Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.N-Carbamoyl-β-alanine amidohydrolase (NCβAA) (EC 3.5.1.6), also known as β-alanine synthase or β-ureidopropionase, catalyzes the third and final step of reductive pyrimidine degradation. In this reaction, N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid is irreversibly hydrolyzed to CO₂, NH₃, and β-alanine or β-aminoisobutyric acid, respectively (43). Eukaryotic NCβAAs have been purified from several sources (10, 25, 33, 39, 42, 44). Nevertheless, only two prokaryotic NCβAAs, belonging to the Clostridium and Pseudomonas genera (4, 29), have been purified to date, although this activity has been inferred for several microorganisms due to the appearance of the reductive pathway of pyrimidine degradation (38, 45). Pseudomonas NCβAA is also able to hydrolyze l-N-carbamoyl-α-amino acids, and indeed, this activity is widespread in the bacterial kingdom (3, 23, 26, 46).β-Amino acids have unique pharmacological properties, and their utility as building blocks of β-peptides, pharmaceutical compounds, and natural products is of growing interest (14). β-Alanine, a natural β-amino acid, is a precursor of coenzyme A and pantothenic acid in bacteria and fungi (vitamin B₅) (7). β-Alanine is widely distributed in the central nervous systems of vertebrates and is a structural analogue of γ-amino-n-butyric acid and glycine, major inhibitory neurotransmitters, suggesting that it may be involved in synaptic transmissions (20). Another important natural β-amino acid is taurine (2-aminoethanesulfonic acid), which plays an important role in several essential processes, such as membrane stabilization, osmoregulation, glucose metabolism, antioxidation, and development of the central nervous system and the retina (9, 28, 33). 2-Aminoethylphosphonate, the most common naturally occurring phosphonate, also known as ciliatine, is an important precursor used in the biosynthesis of phosphonolipids, phosphonoproteins, and phosphonoglycans (5). β-Homoalanine (β-aminobutyric acid) has been used successfully for the design of nonnatural ligands for therapeutic application against autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or autoimmune uveitis (30). Substituted β-amino acids can be denominated β², β³, and β^2,3, depending on the position of the side chain(s) (R) on the amino acid skeleton (18). β²-Amino acids are not yet as readily available as their β³-counterparts, as they must be prepared using multistep procedures (17).We decided to characterize NCβAA (β-carbamoylase) from Agrobacterium tumefaciens C58 (βcar_At) after showing that some dihydropyrimidinases belonging to the Arthrobacter and Sinorhizobium genera are able to hydrolyze different 5- or 6-substituted dihydrouracils to the corresponding N-carbamoyl-β-amino acids (18, 22). If βcar_At could decarbamoylate the reaction products of dihydrouracils, different β-amino acids would be obtained enzymatically in the same way that α-amino acids are produced via the hydantoinase process (6, 21). We therefore describe the physical, biochemical, kinetic, and substrate specificity properties of recombinant βcar_At.     

Gamma Interferon Signaling in Macrophage Lineage Cells Regulates Central Nervous System Inflammation and Chemokine Production

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-γ). Here, we assessed the role of CD4⁺ T cells and IFN-γ on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-γ had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-γ signaling on macrophage lineage cells was assessed using transgenic mice, called “macrophages insensitive to interferon gamma” (MIIG) mice, that express a dominant-negative IFN-γ receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4⁺ T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4⁺ T-cell production of IFN-γ promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.Immune cell recruitment to and infiltration of the central nervous system (CNS) is central to the pathology of a variety of inflammatory neurological diseases, including infectious meningoencephalitis, multiple sclerosis, and cerebral ischemia (59, 60). Chemokines have been shown to be highly upregulated in both human diseases and animal models of neuroinflammation and are thought to be important mediators of immune cell entry into the CNS (59, 60). For example, during experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), the chemokines CCL2 (monocyte chemoattractant protein 1 [MCP-1α]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL5 (regulated upon activation, T-cell expressed and secreted [RANTES]), and CXCL10 (gamma interferon [IFN-γ]-inducible protein 10 [IP-10]) are produced by either resident CNS cells or infiltrating cells (27) and serve to amplify the ongoing inflammatory response (25, 28). However, in some EAE studies, neither CCL3 nor CXCL10 were required for disease (72, 73). During CNS viral infection, CXCL10 and CCL5 are highly produced in several models (2, 41, 48, 82). In addition, mice deficient in CCR5, which binds (among others) CCL3 and CCL5, do not display impaired CNS inflammation after certain viral infections (13). Thus, the role of chemokines in CNS inflammation is likely complex and dissimilar between autoimmune and viral infection models.IFN-γ is present in the CNS during autoimmunity and infection (7, 54, 69). Several studies suggest that IFN-γ can be a potent inducer of CNS chemokine expression. Adenoviral expression of IFN-γ in the CNS strongly induced CCL5 and CXCL10 mRNA and protein, and this induction was dependent on the presence of the IFN-γ receptor (50). In EAE and Toxoplasma infection, mice deficient in IFN-γ or the IFN-γ receptor demonstrated reduced expression of several chemokines, including CCL2, CCL3, CCL5, and CXCL10 (26, 69). However, given the near-ubiquitous expression of the IFN-γ receptor (44), the mechanisms by which IFN-γ regulates CNS chemokine production remain to be elucidated.We studied neuroinflammation and immune-mediated disease using a well-studied mouse model of infection with lymphocytic choriomeningitis virus (LCMV). Intracranial (i.c.) injection of mice with LCMV results in seizures and death 6 to 8 days after inoculation. The onset of symptoms is associated with a massive influx of mononuclear cells into the cerebrospinal fluid (CSF), meninges, choroid plexus, and ependymal membranes (6, 8, 18), as well as the presence of proinflammatory cytokines (7, 38). The immune response is critical for disease, since infection of irradiated or T-cell-depleted mice leads to persistent infection with very high levels of virus in multiple tissues without the development of lethal meningitis (18, 34, 64). i.c. LCMV infection of β₂-microglobulin-deficient mice (β₂m^−/− mice) also results in meningitis and production of proinflammatory cytokines and chemokines; however, meningitis occurs with a later onset and lower severity compared to wild-type mice (17, 24, 53, 57). Interestingly, i.c. LCMV infection of these mice also causes severe anorexia and weight loss (33, 38, 46, 52, 57) that is mediated by major histocompatibility complex (MHC) class II-restricted, CD4⁺ T cells (17, 46, 53, 57). Anorexia and weight loss are also observed in wild-type mice, but they succumb to lethal meningitis shortly thereafter (33), making study of this particular aspect of disease difficult. LCMV-induced weight loss, similar to what we have observed in β₂m^−/− mice also occurs in perforin-deficient mice, which possess CD8⁺ T cells (37). Although some reports have observed weight loss after peripheral LCMV infection (11, 45), we note that these studies used high doses of the clone 13 strain of LCMV, in contrast to our studies which have used the Armstrong strain of LCMV and orders of magnitude less virus (33, 38, 46, 52, 57). Although we cannot exclude a contribution of peripheral cells to weight loss in our i.c. Armstrong infection model, we previously showed that this weight loss does not occur with peripheral infection with LCMV Armstrong (33, 38), indicating that interactions between the CNS and the immune system are contribute substantially to disease.During LCMV infection, there is biphasic production of IFN-γ: a small, early peak of IFN-γ (most likely produced by NK or NKT cells), followed by T-cell-mediated production of IFN-γ (23, 75). Further, both CD4⁺ T cells and CD8⁺ T cells produce large amounts of IFN-γ after LCMV infection and T-cell production of IFN-γ is critical for LCMV-induced weight loss (35). Chemokines, especially CXCL10, CCL5, and CCL2, and their receptors, are upregulated in the brain after i.c. LCMV infection (2, 13). Brain chemokine mRNA expression after i.c. LCMV infection is reduced in IFN-γ-deficient mice and relatively absent in athymic mice (2). However, the mechanism(s) by which T cells and IFN-γ mediate the effects on CNS chemokine expression, cellular infiltration into the CNS, and LCMV-induced anorexic weight loss remain unclear.In the present study, we focused on two major questions. The first question concerned the role of IFN-γ on immune cell recruitment to and chemokine/cytokine production within the CNS? We found that macrophages and myeloid dendritic cells (DCs) require IFN-γ for their accumulation within the CNS. Second, since macrophages and myeloid DCs are the predominant cellular infiltrate, we sought to determine whether IFN-γ signaling on these cells was direct with regard to their recruitment and to chemokine/cytokine production. We found that IFN-γ signaling in macrophage lineage cells contributes significantly to their recruitment, to chemokine production in the CNS, and to anorexic weight loss. Together, these data suggest that much of the proinflammatory effects of IFN-γ in the CNS are mediated by the effects of IFN-γ on CD68-bearing cells.     

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ^−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα^−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ^−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.Adaptation of pancreatic islet mass and function relative to metabolic demand maintains glucose homeostasis and may prevent the development of type 2 diabetes. β-Cell proliferation, apoptosis, growth, and function are tightly regulated by various extracellular factors and intracellular signaling pathways (23, 24, 34). In β-cells, insulin receptor substrate 2 (IRS2) controls maintenance and expansion of islet mass (29, 31, 42). In fact, IRS2-deficient mice are insulin resistant, show β-cell failure and hyperglycemia, and finally develop diabetes (26, 42). In contrast, deficiency of IRS1 only causes insulin resistance without the development of diabetes due to a compensatory increase in functional β-cell mass (1, 38). These observations indicated that IRS2, but not IRS1, is necessary for maintenance and compensatory increase of β-cell mass. Furthermore, experiments with isolated islets revealed that overexpression of IRS2, but not of IRS1, can increase β-cell proliferation and protect cells against high-glucose-induced apoptosis (29). Downstream of IRS2, phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) signaling is considered to be the critical pathway for the regulation of β-cell mass and function (12, 15, 16, 27). The serine-threonine kinase PKB, also known as Akt, is required for various cellular processes, from the regulation of cell cycle, survival, and growth to glucose and protein metabolism. In mammals, three PKB/Akt isoforms have been characterized and named PKBα/Akt1, PKBβ/Akt2, and PKBγ/Akt3. Although encoded by different genes on different chromosomes, the three isoforms display high homology at the protein level with 80 to 85% identical residues and the same structural organization (43). However, they differ in terms of tissue-specific expression. PKBα is expressed in most tissues and PKBβ is highly expressed in insulin-responsive tissues, whereas PKBγ expression is prominent in the brain and testes (17). All three isoforms are expressed in β-cells (30, 37). The roles of PKB in different tissues have been studied in transgenic-mouse models. While Pkbα^−/− and Pkbγ^−/− mice show impaired fetal growth and brain development, respectively, glucose homeostasis is unaffected in both models (9, 11, 14, 39, 46). In contrast, Pkbβ^−/− mice are insulin resistant and mildly glucose intolerant and have less adipose tissue. Depending on the strain and gender, these mice show either late loss of β-cells followed by the development of diabetes and mild growth deficiency or compensatory increase of β-cell mass without age-dependent progression into overt hyperglycemia (10, 17). These studies suggested that PKBβ is the only isoform playing a role in the regulation of energy homeostasis. On the other hand, constitutive activation of PKBα in β-cells is sufficient to increase growth and proliferation (5, 40), and in INS1 cells it prevents free fatty acid (FFA)-induced apoptosis (44). Furthermore, antagonizing total PKB activity in β-cells by ectopic expression of a kinase-dead mutant causes defects in insulin secretion (4), suggesting that in islets PKB is required mainly for normal function of the β-cells. Although these data support the notion that PKB must play a role in pancreatic β-cells, they are not in line with the stronger metabolic phenotype displayed by IRS2-deficient mice. In fact, PKBα and PKBγ appear not to be required to regulate glucose homeostasis (9, 11, 39), and in the case of Pkbβ^−/− mice, even though glucose homeostasis is impaired due to strong peripheral insulin resistance, the overall metabolic phenotype is far less severe than in Irs2^−/− mice (10), indicating that the capacity for β-cell compensation is retained in the absence of PKBβ.The aim of this study was to clarify the role of PKB in the regulation of islet mass and to define the relevance of PKB isoforms for IRS2 action in β-cells. Although it had been shown that PKBα is dispensable for the regulation of glucose homeostasis (9, 11), we found lower blood glucose concentrations in Pkbα^−/− mice. Based on this observation, we assessed in more detail the metabolic and the endocrine pancreatic phenotypes of Pkbα-, Pkbβ-, or Pkbγ-deficient mice. In addition, glucose uptake into fat cells, insulin secretion, and islet cell proliferation were investigated. Contrary to previous assumptions implying that PKBβ is the only (or at least the main) isoform playing a role in the regulation of glucose metabolism, we present evidence that both PKBα and PKBβ isoforms are required in the periphery for regulation of glucose homeostasis. While we confirmed that Pkbβ^−/− mice are insulin resistant and glucose intolerant with compensatory increase of β-cell mass, Pkbα^−/− mice showed lower blood glucose levels, were more insulin sensitive, and revealed higher serum glucagon concentrations accompanied by a mild increase in α-cell mass and proliferation. Moreover, our in vitro experiments showed that PKBα is specifically activated by IRS2 in β-cells and that its activation is required for IRS2-induced proliferation in islets.