plays key roles in the pathogenesis of diabetes in this animal model (31). In conclusion, it can be surmised that Rac1 plays novel roles in the physiological insulin secretion in the pancreatic β-cell. It also appears that metabolic stress and diabetogenic conditions promote sustained activation of Rac1 culminating in the activation of signaling pathways to result in dysfunction and demise of the islet β-cell (15,17).

3. PP2A in islet function and dysfunction

As stated above, GSIS in pancreatic β-cells involves the generation of second messengers, such as cAMP, and lipid hydrolytic products of PLases , which, in turn, regulate a variety of protein kinases leading to phosphorylation of signaling proteins to promote insulin secretion. Despite such compelling evidence on regulatory roles of protein phosphorylation in islet function, relatively limited information is available on the localization of phosphoprotein phosphatases (e.g., PP2A) and their contributory roles in β-cell function (7,10,32). Available evidence on PP2A-mediated regulation of islet function in health and diabetes is briefly highlighted below.

The PP2A family of phosphatases contribute to a variety of cellular events including proliferation, survival and apoptosis. The PP2A accounts for ∼1% of total cellular protein and ∼80% of total serine/threonine phosphatases, thus representing a major class of phosphatases in mammalian cells. The PP2A heterodimer is comprised of a scaffolding A subunit (∼65 kDa) and the catalytic C subunit (∼36 kDa; PP2Ac). At least two different families of A (α/β) and C (α/β) have been described. The A/C dimer interacts with the regulatory B subunit to complete the PP2A holoenzyme assembly. Binding of B subunit to the A/C heterodimer not only stabilizes the holoenzyme, but also influences substrate specificity and/or subcellular localization of a given PP2A holoenzyme (32–25). While the A and C subunits are ubiquitously expressed, B subunits are expressed in a tissue-specific manner during cell development. Unlike the B subunits, PP2Ac is highly conserved, with >70% sequence homology among different species. The six C-terminal amino acid residues (TPDYFL) are conserved in all known PP2Ac subunits, and the three C-terminal residues (YFL) are also conserved in other protein serine/threonine phosphatases, such as protein phosphatase 4 (PP4) and protein phosphatase6 (PP6), thus implicating a role for YFL motif in the catalytic function and regulation of these enzymes (32–35).

A large body of experimental evidence indicates that PP2Ac is subjected to post-translational modifications, such as phosphorylation on serine or tyrosine residues or CML on a C-terminal Leu-309 residue (32). In the context of functional consequences of CML of PP2Ac, Favre and coworkers (36) have suggested that CML of PP2Ac results in modest, but significant increase in PP2A activity in intact human breast cancer cells. Studies from our laboratory in pancreatic β-cells further confirmed functional activation of PP2A following the CML of PP2A (37). Altogether, these findings suggested an important functional relationship between CML of PP2Ac and the catalytic activity of PP2A.

3.1. PP2A in insulin secretion

Earlier observations from our laboratory provided the first evidence in INS-1 832/13 β-cells, normal rodent islets and human islets to suggest that the C-terminal Leu-309 of PP2Ac undergoes reversible CML resulting in increased catalytic function of PP2A (37). OKA (but not 1-nor-okadaone, its inactive analog) inhibited the CML of PP2Ac and PP2A activity in normal rat islets and clonal β-cells as well as in intact rat islets. Glucose elicited such inhibitory effects in intact β-cells. Mannoheptulose, a known inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac (37). Based on these data, we proposed that alterations in the intracellular concentrations of specific intermediates of glucose metabolism leads to inhibition of the holoenzyme assembly and the catalytic function of PP2A. This, in turn, retains putative signaling proteins in their phosphorylated state, which may be necessary for insulin secretion (10,32). Together, this model suggests dual regulatory effects of glucose (and its metabolites) on the phosphorylation status of candidate proteins, namely stimulation and inhibition of protein kinases and phosphatases, respectively (8,10,32). In addition to the above regulatory mechanisms, PP2A is subjected to additional (multifactorial) regulation in the islet (Figure 2). While potential regulatory effects of some of these agents, including insulin secretagogues, are understood to a large degree, very little is known with regard to control mechanisms underlying individual holoenzyme assemblies that appear to dictate the substrate specificity, subcellular localization of PP2A and its sensitivity to various modulators. It may be reasonable to speculate that different holoenzyme assemblies might dictate the sensitivity of PP2A to various modulators such as glucose (or its metabolites), amino acids, ceramide (CER), etc. The reader is referred to reviews in the field further affirming roles of protein phosphatases, including PP2A in islet function (10,16,32).

Figure 2: Multifactorial regulation of PP2A-like enzymes in the islet β-cell.

We proposed earlier that PP2A plays a central role in the islet cell survival and demise. As depicted in the figure, functional regulation of PP2A is under the control of many modulators. Note that most, but not all, of these modulators are known to regulate PP2A function via their effects (stimulation or inhibition based on conditions) on the CML of PP2Ac. Chronic exposure to glucose has been shown to increase the CML of PP2Ac leading to its activation. CER has been shown to activate PP2A without affecting the CML of PP2Ac. Effects of inositol phosphates and sulfonylureas on CML remains to be studied. Metabolites of glucose have been shown to inhibit CML of PP2Ac leading to its inactivation. Relevant to this article are effects of two regulatory proteins, namely SET and α4 of PP2A function. Overall, the functional regulation of PP2A is complex due to its subunit composition, subcellular distributions as well as properties of its regulatory proteins/factors (see text for additional details).

3.2. PP2A in β-cell dysfunction: Evidence from in vitro and in vivo models of metabolic stress and diabetes

Recent studies by Arora and coworkers have reported sustained activation of PP2A in human islets, rat islets and insulin-secreting INS-1 832/13 cells under the duress of chronic hyperglycemic conditions (19). siRNA-mediated knockdown of PP2Ac significantly attenuated glucose-induced activation of PP2A. Exposure of these cells to high glucose significantly increased the CML of PP2Ac, thus suggesting a novel role for this posttranslational modification in sustained activation of PP2A under metabolic stress conditions. siRNA-mediated depletion of the cytosolic leucine carboxymethyl transferase 1 (LCMT1), which promotes CML of PP2Ac, significantly attenuated PP2A activation under high glucose conditions. Interestingly, glucotoxic conditions significantly promoted the expression of B55α, a regulatory subunit of PP2A, which has been implicated in islet dysfunction under conditions of oxidative stress and diabetes. Lastly, hyperglycemic conditions significantly promoted PP2A activity in INS-1 832/13 cells (∼3-fold), rodent islets (∼1.8-fold) and human islets (∼2.2-fold; ¹⁹). Sustained activation of PP2A was also seen in islets derived from the prediabetic (7 weeks; ∼1.4 fold) and diabetic (13 weeks; ∼2-fold) Zucker diabetic rats (38). Based on these findings, we concluded that exposure of the islet β-cell to diabetogenic conditions leads to accelerated PP2A signaling pathway, culminating in the loss of GSIS and metabolic dysfunction of the islet β-cell (18,19,38).

Germane to the above observations are studies by Yan and coworkers (20), which established a link between PP2A holoenzyme containing the B55α regulatory subunit, with nuclear import of FOXO1 in pancreatic islet β-cells under oxidative stress. Using a variety of experimental approaches, they reported association of FOXO1 with a PP2A holoenzyme composed of AB55αC subunits. They documented that knockdown of B55α in INS-1 cells reduced FOXO1 dephosphorylation, inhibited FOXO1 nuclear translocation, and attenuated oxidative stress-induced cell death. These findings (i.e., increased expression of both B55α and nuclear FOXO1) were replicated in islets derived from db/db mouse, an animal model of T2DM (20). In a recent study, Chen et al (39) investigated potential beneficial effects of S-Equol, a potential anti-diabetic agent, on insulin secretory defects in in vitro and in vivo models of glucotoxicity and diabetes. Specifically, they investigated the ability of Chrebp/Txnip signaling module in promoting insulin secretory defects and subsequent dysfunction and demise of the islet β-cell under metabolic stress conditions. Data from these investigations provided compelling evidence to suggest that S-Equanol treatment prevents loss in insulin secretion mediated via accelerated Chrebp/Txnip signaling pathway by activating protein kinase A and inhibiting PP2A activities (39).

Based on the above discussion, it may be concluded that increased intracellular oxidative stress plays key regulatory roles in the hyperactivation of PP2A in pancreatic β-cells. Chronic exposure of these cells to metabolic stress conditions (e.g., glucotoxicity, lipotoxicity, glucolipotoxicity, exposure to proinflammatory cytokines and biologically active sphingolipids) leads to increased superoxide-derived oxidative stress, which may, in part, be due to activation of NADPH oxidases endogenous to the β-cell. The superoxides, in turn, might elicit stimulatory effects on the CML of PP2Ac, thus promoting conditions conducible for PP2A holoenzyme assembly and functional activation of PP2A. It is likely that increased intracellular oxidative stress promotes expression of specific B subunits that are necessary of the PP2A holoenzyme (18–20,38). Based on the existing data on the hyperactivation of PP2A under metabolic stress conditions, we propose that hyperactivation of PP2A results in dephosphorylation and inactivation of pro-survival proteins (e.g., BCl₂, Akt, ERK1/2, FOXO1) and the onset of metabolic dysfunction and cell demise.

4. Identification of protein interaction partners (PIPs) of Rac1 and PP2Ac in β-cells

Proteomics-based investigations in multiple cell types exposed to normal and/or a variety of pathological conditions have been utilized to detect potential protein interacting partners (PIPs) for various candidate proteins. From a methodological standpoint, co-immunoprecipitation followed by mass spectrometry-based proteomics are widely accepted as a powerful experimental approaches for exploration of PIPs. However, the majority of these investigations have employed protein overexpression and/or epitope-tagged bait proteins. To refine this process further, we utilized a straightforward, label-free approach combining co-immunoprecipitation with HPLC-ESI-MS/MS, without the use of protein overexpression or protein tags, to investigate changes in the abundance of endogenous proteins associated with a bait protein (40,41). We applied this proteomics approach to identify PIPs of Rac1 or PP2Ac in pancreatic islet β-cells exposed to basal and glucotoxic conditions. We have been able to validate our proteomics findings via Western blotting. Indeed, data accrued from these investigations provided fresh insights into potential candidate proteins that could be involved in bridging the Rac1 and PP2A signaling pathways in eliciting positive (insulin secretion) and negative (metabolic dysregulation) modulatory effects on islet β-cells. Salient findings are briefly highlighted below.

Using aforestated approaches we identified 324 PIPs of Rac1 in pancreatic β-cells, out of which 27 showed a significant increase in the interaction with Rac1 under hyperglycemic conditions (25). Some of the significantly enriched pathways included members of the actin cytoskeleton signaling, Rho GDI signaling, calcium signaling, Pak signaling and Rho GTPase-mediated signaling pathways. Germane to the current Commentary are our findings of identification of ARHGEF6 (α-PIX), ARHGEF7 (β-PIX) and Pak1 as interaction partners for Rac1. Furthermore, in support of proteomics data, we reported a significant increase in association between β-PIX and Rac1 in pancreatic β-cells exposed to hyperglycemic conditions (25). These findings, in addition to the existing evidence that β-PIX, a known GEF for Rac1/Cdc42, also associates with α4 (42), raise an interesting possibility that α4/β-PIX module could contribute to aberrant Rac1/Cdc42 activation in the islet β-cell under metabolic stress (see below for additional discussion). Lastly, our proteomics studies identified importin (also known as karyopherin) as a glucose-responsive interacting partner for Rac1 in pancreatic β-cells (25). Published evidence suggests that importin mediates nuclear transport of SET, a known endogenous inhibitor of PP2A (43). Furthermore, SET has been implicated in regulating Rac1 activation pathway (see the following sections for additional discussion).

Using above approaches, we also determined the identity of glucose-responsive PIPs of PP2Ac in pancreatic β-cells exposed to basal and have identified a total of 514 interaction partners for PP2Ac, out of which observed that 13 protein interaction partners of PP2Ac with a fold change greater than 5 in response to high glucose treatment (44). Some of these PIPs of PP2Ac included Rac1 and a variety of proteins involved in protein sorting and trafficking (GGA2 and SRP72) and vesicle trafficking (VPS52, VPS37A, Rab10, Rab5C etc.). Furthermore, we identified immunoglobulin-binding protein (Igbp1) as one of the interaction partners of PP2Ac. Igbp1 is also referred to as α4 (or Tap42 in yeast), and has been shown to serve the function of a non-canonical adaptor subunit of PP2A (45; one of the foci of this Commentary ). Expression of α4 was confirmed immunologically in INS-1 832/13 cells, normal rodent islets and human islets (38). In addition to forming complexes with the A and B subunits, PP2Ac has been shown to interact with other adaptor regulatory subunits, including α4. It is noteworthy that, in these studies, we observed a significant increase (2.3-fold) in the interaction between PP2Ac and B55α subunit in pancreatic β-cells exposed to hyperglycemic conditions (44). As mentioned above, studies of Yan and associates implicated B55α subunit in the onset of metabolic dysfunction of the islet β-cell. They also observed increased expression of B55α subunits in islets derived from db/db mouse, a model for T2DM (20). Compatible with these observations, we reported a significant increase (1.5-fold) in the expression of B55α subunit in pancreatic β-cells exposed to hyperglycemic conditions (19). Taken together, our proteomics studies not only identified novel interaction partners for PP2Ac and Rac1 in pancreatic β-cells, they also provided fresh insights for future investigations to further assess potential cross-talk between Rac1 and PP2Ac signaling pathways in the cascade of events leading to metabolic dysregulation of the islet β-cell under diabetogenic conditions.

5. Proposed roles and key players of Rac1-PP2A signaling axis in the onset of beta-cell dysfunction under diabetogenic conditions

Based on the above discussion, several signaling proteins emerge as contributors to metabolic dysregulation of the islet β-cell by bridging and accelerating the PP2A and Rac1 signaling pathways (Figure 3). Potential roles of these proteins/factors in the pathology of islet defects are briefly highlighted below.

Figure 3: Potential contributors to accelerated signaling of PP2A-Rac1 module in metabolic stress induced dysfunction of the islet β-cell.

The proposed model identifies at least two major contributors, namely α4 and SET, that mediate functional activation of PP2A and Rac1 signaling in pancreatic β-cells subjected to metabolic stress conditions. α4 contributes to PP2A activation via multiple pathways including prevention of PP2Ac degradation, CML of PP2Ac and holoenzyme assembly. Mechanisms underlying its ability to activate Rac1 still remain to be elucidated. SET is inhibitory to PP2A function. Therefore, it is likely that metabolic stress promotes its dissociation from PP2Ac from the SET-PP2Ac complex by preventing its requisite post-translational modification steps, including phosphorylation. SET could contribute to Rac1 activation via its nuclear-cytoplasmic shuttling, and/or yet unidentified mechanism(s). These need to be assessed in the islet β-cell. Lastly, in addition to SET and α4, stress-induced dysfunction might require the interplay between other proteins/factors including nm-23 and Pak1. Again, a methodical investigation of these individual signaling steps might be necessary to assign roles for these molecules in the pathogenesis of β-cell dysfunction in diabetes (see text for additional details).

5.1. IGBP-1/α4

As stated above, in addition to forming complexes with the A and B subunits in the formation of PP2A holoenzyme, PP2Ac has been shown to interact with other adaptor regulatory subunits, including α4, which has been implicated in PP2A biogenesis, stability and activation (33,34,45,46). Furthermore, α4 has been shown to play key regulatory roles in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. Studies by McConnell and coworkers have identified a novel ubiquitin-interacting motif (UIM) within α4 (amino acid residues 46–60) and suggested that α4 is monoubiquitinated. Deletion of the UIM within α4 leads to its association with polyubiquitinated proteins. Interestingly, the WT α4, but not an α4 UIM deletion mutant, suppressed PP2Ac polyubiquitination. Together, these observations suggested direct regulation of PP2Ac polyubiquitination by a novel UIM within α4 (46). Besides PP2A, α4 interacts with other phosphatases including PP4 and PP6 (47). Bimolecular fluorescence complementation analysis studies by Mo and coworkers have provided compelling evidence to indicate that α4 also promotes formation of the AC core dimer (48). Published evidence, albeit limited, appears to suggest direct regulatory roles of α4 in promoting the CML of PP2Ac, and functional activation of PP2A. For example, Nien and coworkers (49) have demonstrated that transient transfection of α4 into COS-1 cells markedly increased the CML of PP2Ac (more than 2-fold), which is accompanied by a significant increase in the catalytic activity of PP2A (∼ 2.5-fold). While these observations raise an interesting possibility that α4 mediates CML of PP2Ac and catalytic activation of PP2A, additional studies are needed to further validate this postulation. Based on above discussion it can be concluded that α4 plays key regulatory roles in PP2A function, including stability of subunits and catalytic activation.

α4 has also been shown to play roles in the activation of Rac1. For example, in 2007, Kong et al. have reported novel modulatory roles of α4 in cell spreading and migration (50). They reported a tight correlation between α4 expression and the levels of active (GTP-bound) Rac1 in a T-cell-specific α4 transgenic mouse model. Significantly lower levels of active Rac1 were detected in α4-deficient cells. In further support of the role of α4-Rac1 axis in cell spreading, these researchers observed a significant rescue of loss in cell spreading in α4-depleted cells following overexpression of a constitutively active mutant of Rac1. Lastly, inhibition of Rac1 significantly attenuated α4-mediated cell spreading and migration; these data implicate novel roles for α4-Rac1 signaling module in cell spreading and migration (50).

Despite the available evidence in other cell types suggesting key regulatory roles of α4 in the modulation of PP2A-Rac1 signaling modules, putative roles of α4 in the islet β-cell function remain elusive. Our immunological evidence in clonal β-cells, normal rat islets and human islets suggested that α4 is expressed in a variety of insulin-secreting cells (38). Our proteomics investigations indicated that α4 is an interacting partner of PP2Ac in pancreatic β-cells (44). Lastly, siRNA-mediated knockdown of endogenous expression of α4 resulted in significant inhibition (∼60%) of high glucose-induced PP2A activity in INS-1 832/13 cells suggesting key regulatory roles for this scaffolding protein in the sustained activation of PP2A under glucotoxic conditions (38). Further studies are needed to define putative signaling mechanisms underlying α4-mediated regulation of PP2A activation including its roles in facilitating post-translational modifications of PP2Ac and the holoenzyme assembly of PP2A. Also, it will be important to further define the roles of PP2A-α4-Rac1 signaling module in the islet β-cell function in health and diabetes (see Working Model; Figure 4 below).

Figure 4: A working model depicting potential signaling steps involved in PP2A-Rac1 signaling modules leading to metabolic dysfunction of the islet β-cell under metabolic stress.

Metabolic stress induces the CML of PP2Ac, recruitment of regulatory B subunits, holoenzyme assembly and activation of PP2A leading to dephosphorylation and inactivation of key survival proteins. In addition, metabolic stress conditions also promote activation of Rac1 leading to stimulation of NADPH oxidases, and associated increase in the intracellular oxidative stress. This, in turn, promotes activation of stress kinases (p38 MAPK and JNK1/2) leading to mitochondrial dysregulation culminating in loss of mitochondrial membrane permeability pore transition and release of cytochrome C into the cytosolic compartment and associated increase in caspase-3 activity. Earlier studies from our laboratory have documented significant alterations in nuclear compartment in β-cells following exposure to metabolic stress, including caspase-3 mediated degradation of nuclear lamin B and loss of nuclear integrity. Furthermore, we have recently reported that sustained activation of Rac1 under metabolic stress conditions leads to inappropriate movement of Rac1 to the nuclear fraction in clonal β-cells, normal rodent islets and human islets to result in activation of apoptotic factors, including p53. It is proposed that α4 and SET play significant roles in promoting PP2A/Rac1 signaling modules leading to islet β-cell dysfunction under these conditions. The author realizes that this model might be construed as simplified (and relatively linear) since published evidence implicates several competing mechanisms that might underlie β-cell dysfunction including inflammation, lipotoxicity, ER stress and accumulation of amyloid toxic oligomers. However, at least at the outset, it might be worthwhile to test this model to precisely define the molecular/cellular mechanisms involved in Rac1-PP2A signaling axis in islet β-cell dysfunction under metabolic stress. Successful outcome from these studies might pave way toward identification of key targets involved in islet β-cell dysfunction under diabetogenic conditions (see text for additional details).

5.2. SET proteins

SET (also known as I2PP2A or TAF-1β) is a well-studied oncoprotein and an endogenous inhibitor of PP2A. It directly binds to PP2Ac, thus eliciting its inhibitory effects on PP2A. Mechanistically, SET has been shown to inhibits PP2A in cell-free assays without eliciting significant inhibitory effects on other phosphatases. Interestingly, however, in addition to its roles as an inhibitor of PP2A, SET has been implicated in the regulation of cellular functions, including gene expression, via promoting alterations in histone acetylation (51–53). Yu et al (54) have reported casein kinase II-mediated phosphorylation of SET at Ser-9, which appears to be critical for its subcellular distribution. Irie and associates have identified protein kinase D2 as the putative kinase that mediates the phosphorylation at Ser171 of SET to diminish its biological activity to inhibit PP2A (55).

In addition to its roles in the regulation of PP2A function and gene expression, studies by Klooster and associates (56) have demonstrated that SET plays novel roles in cell migration via regulation of a host of signaling steps, including activation of and subcellular targeting of Rac1. Briefly, they demonstrated that: (i) the hypervariable domain of Rac1 interacts with the nucleosome assembly protein domain of SET; (ii) SET translocates to plasma membrane upon Rac1 activation; (iii) serine phosphorylation of SET regulates Rac1 binding and membrane targeting, and cell migration. Based on this compelling experimental evidence these investigators proposed a model, which suggests that dephosphorylated SET is retained in the nuclear compartment as a dimer. Phosphorylation of SET at Ser-9 results in dissociation of the dimer culminating in distribution of SET in the nuclear as well as the cytosolic compartments, thus favoring conditions conducive for interaction of Rac1 with SET in the cytosol. They also proposed that following activation of Rac1 by β-PIX, SET translocates to the plasma membrane where active Rac1 promotes activation of downstream signaling kinase-mediated signaling events. Interestingly, it appears that optimal regulation of this signaling axis requires SET-mediated inhibition of PP2A. It is noteworthy that phosphorylation of Ser-9 appears to promote binding of SET to the hypervariable C-terminal domain of Rac1 (57). In a recent investigation, Mody et al (58) assessed the contributory roles of SET (specifically, SET-2 isoform) in epithelial-mesenchymal transition (EMT) of pancreatic cancer. In this model, they proposed that SET-mediated increase in EMT may, in part, be due to increase in the expression of N-cadherin. This postulation confirmed SET-mediated increase in the transcriptional activation of N-cadherin via accelerated Rac1-JNK-c-Jun signaling axis in cells following overexpression of SET. In summary, SET mediates cross talk between Rac1 and PP2A signaling pathways. However, putative roles of SET in promoting/accelerating the PP2A-Rac1 axis in β-cells remain elusive and requires an in-depth investigation.

Published evidence also suggests that SET plays a significant role in cancer progression via inhibiting the tumor suppressor PP2A and the metastasis suppressor nm23-H1. Using COG112, a novel ApoE-based peptide, Switzer and coworkers examined potential cross-talk between SET with PP2A, nm23-H1 and Rac1 (59). Using complementary experimental approaches, they demonstrated that COG112 binds to SET protein leading to disruption of association between PP2Ac and SET culminating in the functional activation of PP2A and inhibition of Akt signaling. They also provided evidence for dissociation of SET-nm23-H1 complex by COG112 following association of this peptide with SET. Lastly, data were also presented to suggest prevention of SET-mediated inhibition of Rac1-mediated cell migration and invasion following binding of COG122 to SET. Altogether, these findings provide compelling evidence for regulation of multiple signaling pathways, namely PP2A, nm-23 H1 and Rac1 by SET oncoprotein. It is likely that SET could exert additional regulatory effects on β-cell function including regulation of nm23-H1 exonuclease. This needs to be validated in the islet β-cell. We have extensively studied nm23 family of exonucleases/histidine kinases in the islet β-cell including their roles in G protein (Rac1) activation and insulin secretion (6). Therefore, nm-23 H1 might represent one of the regulatory proteins that could contribute to PP2A-Rac1 signaling in the islet β-cell. These aspects of β-cell biology are discussed in the following sections. Lastly, our proteomics findings indicated significant increase in the interaction between Rac1 and nuclear importins (karyopherins), which are known to regulate nucleo-cytoplasmic shuttling of SET proteins (25). Based on the evidence in other cell types (and limited information in the islet β-cell), we propose that metabolic stress conditions promote dissociation of SET from PP2Ac and Rac1 thereby relieving its inhibitory effects on these signaling modules. It is likely that metabolic stress conditions promote inhibition of SET functions via phosphorylation of specific amino acid residues. Future studies will address some of these important questions (Figure 4).

5.3. nm23 class of signaling proteins:

As described in the above sections, SET appears to regulate functions of nm23-H1 by preventing its exonuclease functions by directly binding to nm23-H1. Furthermore, COG112 has been shown to relieve the inhibitory effects of SET on nm-23 H1 by directly binding to SET. Several lines of experimental evidence suggest noncanonical activation of small G proteins (Rac1) as well as trimeric G proteins via transfer of high-energy phosphates by nm23 class of proteins (nucleoside diphosphate kinases; NDPKs). This represents a novel mode of regulation of G proteins via a GPCR-independent mechanism (6). Briefly, NDPK catalyzes the trans-phosphorylation of nucleotide diphosphate (e.g., GDP) to nucleotide triphosphates (e.g., GTP). Using a variety of biochemical techniques, we previously characterized NDPK activity in clonal insulin-secreting pancreatic β-cells and primary rodent and human islets. In follow-up investigations, we reported expression of three isoforms of NDPK in the islet β-cell. They are NDPK-A (nm23-H1), which is predominantly cytosolic; NDPK-B (nm-23 H2), which is cytosolic as well as membranous in distribution; and NDPK-D (nm-23 H4), which is predominantly mitochondrial in distribution. Earlier investigations from our laboratory have studied and demonstrated roles of nm23-H1 in GSIS from pancreatic β-cells. They also provided evidence for potential regulation of glucose-induced G protein (Arf6 and Rac1) activation, by nm23-H1 in pancreatic β-cells. Based on these observations, we concluded that the nm23-H1 activation step is upstream of Arf6 activation in signaling events leading to GSIS. The reader is referred to several recent reviews on potential regulatory roles of nm-23-like proteins in G protein activation and function in pancreatic β-cells (6). In the context of regulation of small G protein functions by nm-23 H1, Mizrahi et al. (60) proposed a similar mechanism for the activation of Nox2 involving Rac1, which is a member of the Nox2 holoenzyme. They proposed a link between GEF-induced dissociation of GDP from GDP-bound Rac1, which is then trans-phosphorylated to GTP by NDPK in the presence of ATP. Such “newly formed” GTP is then bound to Rac1 to yield its active GTP-bound conformation for association with core subunits and activation of Nox2 enzyme (60). Additional studies are needed to critically evaluate non-canonical activation of G proteins by nm23 class of proteins (6). It should also be noted, however, potential crosstalk between SET-nm-23-Rac1 signaling pathways in the pathology of islet dysfunction under metabolic stress conditions remain an open area of investigation in the field of islet biology.

5.4. p-21 activated kinase (Pak1)

We have recently identified Pak1 as one of the interacting partners for Rac1 in pancreatic β-cells (25). Several lines of experimental evidence implicate regulatory roles for Pak1 in islet function, including insulin secretion and glucose homeostasis. Studies by Wang and coworkers have demonstrated significant defects in second phase insulin secretion in islets derived from Pak1(^−/−) knockout mice (61). They also demonstrated critical regulatory roles for Pak1 signaling pathway in insulin secretion from human islets. Furthermore, islets from Pak1 (−/−) knockout mice exhibited whole body glucose intolerance in vivo and peripheral insulin resistance. Based on these findings, these researchers concluded that Pak1 plays essential roles in whole body glucose homeostatis. Along these lines, using islets derived from Pak1-knockout mice or Pak1-depleted insulin-secreting MIN6 cells, Chen and coworkers have implicated novel roles for Pak1 in regulating survivin protein stability in the β-cell, and postulated that Pak1 might sub serve the roles of a potential molecular target for the restoration of β-cell mass (62). By employing Pak1 knockout animal models, Ahn and coworkers have provided compelling evidence for involvement of Pak1 in diet-induced beta cell mass and expansion and postulated that Pak1 deficiency could lead to increased susceptibility to diabetes (63). Lastly, Veluthakal et al (64) have recently reported rescue of defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion in T2DM human islets by an Epac (a GEF) activator leading to restoration of optimal GSIS. Together, these data provide compelling evidence for direct regulatory roles of Pak1 in islet function, including insulin secretion. Furthermore, they also identify potential defects in this signaling pathway in T2DM islets. Despite these advances in this field, potential roles of PP2A-Pak1 signaling pathways in the onset of metabolic dysregulation of the islet remain an under studied area of investigation of islet biology. Together, these findings clearly implicate novel regulatory roles for Pak1 in islet function and whole-body glucose homeostasis.

What then are potential crosstalk mechanisms that underlie between PP2A-Pak1-G protein signaling pathways? Several recent investigations, specifically from the Solaro laboratory have highlighted potential contributory roles for Pak proteins in cardiac excitation and contraction (65,66). More importantly, they have implicated PP2A and small G proteins (Cdc42 and Rac1) in Pak1 mediated effects on cardiac function. They recently overviewed (65,66) functional studies of Pak1 and its potential role as an upstream signal for PP2A in the heart. Evidence was also presented for direct activation of Pak1 by Cdc42 and Rac1. From a mechanistic standpoint, these investigators suggested that Pak1 forms a signaling module with PP2A, and subsequent activation of upstream G protein (Cdc42/Rac1) signaling pathway leads to autophosphorylation of Pak1. Consequentially, the associated PP2A becomes auto-dephosphorylated leading to accelerated dephosphorylation of cardiac regulatory proteins. Based on data accrued in complementary investigations, the authors surmised that coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function but is likely to be important in pathophysiological conditions (65,66). Recent investigations by Varshney and Dey (67) in neuronal cells have demonstrated novel roles for Pak2 in glucose uptake and insulin sensitivity in neuronal cells. They observed significant inhibition of Pak2 activity in these cells following stimulation with insulin, which is followed by a marked increase in GLUT4 translocation and glucose uptake. Pharmacological investigations revealed involved potential involvement of Akt and PI3-kinase as mediators of insulin effects on Pak2 and glucose uptake. Interestingly, inhibition of Rac1 activity resulted in inhibition of Pak2 activity while suppression of PP2A activity augmented Pak2 activity with corresponding changes in glucose uptake. Based on these data these researchers proposed an inhibitory role of insulin signaling (via Akt-PI3 kinase pathway) and PP2A on Pak2 activity. They also proposed Pak2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity. Together, these studies established potential cross talk between PP2A-Rac1 signaling pathways in insulin-mediated glucose homeostasis in neuronal cells. These aspects of β-cell biology, specifically PP2A-Pak1 module, remain unexplored, and will need further investigation.

6. Proposed model

Based on the above discussion, I propose a model for potential crosstalk between Rac1 and PP2A signaling pathways in the onset of metabolic stress-induced dysfunction of the islet β-cell (Figure 4). Metabolic stress induces CML of PP2Ac, thus favoring recruitment of regulatory B subunits, holoenzyme assembly culminating in the catalytic activation of PP2A (18–20). This, in turn, leads to dephosphorylation and inactivation of key signaling proteins involved in insulin secretion, cell proliferation and survival, resulting in the apoptotic demise of the islet β cell. Metabolic stress also promotes functional activation of Rac1 leading to stimulation of Nox2 (30) resulting in increased ROS generation. Increased intracellular oxidative stress and stress kinase (p38 and JNK1/2) activation leads to mitochondrial dysregulation (loss in membrane permeability pore transition, release of cytochrome C) leading to the activation of executioner caspases (caspase-3), and degradation and inactivation of key enzymes involved in G protein prenylation, such as the common α-subunit of FTase/GGTase and nuclear lamins (6,15). Accumulating evidence also suggests that sustained activation of Rac1 under metabolic stress conditions leads to inappropriate movement of this G protein to the nuclear fraction to promote activation of apoptotic factors (24). In addition, it appears that Rac1-Nox2-derived increase in intracellular oxidative stress also contributes to increased expression of LCMT-1 (PP2Ac methylating enzyme) and B56α subunit of PP2A, to accelerate the PP2A holoenzyme formation and functional activation of the enzyme (19,20). Lastly, sustained activation of PP2A could lead to mitochondrial dysfunction via dephosphorylation and inactivation of key signaling proteins involved in mitochondrial function (Bcl2). Together, these signaling events establish a potential crosstalk between the PP2A-Rac1 modules to induce islet β-cell dysregulation. Based on available evidence in other cell types (and limited, but compelling evidence in the islet β-cell), I propose that α4 and SET proteins play key regulatory roles in metabolic stress-induced activation of PP2A-Rac1 signaling pathways leading to cell dysfunction. Other putative accessory proteins (nm-23 H1, Pak-1) might also play indirect or direct roles in the acceleration of PP2A-Rac1 signaling pathways (Figure 3). Methodical investigations to address the regulatory roles of these signaling proteins (Figure 3) in the acceleration of PP2A-Rac1 signaling module in a stress-challenged β-cell should provide fresh insights for defining the molecular/cellular mechanisms underlying these cellular events. More importantly, such investigations will identify key therapeutic targets involved in islet β-cell dysfunction under the duress of metabolic stress.

7. Knowledge gaps and future directions

The concept of glucotoxicity was first introduced by Unger and associates suggesting that chronic stimulation of the β-cell by high glucose could result in exhaustion of insulin stores, worsening of hyperglycemia, and eventual functional deterioration and demise of the effete β-cell (11). Potential contributory roles of these pathological insults in the genesis of islet dysmetabolism and demise are highlighted in reviews by Poitout and Robertson (14) and Prentki (12). However, putative molecular and cellular mechanisms underlying the loss of functional β-cell mass under metabolic stress remain only partially understood. Several lines of evidence suggest key regulatory roles for sustained activation of PP2A-Rac1 signaling modules in islet β-cell dysfunction under metabolic stress in a variety of insulin-secreting cells, including clonal β-cells, normal rodent islets and human islets. This Commentary highlighted potential contributory roles for specific signaling proteins that might be involved in acceleration of PP2A-Rac1 signaling pathways in islet β-cells exposed to diabetogenic conditions (Figures 3 and 4). A clear understanding of roles of these regulatory molecules in islet dysfunction should aid in the identification of novel drug targets for the development of therapeutics for the treatment and prevention of diabetes. Based on the above discussion, several knowledge gaps are identified (see below) that need to be filled in to gain a better picture of regulatory control of PP2A-Rac1 signaling pathway by the regulatory proteins discussed above (Figure 3). Four potential knowledge gaps are highlighted below.

First, it will be interesting to investigate potential regulatory roles of CER on PP2A-Rac1 signaling axis in the onset of cell dysfunction. For example, sustained activation of Rac1 and PP2A were observed not only in chronic hyperglycemic conditions, but they were also demonstrable under conditions of exposure to biologically-active sphingolipids, such as CER. Experimental evidence from in vitro and in vivo studies implicates intracellular generation of CER as a trigger for the onset of β-cell demise under above pathological conditions. Earlier investigations have reported a CER-activated protein phosphatase 2A (CAPP) in insulin-secreting cells. Cell-permeable CER stimulated OKA-sensitive phosphatase activity (16). Along these lines, observations from studies of Bharath and coworkers have demonstrated association of CER with SET in endothelial cells leading to translocation of catalytically active PP2A to the plasma membrane fraction and perturbing the downstream signaling steps culminating in arterial dysfunction in animal models of diet-induced obesity (68). Earlier studies have reported CER-induced activation of Rac1 in pancreatic β-cells. These findings were further confirmed using L-threo-C6-pyridinium-ceramide bromide, a novel water-soluble cationic CER, which also induced mitochondrial dysfunction and loss in cell viability in insulin-secreting INS 832/13 cells (16). Additional studies are needed to further characterize CAPP in pancreatic β-cells including determination of the composition of the CAAP holoenzyme, and potential regulation of this pathway by Rac1 signaling cascade. Similar mechanisms have been proposed by Hsu et al for CAPP-mediated mitochondrial dysregulation in amyloid beta-peptide-induced cerebrovascular degeneration. They reported CAPP-mediated dephosphorylation of Akt and FKHRL1 resulting translocation of Bad to the mitochondria and transactivation of Bim to induce mitochondrial dysfunction in these cells following exposure to amyloid β-peptide (69). It should be noted, however, that experimental evidence is also accumulating to suggest inhibition of PP2A under oxidative stress conditions in cancerous cells (70,71). In addition, in contrast to the currently proposed model in the islet β-cell, published evidence, also in cancer cells, suggest attenuation of PP2A activity by Rac1-mediated, redox dependent phosphorylation of Bcl2 (72). These aspects need to be taken into consideration while assessing the overall impact of intracellular oxidative stress on PP2A-Rac1 signaling pathway.

Second, in the context of Rac1 activation in pancreatic β-cells under the duress of metabolic stress, it appears that subcellular distribution of Rac1 plays key roles in eliciting the apoptotic effects. We have demonstrated that constitutively active Rac1 translocates to the nuclear fraction in clonal β-cells and normal rodent and human islets in a prenylation-independent manner (24). These findings need to be validated further in the context of potential impact of other post-translational modifications of this protein including acylation, phosphorylation, palmitoylation, and SUMOylation (22; Figure 2).

Third, studies by Switzer and coworkers (59) involving ApoE peptides, such as COG112, have provided fresh insights into potential regulatory roles of SET in modulating the functions of three signaling pathways, namely PP2A, Rac1 and nm-23 H1 in cell function. Indeed, it would be interesting to test these peptides in pancreatic β-cells to assess their ability to specifically bind to SET and relieve the inhibitory effects of the later on PP2A, Rac1 and nm-23H1. Indeed, such an experimental approach will provide additional insights into regulation of the PP2A-Rac1 signaling module in islet dysfunction as depicted in our model (Figure 4). Furthermore, it will be interesting to determine if this peptide is able to release nm23-H1 from the SET-nm23-H1 complex, and if so, whether it translocates to the nuclear fraction to perform the exonuclease functions.

Fourth, recent studies by Plecitá-Hlavatá and coworkers (73) have provided compelling evidence for a key regulatory role of NADPH oxidase 4 (Nox4) in GSIS. They reported loss of fast phase of GSIS in Nox4-null and β-cell specific Nox4-knockout mice. Interestingly, both mouse models exhibited impaired glucose intolerance as well as peripheral insulin resistance. While critical regulatory roles of Rac1 for other forms of Nox, namely Nox1, Nox2, and Nox3 are reported, potential regulation of Nox4 by Rac1 remains to be examined (74). Furthermore, even though modulatory roles of Nox2 in the induction of islet β-cell dysfunction are reported (30), putative roles of other Nox forms (Nox1, Nox3, Nox4 and others) remain to be determined in the pancreatic islet β-cell.

Lastly, as reviewed recently in (6) abundant evidence implicates coupling of small G proteins (Rac1, Rap1 and Cdc42) to GPCRs in pancreatic β-cells. Despite the evidence in other cell types of potential regulation of cellular function by PP2A mediated via GPCRs (below), very little is known about such regulatory mechanisms in pancreatic β-cells. Several lines of published evidence, specifically in the heart, suggests novel regulatory roles of PP2A activity via activation of G protein-coupled receptors (GPCRs). For example, it has been documented that stimulation of adenosine type 1 receptors (A1.Rs) promotes the CML of PP2Ac leading to its translocation to the membranous compartment in adult rat ventricular myocytes (ARVM; ⁷⁵). It was also demonstrated in these studies that stimulation of G_i-coupled A1.Rs (with N6-cyclopentyladenosine; CPA), Gi-coupled muscarinic M2 receptors (with carbachol) and angiotensin II AT2 receptors (with CGP42112) in ARVM, induced the CML of PP2AC (73). In mechanistic studies, these investigators reported that exposure of ARVM to CPA increased the interaction between PP2Ac and LCMT-1, but not PME-1. Based on data from complementary pharmacological and molecular biological investigations, they proposed that in ARVM, A1.R-induced PP2AC translocation to the particulate fraction occurs through a G_iPCR-Gβγ-PI3K mediated intracellular signaling pathway, which may involve increased CML of PP2AC. Compatible with these observations are findings by Lecuona et al demonstrating that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K-ATPase α1-subunit at the serine 18 residue by PP2A in alveolar cells (76). Investigations by Evans and coworkers (77) have observed physical association of PP2Ac with the C-terminal cytoplasmic domain of GPR54, and proposed that such an interaction might, in turn, regulate the phosphorylation of critical signaling intermediates. Studies of Vasudevan et al have reported that PI3-Kγ is activated by GPCRs. They demonstrated that PI3-Kγ inhibits PP2A at the β-adrenergic receptor complex, thereby affecting the G protein coupling.

Using a variety of experimental techniques, they reported precise talk between PI3-Kγ, I2PP2A (SET) and PP2A in the regulation of cellular events downstream to β-adrenergic receptor activation (78). Lastly, Mohan and coworkers have reported noncanonical regulation of insulin-mediated ERK activation by PI3-Kγ (79). Findings from their investigations have suggested that, instead of its known regulation of its functions as a kinase, PI3-Kγ inhibited PP2A by scaffolding and sequestering, thereby retaining the function of ERK, a key regulatory enzyme in a variety of cellular functions. Despite this evidence in other cells, very little is understood about coupling of PP2A-Rac1 signaling module via GPCR activation. This remains a fertile area of investigation.

8. Conclusions

This Commentary overviewed potential cross-talk between PP2A and Rac1 signaling modules in the onset of metabolic dysregulation of the islet β-cell leading to impaired GSIS, loss of β-cell mass and the onset of diabetes. Published evidence in animal models (global/conditional deletion as well as models of impaired insulin secretion and diabetes) is also highlighted to reflect potential abnormalities in the expression and function of some of these proteins (Rac1, PP2A and Pak1) indicating their involvement in islet functions, including insulin secretion and overall glucose homeostasis. It also identified potential knowledge gaps, and highlighted directions for future research in this fertile area of translational islet biology. The author is hopeful that this commentary will provide a basis for future studies toward a better understanding of roles of PP2A-Rac1 signaling module in pancreatic β-cell dysfunction, and identification of therapeutic targets for the treatment of islet β-cell dysfunction in diabetes.

Abbreviations used

Arf6

ADP-ribosylation factor 6

ARNO

ADP-ribosylation factor nucleotide binding site opener

ATM kinase

Ataxia telangiectasia mutated kinase

Cdc42

cell division control protein 42

CER

ceramide

CML

carboxylmethylation

Epac

exchange protein directly activated by cAMP

ER stress

endoplasmic reticulum stress

ERK1/2

extracellular signal-regulated kinases 1/2

FPP

farnesyl pyrophosphate

FTase

farnesyltransferase

GAP

GTPase-activating protein

GDI

guanine nucleotide dissociation inhibitor

GEF

guanine nucleotide exchange factor

GGTase

geranylgeranyltransferase

GPCR

G protein-coupled receptor

GSIS

glucose-stimulated insulin secretion

IGBP1

immunoglobulin-binding protein1 (also known as α4)

JNK1/2

Jun NH2-terminal kinases 1/2

LCMT-1

leucine carboxymethyl transferase-1

NDPK

nucleoside diphosphate kinase

Nox2

NADPH oxidase

NSC23766

N6-[2-[4-(Diethylamino)-1-methylbu-tyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-qu-inolinediamine trihydrochloride

OKA

okadaic acid

p38MAPK

p38 mitogen-activated protein kinase

PI-3-kinase

Phosphoinositide 3-kinase

PIPs

protein interaction partners

PLases

phospholipases

PP2A

protein phosphatase 2A

PP2Ac

catalytic subunit of PP2A

PP4

protein phosphatase 4

PP6

protein phosphatase 6

Rac1

Ras-related C3 botulinum toxin substrate 1

ROS

reactive oxygen species

siRNA

small interfering RNA

Tiam1

T-cell lymphoma invasion and metastasis-inducing protein 1

UPLC-ESI-MS/MS

ultra-performance liquid chromatography-electrospray tandem mass spectrometry

Vav2

Vav guanine nucleotide exchange factor 2