Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt?

The control of pancreatic beta-cell growth and survival in the adult plays a pivotal role in the pathogenesis of type 2 diabetes. In certain insulin-resistant states, such as obesity, the increased insulin-secretory demand can often be compensated for by an increase in beta-cell mass, so that the onset of type 2 diabetes is avoided. This is why approximately two-thirds of obese individuals do not progress to type 2 diabetes. However, the remaining one-third of obese subjects that do acquire type 2 diabetes do so because they have inadequate compensatory beta-cell mass and function. As such, type 2 diabetes is a disease of insulin insufficiency. Indeed, it is now realized that, in the vast majority of type 2 diabetes cases, there is a decreased beta-cell mass caused by a marked increase in beta-cell apoptosis that outweighs rates of beta-cell mitogenesis and neogenesis. Thus a means of promoting beta-cell survival has potential therapeutic implications for treating type 2 diabetes. However, understanding the control of beta-cell growth and survival at the molecular level is a relatively new subject area of research and still in its infancy. Notwithstanding, recent advances have implicated signal transduction via insulin receptor substrate-2 (IRS-2) and downstream via protein kinase B (PKB, also known as Akt) as critical to the control of beta-cell survival. In this review, we highlight the mechanism of IRS-2, PKB, and anti-apoptotic PKB substrate control of beta-cell growth and survival, and we discuss whether these may be targeted therapeutically to delay the onset of type 2 diabetes.     

TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.     

Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.     

The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway

Phosphoinositide-3 kinase (PI-3 kinase) and its downstream signaling molecules PDK-1 and Akt were analyzed in SK-N-SH and SK-N-BE(2) human neuroblastoma cell lines. When cells were stimulated with insulin, PI-3 kinase was activated in both cell lines, whereas the translocation of PDK-1 to the membrane fraction and phosphorylated Akt were observed only in SK-N-SH cells. Analyses of the insulin-mediated reactive oxygen species (ROS) generation and Phosphatase and Tensin homolog (PTEN) oxidation indicate that PTEN oxidation occurred in SK-N-SH cells, which can produce ROS, but not in SK-N-BE(2) cells, which cannot increase ROS in response to insulin stimulation. When SK-N-SH cells were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride before insulin stimulation, insulin-mediated translocation of PDK-1 to the membrane fraction and phosphorylation of Akt were remarkably reduced, whereas PI-3 kinase activity was not changed significantly. These results indicate that not only PI-3 kinase activation but also inhibition of PTEN by ROS is needed to increase cellular level of phosphatidylinositol 3,4,5-trisphosphate for recruiting downstream signaling molecules such as PDK-1 and Akt in insulin-mediated signaling. Moreover, the ROS generated by insulin stimulation mainly contributes to the inactivation of PTEN and not to the activation of PI-3 kinase in the PI-3 kinase/Akt pathway.     

Renal activity of Akt kinase in obese Zucker rats

Insulin resistance (IR) and consequent hyperinsulinemia are hallmarks of Type 2 diabetes (DM2). Akt kinase (Akt) is an important molecule in insulin signaling, implicated in regulation of glucose uptake, cell growth, cell survival, protein synthesis, and endothelial nitric oxide (NO) production. Impaired Akt activation in insulin-sensitive tissues contributes to IR. However, Akt activity in other tissues, particularly those affected by complications of DM2, has been less studied. We hypothesized that hyperinsulinemia could have an impact on activity of Akt and its effectors involved in regulation of renal morphology and function in DM2. To address this issue, renal cortical Akt was determined in obese Zucker rats (ZO), a model of DM2, and lean controls (ZL). We also studied expression and phosphorylation of the mammalian target of rapamycin (mTOR) and endothelial NO synthase (eNOS), molecules downstream of Akt in the insulin signaling cascade, and documented modulators of renal injury. Akt activity was measured by a kinase assay with GSK-3 as a substrate. Expression of phosphorylated (active) and total proteins was measured by immunoblotting and immunohistochemistry. Renal Akt activity was increased in ZO as compared to ZL rats, in parallel with progressive hyperinsulinemia. No differences in Akt were observed in the skeletal muscle. Corresponding to increases in Akt activity, ZO rats demonstrated enhanced phosphorylation of renal mTOR. Acute PI3K inhibition with wortmannin (100 mug/kg) attenuated renal Akt and mTOR activities in ZO, but not in ZL rats. In contrast to mTOR, eNOS phosphorylation was similar in ZO and ZL rats, despite higher total eNOS expression. In conclusion, ZO rats demonstrated increases in renal Akt and mTOR activity and expression. However, eNOS phosphorylation did not follow this pattern. These data suggest that DM2 is associated with selective IR in the kidney, allowing pro-growth signaling via mTOR, whereas potentially protective effects mediated by eNOS are blunted.     

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.     

Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway

Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF did not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) were required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt was found to inhibit insulin signaling. Moreover, this inhibition required the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt were inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade.     

Phosphatidylinositol 3 kinase–Akt signaling serves as a circadian output in the retina

The daily rhythm of L-type voltage-gated calcium channels (L-VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L-VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase–protein kinase B (PI3K–Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor-induced ion channel trafficking and membrane insertion. Here, we report that PI3K–Akt signaling participates in the circadian phase-dependent modulation of L-VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L-VGCC current amplitudes and the expression of membrane-bound L-VGCCα1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L-VGCCα1D subunit compared with non-transfected photoreceptors. Interestingly, both PI3K–Akt and extracellular signal-related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L-VGCC rhythms. Inhibition of either pathway abolished the L-VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L-VGCC rhythms in retina photoreceptors.     

mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif

The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.     

Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1β-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1β for 24 h induced a 2–3-fold increase in GRK2 and decreased C–C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/− animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.     

Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC-alpha-dependent Akt activation in endosomes

Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.     

Phosphatase and tensin homolog regulation of islet growth and glucose homeostasis

The Irs2 branch of the insulin/insulin-like growth factor signaling cascade activates the phosphatidylinositol 3-kinase --> Akt --> Foxo1 cascade in many tissues, including hepatocytes and pancreatic beta-cells. The 3'-lipid phosphatase Pten ordinarily attenuates this cascade; however, its influence on beta-cell growth or function is unknown. To determine whether decreased Pten expression could restore beta-cell function and prevent diabetes in Irs2(-/-) mice, we generated wild type or Irs2 knock-out mice that were haploinsufficient for Pten (Irs2(-/-)::Pten(+/-)). Irs2(-/-) mice develop diabetes by 3 months of age as beta-cell mass declined progressively until insulin production was lost. Pten insufficiency increased peripheral insulin sensitivity in wild type and Irs2(-/-) mice and increased Akt and Foxo1 phosphorylation in the islets. Glucose tolerance improved in the Pten(+/-) mice, although beta-cell mass and circulating insulin levels decreased. Compared with Irs2(-/-) mice, the Irs2(-/-)::Pten(+/-) mice displayed nearly normal glucose tolerance and survived without diabetes, because normal but small islets produced sufficient insulin until the mice died of lymphoproliferative disease at 12 months age. Thus, steps to enhance phosphatidylinositol 3-kinase signaling can promote beta-cell growth, function, and survival without the Irs2 branch of the insulin/insulin-like growth factor signaling cascade.     

Modulation of total Akt kinase by increased expression of a single isoform: requirement of the sphingosine-1-phosphate receptor, Edg3/S1P3, for the VEGF-dependent expression of Akt3 in primary endothelial cells

Akt kinase is an important downstream effector of VEGF in primary endothelial cells (EC), promoting angiogenesis by increased cellular survival, motility and tubulogenesis. Akt1 is the founding member of a family of serine threonine kinases thought to have overlapping function. We sought to determine if other Akt family members were also regulated by VEGF in EC. We show that treatment of EC with the angiogenic inducers VEGF or sphingosine-1-phosphate (S1P) results in an increased stabilization of Akt3 mRNA, concurrent with a PI3 kinase-dependent, Akt1-independent increase in both the protein and its phosphorylation. Given the similarity of Akt3 regulation by VEGF and S1P, the sensitivity of VEGF stimulation to the Gi-protein uncoupling reagent, pertussis toxin was tested and shows that VEGF stimulation requires Gi-protein signaling. We show that the VEGF stimulates the expression of Edg3/S1P3 (S1P3) and that expression of this Gi-protein-coupled receptor is both sufficient and necessary for the expression of Akt3. Blockade of a single isoform does not overtly affect cellular function, whereas inhibition of both kinases results in an increase in apoptosis and a down-regulation of cyclin D3. These results suggest a model whereby extracellular cues maintain total Akt kinase levels through the regulation of specific isoform expression providing a fail-safe mechanism to maintain necessary levels of Akt kinase activity.     

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.     

Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol 3-kinase and Rheb signaling

The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.