研究者詳細 - 池田　真一

論文 - 池田　真一

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Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1

Chen J., Ikeda S.I., Yang Y., Zhang Y., Ma Z., Liang Y., Negishi K., Tsubota K., Kurihara T.

Molecular Medicine 30 （ 1 ） 25 2024年12月

ISSN 10761551

Background: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. Method: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon’s injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. Results: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. Conclusion: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

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Ginkgo biloba extracts improve choroidal circulation leading to suppression of myopia in mice

Hou J., Mori K., Ikeda S.i., Jeong H., Torii H., Negishi K., Kurihara T., Tsubota K.

Scientific Reports 13 （ 1 ） 3772 - 3772 2023年12月

Myopia is becoming more common across the world, necessitating the development of preventive methods. We investigated the activity of early growth response 1 (EGR-1) protein and discovered that Ginkgo biloba extracts (GBEs) activated EGR-1 in vitro. In vivo, C57BL/6 J mice were fed either normal or 0.0667% GBEs (200 mg/kg) mixed chow (n = 6 each), and myopia was induced with − 30 diopter (D) lenses from 3 to 6 weeks of age. Refraction and axial length were measured by an infrared photorefractor and an SD-OCT system, respectively. In lens-induced myopia mice, oral GBEs significantly improved refractive errors (− 9.92 ± 1.53 D vs. − 1.67 ± 3.51 D, p < 0.001) and axial elongation (0.22 ± 0.02 mm vs. 0.19 ± 0.02 mm, p < 0.05). To confirm the mechanism of GBEs in preventing myopia progression, the 3-week-old mice were divided into normally fed with either myopic-induced or non-myopic-induced groups and GBEs fed with either myopic-induced or non-myopic-induced groups (n = 10 each). Choroidal blood perfusion was measured with optical coherence tomography angiography (OCTA). In both non-myopic induced groups, compared to normal chow, oral GBEs significantly improved choroidal blood perfusion (8.48 ± 15.75%Area vs. 21.74 ± 10.54%Area, p < 0.05) and expression of Egr-1 and endothelial nitric oxide synthase (eNOS) in the choroid. In both myopic-induced groups, compared to normal chow, oral GBEs also improved choroidal blood perfusion (− 9.82 ± 9.47%Area vs. 2.29 ± 11.84%Area, p < 0.05) and was positively correlated with the change in choroidal thickness. These findings suggest that GBEs may inhibit the progression of myopia by improving choroidal blood perfusion.

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Establishment of a novel ER-stress induced myopia model in mice

Kang L., Ikeda S.i., Yang Y., Jeong H., Chen J., Zhang Y., Negishi K., Tsubota K., Kurihara T.

Eye and Vision 10 （ 1 ） 44 - 44 2023年12月

ISSN 2326-0254

Background: Recent studies have indicated a strong correlation between endoplasmic reticulum (ER) stress and myopia and that eyedrops containing the ER stress inducer tunicamycin (Tm) can induce myopic changes in C57BL/6 J mice. Therefore, this study aimed to create a new myopia model using Tm eyedrops and to explore the mechanism of ER stress-mediated myopia development. Methods: Three-week-old C57BL/6 J mice were treated with different concentrations (0, 25, 50, and 100 μg/mL) and/or number of applications (zero, one, three, and seven) of Tm eyedrops. Refraction and axial length (AL) were measured before and one week after Tm treatment. Scleral collagen alterations were evaluated under polarised light after picrosirius red staining. ER stress-related indicators, such as the expression of collagen I and cleaved collagen were detected using Western blotting. Results: Compared with the control group, mice administered eyedrops with 50 μg/mL Tm only once showed the greatest myopic shifts in refraction and AL elongation and reduced scleral expression of collagen I. Picrosirius red staining showed a lower percentage of bundled collagen in the Tm group. Expression of ER-stress indicators increased in the Tm groups. Furthermore, optimised administration of Tm induced matrix metalloproteinase-2 (MMP2) expression in the sclera, which plays a major role in collagen degradation. Conclusions: We have demonstrated that ER stress in the sclera is involved in myopia progression. Tm eyedrops induced myopic changes, loosening of the scleral collagen and decreased expression of collagen I. This process may be associated with ER stress in the sclera, which upregulates the expression of MMP2 leading to collagen degradation.

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The orientation of choroidal macrophage polarization significantly influences the development of myopia in murine models

Jing Hou, Shin-ichi Ikeda, Kiwako Mori, Heonuk Jeong, Hidemasa Torii, Kazuno Negishi, Kazuo Tsubota, Toshihide Kurihara

（Cold Spring Harbor Laboratory） 2023年06月

Abstract

Myopia is a primary contributor to visual impairment and has emerged as a global public health concern. Evidence indicates that one of the main structural features of myopia is the corresponding decrease in choroidal thickness, and choroidal macrophages play an important role in maintaining the choroidal thickness. Nevertheless, the effect of choroidal macrophages on myopia remains unclear. Here, we discovered that the continuous intraperitoneal injection of clodronate liposomes depleted choroidal macrophages and leads to myopia, which confirmed that the presence of choroidal macrophages plays an important role in myopia development.

Subsequently, based on the phenotypic characteristics of macrophages, experiments were designed to study the effects of different polarization directions of macrophages on myopia development. We found that lipopolysaccharides (LPS) injection can induce the polarization of choroidal M1 macrophages and result in myopia. Conversely, IL-4 or IL-13 injection causes choroidal M2 macrophage polarization and suppresses the progression of myopia. Additionally, we demonstrated that the opposite effects of M1 and M2 macrophages on myopia development may be related to their impacts on inflammation and oxidative stress response. These findings establish that choroidal macrophages are critically important in the development of myopia and provide new strategies for the development of myopic therapies.

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Contralateral effect in progression and recovery of lens-induced myopia in mice

Ma Z., Jeong H., Yang Y., Jiang X., Ikeda S.i., Negishi K., Kurihara T., Tsubota K.

Ophthalmic and Physiological Optics 43 （ 3 ） 558 - 565 2023年05月

ISSN 02755408

Purpose: Apart from genetic factors, recent animal studies on myopia have focused on localised mechanisms. In this study, we aimed to examine the contralateral effects of monocular experimental myopia and recovery, which cannot be explained by a mere local mechanism. Methods: One eye of 3-week-old C57BL/6 male mice was fitted with a −30 dioptre (D) lens. The mice were distributed into two groups based on different conditions in the contralateral eye: either no lens (NLC) (n = 10) or a Plano lens on the contralateral eye (PLC) group (n = 6). Mice receiving no treatment on either eye were set as a control group (n = 6). Lenses were removed after 3 weeks of myopia induction. All mice were allowed to recover for 1 week in the same environment. Refractive status, axial length (AL) and choroidal thickness were measured before myopia induction, after 1 and 3 weeks of lens wear and after 1 week of recovery. Results: One week after removing the lenses, complete recovery was observed in the eyes that wore the −30 D lenses. In both the PLC and NLC groups, the refractive status showed a myopic shift after lens removal. Additionally, the choroid was significantly thinned in these eyes. The −30 D wearing eye showed a significant increase in AL after 3 weeks of lens wear. While the AL of the −30 D wearing eye ceased to grow after the lens was removed, the AL in the PLC and NLC contralateral eyes increased, and the binocular ALs gradually converged. Conclusions: Recovery of lens-induced myopia was observed in mouse models. In the fellow eyes, the effects, including thinning of the choroid and changes in refractive status, were triggered by contralateral visual cues.

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Suppressive effects of violet light transmission on myopia progression in a mouse model of lens-induced myopia

Jeong H., Kurihara T., Jiang X., Kondo S., Ueno Y., Hayashi Y., Lee D., Ikeda S.I., Mori K., Torii H., Negishi K., Tsubota K.

Experimental Eye Research （Elsevier BV） 228 109414 - 109414 2023年03月

ISSN 00144835

The prevalence of myopia has been steadily increasing for several decades, and this condition can cause extensive medical and economic issues in society. Exposure to violet light (VL), a short wavelength (360–400 nm) of visible light from sunlight, has been suggested as an effective preventive and suppressive treatments for the development and progression of myopia. However, the clinical application of VL remains unclear. In this study, we aimed to investigate the preventive and suppressive effects of VL on myopia progression. Various transmittances of VL (40%, 70%, and 100%) were tested in C57BL/6J mice with lens-induced myopia (LIM). Changes in the refractive error, axial length, and choroid thickness during the 3-week LIM were measured. The myopic shift in refractive error and difference in axial length between the 0 and -30 diopter lens was lessened in a transmission-dependent manner. Choroidal thinning, which was observed in myopic conditions, was suppressed by VL exposure and affected by its transmission. The results suggest that myopia progression can be managed using VL transmittance. Therefore, these factors should be considered for the prevention and treatment of myopia.

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Contralateral effect in progression and recovery of lens-induced myopia in mice

Ziyan Ma, Heonuk Jeong, Yajing Yang, Xiaoyan Jiang, Shin-ichi Ikeda, Kazuno Negishi, Toshihide Kurihara, Kazuo Tsubota

OPHTHALMIC AND PHYSIOLOGICAL OPTICS （WILEY） 2023年03月

ISSN 0275-5408

Purpose: Apart from genetic factors, recent animal studies on myopia have focused on localised mechanisms. In this study, we aimed to examine the contralateral effects of monocular experimental myopia and recovery, which cannot be explained by a mere local mechanism.Methods: One eye of 3-week-old C57BL/6 male mice was fitted with a -30 dioptre (D) lens. The mice were distributed into two groups based on different conditions in the contralateral eye: either no lens (NLC) (n = 10) or a Plano lens on the contralateral eye (PLC) group (n = 6). Mice receiving no treatment on either eye were set as a control group (n = 6). Lenses were removed after 3 weeks of myopia induction. All mice were allowed to recover for 1 week in the same environment. Refractive status, axial length (AL) and choroidal thickness were measured before myopia induction, after 1 and 3 weeks of lens wear and after 1 week of recovery.Results: One week after removing the lenses, complete recovery was observed in the eyes that wore the -30 D lenses. In both the PLC and NLC groups, the refractive status showed a myopic shift after lens removal. Additionally, the choroid was significantly thinned in these eyes. The -30 D wearing eye showed a significant increase in AL after 3 weeks of lens wear. While the AL of the -30 D wearing eye ceased to grow after the lens was removed, the AL in the PLC and NLC contralateral eyes increased, and the binocular ALs gradually converged.Conclusions: Recovery of lens-induced myopia was observed in mouse models. In the fellow eyes, the effects, including thinning of the choroid and changes in refractive status, were triggered by contralateral visual cues.

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Physical inactivity induces insulin resistance in plantaris muscle through protein tyrosine phosphatase 1B activation in mice

Kakehi S., Tamura Y., Ikeda S.I., Kaga N., Taka H., Nishida Y., Kawamori R., Watada H.

Frontiers in Physiology 14 2023年

Inactivity causes insulin resistance in skeletal muscle and exacerbates various lifestyle-related diseases. We previously found that 24-h hindlimb cast immobilization (HCI) of the predominantly slow-twitch soleus muscle increased intramyocellular diacylglycerol (IMDG) and insulin resistance by activation of lipin1, and HCI after a high-fat diet (HFD) further aggravated insulin resistance. Here, we investigated the effects of HCI on the fast-twitch–predominant plantaris muscle. HCI reduced the insulin sensitivity of plantaris muscle by approximately 30%, and HCI following HFD dramatically reduced insulin sensitivity by approximately 70% without significant changes in the amount of IMDG. Insulin-stimulated phosphorylation levels of insulin receptor (IR), IR substrate-1, and Akt were reduced in parallel with the decrease in insulin sensitivity. Furthermore, tyrosine phosphatase 1B (PTP1B), a protein known to inhibit insulin action by dephosphorylating IR, was activated, and PTP1B inhibition canceled HCI-induced insulin resistance. In conclusion, HCI causes insulin resistance in the fast-twitch–predominant plantaris muscle as well as in the slow-twitch–predominant soleus muscle, and HFD potentiates these effects in both muscle types. However, the mechanism differed between soleus and plantaris muscles, since insulin resistance was mediated by the PTP1B inhibition at IR in plantaris muscle.

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Identification of Potential Therapeutic Targets for Myopic Choroidal Neovascularization via Discovery-Driven Data Mining

Chen J., Ikeda S.i., Negishi K., Tsubota K., Kurihara T.

Current Eye Research 48 （ 12 ） 1160 - 1169 2023年

ISSN 02713683

Purpose: Myopic choroidal neovascularization (mCNV) is a prevalent cause of vision loss. However, the development of effective therapeutic targets for mCNV has been hindered by the paucity of suitable animal models. Therefore, the aim of this study is to identify potential genes and pathways associated with mCNV and to unearth prospective therapeutic targets that can be utilized to devise efficacious treatments. Methods: Text data mining was used to identify genes linked to choroid, neovascularization, and myopia. g: Profiler was utilized to analyze the biological processes of gene ontology and the Reactome pathways. Protein interaction network analysis was performed using strings and visualized in Cytoscape. MCODE and cytoHubba were used for further screening. Results: Discovery-driven text data mining identified 55 potential genes related to choroid, neovascularization, and myopia. Gene enrichment analysis revealed 11 biological processes and seven Reactome pathways. A protein-protein interaction network with 47 nodes was constructed and analyzed using centrality ranking. Key clusters were identified through algorithm tools. Finally, 14 genes (IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1), in addition to VEGFA, were evaluated as targets with potential as future therapeutics. Conclusions: This study provides new potential therapeutic targets for mCNV, including IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1, which correspond to seven potential enriched pathways. These findings provide a basis for further research and offer new possibilities for developing therapeutic interventions for this condition.

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Bisphenol A exposure triggers endoplasmic reticulum stress pathway leading to ocular axial elongation in mice

Chen J., Ikeda S.I., Kang L., Negishi K., Tsubota K., Kurihara T.

Frontiers in Medicine 10 1255121 2023年

ISSN 2296-858X

Background: Ocular axial elongation is one of the features of myopia progression. Endoplasmic reticulum (ER) stress-associated scleral remodeling plays an important role in ocular axial elongation. Bisphenol A (BPA) is one of the most common environmental pollutants and is known to affect various human organs through ER stress. However, whether BPA exerts an effect on scleral remodeling remains unknown. The purpose of this study was to determine the effect of BPA on the development of myopia and scleral ER stress. Methods: BPA was administered by intraperitoneal injection. 4-PBA was administered as an endoplasmic reticulum stress inhibitor by eye drops. Refraction and axial length were measured by refractometer and SD-OCT system. Western blot was performed to detect the expression level of ER stress-related proteins. Results: BPA-administered mice exhibit axial elongation and myopic refractive shift with endoplasmic reticulum stress in the sclera. BPA administration activated scleral PERK and ATF6 pathways, and 4-PBA eye drops attenuated ER stress response and suppressed myopia progression. Conclusion: BPA controlled axial elongation during myopia development in a mouse model by inducing scleral ER stress and activation of the PERK/ATF6 pathway. 4-PBA eye drops as ER stress inhibitor suppressed BPA-induced myopia development.

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Scleral PERK and ATF6 as targets of myopic axial elongation of mouse eyes

Ikeda S.i., Kurihara T., Jiang X., Miwa Y., Lee D., Serizawa N., Jeong H., Mori K., Katada Y., Kunimi H., Ozawa N., Shoda C., Ibuki M., Negishi K., Torii H., Tsubota K.

Nature Communications 13 （ 1 ） 5859 - 5859 2022年12月

Axial length is the primary determinant of eye size, and it is elongated in myopia. However, the underlying mechanism of the onset and progression of axial elongation remain unclear. Here, we show that endoplasmic reticulum (ER) stress in sclera is an essential regulator of axial elongation in myopia development through activation of both PERK and ATF6 axis followed by scleral collagen remodeling. Mice with lens-induced myopia (LIM) showed ER stress in sclera. Pharmacological interventions for ER stress could induce or inhibit myopia progression. LIM activated all IRE1, PERK and ATF6 axis, and pharmacological inhibition of both PERK and ATF6 suppressed myopia progression, which was confirmed by knocking down above two genes via CRISPR/Cas9 system. LIM dramatically changed the expression of scleral collagen genes responsible for ER stress. Furthermore, collagen fiber thinning and expression of dysregulated collagens in LIM were ameliorated by 4-PBA administration. We demonstrate that scleral ER stress and PERK/ATF6 pathway controls axial elongation during the myopia development in vivo model and 4-PBA eye drop is promising drug for myopia suppression/treatment.

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Vascular endothelial growth factor from retinal pigment epithelium is essential in choriocapillaris and axial length maintenance

Zhang Y., Jeong H., Mori K., Ikeda S.I., Shoda C., Miwa Y., Nakai A., Chen J., Ma Z., Jiang X., Torii H., Kubota Y., Negishi K., Kurihara T., Tsubota K.

PNAS Nexus 1 （ 4 ） pgac166 2022年09月

Myopia,which prevalence is rapidly increasing, causes visual impairment; however, the onset mechanism of pathological axial length (AL) elongation remains unclear. A highly vascularized choroid between the retinal pigment epithelium (RPE) and sclera not only maintains physiological activities, but also contributes to ocular development and growth regulation. Vascular endothelial growth factor (VEGF) secreted from the RPE to the choroid is essential for retinal function and maintenance of the choriocapillaris. Herein, we demonstrated that the loss of VEGF secreted fromthe RPE caused abnormal choriocapillaris development and AL elongation, with features similar to those of the lens-induced myopia (LIM) mouse model, whereas VEGF overexpression by knocking-out von Hippel Lindau (VHL) specific to the RPE expands the choriocapillaris and shortens the AL. Additionally, LDL Receptor Related Protein 2 (LRP2) deletion in the RPE downregulated VEGF expression and leads to pathological AL elongation. Furthermore, high-myopia patientswithout choriocapillaris demonstrated longer ALs than did those with preserved choriocapillaris. These results suggest that physiological secretion of VEGF from the RPE is required for proper AL development by maintaining the choriocapillaris. The pinpoint application of VEGF to the choriocapillaris may become a potential intervention for the prevention and treatment of axial myopia progression.

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Retinal degeneration induced in a mouse model of ischemia–reperfusion injury and its management by pemafibrate treatment

Lee D., Nakai A., Miwa Y., Tomita Y., Kunimi H., Chen J., Ikeda S.I., Tsubota K., Negishi K., Kurihara T.

FASEB Journal 36 （ 9 ） e22497 2022年09月

ISSN 08926638

Retinal ischemia–reperfusion (I/R) injury is a common cause of visual impairment. To date, no effective treatment is available for retinal I/R injury. In addition, the precise pathological mechanisms still need to be established. Recently, pemafibrate, a peroxisome proliferator-activated receptor α (PPARα) modulator, was shown to be a promising drug for retinal ischemia. However, the role of pemafibrate in preventing retinal I/R injury has not been documented. Here, we investigated how retinal degeneration occurs in a mouse model of retinal I/R injury by elevation of intraocular pressure and examined whether pemafibrate could be beneficial against retinal degeneration. Adult mice were orally administered pemafibrate (0.5 mg/kg/day) for 4 days, followed by retinal I/R injury. The mice were continuously administered pemafibrate once every day until the end of the experiments. Retinal functional changes were measured using electroretinography. Retina, liver, and serum samples were used for western blotting, quantitative PCR, immunohistochemistry, or enzyme linked immunosorbent assay. Retinal degeneration induced by retinal inflammation was prevented by pemafibrate administration. Pemafibrate administration increased the hepatic PPARα target gene expression and serum levels of fibroblast growth factor 21, a neuroprotective molecule in the eye. The expression of hypoxia-response and pro-and anti-apoptotic/inflammatory genes increased in the retina following retinal I/R injury; however, these changes were modulated by pemafibrate administration. In conclusion, pemafibrate is a promising preventive drug for ischemic retinopathies.

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Lipidomic analysis revealed n-3 polyunsaturated fatty acids suppressed choroidal thinning and myopia progression in mice

Mori K., Kuroha S., Hou J., Jeong H., Ogawa M., Ikeda S.i., Kang J.X., Negishi K., Torii H., Arita M., Kurihara T., Tsubota K.

FASEB Journal （FASEB Journal） 36 （ 6 ） e22312 2022年06月

ISSN 08926638

Myopia is increasing worldwide and its preventable measure should urgently be pursued. N-3 polyunsaturated fatty acids (PUFAs) have been reported to have various effects such as vasodilative and anti-inflammatory, which myopia may be involved in. This study is to investigate the inhibitory effect of PUFAs on myopia progression. A lens-induced myopia (LIM) model was prepared using C57B L6/J 3-week-old mice, which were equipped with a −30 diopter lens to the right eye. Chows containing two different ratios of n-3/n-6 PUFA were administered to the mice, and myopic shifts were confirmed in choroidal thickness, refraction, and axial length in the n-3 PUFA-enriched chow group after 5 weeks. To exclude the possibility that the other ingredients in the chow may have taken the suppressive effect, fat-1 transgenic mice, which can produce n-3 PUFAs endogenously, demonstrated significant suppression of myopia. To identify what elements in n-3 PUFAs took effects on myopia suppression, enucleated eyes were used for targeted lipidomic analysis, and eicosapentaenoic acid (EPA) were characteristically distributed. Administration of EPA to the LIM model confirmed the inhibitory effect on choroidal thinning and myopia progression. Subsequently, to identify the elements and the metabolites of fatty acids effective on myopia suppression, targeted lipidomic analysis was performed and it demonstrated that metabolites of EPA were involved in myopia suppression, whereas prostaglandin E2 and 14,15-dihydrotestosterone were associated with progression of myopia. In conclusion, EPA and its metabolites are related to myopia suppression and inhibition of choroidal thinning.

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Short-term physical inactivity induces diacylglycerol accumulation and insulin resistance in muscle via lipin1 activation

Kakehi S., Tamura Y., Ikeda S.I., Kaga N., Taka H., Ueno N., Shiuchi T., Kubota A., Sakuraba K., Kawamori R., Watada H.

American Journal of Physiology - Endocrinology and Metabolism （American Journal of Physiology - Endocrinology and Metabolism） 321 （ 6 ） E766 - E781 2021年12月

ISSN 01931849

Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization (HCI) to mice with normal or high-fat diet (HFD) and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. Although 2-wk HFD alone did not alter intramyocellular diacylglycerol (IMDG) accumulation, HCI alone increased it by 1.9-fold and HCI after HFD further increased it by 3.3-fold. Parallel to this, we found increased protein kinase C ϵ (PKCϵ) activity, reduced insulin-induced 2-deoxyglucose (2-DOG) uptake, and reduced phosphorylation of insulin receptor β (IRβ) and Akt, key molecules for insulin signaling pathway. Lipin1, which converts phosphatidic acid to diacylglycerol, showed increase of its activity by HCI, and dominant-negative lipin1 expression in muscle prevented HCI-induced IMDG accumulation and impaired insulin-induced 2-DOG uptake. Furthermore, 24-h leg cast immobilization in human increased lipin1 expression. Thus, even short-term immobilization increases IMDG and impairs insulin sensitivity in muscle via enhanced lipin1 activity.

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Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Jiang X., Pardue M.T., Mori K., Ikeda S.I., Torii H., D'Souza S., Lang R.A., Kurihara T., Tsubota K.

Proceedings of the National Academy of Sciences of the United States of America （Proceedings of the National Academy of Sciences of the United States of America） 118 （ 22 ） e2018840118 - e2018840118 2021年06月

ISSN 00278424

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus-induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

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Endurance runners with intramyocellular lipid accumulation and high insulin sensitivity have enhanced expression of genes related to lipid metabolism in muscle

Kakehi S., Tamura Y., Takeno K., Ikeda S.I., Ogura Y., Saga N., Miyatsuka T., Naito H., Kawamori R., Watada H.

Journal of Clinical Medicine （Journal of Clinical Medicine） 9 （ 12 ） 1 - 12 2020年12月

Context: Endurance-trained athletes have high oxidative capacities, enhanced insulin sensitivities, and high intracellular lipid accumulation in muscle. These characteristics are likely due to altered gene expression levels in muscle. Design and setting: We compared intramyocellular lipid (IMCL), insulin sensitivity, and gene expression levels of the muscle in eight nonobese healthy men (control group) and seven male endurance athletes (athlete group). Their IMCL levels were measured by proton-magnetic resonance spectroscopy, and their insulin sensitivity was evaluated by glucose infusion rate (GIR) during a euglycemic–hyperinsulinemic clamp. Gene expression levels in the vastus lateralis were evaluated by quantitative RT-PCR (qRT-PCR) and microarray analysis. Results: IMCL levels in the tibialis anterior muscle were approximately 2.5 times higher in the athlete group compared to the control group, while the IMCL levels in the soleus muscle and GIR were comparable. In the microarray hierarchical clustering analysis, gene expression patterns were not clearly divided into control and athlete groups. In a gene set enrichment analysis with Gene Ontology gene sets, “RESPONSE TO LIPID” was significantly upregulated in the athlete group compared with the control group. Indeed, qRT-PCR analysis revealed that, compared to the control group, the athlete group had 2–3 times higher expressions of proliferator-activated receptor gamma coactivator-1 alpha (PGC1A), adiponectin receptors (AdipoRs), and fatty acid transporters including fatty acid transporter-1, plasma membrane-associated fatty acid binding protein, and lipoprotein lipase. Conclusions: Endurance runners with higher IMCL levels have higher expression levels of genes related to lipid metabolism such as PGC1A, AdipoRs, and fatty acid transporters in muscle.

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Oral bovine milk lactoferrin administration suppressed myopia development through matrix metalloproteinase 2 in a mouse model

Ikeda S.I., Kurihara T., Toda M., Jiang X., Torii H., Tsubota K.

Nutrients （Nutrients） 12 （ 12 ） 1 - 9 2020年12月

Recent studies have reported an association between myopia development and local ocular inflammation. Lactoferrin (LF) is an iron-binding protein present in saliva, tears, and mother’s milk. Furthermore, sequestering iron by LF can cause its antibacterial property. Moreover, LF has an anti-inflammatory effect. We aimed to determine the suppressive effect of LF against the development and progress of myopia using a murine lens-induced myopia (LIM) model. We divided male C57BL/6J mice (3 weeks old) into two groups. While the experimental group was orally administered LF (1600 mg/kg/day, from 3-weeks-old to 7-weeks-old), a similar volume of Ringer’s solution was administered to the control group. We subjected the 4-week-old mice to −30 diopter lenses and no lenses on the right and left eyes, respectively. We measured the refraction and the axial length at baseline and 3 weeks after using a refractometer and a spectral domain optical coherence tomography (SD-OCT) system in both eyes. Furthermore, we determined the matrix metalloproteinase-2 (MMP-2) activity, and the amount of interleukin-6 (IL-6), MMP-2, and collagen 1A1 in the choroid or sclera. The eyes with a minus lens showed a refractive error shift and an axial length elongation in the control group, thus indicating the successful induction of myopia. However, there were no significant differences in the aforementioned parameters in the LF group. While LIM increased IL-6 expression and MMP-2 activity, it decreased collagen 1A1 content. However, orally administered LF reversed these effects. Thus, oral administration of LF suppressed lens-induced myopia development by modifying the extracellular matrix remodeling through the IL-6–MMP-2 axis in mice.

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A murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

Lee D., Miwa Y., Jeong H., Ikeda S.I., Katada Y., Tsubota K., Kurihara T.

Journal of Visualized Experiments （Journal of Visualized Experiments） 2020 （ 165 ） 1 - 19 2020年11月

ISSN 1940087X

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

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Estimation of the minimum eﬀective dose of dietary supplement crocetin for prevention of myopia progression in mice

Mori K., Kurihara T., Jiang X., Ikeda S., Yotsukura E., Torii H., Tsubota K.

Nutrients （Nutrients） 12 （ 1 ） 2020年

© 2020, MDPI AG. All rights reserved. The natural carotenoid crocetin has been reported to suppress phenotypes of an experimental myopia model in mice. We investigated the minimum effective dose to prevent myopia progression in a murine model. Three-week-old male mice (C57B6/J) were equipped with a −30 diopter (D) lens to induce myopia, and fed with normal chow, 0.0003%, or 0.001% of crocetin-containing chow. Changes in refractive errors and axial lengths (AL) were evaluated after three weeks. Pharmacokinetics of crocetin in the plasma and the eyeballs of mice was evaluated with specific high sensitivity quantitative analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the minimum effective dosage. A concentration of 0.001% of crocetin-containing chow showed a significant (p < 0.001) suppressive effect against both refractive and AL changes in the murine model. Meanwhile, there was no significant difference of AL change between the 0.0003% and the normal chow groups. The concentration of crocetin in the plasma and the eyeballs from mice fed with 0.001% crocetin-containing chow was significantly higher than control and 0.0003% crocetin-containing chow. In conclusion, we suggest 0.001% of crocetin-containing extract is the minimum effective dose showing a significant suppressive effect against both refractive and AL changes in the murine model.

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Oral crocetin administration suppressed refractive shift and axial elongation in a murine model of lens-induced myopia

Mori K., Kurihara T., Miyauchi M., Ishida A., Jiang X., Ikeda S., Torii H., Tsubota K.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

© 2019, The Author(s). Increased global incidence of myopia necessitates establishment of therapeutic approaches against its progression. To explore agents which may control myopia, we screened 207 types of natural compounds and chemical reagents based on an activity of a myopia suppressive factor, early growth response protein 1 (Egr-1) in vitro. Among the candidates, crocetin showed the highest and dose-dependent activation of Egr-1. For in vivo analysis, experimental myopia was induced in 3-week-old C57BL/6 J mice with −30 diopter (D) lenses for 3 weeks. Animals were fed with normal or mixed chow containing 0.003% (n = 19) and 0.03% (n = 7) of crocetin during myopia induction. Refraction and axial length were measured at 3-week-old and the 6-week-old with an infrared photorefractor and a SD-OCT system. Compared to controls (n = 14), crocetin administration showed a significant smaller change of refractive errors (−13.62 ± 8.14 vs +0.82 ± 5.81 D for 0.003%, p < 0.01, −2.00 ± 4.52 D for 0.03%, p < 0.01) and axial elongation (0.27 ± 0.03 vs 0.22 ± 0.04 mm for 0.003%, p < 0.01, 0.23 ± 0.05 mm for 0.03%, p < 0.05). These results suggest that a dietary factor crocetin may have a preventive effect against myopia progression.

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Effects of hyperoxia on the refraction in murine neonatal and adult models

Mori K., Kurihara T., Jiang X., Ikeda S., Ishida A., Torii H., Tsubota K.

International Journal of Molecular Sciences （International Journal of Molecular Sciences） 20 （ 23 ） 2019年12月

ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Whether hyperoxia affects the refraction in neonatal and adult mice is unknown. The mice exposed to 85% oxygen at postnatal 8 days (P8d) for 3 days and the mice exposed to normal air were assigned to the neonatal hyperoxia and normoxia groups, respectively. The refraction, the corneal curvature radius (CR) and the axial length (AL) were measured at P30d and P47d. Postnatal 6 weeks (P6w) adult mice were divided into the adult hyperoxia and normoxia groups. These parameters were measured before oxygen exposure, after 1 and 6 weeks, and every 7 weeks. The lens elasticity was measured at P7w and P26w by enucleation. The neonatal hyperoxia group showed a significantly larger myopic change than the neonatal normoxia group (P47d −6.56 ± 5.89 D, +4.11 ± 2.02 D, p < 0.001), whereas the changes in AL were not significantly different (P47d, 3.31 ± 0.04 mm, 3.31 ± 0.05 mm, p = 0.852). The adult hyperoxia group also showed a significantly larger myopic change (P12w, −7.20 ± 4.09 D, +7.52 ± 2.54 D, p < 0.001). The AL did not show significant difference (P12w, 3.44 ± 0.03 mm, 3.43 ± 0.01 mm, p = 0.545); however, the CR in the adult hyperoxia group was significantly smaller than the adult normoxia group (P12w, 1.44 ± 0.03 mm, 1.50 ± 0.03 mm, p = 0.003). In conclusion, hyperoxia was demonstrated to induce myopic shift both in neonatal and adult mice, which was attributed to the change in the CR rather than the AL. Elucidation of the mechanisms of hyperoxia and the application of this result to humans should be carried out in future studies.

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Ocular-component-specific miRNA expression in a murine model of lens-induced myopia

Tanaka Y., Kurihara T., Hagiwara Y., Ikeda S., Mori K., Jiang X., Torii H., Tsubota K.

International Journal of Molecular Sciences （International Journal of Molecular Sciences） 20 （ 15 ） 2019年08月

ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. To identify tissues and molecules involved in refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed a comprehensive analysis of microRNA (miRNA) expression. Three weeks after negative 30 diopter lens fixation on three-week-old C57BL/6J mice, total RNA was extracted from individual ocular components including cornea, iris, lens, retina, retinal pigment epithelium (RPE)/choroid, and sclera tissue. The miRNA expression analysis was pooled from three samples and carried out using Agilent Mouse miRNA Microarray (8 × 60 K) miRBase21.0. The expression ratio was calculated, and differentially expressed miRNAs were extracted, using GeneSpring GX 14.5. Myopic induction showed a significant myopic refractive change, axial elongation, and choroidal thinning. Through the comprehensive miRNA analysis, several upregulated miRNAs (56 in cornea tissue, 13 in iris tissue, 6 in lens tissue, 0 in retina tissue, 29 in RPE/choroid tissue, and 30 in sclera tissue) and downregulated miRNAs (7 in cornea tissue, 28 in iris tissue, 17 in lens tissue, 9 in retina tissue, 7 in RPE/choroid tissue, and 40 in sclera tissue) were observed. Overlapping expression changes in miRNAs were also found in different ocular components. Some of this miRNA dysregulation may be functionally involved in refractive myopia shift and axial length elongation.

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The effect of dietary supplementation of crocetin for myopia control in children: A randomized clinical trial

Mori K., Torii H., Fujimoto S., Jiang X., Ikeda S.I., Yotsukura E., Koh S., Kurihara T., Nishida K., Tsubota K.

Journal of Clinical Medicine （Journal of Clinical Medicine） 8 （ 8 ） 2019年08月

The prevalence of myopia has been increasing in recent years. The natural carotenoid crocetin has been reported to suppress experimental myopia in mice. We evaluated the effects of crocetin on myopia suppression in children. A multicenter randomized double-blind placebo-controlled clinical trial was performed with 69 participants aged 6 to 12 years, whose cycloplegic spherical equivalent refractions (SER) were between −1.5 and −4.5 diopter (D). The participants were randomized to receive either a placebo or crocetin and followed up for 24 weeks. Axial length (AL) elongation and changes in SER were evaluated for 24 weeks. Both written informed assent from the participants and written informed consent from legal guardians were obtained in this study because the selection criteria of this trial included children aged between 6 and 12 years old. This trial was approved by the institutional review boards. A mixed-effects model was used for analysis, using both eyes. Two participants dropped out and 67 children completed this trial. The change in SER in the placebo group, −0.41 ± 0.05 D (mean ± standard deviation), was significantly more myopic compared to that in the crocetin group, −0.33 ± 0.05 D (p = 0.049). The AL elongation in the placebo group, 0.21 ± 0.02 mm, was significantly bigger than that in the crocetin group, 0.18 ± 0.02 mm (p = 0.046). In conclusion, dietary crocetin may have a suppressive effect on myopia progression in children, but large-scale studies are required in order to confirm this effect.

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バイオレットライトを使用した前向き研究における成人強度近視2症例の眼軸長伸長量

鳥居秀成, 戸田郁子, 大野綾香, 近藤眞一郎, 四倉絵里沙, 森紀和子, 小橋英長, 姜効炎, 池田真一, 栗原俊英, 坪田一男

日本眼科学会雑誌（(公財)日本眼科学会） 123 （臨増） 182 - 182 2019年03月

ISSN 0029-0203

マウス近視モデルにおけるバイオレットライト(VL)近視抑制効果の波長特異性

姜效炎, 栗原俊英, 池田真一, 森紀和子, 鳥居秀成, 坪田一男

日本眼科学会雑誌（(公財)日本眼科学会） 123 （臨増） 304 - 304 2019年03月

ISSN 0029-0203

マウス近視モデルにおけるクロセチンの近視抑制効果

森紀和子, 栗原俊英, 石田文子, 姜效炎, 池田真一, 鳥居秀成, 坪田一男

日本眼科学会雑誌（(公財)日本眼科学会） 123 （臨増） 305 - 305 2019年03月

ISSN 0029-0203

ブラジル赤道部付近における学童の1年間の屈折値変化量と眼軸長変化量

四倉絵里沙, 鳥居秀成, 内野美樹, 白石哲郎, 岡直幸, 森紀和子, 姜効炎, 池田真一, 栗原俊英, 根岸一乃, 坪田一男

日本眼科学会雑誌（(公財)日本眼科学会） 123 （臨増） 183 - 183 2019年03月

ISSN 0029-0203

Inducement and evaluation of a murine model of experimental myopia

Jiang X., Kurihara T., Ikeda S., Kunimi H., Mori K., Torii H., Tsubota K.

Journal of Visualized Experiments （Journal of Visualized Experiments） 2019 （ 143 ） 2019年01月

ISSN 1940087X

© 2019 Journal of Visualized Experiments. Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in animals is to put clear minus lenses in front of eyes for weeks (lens-induced myopia, LIM). However, extant protocols for inducement and evaluation vary from laboratory to laboratory. Here, we described a highly practical and reproducible method to induce LIM in mice using newly designed eyeglasses. The method fixes the lens stably in front of the mouse eye while allows the lens to be taken off for cleaning or topical drug administration. The phenotype is robust and efficient, and the variance is small. The method described here can be applied to mice right after weaning which extends the possible duration for experiments. We also gave technical advises for achieving reproducible results in refraction and axial length measurements. We hope the step-by-step protocol described here and the detailed article can help researchers perform myopia experiments with myopia more smoothly and make the data comparable across laboratories.

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Current Prevalence of Myopia and Association of Myopia with Environmental Factors among Schoolchildren in Japan

Yotsukura E., Torii H., Inokuchi M., Tokumura M., Uchino M., Nakamura K., Hyodo M., Mori K., Jiang X., Ikeda S., Kondo S., Negishi K., Kurihara T., Tsubota K.

JAMA Ophthalmology （JAMA Ophthalmology） 137 （ 11 ） 1233 - 1239 2019年

ISSN 21686165

© 2019 American Medical Association. All rights reserved. Importance: Given the estimates of increasing prevalence of myopia, especially in Asia, it is important to determine the current prevalence of myopia among populations of schoolchildren in Japan. Objective: To investigate the current prevalence rate of myopia and the association between environmental factors and myopia in Japanese schoolchildren. Design, Setting, and Participants: This cross-sectional study assessed 1478 participants, including 726 elementary school students and 752 junior high school students, at 2 schools in Tokyo, Japan, who underwent eye examinations from April 1 to May 31, 2017, that included measurement of the refractive errors by autorefractometry with noncycloplegic refraction and ocular biometric factors. After excluding those who had been treated with atropine or orthokeratology (n = 11), had a history of eye disease (n = 2), had no parental consent (n = 41), and were absent (n = 8), 1416 schoolchildren were analyzed. Main Outcomes and Measures: The primary outcome was the prevalence of myopia and high myopia. Secondary outcomes were environmental factors that were associated with myopia. Results: A total of 1416 schoolchildren (mean [SD] age, 10.8 [2.7] years; 792 [55.9%] male) were studied. The prevalence rates of myopia (spherical equivalent ≤-0.5 diopters [D]) were 76.5% (95% CI, 73.4%-79.7%) among the elementary school students and 94.9% (95% CI, 93.3%-96.5%) among the junior high school students. The prevalence rates of high myopia (spherical equivalent ≤-6.0 D) were 4.0% (95% CI, 2.5%-5.4%) among the elementary school students and 11.3% (95% CI, 8.8%-13.7%) among the junior high school students. The prevalence rates of high myopia classified based on axial length of 26.0 mm or longer were 1.2% (95% CI, 0.4%-2.0%) among elementary school students and 15.2% (95% CI, 12.5%-17.8%) among junior high school students. Multiple regression analysis showed that higher-order aberrations and dry eye disease were associated with refractive error in elementary school students (spherical aberration: β = 6.152; 95% CI, 3.161-9.143; P <.001; dry eye disease: β = -0.626; 95% CI, -1.189 to -0.063; P =.03) and with axial length in junior high school students (spherical-like aberration: β = 26.546; 95% CI, 18.708-34.385; P <.001; dry eye disease: β = 0.354; 95% CI, 0.131-0.578; P <.002). Conclusions and Relevance: Although the use of noncycloplegic autorefraction with a cutoff of -0.50 D could lead to overestimation of results, these findings suggest that the current prevalence rates of myopia among elementary and junior high school students in Asia are high, especially if the results from these 2 schools are generalizable to all schoolchildren in Japan and Asia..

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A highly efficient murine model of experimental myopia

Jiang X., Kurihara T., Kunimi H., Miyauchi M., Ikeda S., Mori K., Tsubota K., Torii H., Tsubota K.

Scientific Reports （Scientific Reports） 8 （ 1 ） 2018年12月

ISSN 2045-2322

© 2018 The Author(s). Despite the global pandemic of myopia, the precise molecular mechanism of the onset of myopia remains largely unknown. This is partially because of the lack of efficient murine myopic models that allow genetic manipulation at low cost. Here we report a highly practical and reproducible lens-induced myopia model by specially designed frames and lenses for mice. A lens power dependent myopic induction in mice was shown until minus 30 diopter lenses. The phenotype was significantly stronger than form-deprivation myopia. We presented the protocol for precise evaluations of the state of myopia, including refraction, corneal curvature and axial length using up-to-date devices. We also found that myopic mouse eyes showed decreased visual acuity on optokinetic response examination. Finally, we confirmed the anti-myopic effect of 1% atropine using this model, which showed its potential in drug screening. The strong phenotype, stable evaluation and the potential for gene manipulation utilizing the presented method in mice will accelerate the translational research of myopia.

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Exercise-induced increase in IL-6 level enhances GLUT4 expression and insulin sensitivity in mouse skeletal muscle

8. Shin-ichi Ikeda, Yoshifumi Tamura, Saori Kakehi, Kageumi Takeno, Yoshio Fujitani, Ryuzo Kawamori and Hirotaka Watada

Biochemical and Biophysical Research Communication （Biochemical and Biophysical Research Communication） 473 （ 4 ） 947 - 952 2016年05月

ISSN 0006-291X

A single bout of exercise is known to increase the insulin sensitivity of skeletal muscle; however, the underlying mechanism of this phenomenon is not fully understood. Because a single bout of exercise induces a transient increase in blood interleukin-6 (IL-6) level, we hypothesized that the enhancement of insulin sensitivity after a single bout of exercise in skeletal muscle is mediated at least in part through IL-6-dependent mechanisms. To test this hypothesis, C57BL6J mice were intravenously injected with normal IgG or an IL-6 neutralizing antibody before exercise. Twenty-four hours after a single bout of exercise, the plantaris muscle was harvested to measure insulin sensitivity and glucose transporter (GLUT)-4 expression levels by ex-vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake and Western blotting, respectively. Compared with sedentary mice, mice that performed exercise showed enhanced IL-6 concentration, insulin-stimulated 2-DG uptake, and GLUT-4 expression in the plantaris muscle. The enhanced insulin sensitivity and GLUT4 expression were canceled by injection of the IL-6 neutralizing antibody before exercise. In addition, IL-6 injection increased GLUT4 expression, both in the plantaris muscle and the soleus muscle in C57BL6J mice. Furthermore, a short period of incubation with IL-6 increased GLUT4 expression in differentiated C2C12 myotubes. In summary, these results suggested that IL-6 increased GLUT4 expression in muscle and that this phenomenon may play a role in the post-exercise enhancement of insulin sensitivity in skeletal muscle. (C) 2016 Elsevier Inc. All rights reserved.

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Increased intramyocellular lipid/impaired insulin sensitivity is associated with altered lipid metabolic genes in muscle of high responders to a high fat diet

Saori Kakehi, Yoshifumi Tamura, Kageumi Takeno, Yuko Sakurai, Minako Kawaguchi, Takashi Watanabe, Takashi Funayama, Fumihiko Sato, Shin-ichi Ikeda, Akio Kanazawa, Yoshio Fujitani, Ryuzo Kawamori, Hirotaka Watada

American Journal of Physiology-Endocrinology and Metabolism （American Physiological Society） 310 （ 1 ） 32 - 40 2016年01月

査読有り, ISSN 0193-1849

The accumulation of intramyocellular lipid (IMCL) is recognized as an important determinant of insulin resistance, and is increased by a high-fat diet (HFD). However, the effects of HFD on IMCL and insulin sensitivity are highly variable. The aim of this study was to identify the genes in muscle that are related to this inter-individual variation. Fifty healthy men were recruited for this study. Before and after HFD for 3 days, IMCL levels in the tibialis anterior were measured by H-1 magnetic resonance spectroscopy, and peripheral insulin sensitivity was evaluated by glucose infusion rate (GIR) during the euglycemic-hyperinsulinemic clamp. Subjects who showed a large increase in IMCL and a large decrease in GIR by HFD were classified as high responders (HRs), and subjects who showed a small increase in IMCL and a small decrease in GIR were classified as low responders (LRs). In five subjects from each group, the gene expression profile of the vastus lateralis muscle was analyzed by DNA microarray analysis. Before HFD, gene expression profiles related to lipid metabolism were comparable between the two groups. Gene Set Enrichment Analysis demonstrated that five gene sets related to lipid metabolism were upregulated by HFD in the HR group but not in the LR group. Changes in gene expression patterns were confirmed by qRT-PCR using more samples (LR, n = 9; HR, n = 11). These results suggest that IMCL accumulation/impaired insulin sensitivity after HFD is closely associated with changes in the expression of genes related to lipid metabolism in muscle.

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Telogen elongation in the hair cycle of ob/ob mice

Natsumi Tasaki, Takeo Minematsu, Shin-ichi Ikeda, Yuko Mugita, Gojiro Nakagami, Hiromi Sanada

Bioscience, Biotechnology, and Biochemistry （Bioscience, Biotechnology, and Biochemistry） 4 （ 1 ） 1 - 6 2015年08月

査読有り

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Pseudomonas aeruginosa quorum-sensing signaling molecule N-3-oxododecanoyl homoserine lactone induces matrix metalloproteinase 9 expression via the AP1 pathway in rat fibroblasts

Gojiro Nakagami, Takeo Minematsu, Tomohiro Morohoshi, Takumi Yamane, Toshiki Kanazawa, Lijuan Huang, Mayumi Asada, Takashi Nagase, Shin-ichi Ikeda, Tsukasa Ikeda, Hiromi Sanada

Bioscience, Biotechnology, and Biochemistry （Bioscience, Biotechnology, and Biochemistry） 22 （ 10 ） 1 - 5 2015年06月

査読有り

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Effects of sitagliptin on ectopic fat contents and glucose metabolism in type 2 diabetic patients with fatty liver

Takahiro Watanabe, Yoshifumi Tamura, Saori Kakehi, Takashi Funayama, Amalia Gastaldelli, Kageumi Takeno, Minako Kawaguchi, Risako Yamamoto, Fumihiko Sato, Shin-ichi Ikeda, Hikari Taka, Tsutomu Fujimura, Yoshio Fujitani, Ryuzo Kawamori and Hirotaka Watada

Journal of Diabetes Investigation （Journal of Diabetes Investigation） 6 （ 2 ） 164 - 172 2015年03月

査読有り

Effects of sitagliptin on ectopic fat contents and glucose metabolism in type 2 diabetic patients with fatty liver: A pilot study

JOURNAL OF DIABETES INVESTIGATION （WILEY-BLACKWELL） 6 （ 2 ） 164 - 172 2015年03月

査読有り, ISSN 2040-1116

Aims/IntroductionRecent data have shown that ectopic fat accumulation in the liver worsens hepatic glucose metabolism, suggesting that fatty liver in patients with type2 diabetes is a therapeutic target. Glucagon-like peptide (GLP)-1 improves fatty liver, but the effect of dipeptidyl peptidase-4 inhibitor on fatty liver is still unclear. The present pilot study determined the effects of 12-week treatment with sitagliptin, a dipeptidyl peptidase-4 inhibitor, on liver fat content in type 2 diabetes with fatty liver. We also evaluated intramyocellular lipid (IMCL) and glucose kinetics during oral glucose tolerance test (OGTT) before and after the treatment.
Materials and MethodsThe study participants were seven type 2 diabetes patients with fatty liver who were studied at baseline and 12weeks after sitagliptin treatment. Intrahepatic lipid (IHL) and IMCL were assessed by H-1 magnetic resonance spectroscopy. Glucose kinetics was assessed during double-tracer OGTT (U-[C-13]-glucose orally and 6,6-[H-2(2)]-glucose intravenously).
ResultsSitagliptin significantly reduced glycated hemoglobin (from 7.10.2 to 6.5 +/- 0.3%, P<0.005), but had no effects on IHL and IMCL. The glucose level diminished, whereas GLP-1 concentration increased during OGTT at the end of treatment. These changes were not accompanied by significant changes in insulin or glucagon levels. However, long-term sitagliptin treatment partially decreased the rate of appearance of oral glucose during OGTT, but did not affect endogenous glucose production or the rate of disappearance.
ConclusionsIt was found that 12-week sitagliptin treatment improved glycated hemoglobin and glucose excursion during OGTT in type 2 diabetes with fatty liver, independent of changes in lipid accumulation in the liver. This trial was registered with the Japan Clinical Trials Registry (UMIN-CTR000005666).

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A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice

Dong-mei Zheng, Zehua Bian, Norihiko Furuya, Mitsue Takeda-Ezaki, Juan Alejandro Oliva Trejo, Katsuyuki Takahashi, Yuka Hiraoka, Reiko Mineki, Hikari Taka, Shin-ichi Ikeda, Masaaki Komatsu, Tsutomu Fujimura, Takashi Ueno and Junji Ezaki

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 456 （ 1 ） 519 - 526 2015年01月

査読有り, ISSN 0006-291X

It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24 h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12 m/min, 2 h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice. (C) 2014 Elsevier Inc. All rights reserved.

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Association between expression of FABPpm in skeletal muscle and insulin sensitivity in intramyocellular-lipid-accumulated non-obese men

Minako Kawaguchi, Yoshifumi Tamura, Saori Kakehi, Kageumi Takeno, Yuko Sakurai, Takahiro Watanabe, Takashi Funayama, Fumihiko Sato, Shin-ichi Ikeda, Yuji Ogura, Norio Saga, Hisashi Naito, Yoshio Fujitani, Akio Kanazawa, Ryuzo Kawamori, and Hirotaka Watada

The Journal of Clinical Endocrinology & Metabolism （The Journal of Clinical Endocrinology & Metabolism） 99 （ 9 ） 3343 - 3352 2014年09月

査読有り, ISSN 0021-972X

Context: Intramyocellular lipid (IMCL) accumulation is observed in both insulin-resistant subjects and insulin-sensitive endurance athletes (athlete's paradox). We hypothesized that the expression pattern of fatty acid transporters may influence oxidative capacity and determine the association between IMCL and insulin resistance.
Objective: The objective of the study was to investigate the muscle expression of fatty acid transporters and their function related to insulin sensitivity in IMCL-accumulated subjects.
Design and Setting: The study subjects were 36 nonobese healthy men. Their IMCL levels were measured by H-1-magnetic resonance spectroscopy, and their insulin sensitivity was evaluated by steady-state glucose infusion rate (GIR) during a euglycemic-hyperinsulinemic clamp. Gene expression levels in the vastus lateralis were evaluated by quantitative RT-PCR. We compared the clinical phenotypes and the expression levels of genes involved in lipid metabolism in skeletal muscle between IMCL-accumulated high-GIR (H-GIR) subjects (n = 8) and low-GIR subjects (n = 9). The functions of candidate fatty acid transporters were determined by in vitro analyses.
Results: Compared with the low-GIR group, body fat was lower and maximum oxygen uptake was higher in the H-GIR group. Several lipid oxidation genes in muscle were up-regulated in the H-GIR group, and this was associated with increased expression of higher plasma membrane-associated fatty acid-binding protein (FABPpm) and decreased expression of fatty acid transport protein (FATP)-1. Overexpression of FABPpm in C2C12 myotubes increased fatty acid oxidation coupled with the elevated expression of genes related to fatty acid oxidation. These changes were not observed in FATP1-overexpressed myotubes.
Conclusions: Differences in the gene expression of fatty acid transporters may, at least in part, affect insulin sensitivity in IMCL-accumulated nonobese men.

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PARK2/Parkin-mediated mitochondrial clearance contributes to the proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Norihiko Furuya, Shin-Ichi Ikeda, Shigeto Sato, Sanae Soma, Junji Ezaki, Juan Alejandro Oliva Trejo, Mitsue Takeda-Ezaki, Tsutomu Fujimura, Eri Arikawa-Hirasawa, Norihiro Tada, Masaaki Komatsu, Keiji Tanaka, Eiki Kominami, Nobutaka Hattori and Takashi Ueno

Autophagy （Autophagy） 10 （ 4 ） 631 - 641 2014年04月

査読有り

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

AUTOPHAGY （LANDES BIOSCIENCE） 10 （ 4 ） 631 - 641 2014年04月

査読有り, ISSN 1554-8627

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin-proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.

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Exercise-induced enhancement of insulin sensitivity is associated with accumulation of M2-polarized macrophages in mouse skeletal muscle

Shin-ichi Ikeda, Yoshifumi Tamura, Saori Kakehi, Kageumi Takeno, Minako Kawaguchi, Takahiro Watanabe, Fumihiko Sato, Takeshi Ogihara, Akiko Kanazawa, Yoshio Fujitani, Ryuzo Kawamori, Hirotaka Watada

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 441 （ 1 ） 36 - 41 2013年11月

査読有り, ISSN 0006-291X

Exercise enhances insulin sensitivity in skeletal muscle, but the underlying mechanism remains obscure. Recent data suggest that alternatively activated M2 macrophages enhance insulin sensitivity in insulin target organs such as adipose tissue and liver. Therefore, the aim of this study was to determine the role of anti-inflammatory M2 macrophages in exercise-induced enhancement of insulin sensitivity in skeletal muscle. C57BL6J mice underwent a single bout of treadmill running (20 m/min, 90 min). Twenty-four hours later, ex vivo insulin-stimulated 2-deoxy glucose uptake was found to be increased in plantaris muscle. This change was associated with increased number of CD163-expressing macrophages (i.e. M2-polarized macrophages) in skeletal muscle. Systemic depletion of macrophages by pretreatment of mice with clodronate-containing liposome abrogated both CD163-positive macrophage accumulation in skeletal muscle as well as the enhancement of insulin sensitivity after exercise, without affecting insulin-induced phosphorylation of Akt and AS160 or exercise-induced GLUT4 expression. These results suggest that accumulation of M2-polarized macrophages is involved in exercise-induced enhancement of insulin sensitivity in mouse skeletal muscle, independently of the phosphorylation of Akt and AS160 and expression of GLUT4. (C) 2013 Elsevier Inc. All rights reserved.

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Determinants of intramyocellular lipid accumulation after dietary fat loading in non-obese men

Yuko Sakurai, Yoshifumi Tamura, Kageumi Takeno, Naoki Kumashiro, Fumihiko Sato, Saori Kakehi, Shin-ichi Ikeda, Yuji Ogura, Norio Saga, Hisashi Naito, Shizuo Katamoto, Yoshio Fujitani, Takahisa Hirose, Ryuzo Kawamori, Hiritaka Watada

Journal of Diabetes Investigation （Journal of Diabetes Investigation） 2 （ 4 ） 310 - 317 2011年08月

査読有り

UNLABELLED: Aims/Introduction: Accumulation of intramyocellular lipid (IMCL) is associated with insulin resistance. However, the factors affecting the change in IMCL remain to be elucidated. The aim of the present study was to determine the factors that influence the change in IMCL level after high-fat loading. MATERIALS AND METHODS: The study subjects were 37 non-obese men. Each subject consumed a high-fat diet for 3 days after a normal-fat diet for 3 days. After each diet program, IMCL levels in the tibialis anterior (TA-IMCL) and soleus (SOL-IMCL) were measured by proton magnetic resonance spectroscopy. Glucose infusion rate (GIR) was evaluated by euglycemic hyperinsulinemic clamp as an index of peripheral insulin sensitivity. RESULTS: The high-fat diet significantly increased TA-IMCL and SOL-IMCL by ∼30 and ∼20%, respectively (P < 0.05), whereas it did not significantly alter GIR. The increase in SOL-IMCL, but not in TA-IMCL, negatively correlated with serum high molecular weight (HMW)-adiponectin (r = -0.36, P < 0.05) and HMW-/total-adiponectin ratio (r = -0.46, P < 0.05). Although high-fat diet-related changes in SOL-IMCL showed high inter-individual variations, in subjects doing exercise, changes in SOL-IMCL (r = 0.55, P < 0.05) and TA-IMCL (r = 0.61, P < 0.05) positively correlated with daily physical activity level. In contrast, in sedentary subjects, changes in SOL-IMCL (r = -0.50, P < 0.01) and TA-IMCL (r = -0.48, P < 0.05) negatively correlated with daily physical activity. CONCLUSIONS: HMW-adiponectin and daily physical activity are determinants of IMCL accumulation by a high-fat diet. Intriguingly, the effect of daily physical activity on the change in IMCL depends on the level of regular exercise. (J Diabetes Invest,doi: 10.1111/j.2040-1124.2010.00091.x, 2011).

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Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

Shin-ichi Ikeda, Takako Kizaki, Shuko Haga, Hideki Ohno, Tohru Takemasa

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 368 （ 2 ） 323 - 328 2008年04月

査読有り, ISSN 0006-291X

Peroxisome proliferator-activated receptor 7 coactivator-lot (PGC-1 alpha) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1 alpha and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1 alpha protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1 alpha expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1 alpha and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions. (C) 2008 Elsevier Inc. All rights reserved.

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Muscle type-specific response of PGC-1 alpha and oxidative enzymes during voluntary wheel running in mouse skeletal muscle

Shin-ichi Ikeda, H. Kawamoto, K. Kasaoka, Y. Hitomi, Takako Kizaki, Y. Sankai, Hideki Ohno, Shuko Haga, Tohru Takemasa

Acta Physiologica （Acta Physiologica） 188 （ 3-4 ） 217 - 223 2006年11月

査読有り, ISSN 1748-1708

Aim: It is generally accepted that endurance exercise increases the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha), which governs the expression of oxidative metabolic enzymes. A previous report demonstrated that the regulation of mitochondrial protein expression in skeletal muscles in response to cold exposure depends on muscle fibre type. Cold exposure and endurance exercise are both metabolic challenges that require adjustments in mitochondrial energy metabolism, we hypothesized that the exercise-induced increase in oxidative enzymes and PGC-1 alpha expression is higher in fast-type than in slow-type muscle.
Methods: Female ICR mice were individually housed in cages equipped with running wheel for 1, 2, 4, 6 or 8 weeks. The soleus, plantaris (PLA) and tibialis anterior (TA) muscles were then prepared from each mouse. The expression levels of PGC-1 alpha, mitochondrial proteins and GLUT4 were evaluated by Western blotting.
Results: The expression level of PGC-1 alpha was increased only in the PLA muscle. Furthermore, the expression levels of all mitochondrial proteins and GLUT4 in the PLA muscle were increased. In the TA muscle, although there was no increase in PGC-1 alpha expression, the expression levels of mitochondrial proteins and GLUT4 were increased.
Conclusion: These results suggest that muscle type-specific responses occur during endurance exercise, and that the increase in PGC-1 alpha expression is not the only factor that promotes oxidative capacity as a result of endurance exercise.

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Simple method for the identification of oxidative fibers in skeletal muscle

Tohru Takemasa, K. Sugimoto, Mitsunori Miyazaki, Masanao Machida, Shin-ichi Ikeda, Y. Hitomi, Takako Kizaki, Hideki Ohno, K. Yamashita, Shuko Haga

European Journal of Applied Physiology （ European Journal of Applied Physiology） 91 （ 2-3 ） 357 - 359 2004年03月

査読有り, ISSN 1439-6319

Skeletal muscle is composed of several different types of myofiber: slow oxidative (SO), fast glycolytic oxidative and fast glycolytic. However, the classification is usually determined by myosin heavy chain typing rather than by metabolic index. In this study, the oxidative metabolic index was investigated as a possible method of myofiber typing. Myoglobin, which is involved in oxygen transport and storage in myofibers, and mitochondria, which are the central organelles for oxidative metabolism, were studied. High levels of myoglobin and mitochondria are believed to exist in SO fibers, but the current study showed that they are considerably richer in some fast type fibers. As myofiber typing using the oxidative metabolic index is important physiologically, an attempt was made to find a simple method for this purpose. Some mitochondrial proteins have been observed to auto-fluoresce but until now this effect was too faint to detect easily. Owing to the recent advances in cooling charge-coupled device technology, such auto-fluorescence can now be used for myofiber typing, and the simple and rapid method for doing so is reported here.

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