Nuclear lamins form the lamina on the interior of the nuclear envelope, and are involved in the regulation of various cellular processes, including DNA replication and chromatin organization. Despite this evidence, little is known about potential alterations in nuclear metabolism, specifically lamin structure and integrity in isolated β-cells subjected to stress conditions, including chronic exposure to hyperglycemia [i.e., glucotoxicity]. Herein, we investigated effects of glucotoxic conditions on the catalytic activation of caspase 3 and the associated degradation of one of its substrate proteins, namely lamin-B. We report that incubation of insulin-secreting INS-1 832/13 cells, normal rat islets or human islets under glucotoxic conditions [20 mM; 12–48 hr] results in the degradation of native lamin B leading to accumulation of the degraded products in non-relevant cellular compartments, including cytosol. Moreover, the effects of high glucose on caspase 3 activation and lamin B degradation were mimicked by thapsigargin, a known inducer of endoplasmic reticulum stress [ER stress]. Nifedipine, a known blocker of calcium channel activation, inhibited high glucose-induced caspase 3 activation and lamin B degradation in these cells. 4-phenyl butyric acid, a known inhibitor of ER stress, markedly attenuated glucose-induced CHOP expression [ER stress marker], caspase 3 activation and lamin B degradation. We conclude that glucotoxic conditions promote caspase 3 activation and lamin B degradation, which may, in part, be due to increased ER stress under these conditions. We also provide further evidence to support beneficial effects of calcium channel blockers against metabolic dysfunction of the islet β-cell induced by hyperglycemic conditions.
Caspase 3; Glucotoxicity; Nuclear lamin B; Pancreatic islet β-cell; nifedipine
A growing body of evidence implicates novel roles for nm23-like proteins in the regulation of cellular functions. However, roles of these proteins in islet function and glucose-stimulated insulin secretion (GSIS) remain largely unknown.
siRNA-nm23-H1 and nucleoside diphosphate kinase and histidine kinase-defcient mutants of nm23-H1 (K12Q and H118F) were used to assess roles of nm23-H1 in GSIS.
siRNA-mediated knockdown of the expression of nm23-H1 markedly inhibited GSIS in INS-1 832/13 cells. Nm23-H1 knockdown also resulted in signifcant inhibition of glucose-mediated activation of Arf6, a small G-protein, which has been implicated in GSIS. Expression of K12Q and H118F mutants of nm23-H1 in INS-1 832/13 cells led to inhibition of glucose-induced translocation and membrane association of Rac1, another small G-protein, which is downstream to Arf6 in the signaling events leading to GSIS. A signifcant inhibition of GSIS was also seen in these cells expressing K12Q and H118F.
We conclude that the nm23-H1 activation step is upstream of Arf6 activation in signaling events leading to GSIS. NDP kinase and histidine kinase functions of nm23-H1 are necessary for glucose-induced membrane association of Rac1 and ensuing insulin secretion. We present the frst evidence for regulation of GSIS by nm23-H1 in pancreatic β-cells.
nm23-H1; Pancreatic β-cell; Insulin secretion; Rac1 and Arf6
Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet β-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1β+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic β-cell.
Pancreatic β-cell; NADPH-oxidase; 2-Bromopalmitate; Cytokines; Oxidative stress; Nitrosative stress
Emerging evidence implicates novel roles for post-translational prenylation [i.e., farnesylation and geranylgeranylation] of various signaling proteins in a variety of cellular functions including hormone secretion, survival and apoptosis. In the context of cellular apoptosis, it has been shown previously that caspase-3 activation, a hallmark of mitochondrial dysregulation, promotes hydrolysis of several key cellular proteins. We report herein that exposure of insulin-secreting INS 832/13 cells or normal rat islets to etoposide leads to significant activation of caspase-3 and subsequent degradation of the common α-subunit of farnesyl/geranylgeranyl transferases [FTase/GGTase]. Furthermore, the above stated signaling steps were prevented by Z-DEVD-FMK, a known inhibitor of caspase-3. In addition, treatment of cell lysates with recombinant caspase-3 also caused FTase/GGTase α-subunit degradation. Moreover, nifedipine, a calcium channel blocker, markedly attenuated etoposide-induced caspase-3 activation, FTase/GGTase α-subunit degradation in INS 832/13 cells and normal rat islets. Further, nifedipine significantly restored etoposide-induced loss in metabolic cell viability in INS 832/13 cells. Based on these findings, we conclude that etoposide induces loss in cell viability by inducing mitochondrial dysfunction, caspase-3 activation and degradation of FTase/GGTase α-subunit. Potential significance of these findings in the context of protein prenylation and β-cell survival are discussed.
Etoposide; caspase-3; farnesyl transferase; geranylgeranyl transferase; nifedipine
The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on β-cells, but it is unable to protect β-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1β-induced damage to isolated β-cells. We report that three novel compounds (THS-73–44, THS-72–5 and THS-78–5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated β-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78–5 protected against IL-1β-mediated loss in β-cell viability. THS-78–5 was also able to attenuate IL-1β-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78–5 against IL-1β-mediated effects may, in part, be due to inhibition of IL-1β-induced transactivation of nuclear factor κB (NF-κB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1β-mediated effects on isolated β-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.
HDAC inhibitors; pancreatic β-cell; inducible nitric oxide synthase; NO release; NF-κB
Hyperactivation of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase is prevalent in human lung cancer and its inhibition by the tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, initially controls tumor growth. However, most patients ultimately relapse due to the development of drug resistance. In this study, we have discovered a STAT3-dependent Akt activation that impairs the efficacy of gefitinib. Mechanistically, gefitinib increased association of EGFR with STAT3, which de-repressed STAT3 from SOCS3, an upstream suppressor of STAT3. Such a de-repression of STAT3 in turn fostered Akt activation. Genetic or pharmacological inhibition of STAT3 abrogated Akt activation and combined gefitinib with STAT3 inhibition synergistically reduced the growth of the tumor cells. Taken together, this study suggests that activation of STAT3 is an intrinsic mechanism of drug resistance in response to EGFR TKIs. Combinational targeting on both EGFR and STAT3 may enhance the efficacy of gefitinib or other EGFR TKIs in lung cancer.
Gefitinib; EGFR; STAT3; Akt recovery
A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.
Geranylgeranylation; Rab G-proteins; Rab escort proteins; insulin secretion; pancreatic β-cells
Several lines of recent evidence implicate regulatory roles for reactive oxygen species (ROS) in islet function and insulin secretion. The phagocyte-like NADPH oxidase (Nox2) has recently been shown to be one of the sources of ROS in the signaling events leading to glucose stimulated insulin secretion (GSIS). We recently reported inhibition of glucose- or mitochondrial fuel-induced Nox2-derived ROS by a specific inhibitor of protein farnesyl transferse (FTase; FTI-277), suggesting that activation of FTase might represent one of the upstream signaling events to Nox2 activation. Furthermore, FTase inhibitors (FTI-277 and FTI-2628) have also been shown to attenuate GSIS in INS 832/13 cells and normal rodent islets. Herein, we provide further evidence to suggest that inhibition of FTase either by pharmacological (e.g., FTI-277) or gene silencing (siRNA-FTase) approaches markedly attenuates mitochondrial fuel-stimulated insulin secretion (MSIS) in INS 832/13 cells. Together, our findings further establish a link between nutrient-induced Nox2 activation, ROS generation and insulin secretion in the pancreatic β-cell.
insulin secretion; mitochondrial fuels; pancreatic β-cells; protein farnesylation
To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.
RESEARCH DESIGN AND METHODS
Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.
Levels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.
We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.
In the majority of cell types, including the islet β-cell, transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation G proteins and their effector modules. The islet β-cell is unusual in that glucose lacks an extracellular receptor. Instead, events consequent to glucose metabolism promote insulin secretion via the generation of diffusible second messengers and mobilization of calcium. A selective increase in intracellular calcium has been shown to regulate the phosphorylation status key islet proteins thereby facilitating insulin secretion. In addition to classical protein kinases [e.g., protein kinases A and C], recent studies from our laboratory have focused on the expression and function of various forms of NDPK/nm23-like histidine kinases in clonal β-cells, normal rodent, and human islets. Further, we recently reported localization of a cytosolic protein histidine phosphatase [PHP] in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knock down of nm23-H1 and PHP in insulin-secreting INS 832/13 cells significantly attenuated glucose-induced insulin secretion. We also observed significant alterations in the expression and function of nm23-H1/PHP in β-cells chronically exposed to elevated levels of glucose and saturated fatty acids, such as palmitate (i.e., glucolipotoxicity). Similar changes were also noted in islets from the Goto-Kakizaki and Zucker Diabetic Fatty rats, two known models for type 2 diabetes. It is concluded that protein histidine phosphorylation–dephosphorylation cycles play novel regulatory roles in G protein-mediated physiological insulin secretion and that abnormalities in this signaling axis lead to impaired insulin secretion in glucolipo-toxicity and type 2 diabetes.
Pancreatic islet; Insulin secretion; Histidine kinases; Histidine phosphatases; nm23
Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E2, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous Protein kinase-B inhibitory protein [Probin] in islet function are discussed.
Pancreatic β-cells; protein farnesylation; Akt; FoxO; β-cell survival
Glucose-stimulated insulin secretion [GSIS] involves a sequence of metabolic events leading to small G-protein [e.g., Rac1]-mediated cytoskeletal remodeling to promote granule mobilization toward the plasma membrane for fusion and release of insulin. Existing evidence supports a positive modulatory role for Rac1 in GSIS. Specific regulatory factors of Rac1 function, including the guanine nucleotide exchange factors [e.g., Tiam1] have also been identified and studied in the islet. Inhibition of Tiam1/Rac1 signaling axis attenuates GSIS suggesting its pivotal role in insulin secretion. In addition to its positive [i.e., friendly] roles in GSIS, Rac1 also plays “non-friendly” role[s] in the islet function. For example, it up-regulates the intracellular reactive oxygen species [ROS] levels via activation of phagocyte-like NADPH oxidase [Nox]. Despite the emerging evidence that a tonic increase in intracellular ROS is necessary for GSIS, experimental evidence also suggests that chronic exposure of β-cells to high glucose, palmitate or cytokines results in the onset of oxidative stress leading to reduction in mitochondrial membrane potential, cytosolic accumulation of cytochrome C and activation of caspase-3 leading to β-cell apoptosis. Pharmacological and molecular biological inhibition of Rac1 activation affords partial protection against Nox-induced oxidative stress and mitochondrial dysfunction induced by elevated glucose, lipids or cytokines. Herein, we overview the existing evidence to suggest positive as well as negative modulatory roles of Rac1 in islet function. Potential avenues for future research including development of inhibitors to halt the Rac1-Nox activation and generation of oxidative stress leading to the metabolic dysfunction of the β-cell are discussed.
Rac1; Tiam1; NADPH-oxidase; Pancreatic islet; mitochondrial dysfunction and insulin secretion
Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. secinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. secinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.
Insulin secretion; pancreatic islet; ARNO; Arf6; Rac1; secinH3
Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western-blot analysis indicated that ICMT is expressed and predominantly membrane associated in INS 832/13 β-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic β-cells.
Rac1; ROS; pancreatic islet; carboxymethylation and insulin secretion
The phagocytic NADPH-oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.
NADPH Oxidase; Rac1; Tiam1; palmitate; ceramide; oxidative stress; pancreatic β-cells
Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Raf-1/extracellular signal–related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic β-cell.
RESEARCH DESIGN AND METHODS
These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]–mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.
Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase β-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion.
Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin.
A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet β-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, subcellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 β-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the α-subunits of FTase/GGTase-1, but not the β-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5–4.0 fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.
Prenylation; farnesylation; geranylgeranylation; islet β-cell; insulin secretion and G-proteins
Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion [GSIS]. However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor [GEF] for Rac1, in GSIS in pancreatic β-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation [i.e., GTP-bound form] and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.
NSC23766; Tiam1; Rac1; Pancreatic β-cells; Glucose-stimulated insulin secretion; calcium-induced insulin secretion
Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the β cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The “newly-formed” phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the β cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet β cell; [iii] propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet β cell function.
Pancreatic β cells; Protein Phosphatase 2A; Glucose-Stimulated Insulin Secretion; G-Proteins; Phospholipid Methylation; Protein Carboxylmethylation