Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Post-stroke treatment with K-134, a phosphodiesterase 3 inhibitor, improves stroke outcomes in the stroke-prone spontaneously hypertensive rat model-A comparative evaluation of antiplatelet drugs.

Post-stroke antiplatelet therapy has been proved to reduce the risk of recurrent stroke; however, it may also increase the incidence of intracranial hemorrhage that could offset any benefits. Therefore, the balance between the benefits and risks of antiplatelet drugs is a critical issue to consider. In the present study, we have compared the effects of post-stroke administration of antiplatelet agents on functional outcomes in the stroke-prone spontaneously hypertensive rat (SHRSP), an established animal model that mimics human lacunar stroke and cerebral small vessel disease. We confirmed that a potent phosphodiesterase 3 (PDE3) inhibitor, K-134, significantly improved post-stroke survival rate and survival time, attenuated stroke-induced neurological deficits, and decreased the incidence of cerebral lesion caused by intracerebral hemorrhage and softening. Similarly, cilostazol showed beneficial effects, though to a lower extent with respect to the survival outcome and neurological symptoms. On the other hand, a P2Y12 inhibitor, clopidogrel significantly improved survival outcomes at the higher dose but caused massive bleeding in the brain at both low and high doses. In contrast, no hemorrhagic lesion was observed in K-134-treated SHRSPs despite its antiplatelet activity. Our findings indicate that K-134 may have a superior post-stroke therapeutic outcome in comparison to other antiplatelet drugs.

Dimerization of sortilin regulates its trafficking to extracellular vesicles.

Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids, and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretory or endocytic compartments of cells, is found in both EVs and cells. In many human diseases, including cancer and cardiovascular disorders, sortilin expression levels are atypically high. To elucidate the relationship between cardiovascular disease, particularly vascular calcification, and sortilin expression levels, we explored the trafficking of sortilin in both the intracellular and extracellular milieu. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Although recent reports have noted that sortilin is regulated by multiple post-translational modifications, the precise mechanisms of sortilin trafficking still need to be determined. Here, we show that sortilin forms homodimers with an intermolecular disulfide bond at the cysteine 783 (Cys783) residue, and because Cys783 can be palmitoylated, it could be shared via palmitoylation and an intermolecular disulfide bond. Formation of this intermolecular disulfide bond leads to trafficking of sortilin to EVs by preventing palmitoylation, which further promotes sortilin trafficking to the Golgi apparatus. Moreover, we found that sortilin-derived propeptide decreased sortilin homodimers within EVs. In conclusion, sortilin is transported to EVs via the formation of homodimers with an intermolecular disulfide bond, which is endogenously regulated by its own propeptide. Therefore, we propose that inhibiting dimerization of sortilin acts as a new therapeutic strategy for the treatment of EV-associated diseases, including vascular calcification and cancer.

Runx/Cbfb signaling regulates postnatal development of granular convoluted tubule in the mouse submandibular gland.

BACKGROUND: The rodent salivary gland is not fully developed at birth and the cellular definitive differentiation takes place postnatally. However, little is known about its molecular mechanism. RESULTS: Here we provide the loss-of-function genetic evidence that Runx signaling affects postnatal development of the submandibular gland (SMG). Core binding factor ß (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb was specifically expressed in the duct epithelium, specifically in the SMG. Epithelial Cbfb deficiency resulted in decrease in the size of the SMG and in the saliva secretion on postnatal day 35. The Cbfb mutant SMG specifically exhibited involution of the granular convoluted tubules (GCT), with a down-regulated expression of its marker genes, such as Klk1, Ngf, and Egf. The induction of GCT is under the control of androgens, and the Cbfb mutant SMG demonstrated down-regulated expression of Crisp3, an androgen-dependent transcript. Because the circulating testosterone or tissue dihydrotestosterone levels were not affected in the Cbfb mutants, it appears that Runx/Cbfb signaling regulate androgen receptor pathway, but does not affect the circulating testosterone levels or the enzymatic conversion to DHT. CONCLUSIONS: Runx signaling is important in the postnatal development of androgen-dependent GCT in the SMG.

Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake.

Adiponectin has been shown to stimulate fatty acid oxidation and enhance insulin sensitivity through the activation of AMP-activated protein kinase (AMPK) in the peripheral tissues. The effects of adiponectin in the central nervous system, however, are still poorly understood. Here, we show that adiponectin enhances AMPK activity in the arcuate hypothalamus (ARH) via its receptor AdipoR1 to stimulate food intake; this stimulation of food intake by adiponectin was attenuated by dominant-negative AMPK expression in the ARH. Moreover, adiponectin also decreased energy expenditure. Adiponectin-deficient mice showed decreased AMPK phosphorylation in the ARH, decreased food intake, and increased energy expenditure, exhibiting resistance to high-fat-diet-induced obesity. Serum and cerebrospinal fluid levels of adiponectin and expression of AdipoR1 in the ARH were increased during fasting and decreased after refeeding. We conclude that adiponectin stimulates food intake and decreases energy expenditure during fasting through its effects in the central nervous system.

Development of dentition: From initiation to occlusion and related diseases.

BACKGROUND: The development of dentition begins in the embryonic oral cavity and progresses in the branchial arches and alveolar bone. Continuous cellular and molecular crosstalk occurs during crown formation, after which the tooth germ begins to migrate apically through the alveolar process into the oral cavity. It eventually comes in contact with its antagonist in the contralateral jaw to establish functional occlusion. Any defect in either step can result in delayed tooth development, the spectrum of which varies from a congenitally missing tooth to an impacted tooth (infraocclusion) with an eruption problem, both of which can impair oral function. HIGHLIGHT: Congenitally missing teeth or eruption problems may result from genetic mutations. Several different mutations have been identified, each causing a distinct phenotype. Thus, it is imperative that medical providers understand the fundamentals of these genetic principles that govern such dental diseases. CONCLUSION: In this review, we focus on several diseases, including congenitally missing teeth and tooth eruption problems. We review these diseases with aspect to their association with a particular syndrome, as well as independently in a non-syndromic capacity. We also review previously identified genetic mutations and discuss the possible mechanisms that cause individual phenotypes by analyzing previous investigations. We also discuss future prospects of how genetic diagnosis and precision medicine could impact the clinical environment in the field of dentistry. ETHICAL APPROVAL: Present study has been carried out in accordance with The Code of Ethics of the World Medical Association and approved by Institutional Review Board of Osaka University Graduate School of Dentistry.

Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism.

Dynamic metabolic changes occur in the liver during the transition between fasting and feeding. Here we show that transient ER stress responses in the liver following feeding terminated by Sdf2l1 are essential for normal glucose and lipid homeostasis. Sdf2l1 regulates ERAD through interaction with a trafficking protein, TMED10. Suppression of Sdf2l1 expression in the liver results in insulin resistance and increases triglyceride content with sustained ER stress. In obese and diabetic mice, Sdf2l1 is downregulated due to decreased levels of nuclear XBP-1s, whereas restoration of Sdf2l1 expression ameliorates glucose intolerance and fatty liver with decreased ER stress. In diabetic patients, insufficient induction of Sdf2l1 correlates with progression of insulin resistance and steatohepatitis. Therefore, failure to build an ER stress response in the liver may be a causal factor in obesity-related diabetes and nonalcoholic steatohepatitis, for which Sdf2l1 could serve as a therapeutic target and sensitive biomarker.

Molecular mechanism of moderate insulin resistance in adiponectin-knockout mice.

Adiponectin has been proposed to act as an antidiabetic adipokine, suppressing gluconeogenesis and stimulating fatty acid oxidation in the liver and skeletal muscle. Although adiponectin-knockout (adipo(-/-)) mice are known to exhibit insulin resistance, the degrees of insulin resistance and glucose intolerance are unexpectedly only moderate. In this study, the adipo(-/-) mice showed hepatic, but not muscle, insulin resistance. insulin-stimulated phosphorylation of IRS-1 and IRS-2 was impaired, the IRS-2 protein level was decreased, and insulin-stimulated phosphorylation of Akt was decreased in the liver of the adipo(-/-) mice. However, the triglyceride content in the liver was not increased in these mice, despite the decrease in the PPARalpha expression involved in lipid combustion, since the expressions of lipogenic genes such as SREBP-1 and SCD-1 were decreased in association with the increased leptin sensitivity. Consistent with this, the down-regulation SREBP-1 and SCD-1 observed in the adipo(-/-) mice was no longer observed, and the hepatic triglyceride content was significantly increased in the adiponectin leptin double-knockout (adipo(-/-)ob/ob) mice. On the other hand, the triglyceride content in the skeletal muscle was significantly decreased in the adipo(-/-) mice, probably due to up-regulated AMPK activity associated with the increased leptin sensitivity. In fact, these phenotypes in the skeletal muscle of these mice were no longer observed in the adipo(-/-)ob/ob mice. In conclusion, adipo(-/-) mice showed impaired insulin signaling in the liver to cause hepatic insulin resistance, however, no increase in the triglyceride content was observed in either the liver or the skeletal muscle, presumably on account of the increased leptin sensitivity.

Generation and Analysis of Cartilage-Specific CCN2 Overexpression in Transgenic Mice.

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

Topical application of lithium chloride on the pulp induces dentin regeneration.

We herein describe a novel procedure for dentin regeneration that mimics the biological processes of tooth development in nature. The canonical Wnt signaling pathway is an important regulator of the Dentin sialophosphoprotein (Dspp) expression. Our approach mimics the biological processes underlying tooth development in nature and focuses on the activation of canonical Wnt signaling to trigger the natural process of dentinogenesis. The coronal portion of the dentin and the underlying pulp was removed from the first molars. We applied lithium chloride (LiCl), an activator of canonical Wnt signaling, on the amputated pulp surface to achieve transdifferentiation toward odontoblasts from the surrounding pulpal cells. MicroCT and microscopic analyses demonstrated that the topical application of LiCl induced dentin repair, including the formation of a complete dentin bridge. LiCl-induced dentin is a tubular dentin in which the pulp cells are not embedded within the matrix, as in primary dentin. In contrast, a dentin bridge was not induced in the control group treated with pulp capping with material carriers alone, although osteodentin without tubular formation was induced at a comparatively deeper position from the pulp exposure site. We also evaluated the influence of LiCl on differentiation toward odontoblasts in vitro. In the mDP odontoblast cell line, LiCl activated the mRNA expression of Dspp, Axin2 and Kallikrein 4 (Klk4) and downregulated the Osteopontin (Osp) expression. These results provide a scientific basis for the biomimetic regeneration of dentin using LiCl as a new capping material to activate dentine regeneration.

Long-Term Stability of Residual Stress Improvement by Water Jet Peening Considering Working Processes.

To prevent primary water stress corrosion cracking (PWSCC), water jet peening (WJP) has been used on the welds of Ni-based alloys in pressurized water reactors (PWRs). Before WJP, the welds are machined and buffed in order to conduct a penetrant test (PT) to verify the weld qualities to access, and microstructure evolution takes place in the target area due to the severe plastic deformation. The compressive residual stresses induced by WJP might be unstable under elevated temperatures because of the high dislocation density in the compressive stress layer. Therefore, the stability of the compressive residual stresses caused by WJP was investigated during long-term operation by considering the microstructure evolution due to the working processes. The following conclusions were made: The compressive residual stresses were slightly relaxed in the surface layers of the thermally aged specimens. There were no differences in the magnitude of the relaxation based on temperature or time. The compressive residual stresses induced by WJP were confirmed to remain stable under elevated temperatures. The stress relaxation at the surface followed the Johnson-Mehl equation, which states that stress relaxation can occur due to the recovery of severe plastic strain, since the estimated activation energy agrees very well with the self-diffusion energy for Ni. By utilizing the additivity rule, it was indicated that stress relaxation due to recovery is completed during the startup process. It was proposed that the long-term stability of WJP under elevated temperatures must be assessed based on compressive stresses with respect to the yield stress. Thermal elastic-plastic creep analysis was performed to predict the effect of creep strain. After 100 yr of simulated continuous operation at 80% capacity, there was little change in the WJP compressive stresses under an actual operating temperature of 623 K. Therefore, the long-term stability of WJP during actual operation was analytically predicted.

Cartilage-specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/CTGF) stimulates insulin-like growth factor expression and bone growth.

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth.

CCN family member 2/connective tissue growth factor (CCN2/CTGF) has anti-aging effects that protect articular cartilage from age-related degenerative changes.

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.

K-134, a phosphodiesterase 3 inhibitor, improves gait disturbance and hindlimb blood flow impairment in rat peripheral artery disease models.

K-134, a phosphodiesterase 3 (PDE3) inhibitor with anti-thrombotic and anti-hyperplastic activity, is being developed for the treatment of intermittent claudication. We assessed the efficacy of K-134 against gait disturbance in two rat experimental peripheral arterial disease (PAD) models: the bilateral laurate-induced PAD model and femoral artery ligation model. In the laurate-induced peripheral arterial disease model, 1 week of repeated oral administration of K-134 significantly improved gait disturbance. Cilostazol and clopidogrel did not significantly improve gait disturbance. Repeated oral administration of K-134 and cilostazol significantly improved gait disturbance in the femoral artery ligation model. We evaluated the effects of K-134 and cilostazol treatment on hindlimb blood flow pre- and post-treadmill exercise in this model by laser Doppler perfusion imaging. Both drugs increased hindlimb blood flow both pre- and post-treadmill exercise after 1 week of treatment. After 4 weeks of drug treatment, without preceding drug administration which is supposed to exert acute effects on vessel walls, both drugs significantly increased hindlimb blood flow after exercise. Moreover, K-134 at 30 mg/kg significantly prolonged walking distance. These results suggest that K-134 may be useful for treating intermittent claudication.

K-134, a phosphodiesterase 3 inhibitor, prevents brain damage by inhibiting thrombus formation in a rat cerebral infarction model.

BACKGROUND: K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol. OBJECTIVES: This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model. METHODS AND RESULTS: We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (n = 12, 87.5±5.6 vs. 126.8±7.5 mm(3), P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol. CONCLUSIONS: These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability.

A phosphodiesterase 3 inhibitor, K-134, improves hindlimb skeletal muscle circulation in rat models of peripheral arterial disease.

OBJECTIVE: Cilostazol is a phosphodiesterase (PDE)3 inhibitor used to treat peripheral arterial disease with intermittent claudication, as there is clinical evidence that it improves treadmill exercise capacity. However, details of the mechanism underlying this enhanced walking capacity remain to be elucidated. METHODS: Based on the hypothesis that PDE3 inhibitors improve peripheral microcirculation in the hindlimbs via vascular smooth muscle relaxation and antiplatelet effects, we examined the effects of a more potent and selective PDE3 inhibitor, K-134, in rat models of peripheral arterial disease (PAD). RESULTS: In a hindlimb ischemia model established by bilateral femoral artery occlusion, oral administration of K-134 for 27 days significantly increased blood flow in hindlimb skeletal muscle after exercise induced by electrical stimulation of the sciatic nerve. Moreover, K-134 enlarged the luminal area of intramuscular arteries and prevented rarefaction of capillary density in the gastrocnemius muscle. These effects were observed without pre-administration on the day following the last administration, suggesting that vasodilatory, antiplatelet and angiogenic activities of K-134 were indirectly responsible for the long-term beneficial effects. In fact, K-134 dose-dependently induced relaxation of rat femoral arteries in vitro, and inhibited rat platelet aggregation ex vivo. Interestingly, in a laurate-induced peripheral vascular injury model, oral administration of K-134 for 6 days prevented progression of hindlimb necrosis. CONCLUSION: These findings suggest that the beneficial effects of PDE3 inhibitors on walking capacity are due to increased hindlimb skeletal muscle blood flow via intramuscular artery enlargement, and that K-134 is a promising drug for PAD associated with platelet hyperaggregability.

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle.

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Dynamic functional relay between insulin receptor substrate 1 and 2 in hepatic insulin signaling during fasting and feeding.

Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.