Yoshiyuki Soeda

Abstract Prior to the formation of amyloid fibrils, the pathological hallmark in tau-related neurodegenerative disease, tau monomers aggregate into a diverse range of oligomers. Granular tau oligomers, consisting of approximately 40 tau protein molecules, are present in the prefrontal cortex of patients at Braak stages I-II, preclinical stages of Alzheimer’s disease (AD). Antibodies to granular tau oligomers as antigens have not been reported. Therefore, we generated new rat monoclonal antibodies by immunization with granular tau oligomers. Three antibodies from different hybridoma clones showed stronger immunoreactivity to granular tau oligomers and tau fibrils compared with monomeric tau. Of the three antibodies, 2D6-2C6 showed 3000-fold greater immunoreactivity in P301L-tau transgenic (rTg4510) mice than in non-transgenic mice, while MC1 antibody, which detects pathological conformations of tau, showed a 5.5-fold increase. These results suggest that 2D6-2C6 recognizes aggregates more specifically than MC1. In AD subjects, 2D6-2C6 recognized neurofibrillary tangles and pretangles, and co-localized within AT8-positive cells containing phosphorylated tau aggregates. The epitope of 2D6-2C6 is the 423–430 amino acid (AA) sequence of C-terminal regions. Taken together, a novel monoclonal antibody, 2D6-2C6, generated by immunization with granular tau oligomers binds to tau aggregates at the 423–430 AA sequence.

Tauopathy is a group of diseases characterized by fibrillary tau aggregate formation in neurons and glial cells of the brain. Tau aggregation originates in the brainstem and entorhinal cortex and then spreads throughout the brain in Alzheimer’s disease (AD), which is the most prevalent form of tauopathy. Understanding the mechanism by which locally formed tau pathology propagates throughout the brain is crucial for comprehending AD pathogenesis. Therefore, a novel model of tau pathology that artificially induces tau aggregation in targeted cells at specific times is essential. This study reports a novel optogenetic module, OptoTau, human tau with the P301L mutation fused with a photosensitive protein CRY2Olig, which induced various forms of tau according to the temporal pattern of blue light illumination pattern. Continuous blue light illumination for 12 h to Neuro2a cells stably expressing OptoTau (OptoTauKI cells) formed clusters along microtubules, many of which eventually accumulated in aggresomes. Conversely, stable tau aggregates were formed when alternating light exposure and darkness in 30-min cycles for 8 sets per day were repeated over 8 days. Stable tau aggregates were induced more rapidly by repeating cycles of 5-min illumination followed by 25 min of darkness over 24 h. These results indicate that OptoTau induced various tau aggregation stages based on the temporal pattern of blue light exposure. Thus, this technique demonstrates potential as a novel approach to developing specific tau aggregation in targeted cells at desired time points. Significance This study developed a technique to manipulate tau aggregation in a blue light-dependent manner using cells that stably express OptoTau, which is an optogenetic tool based on the CRY2Olig module. Tau accumulation in aggresomes or insoluble tau aggregation were specifically induced in response to blue light illumination conditions. These results are crucial as they provide a new technological basis for developing a singular point of tau aggregation in specific targeted cells at a specific time.

Alzheimer's disease is a devastating disease that is accompanied by dementia, and its incidence increases with age. However, no interventions have exhibited clear therapeutic effects. We aimed to develop and characterize behavioural tasks that allow the earlier identification of signs preceding dementia that would facilitate the development of preventative and therapeutic interventions for Alzheimer's disease. To this end, we developed a 3D virtual reality task sensitive to the activity of grid cells in the entorhinal cortex, which is the region that first exhibits neurofibrillary tangles in Alzheimer's disease. We investigated path integration (assessed by error distance) in a spatial navigation task sensitive to grid cells in the entorhinal cortex in 177 volunteers, aged 20-89 years, who did not have self-reported dementia. While place memory was intact even in old age, path integration deteriorated with increasing age. To investigate the relationship between neurofibrillary tangles in the entorhinal cortex and path integration deficit, we examined a mouse model of tauopathy (P301S mutant tau-overexpressing mice; PS19 mice). At 6 months of age, PS19 mice showed a significant accumulation of phosphorylated tau only in the entorhinal cortex, associated with impaired path integration without impairments in spatial cognition. These data are consistent with the idea that path integration deficit is caused by the accumulation of phosphorylated tau in the entorhinal cortex. This method may allow the early identification of individuals likely to develop Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of extracellular amyloid-beta peptides (Aβ) resulting in senile plaques and intracellular hyperphosphorylated tau protein resulting in neurofibrillary tangles (NFTs). Mucuna beans (Mucuna pruriences (L.) DC. var. utilis) are unique plants containing 3-9% L-3,4-dihydroxyphenylalanine (L-DOPA). Here we investigated the effect of the administration of Mucuna beans on AD prevention by feeding triple-transgenic mice (3 × Tg-AD mice) with a diet containing Mucuna beans for 13 months. The levels of Aβ oligomers and detergent-insoluble phosphorylated tau decreased in the brain of mice fed with Mucuna beans (Mucuna group) compared to those of the Control group. Aβ accumulation and phosphorylated tau accumulation in the brain in the Mucuna group were also reduced. In addition, administration of Mucuna beans improved cognitive function. These results suggest that administration of Mucuna beans may have a preventive effect on AD development in 3 × Tg-AD mice.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) as senile plaques and cerebral amyloid angiopathy, and hyperphosphorylated tau (hp-tau) as neurofibrillary tangles in the brain. The AD-related pathology has been reported in several non-human animals, and most animals develop only the Aβ or tau pathology. We herein describe the Aβ and hp-tau pathology in the brains of aged pinniped species (seal, sea lion, and walrus). Molecular analyses revealed that the sequence of pinniped Aβ was identical to that of human Aβ. Histopathological examinations detected argyrophilic plaques composed of Aβ associated with dystrophic neurites in the cerebral cortex of aged pinnipeds. Astrogliosis and microglial infiltration were identified around Aβ plaques. Aβ deposits were observed in the blood vessel walls of the meninges and cerebrum. Pinniped tau protein was physiologically subjected to alternative splicing at exons 2, 3, and 10, and presented as five isoforms: two 3-repeat tau isoforms (1N3R, 2N3R) and three 4-repeat tau isoforms (0N4R, 1N4R, 2N4R); 0N3R tau isoform was absent. Histopathological examinations revealed argyrophilic fibrillar aggregates composed of hp-tau in the neuronal somata and neurites of aged pinniped brains. Few hp-tau aggregates were found in oligodendrocytes and microglia. Biochemically, hp-tau of the 3-repeat and 4-repeat isoforms was detected in brain sarkosyl-insoluble fractions. Aβ and hp-tau both predominantly accumulated in the neocortex, particularly the frontal cortex. Furthermore, the activation of GSK-3β was detected within cells containing hp-tau aggregates, and activated GSK-3β was strongly expressed in cases with severe hp-tau pathologies. The present results suggest that, in association with Aβ deposition, the activation of GSK-3β contributes to hp-tau accumulation in pinniped brains. Here, we report that pinniped species naturally accumulate Aβ and tau with aging, similar to the human AD pathology.

Glycogen synthase kinase 3β (GSK-3β) is a therapeutic target for various age-related neurodegenerative diseases. It is linked to the two main pathological features of Alzheimer's disease (AD), tau and amyloid β (Aβ); GSK-3β is a major candidate to pathologically hyperphosphorylate tau and modulate Aβ production. However, inhibition of GSK-3β in clinical studies in humans has been found to not significantly improve cognitive function of AD patients, prompting us to study the physiological role of GSK-3β in old mice. Using a contextual fear-conditioning paradigm, we now report that old gsk-3β+/- mice are deficient in both short-term and long-term memory formation, suggesting that GSK-3β is required for memory formation at old age. Biochemical and immunohistochemical analyses showed that the number of synapses does not differ between gsk-3β+/- and age-matched wild-type (wt) littermate mice. Based on these observations, we propose that, GSK-3β may contribute to help maintain brain function during aging. Our results may explain the poor efficacy of GSK-3β inhibitors in preserving memory capacity in AD patients.

Microtubule-associated protein tau is characterized by the fact that it is an intrinsically disordered protein due to its lack of a stable conformation and high flexibility. Intracellular inclusions of fibrillar forms of tau with a β-sheet structure accumulate in the brain of patients with Alzheimer's disease and other tauopathies. Accordingly, detachment of tau from microtubules and transition of tau from a disordered state to an abnormally aggregated state are essential events preceding the onset of tau-related diseases. Many reports have shown that this transition is caused by post-translational modifications, including hyperphosphorylation and acetylation. The misfolded tau is self-assembled and forms a tau oligomer before the appearance of tau inclusions. Animal and pathological studies using human samples have demonstrated that tau oligomer formation contributes to neuronal loss. During the progression of tauopathies, tau seeds are released from cells and incorporated into other cells, leading to the propagation of pathological tau aggregation. Accumulating evidence suggests several potential approaches for blocking tau-mediated toxicity: (1) direct inhibition of pathological tau aggregation and (2) inhibition of tau post-translational modifications that occur prior to pathological tau aggregation, (3) inhibition of tau propagation and (4) stabilization of microtubules. In addition to traditional low-molecular-weight compounds, newer drug discovery approaches such as the development of medium-molecular-weight drugs (peptide- or oligonucleotide-based drugs) and high-molecular-weight drugs (antibody-based drugs) provide alternative pathways to preventing the formation of abnormal tau. Of particular interest are recent studies suggesting that tau droplet formation by liquid-liquid phase separation may be the initial step in aberrant tau aggregation, as well results that implicate roles for tau in dendritic and nuclear functions. Here, we review the mechanisms through which drugs can target tau and consider recent clinical trials for the treatment of tauopathies. In addition, we discuss the utility of these newer strategies and propose future directions for research on tau-targeted therapeutics.

Alzheimer's disease pathology is characterized by extracellular deposits of amyloid-β (Aβ) and intracellular inclusions of hyperphosphorylated tau. Although genetic studies of familial Alzheimer's disease suggest a causal link between Aβ and disease symptoms, the failure of various Aβ-targeted strategies to slow or halt disease progression has led to consideration of the idea that inhibition of tau aggregation might be a more promising therapeutic approach. Methylene blue (MB), which inhibits tau aggregation and rescue memory deficits in a mouse model of tauopathy, however, lacked efficacy in a recent Phase III clinical trial. In order to gain insight into this failure, the present study was designed to examine the mechanism through which MB inhibits tau aggregation. We found that MB inhibits heparin-induced tau aggregation in vitro, as measured by thioflavin T fluorescence. Further, MB reduced the amount of tau in precipitants recovered after ultracentrifugation of the aggregation mixture. Atomic force microscopy revealed that MB reduces the number of tau fibrils but increases the number of granular tau oligomers. The latter result was confirmed by sucrose gradient centrifugation: MB treatment was associated with higher levels of granular tau oligomers (fraction 3) and lower levels of tau fibrils (fractions 5 and 6). We previously demonstrated that the formation of granular tau oligomers, rather than tau fibrils, is essential for neuronal death. Thus, the fact that MB actions are limited to inhibition of tau fibril formation provides a mechanistic explanation for the poor performance of MB in the recent Phase III clinical trial.

Tau is a microtubule-associated protein, localizing mainly in the axon of mature neurons. Phenotypic analysis of Tau knockout mice has revealed an impairment of synaptic plasticity but without gross changes in brain morphology. Since we previously described the presence of tau mRNA in the somatodendritic compartment, including the postsynapse, and demonstrated that it could be locally translated in response to glutamate, it appears that the regulated translation of synaptic tau can have a direct impact on synaptic function. Using SH-SY5Y cells, we herein confirm that glutamate dose-dependently regulates the translation of tau protein without altering tau mRNA levels. This is supported by the finding that cycloheximide blocks glutamate-stimulated increases in tau protein levels. Our observation that neural excitation can directly upregulate tau mRNA translation helps explain the pathological accumulation of tau in the somatodendrite.

Hippocampal hyperactivity, ascribed to amyloid β (Aβ)-induced imbalances in neural excitation and inhibition, is found in patients with mild cognitive impairment, a prodromal stage of Alzheimer's disease (AD). To better understand the relationship between hippocampal hyperactivity and the molecular triggers of behavioral impairments in AD, we used Mn-enhanced MRI (MEMRI) to assess neuronal activity after subjecting mice to a task requiring spatial learning and memory. Depletion of endogenous tau in an amyloid precursor protein (APP) transgenic (J20) mouse line was shown to ameliorate hippocampal hyperactivity in J20 animals, tau depletion failed to reverse memory deficits associated with APP/Aβ overproduction. On the other hand, deletion of tau alleviated the hyperlocomotion displayed by APP transgenics, suggesting that the functional effects of Aβ-tau interactions reflect the temporal appearance of these molecules in individual brain areas.

Tau is a major component of the neurofibrillary tangles (NFT) that represent a pathological hallmark of Alzheimer's disease (AD). Although generally considered an axonal protein, Tau is found in the somato-dendritic compartment of degenerating neurons and this redistribution is thought to be a trigger of neurodegeneration in AD. Here, we show the presence of tau mRNA in a dendritic ribonucleoprotein (RNP) complex that includes Ca2+-calmodulin dependent protein kinase (CaMK)IIα mRNA and that is translated locally in response to glutamate stimulation. Further, we show that Tau mRNA is a component of mRNP granules that contain RNA-binding proteins, and that it interacts with Myosin Va, a postsynaptic motor protein; these findings suggest that tau mRNA is transported into dendritic spines. We also report that tau mRNA localized in the somato-dendritic component of primary hippocampal cells and that a sub-toxic concentration of glutamate enhances local translation and hyperphosphorylation of tau, effects that are blocked by the gluatamatergic antagonists MK801 and NBQX. These data thus demonstrate that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-d-aspartate (NMDA) stimulation redistributes tau to the somato-dendritic region of neurons where it may trigger neurodegeneration.

Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid β peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease.

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies.

The carboxyl-terminal sequence of tau composes the framework for its intracellular inclusions that appear in diverse neurodegenerative disorders known as tauopathies. However, microtubule-associated protein 2 (MAP2), which contains a homologous carboxyl-terminal sequence of tau, is undetectable in the mature tau inclusions. The mechanisms underlying this phenomenon have remained largely unknown. Here, we show that tau and MAP2 have different aggregation properties: tau aggregates to form filaments but MAP2 remains to be granules. Exchanging (221) YKPV(224) of tau (0N3R) near the PHF6 motif for (340) TKKI(343) of MAP2c profoundly changed aggregation properties, suggesting that the YKPV motif is important for filament formation, whereas the TKKI motif is for granule formation. Thus, these minimal sequences may determine the different fates of tau and MAP2 in the formation of inclusions in tauopathies. Tau and microtubule-associated protein 2 (MAP2) are homologous microtubule-associated proteins in neurons. So far, it is largely unknown why tau but not MAP2 is selectively involved in the filamentous inclusions (neurofibrillary tangles, NFT) formation in tauopathies, including Alzheimer's disease. In this study, we found that the difference of only two amino acids in tau and MAP2 sequences may determine their different fates in tauopathies. These results may lead to the elucidation of tau deregulation in pathological conditions.

In tauopathies, a neural microtubule-associated protein tau (MAPT) is abnormally aggregated and forms neurofibrillary tangle. Therefore, inhibition of the tau aggregation is one of the key approaches for the treatment of these diseases. Here, we have identified a novel tau aggregation inhibitor, PE859. An oral administration of PE859 resulted in the significant reduction of sarkosyl-insoluble aggregated tau along with the prevention of onset and progression of the motor dysfunction in JNPL3 P301L-mutated human tau transgenic mice. These results suggest that PE859 is useful for the treatment of tauopathies.

In an aging society, the number of people with dementia has increased. Since Alzheimer's disease and a part of frontotemporal lober degeneration (FTLD-tau) have abnormal tau pathology in brain, these are called Tauopathy. Previously results showed that occurrence of abnormal tau has been involved in development of cognitive dysfunction and neuronal loss indicating that tau-focused drug (inhibitors of tau hyperphosphorylation, tau aggregation and so on) may be valuable in therapy for Tauopathy. This study collated data of clinical trials that evaluated tau-based drugs to help development of agent for dementia drugs in future. We discovered a novel tau aggregation inhibitor, and elucidated the inhibitory mechanism of the compound on tau aggregation. These results suggest that tau aggregation is an important target for therapy of dementia.

SH2-containing inositol 5′-phosphatase 2 (SHIP2) is a lipid phosphatase that negatively regulates the metabolic signalling of insulin in peripheral tissues; however, the expression of SHIP2 in the hypothalamus and its functional roles are largely unknown. In the present study, immunohistochemical analysis demonstrated that SHIP2 protein exists in neuronal cells expressing neuropeptide Y and pro-opiomelanocortin in the arcuate nucleus of the hypothalamus in C57BL/6J mice. Interestingly, the expression levels of SHIP2 in the hypothalamus were elevated in aged C57BL/6J mice and diabetic db/db mice. To clarify the significance of the increased expression of SHIP2 in the hypothalamus, we examined the central effects of insulin and leptin in transgenic mice overexpressing SHIP2 (SHIP2-Tg). Accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of Akt in the hypothalamus, induced by i.c.v. injection of insulin, were attenuated in SHIP2-Tg compared to wild-type mice, whereas leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the hypothalamus was comparable between them. The suppression of food intake after i.c.v. administration of insulin (but not leptin) was attenuated consistently in SHIP2-Tg. In addition, SHIP2-Tg showed increased food consumption after starvation and become heavier with visceral fat accumulation than wild-type mice, despite normal levels of oxygen consumption and spontaneous movement. These results suggest that SHIP2 contributes to the regulation of food intake mainly via the attenuation of insulin signalling in the hypothalamus of mice. © 2013 British Society for Neuroendocrinology.

Oxidative stress is considered to promote aging and age-related disorders such as tauopathy. Although recent reports suggest that oxidative stress under certain conditions possesses anti-aging properties, no such conditions have been reported to ameliorate protein-misfolding diseases. Here, we used neuronal and murine models that overexpress human tau to demonstrate that mild oxidative stress generated by alloxan suppresses several phenotypes of tauopathy. Alloxan treatment reduced HSP90 levels and promoted proteasomal degradation of tau, c-Jun N-amino terminal kinase, and histone deacetylase (HDAC) 6. Moreover, reduced soluble tau (phosphorylated tau) levels suppressed the formation of insoluble tau in tau transgenic mice, while reduced HDAC6 levels contributed to microtubule stability by increasing tubulin acetylation. Age-dependent decreases in HDAC2 and phospho-tau levels correlated with spatial memory enhancement in alloxan-injected tau mice. These results suggest that mild oxidative stress, through adaptive stress responses, operates counteractively against some of the tauopathy phenotypes.

Impairment of insulin and IGF-I signaling in the brain is one of the causes of dementia associated with diabetes mellitus and Alzheimer's disease. However, the precise pathological processes are largely unknown. In the present study, we found that SH2-containing inositol 5'-phosphatase 2 (SHIP2), a negative regulator of phosphatidylinositol 3,4,5-trisphosphate-mediated signals, is widely expressed in adult mouse brain. When a dominant-negative mutant of SHIP2 was expressed in cultured neurons, insulin signaling was augmented, indicating physiological significance of endogenous SHIP2 in neurons. Interestingly, SHIP2 mRNA and protein expression levels were significantly increased in the brain of type 2 diabetic db/db mice. To investigate the impact of increased expression of SHIP2 in the brain, we further employed transgenic mice overexpressing SHIP2 and found that increased amounts of SHIP2 induced the disruption of insulin/IGF-I signaling through Akt. Neuroprotective effects of insulin and IGF-I were significantly attenuated in cultured cerebellar granule neurons from SHIP2 transgenic mice. Consistently, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay demonstrated that the number of apoptosis-positive cells was increased in cerebral cortex of the transgenic mice at an elderly age. Furthermore, SHIP2 transgenic mice exhibited impaired memory performance in the Morris water maze, step-through passive avoidance, and novel-object-recognition tests. Importantly, inhibition of SHIP2 ameliorated the impairment of hippocampal synaptic plasticity and memory formation in db/db mice. These results suggest that SHIP2 is a potent negative regulator of insulin/IGF-I actions in the brain, and excess amounts of SHIP2 may be related, at least in part, to brain dysfunction in insulin resistance with type 2 diabetes.

Aims/hypothesis: Orexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism. Methods: Orexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice. Results: We found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3β phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice. Conclusions/interpretation: Our results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age. © 2008 Springer-Verlag.

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a 5'-lipid phosphatase hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P(3) to PI(3,4)P(2) in the regulation of insulin signaling, and is shown to be increased in peripheral tissues of diabetic C57BL/KSJ-db/db mice. To clarify the impact of SHIP2 in the pathogenesis of insulin resistance with type 2 diabetes, we generated transgenic mice overexpressing SHIP2. The body weight of transgenic mice increased by 5.0% (P < 0.05) compared with control wild-type littermates on a normal chow diet, but not on a high-fat diet. Glucose tolerance and insulin sensitivity were mildly but significantly impaired in the transgenic mice only when maintained on the normal chow diet, as shown by 1.2-fold increase in glucose area under the curve over control levels at 9 months old. Insulin-induced phosphorylation of Akt was decreased in the SHIP2-overexpressing fat, skeletal muscle, and liver. In addition, the expression of hepatic mRNAs for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was increased, that for sterol regulatory element-binding protein 1 was unchanged, and that for glucokinase was decreased. Consistently, hepatic glycogen content was reduced in the 9-month-old transgenic mice. Structure and insulin content were histologically normal in the pancreatic islets of transgenic mice. These results indicate that increased abundance of SHIP2 in vivo contributes, at least in part, to the impairment of glucose metabolism and insulin sensitivity on a normal chow diet, possibly by attenuating peripheral insulin signaling and by altering hepatic gene expression for glucose homeostasis.