Pharmacological inhibition of tyrosine protein-kinase 2 reduces islet inflammation and delays type 1 diabetes onset in mice.

Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human ß cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced ß cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of ß cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented ß cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for ß cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.

Deoxyhypusine synthase promotes a pro-inflammatory macrophage phenotype.

The metabolic inflammation (meta-inflammation) of obesity is characterized by proinflammatory macrophage infiltration into adipose tissue. Catalysis by deoxyhypusine synthase (DHPS) modifies the translation factor eIF5A to generate a hypusine (Hyp) residue. Hypusinated eIF5A (eIF5AHyp) controls the translation of mRNAs involved in inflammation, but its role in meta-inflammation has not been elucidated. Levels of eIF5AHyp were found to be increased in adipose tissue macrophages from obese mice and in murine macrophages activated to a proinflammatory M1-like state. Global proteomics and transcriptomics revealed that DHPS deficiency in macrophages altered the abundance of proteins involved in NF-κB signaling, likely through translational control of their respective mRNAs. DHPS deficiency in myeloid cells of obese mice suppressed M1 macrophage accumulation in adipose tissue and improved glucose tolerance. These findings indicate that DHPS promotes the post-transcriptional regulation of a subset of mRNAs governing inflammation and chemotaxis in macrophages and contributes to a proinflammatory M1-like phenotype.

The Pancreatic ß-cell Response to Secretory Demands and Adaption to Stress.

Pancreatic ß cells dedicate much of their protein translation capacity to producing insulin to maintain glucose homeostasis. In response to increased secretory demand, ß cells can compensate by increasing insulin production capability even in the face of protracted peripheral insulin resistance. The ability to amplify insulin secretion in response to hyperglycemia is a critical facet of ß-cell function, and the exact mechanisms by which this occurs have been studied for decades. To adapt to the constant and fast-changing demands for insulin production, ß cells use the unfolded protein response of the endoplasmic reticulum. Failure of these compensatory mechanisms contributes to both type 1 and 2 diabetes. Additionally, studies in which ß cells are "rested" by reducing endogenous insulin demand have shown promise as a therapeutic strategy that could be applied more broadly. Here, we review recent findings in ß cells pertaining to the metabolic amplifying pathway, the unfolded protein response, and potential advances in therapeutics based on ß-cell rest.

Adiponectin receptor fragmentation in mouse models of type 1 and type 2 diabetes.

The protein hormone adiponectin regulates glucose and fatty acid metabolism by binding to two PAQR-family receptors (AdipoR1 and AdipoR2). Both receptors feature a C-terminal segment which is released by proteolysis to form a freely circulating C-terminal fragment (CTF) found in the plasma of normal individuals but not in some undefined diabetes patients. The AdipoR1-CTF344-376 is a competitive inhibitor of tumor necrosis factor α cleavage enzyme (TACE) but it contains a shorter peptide domain (AdipoR1 CTF351-362) that is a strong non-competitive inhibitor of insulin-degrading enzyme (IDE). The link between adiponectin receptor fragmentation and diabetes pathology is unclear but could lead to new therapeutic strategies. We therefore investigated physiological variations in the concentrations of CTF in non-obese diabetic (NOD/ShiLtJ) mice and C57BL/6 mice with diet-induced obesity (DIO) as models of diabetes types 1 and 2, respectively. We tested for changes in adiponectin receptor signaling, immune responses, disease progression, and the abundance of neutralizing autoantibodies. Finally, we administered exogenous AdipoR1-CTF peptides either containing or lacking the IDE-binding domain. We observed the more pronounced CTF shedding in the TACE-active NOD mice, which represents an inflammatory autoimmune phenotype, but fragmentation was also observed to a lesser extent in the DIO model. Autoantibodies to CTF were detected in both models. Neither exogenous CTF peptide affected IgG-CTF plasma levels, body weight or the conversion of NOD mice to diabetes. The pattern of AdipoR1 fragmentation and autoantibody production under physiological conditions of aging, DIO, and autoimmune diabetes therefore provides insight into the association adiponectin biology and diabetes.

PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity.

Regulated proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In ß cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. ß cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null ß cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of proinsulin in the ER to support insulin production and ß cell health.

Use of the PET ligand florbetapir for in vivo imaging of pancreatic islet amyloid deposits in hIAPP transgenic mice.

AIMS/HYPOTHESIS: Islet amyloid deposits contribute to beta cell dysfunction and death in most individuals with type 2 diabetes but non-invasive methods to determine the presence of these pathological protein aggregates are currently not available. Therefore, we examined whether florbetapir, a radiopharmaceutical agent used for detection of amyloid-ß deposits in the brain, also allows identification of islet amyloid in the pancreas. METHODS: Saturation binding assays were used to determine the affinity of florbetapir for human islet amyloid polypeptide (hIAPP) aggregates in vitro. Islet amyloid-prone transgenic mice that express hIAPP in their beta cells and amyloid-free non-transgenic control mice were used to examine the ability of florbetapir to detect islet amyloid deposits in vitro, in vivo and ex vivo. Mice or mouse pancreases were subjected to autoradiographic, histochemical and/or positron emission tomography (PET) analyses to assess the utility of florbetapir in identifying islet amyloid. RESULTS: In vitro, florbetapir bound synthetic hIAPP fibrils with a dissociation constant of 7.9 nmol/l. Additionally, florbetapir bound preferentially to amyloid-containing hIAPP transgenic vs amyloid-free non-transgenic mouse pancreas sections in vitro, as determined by autoradiography (16,475 ± 5581 vs 5762 ± 575 density/unit area, p < 0.05). In hIAPP transgenic and non-transgenic mice fed a high-fat diet for 1 year, intravenous administration of florbetapir followed by PET scanning showed that the florbetapir signal was significantly higher in amyloid-laden hIAPP transgenic vs amyloid-free non-transgenic pancreases in vivo during the first 5 min of the scan (36.83 ± 2.22 vs 29.34 ± 2.03 standardised uptake value × min, p < 0.05). Following PET, pancreases were excised and florbetapir uptake was determined ex vivo by γ counting. Pancreatic uptake of florbetapir was significantly correlated with the degree of islet amyloid deposition, the latter assessed by histochemistry (r = 0.74, p < 0.001). CONCLUSIONS/INTERPRETATION: Florbetapir binds to islet amyloid deposits in a specific and quantitative manner. In the future, florbetapir may be useful as a non-invasive tool to identify islet amyloid deposits in humans.

A selective GPR40 (FFAR1) agonist LY2881835 provides immediate and durable glucose control in rodent models of type 2 diabetes.

LY2881835 is a selective, potent, and efficacious GPR40 agonist. The objective of the studies described here was to examine the pharmacological properties of LY2881835 in preclinical models of T2D. Significant increases in insulin secretion were detected when LY2881835 was tested in primary islets from WT mice but not in islets from GPR40 KO mice. Furthermore, LY2881835 potentiated glucose stimulated insulin secretion in normal lean mice. Acute administration of LY2881835 lowered glucose during OGTTs in WT mice but not in GPR40 KO mice. These findings demonstrate that LY2881835 induces GPR40-mediated activity ex vivo and in vivo. LY2881835 was administered orally at 10 mg/kg to diet-induced obese (DIO) mice (an early model of T2D due to insulin resistance) for 14 days. Statistically significant reductions in glucose were seen during OGTTs performed on days 1 and 15. When a study was done for 3 weeks in Zucker fa/fa rats, a rat model of insulin resistance, normalization of blood glucose levels equivalent to those seen in lean rats was observed. A similar study was performed in streptozotocin (STZ)-treated DIO mice to explore glucose control in a late model of T2D. In this model, pancreatic insulin content was reduced ~80% due to STZ-treatment plus the mice were insulin resistant due to their high fat diet. Glucose AUCs were significantly reduced during OGTTs done on days 1, 7, and 14 compared to control mice. In conclusion, these results demonstrate that LY2881835 functions as a GPR40-specific insulin secretagogue mediating immediate and durable glucose control in rodent models of early- and late-stage T2D.

The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of ß Cells.

Although glucose uniquely stimulates proinsulin biosynthesis in ß cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (Xbp1) mRNA splicing in adult primary ß cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective ß cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, ß cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in ß cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with ß cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the ß cell for increased proinsulin synthesis and to limit oxidative stress that leads to ß cell failure.

Phosphorylation of eIF2α at serine 51 is an important determinant of cell survival and adaptation to glucose deficiency.

Various forms of stress induce pathways that converge on the phosphorylation of the alpha (α) subunit of eukaryotic translation initiation factor eIF2 at serine 51 (S51), a modification that results in a global inhibition of protein synthesis. In many cases eIF2α phosphorylation is a biological response that facilitates cells to cope with stressful environments. Glucose deficiency, an important form of stress, is associated with an induction of apoptosis. Herein, we demonstrate that eIF2α phosphorylation is a key step in maintaining a balance between the life and death of a glucose-deficient cell. That is, eIF2α phosphorylation acts as a molecular switch that shifts cells from a proapoptotic to a cytoprotective state in response to prolonged glucose deficiency. This adaptation process is associated with the timely expression of proteins and activation of pathways with significant contributions to cell survival and adaptation including the X-linked inhibitor of apoptosis protein (XIAP). We also show that among the eIF2α kinases GCN2 plays a proapoptotic role whereas PERK and PKR play a cytoprotective one in response to glucose deficiency. Our data demonstrate that eIF2α phosphorylation is a significant determinant of survival and adaptation of glucose-deficient cells with possible important implications in biological processes that interfere with glucose metabolism.

Protein kinase R contributes to immunity against specific viruses by regulating interferon mRNA integrity.

Cytosolic viral RNA recognition by the helicases RIG-I and MDA5 is considered the major pathway for IFN-alpha/beta induction in response to RNA viruses. However, other cytoplasmic RNA sensors, including the double-stranded RNA-binding protein kinase R (PKR), have been implicated in IFN-alpha/beta production, although their relative contribution and mechanism have been unclear. Using cells expressing nonfunctional PKR or reduced levels of kinase, we show that PKR is required for production of IFN-alpha/beta proteins in response to a subset of RNA viruses including encephalomyocarditis, Theiler's murine encephalomyelitis, and Semliki Forest virus, but not influenza or Sendai virus. Surprisingly, although IFN-alpha/beta mRNA induction is largely normal in PKR-deficient cells, much of that mRNA lacks the poly(A) tail, indicating that its integrity is compromised. Our results suggest that PKR plays a nonredundant role in IFN-alpha/beta production in response to some but not all viruses, in part by regulating IFN-alpha/beta mRNA stability.

eIF2alpha phosphorylation tips the balance to apoptosis during osmotic stress.

Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2alpha, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2alpha phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2alpha phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2alpha promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2alpha phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2alpha phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2alpha phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2alpha phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.

Tumor suppression by PTEN requires the activation of the PKR-eIF2alpha phosphorylation pathway.

Inhibition of protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) at Ser(51) occurs as a result of the activation of a family of kinases in response to various forms of stress. Although some consequences of eIF2alpha phosphorylation are cytoprotective, phosphorylation of eIF2alpha by RNA-dependent protein kinase (PKR) is largely proapoptotic and tumor suppressing. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is a tumor suppressor protein that is mutated or deleted in various human cancers, with functions that are mediated through phosphatase-dependent and -independent pathways. Here, we demonstrate that the eIF2alpha phosphorylation pathway is downstream of PTEN. Inactivation of PTEN in human melanoma cells reduced eIF2alpha phosphorylation, whereas reconstitution of PTEN-null human glioblastoma or prostate cancer cells with either wild-type PTEN or phosphatase-defective mutants of PTEN induced PKR activity and eIF2alpha phosphorylation. The antiproliferative and proapoptotic effects of PTEN were compromised in mouse embryonic fibroblasts that lacked PKR or contained a phosphorylation-defective variant of eIF2alpha. Induction of the pathway leading to phosphorylation of eIF2alpha required an intact PDZ-binding motif in PTEN. These findings establish a link between tumor suppression by PTEN and inhibition of protein synthesis that is independent of PTEN's effects on phosphoinositide 3'-kinase signaling.

Translation attenuation through eIF2alpha phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells.

Accumulation of unfolded protein within the endoplasmic reticulum (ER) attenuates mRNA translation through PERK-mediated phosphorylation of eukaryotic initiation factor 2 on Ser51 of the alpha subunit (eIF2alpha). To elucidate the role of eIF2alpha phosphorylation, we engineered mice for conditional expression of homozygous Ser51Ala mutant eIF2alpha. The absence of eIF2alpha phosphorylation in beta cells caused a severe diabetic phenotype due to heightened and unregulated proinsulin translation; defective intracellular trafficking of ER cargo proteins; increased oxidative damage; reduced expression of stress response and beta-cell-specific genes; and apoptosis. However, glucose intolerance and beta cell death in these mice were attenuated by a diet containing antioxidant. We conclude that phosphorylation of eIF2alpha coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production. The finding that increased proinsulin synthesis causes oxidative damage in beta cells may reflect events in the beta cell failure associated with insulin resistance in type 2 diabetes.

Phosphorylation of eukaryotic translation initiation factor 2alpha coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2alpha plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2alpha phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting approximately 90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2alpha, suggesting that this pathway may be broadly utilized by stresses that activate eIF2alpha kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development.

Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of eIF2(alphaP), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to eIF2(alphaP), but the significance of their contribution to its dephosphorylation has not been explored systematically. Here we report that unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, Ppp1r15b(-/-) mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, Eif2a(S51A), that prevents regulated phosphorylation of eIF2alpha. These findings reveal that the essential process of eIF2(alphaP) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development.

Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes.

The progression from insulin resistance to type 2 diabetes is caused by the failure of pancreatic beta cells to produce sufficient levels of insulin to meet the metabolic demand. Recent studies indicate that nutrient fluctuations and insulin resistance increase proinsulin synthesis in beta cells beyond the capacity for folding of nascent polypeptides within the endoplasmic reticulum (ER) lumen, thereby disrupting ER homeostasis and triggering the unfolded protein response (UPR). Chronic ER stress promotes apoptosis, at least in part through the UPR-induced transcription factor C/EBP homologous protein (CHOP). We assessed the effect of Chop deletion in multiple mouse models of type 2 diabetes and found that Chop-/- mice had improved glycemic control and expanded beta cell mass in all conditions analyzed. In both genetic and diet-induced models of insulin resistance, CHOP deficiency improved beta cell ultrastructure and promoted cell survival. In addition, we found that isolated islets from Chop-/- mice displayed increased expression of UPR and oxidative stress response genes and reduced levels of oxidative damage. These findings suggest that CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis in beta cells under conditions of increased insulin demand.

Novel function of PERK as a mediator of force-induced apoptosis.

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.

The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes.

The endoplasmic reticulum (ER) is the entry site into the secretory pathway for newly synthesized proteins destined for the cell surface or released into the extracellular milieu. The study of protein folding and trafficking within the ER is an extremely active area of research that has provided novel insights into many disease processes. Cells have evolved mechanisms to modulate the capacity and quality of the ER protein-folding machinery to prevent the accumulation of unfolded or misfolded proteins. These signaling pathways are collectively termed the unfolded protein response (UPR). The UPR sensors signal a transcriptional response to expand the ER folding capacity, increase degradation of malfolded proteins, and limit the rate of mRNA translation to reduce the client protein load. Recent genetic and biochemical evidence in both humans and mice supports a requirement for the UPR to preserve ER homeostasis and prevent the beta-cell failure that may be fundamental in the etiology of diabetes. Chronic or overwhelming ER stress stimuli associated with metabolic syndrome can disrupt protein folding in the ER, reduce insulin secretion, invoke oxidative stress, and activate cell death pathways. Therapeutic interventions to prevent polypeptide-misfolding, oxidative damage, and/or UPR-induced cell death have the potential to improve beta-cell function and/or survival in the treatment of diabetes.

Glucose activates a protein phosphatase-1-mediated signaling pathway to enhance overall translation in pancreatic beta-cells.

Both the rate of overall translation and the specific acceleration of proinsulin synthesis are known to be glucose-regulated processes in the beta-cell. In this study, we propose that glucose-induced stimulation of overall translation in beta-cells depends on a protein phosphatase-1-mediated decrease in serine-51 phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), a pivotal translation initiation factor. The decrease was rapid and detectable within 15 min and proportional to the range of glucose concentrations that also stimulate translation. Lowered net eIF2alpha phosphorylation was not associated with a detectable decrease in activity of any eIF2alpha kinase. Moreover, okadaic acid blocked glucose-induced eIF2alpha dephosphorylation, suggesting that the net effect was mediated by a protein phosphatase. Experiments with salubrinal on intact cells and nuclear inhibitor of protein phosphatase-1 (PP1) on cell extracts suggested that this phosphatase was PP1. The net effect contained, however, a component of glucose-induced folding load in the endoplasmic reticulum because coincubation with cycloheximide further amplified the effect of glucose on eIF2alpha dephosphorylation. Thus, the steady-state level of eIF2alpha phosphorylation in beta-cells is the result of a balance between folding-load-induced phosphorylation and PP1-dependent dephosphorylation. Because defects in the pancreatic endoplasmic reticulum kinase-eIF2alpha signaling system lead to beta-cell failure and diabetes, deregulation of the PP1 system could likewise lead to cellular dysfunction and disease.

Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis.

As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2alpha)atSer(51) is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2alpha stress kinases. We used gene targeting to replace the wild-type Ser(51) allele with an Ala in the eIF2alpha gene to test the hypothesis that translational control through eIF2alpha phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2alpha mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-alpha, and serum deprivation. TNFalpha treatment induced eIF2alpha phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2alpha kinase PKR. In addition, expression of a phospho-mimetic Ser(51) to Asp mutant eIF2alpha-activated caspase 3, indicating that eIF2alpha phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2alpha phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2alpha kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2alpha. We propose that eIF2alpha phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.