Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum.

Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). In this study, we demonstrate that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori.

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

A Ca2+-binding motif underlies the unusual properties of certain photosynthetic bacterial core light-harvesting complexes.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca2+-dependent. However, it is unknown what structural determinants might contribute to these properties. Here, we present a cryo-EM structure of the reaction center-associated LH1 complex at 2.81 Å resolution, in which we identify multiple pigment-binding α- and ß-polypeptides within an LH1 ring. Of the 16 α-polypeptides, we show that six (α1) bind Ca2+ along with ß1- or ß3-polypeptides to form the Ca2+-binding sites. This structure differs from that of fully Ca2+-bound LH1 from Thermochromatium tepidum, enabling determination of the minimum structural requirements for Ca2+-binding. We also identified three amino acids (Trp44, Asp47, and Ile49) in the C-terminal region of the A. tepidum α1-polypeptide that ligate each Ca ion, forming a Ca2+-binding WxxDxI motif that is conserved in all Ca2+-bound LH1 α-polypeptides from other species with reported structures. The partial Ca2+-bound structure further explains the unusual phenotypic properties observed for this bacterium in terms of its Ca2+-requirements for thermostability, spectroscopy, and phototrophic growth, and supports the hypothesis that A. tepidum may represent a "transitional" species between mesophilic and thermophilic purple sulfur bacteria. The characteristic arrangement of multiple αß-polypeptides also suggests a mechanism of molecular recognition in the expression and/or assembly of the LH1 complex that could be regulated through interactions with reaction center subunits.

Localization of SNARE proteins in the brain and corpus allatum of Bombyx mori.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyx mori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and central complex (CX) of the brain. SNAP-25 was restricted to neurons in the PI and the CX of the brain. Syntaxin 1A was restricted to neurons in the PI and DL of the brain. VAMP2 co-localized with SNAP-25 in the CX, and with Syntaxin 1A in the PI and DL. VAMP2, SNAP-25, and Syntaxin 1A are present in the CA. Bombyxin-immunohistochemical reactivities (IRs) of brain and CA overlapped with VAMP2-, SNAP-25, and Syntaxin 1A-IRs. VAMP2 and Syntaxin 1A are present in the prothoracicotropic hormone (PTTH)-secretory neurons of the brain.

Expression analysis of peptidergic enteroendocrine cells in the silkworm Bombyx mori.

Enteroendocrine cells (ECs) in the insect midgut respond to physiological changes in the intestine by releasing multiple peptides to control food intake, gastrointestinal activity and systemic metabolism. Here, we performed a comprehensive mapping of ECs producing different regulatory peptides in the larval midgut of Bombyx mori. In total, we identified 20 peptide genes expressed in different ECs in specific regions of the midgut. Transcript-specific in situ hybridisation combined with antibody staining revealed approximately 30 subsets of ECs, each producing a unique peptide or a combination of several different peptides. Functional significance of this diversity and specific roles of different enteroendocrine peptides are largely unknown. Results of this study highlight the importance of the midgut as a major endocrine/paracrine source of regulatory molecules in insects and provide important information to clarify functions of ECs during larval feeding and development.

Cryo-EM Structure of the Photosynthetic LH1-RC Complex from Rhodospirillum rubrum.

Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl aG) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αß-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the RC. The geranylgeraniol side chains of BChl aG coordinated by LH1 ß-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αß-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

Structural insights into the targeting specificity of ubiquitin ligase for S. cerevisiae isocitrate lyase but not C. albicans isocitrate lyase.

In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S.cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 Å, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S.cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

Critical Amino Acid Variants in HLA-DRB1 and -DQB1 Allotypes in the Development of Classical Type 1 Diabetes and Latent Autoimmune Diabetes in Adults in the Japanese Population.

The effects of amino acid variants encoded by the human leukocyte antigen (HLA) class II on the development of classical type 1 diabetes (T1D) and latent autoimmune diabetes in adults (LADA) have not been fully elucidated. We retrospectively investigated the HLA-DRB1 and -DQB1 genes of 72 patients with classical T1D and 102 patients with LADA in the Japanese population and compared the frequencies of HLA-DRB1 and -DQB1 alleles between these patients and the Japanese populations previously reported by another institution. We also performed a blind association analysis with all amino acid positions in classical T1D and LADA, and compared the associations of HLA-DRB1 and -DQB1 amino acid positions in classical T1D and LADA. The frequency of DRß-Phe-13 was significantly higher and those of DRß-Arg-13 and DQß-Gly-70 were significantly lower in patients with classical T1D and LADA than in controls. The frequencies of DRß-His-13 and DQß-Glu-70 were significantly higher in classical T1D patients than in controls. The frequency of DRß-Ser-13 was significantly lower and that of DQß-Arg-70 was significantly higher in LADA patients than in controls. HLA-DRß1 position 13 and HLA-DQß1 position 70 could be critical amino acid positions in the development of classical T1D and LADA.

Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell cycle progression.

Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.

Peripheral combination treatment of leptin and an SGLT2 inhibitor improved glucose metabolism in insulin-dependent diabetes mellitus mice.

We investigated whether peripheral combination treatment of a sodium-glucose cotransporter 2 (SGLT2) inhibitor and leptin improves glucose metabolism in insulin-dependent diabetes mellitus (IDDM) model mice. Twelve-week-old male C57BL6 mice were intraperitoneally administered a high dose of streptozotocin to produce IDDM. IDDM mice were then divided into five groups: SGLT2 inhibitor treatment alone, leptin treatment alone, leptin and SGLT2 inhibitor co-treatment, untreated IDDM mice, and healthy mice groups. The blood glucose (BG) level at the end of the dark cycle was measured, and a glucose tolerance test (GTT) was performed and compared between the five groups. Leptin was peripherally administered at 20 µg/day using an osmotic pump, and an SGLT2 inhibitor, ipragliflozin, was orally administered at 3 mg/kg/day. Monotherapy with SGLT2 inhibitor or leptin significantly improved glucose metabolism in mice as evaluated by BG and GTT compared with the untreated group, whereas the co-treatment group with SGLT2 inhibitor and leptin further improved glucose metabolism as compared with the monotherapy group. Notably, glucose metabolism in the co-treatment group improved to the same level as that in the healthy mice group. Thus, peripheral combination treatment with leptin and SGLT2 inhibitor improved glucose metabolism in IDDM mice without the use of insulin.

Thrombomodulin ameliorates transforming growth factor-ß1-mediated chronic kidney disease via the G-protein coupled receptor 15/Akt signal pathway.

Kidney fibrosis is the common consequence of chronic kidney diseases that inexorably progresses to end-stage kidney disease with organ failure treatable only with replacement therapy. Since transforming growth factor-ß1 is the main player in the pathogenesis of kidney fibrosis, we posed the hypothesis that recombinant thrombomodulin can ameliorate transforming growth factor-ß1-mediated progressive kidney fibrosis and failure. To interrogate our hypothesis, we generated a novel glomerulus-specific human transforming growth factor-ß1 transgenic mouse to evaluate the therapeutic effect of recombinant thrombomodulin. This transgenic mouse developed progressive glomerular sclerosis and tubulointerstitial fibrosis with kidney failure. Therapy with recombinant thrombomodulin for four weeks significantly inhibited kidney fibrosis and improved organ function compared to untreated transgenic mice. Treatment with recombinant thrombomodulin significantly inhibited apoptosis and mesenchymal differentiation of podocytes by interacting with the G-protein coupled receptor 15 to activate the Akt signaling pathway and to upregulate the expression of anti-apoptotic proteins including survivin. Thus, our study strongly suggests the potential therapeutic efficacy of recombinant thrombomodulin for the treatment of chronic kidney disease and subsequent organ failure.

A novel CDK-independent function of p27Kip1 in preciliary vesicle trafficking during ciliogenesis.

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

Functional characterization of insect-specific RabX6 of Bombyx mori.

Rab proteins are low-molecular weight (20-25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori.

A Blood-Brain-Barrier-Penetrating Anti-human Transferrin Receptor Antibody Fusion Protein for Neuronopathic Mucopolysaccharidosis II.

Mucopolysaccharidosis II (MPS II) is an X-linked recessive lysosomal storage disease caused by mutations in the iduronate-2-sulfatase (IDS) gene. Since IDS catalyzes the degradation of glycosaminoglycans (GAGs), deficiency in this enzyme leads to accumulation of GAGs in most cells in all tissues and organs, resulting in severe somatic and neurological disorders. Although enzyme replacement therapy with human IDS (hIDS) has been used for the treatment of MPS II, this therapy is not effective for defects in the CNS mainly because the enzyme cannot cross the blood-brain barrier (BBB). Here, we developed a BBB-penetrating fusion protein, JR-141, which consists of an anti-human transferrin receptor (hTfR) antibody and intact hIDS. The TfR-mediated incorporation of JR-141 was confirmed by using human fibroblasts in vitro. When administrated intravenously to hTfR knockin mice or monkeys, JR-141, but not naked hIDS, was detected in the brain. In addition, the intravenous administration of JR-141 reduced the accumulation of GAGs both in the peripheral tissues and in the brain of hTfR knockin mice lacking Ids, an animal model of MPS II. These data provide a proof of concept for the translation of JR-141 to clinical study for the treatment of patients with MPS II with CNS disorders.

Tetraploidy in cancer and its possible link to aging.

Tetraploidy, a condition in which a cell has four homologous sets of chromosomes, is often seen as a natural physiological condition but is also frequently seen in pathophysiological conditions such as cancer. Tetraploidy facilitates chromosomal instability (CIN), which is an elevated level of chromosomal loss and gain that can cause production of a wide variety of aneuploid cells that carry structural and numerical aberrations of chromosomes. The resultant genomic heterogeneity supposedly expedites karyotypic evolution that confers oncogenic potential in spite of the reduced cellular fitness caused by aneuploidy. Recent studies suggest that tetraploidy might also be associated with aging; mice with mutations in an intermediate filament protein have revealed that these tetraploidy-prone mice exhibit tissue disorders associated with aging. Cellular senescence and its accompanying senescence-associated secretory phenotype have now emerged as critical factors that link tetraploidy and tetraploidy-induced CIN with cancer, and possibly with aging. Here, we review recent findings about how tetraploidy is related to cancer and possibly to aging, and discuss underlying mechanisms of the relationship, as well as how we can exploit the properties of cells exhibiting tetraploidy-induced CIN to control these pathological conditions.

Roles of afadin in functional differentiations of hippocampal mossy fiber synapse.

A hippocampal mossy fiber synapse has a complex structure and is implicated in learning and memory. In this synapse, the mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions and wrap around a multiply-branched spine, forming synaptic junctions. We have recently shown using transmission electron microscopy, immunoelectron microscopy and serial block face-scanning electron microscopy that atypical puncta adherentia junctions are formed in the afadin-deficient mossy fiber synapse and that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities and the density of synaptic vesicles docked to active zones are decreased in the afadin-deficient synapse. We investigated here the roles of afadin in the functional differentiations of the mossy fiber synapse using the afadin-deficient mice. The electrophysiological studies showed that both the release probability of glutamate and the postsynaptic responsiveness to glutamate were markedly reduced, but not completely lost, in the afadin-deficient mossy fiber synapse, whereas neither long-term potentiation nor long-term depression was affected. These results indicate that afadin plays roles in the functional differentiations of the presynapse and the postsynapse of the hippocampal mossy fiber synapse.

NGL-3-induced presynaptic differentiation of hippocampal neurons in an afadin-dependent, nectin-1-independent manner.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. ß-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with ß-catenin and γ-catenin in a nectin-1-independent manner.

Roles of afadin in the formation of the cellular architecture of the mouse hippocampus and dentate gyrus.

The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.

PTP1B deficiency improves hypothalamic insulin sensitivity resulting in the attenuation of AgRP mRNA expression under high-fat diet conditions.

Hypothalamic insulin receptor signaling regulates energy balance and glucose homeostasis via agouti-related protein (AgRP). While protein tyrosine phosphatase 1B (PTP1B) is classically known to be a negative regulator of peripheral insulin signaling by dephosphorylating both insulin receptor ß (IRß) and insulin receptor substrate, the role of PTP1B in hypothalamic insulin signaling remains to be fully elucidated. In the present study, we investigated the role of PTP1B in hypothalamic insulin signaling using PTP1B deficient (KO) mice in vivo and ex vivo. For the in vivo study, hypothalamic insulin resistance induced by a high-fat diet (HFD) improved in KO mice compared to wild-type (WT) mice. Hypothalamic AgRP mRNA expression levels were also significantly decreased in KO mice independent of body weight changes. In an ex vivo study using hypothalamic organotypic cultures, insulin treatment significantly increased the phosphorylation of both IRß and Akt in the hypothalamus of KO mice compared to WT mice, and also significantly decreased AgRP mRNA expression levels in KO mice. While incubation with inhibitors of phosphatidylinositol-3 kinase (PI3K) had no effect on basal levels of Akt phosphorylation, these suppressed insulin induction of Akt phosphorylation to almost basal levels in WT and KO mice. The inhibition of the PI3K-Akt pathway blocked the downregulation of AgRP mRNA expression in KO mice treated with insulin. These data suggest that PTP1B acts on the hypothalamic insulin signaling via the PI3K-Akt pathway. Together, our results suggest a deficiency of PTP1B improves hypothalamic insulin sensitivity resulting in the attenuation of AgRP mRNA expression under HFD conditions.