Details of a Researcher - OOSAKI Yuuki

Papers - OOSAKI Yuuki

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Pathological significance of intranuclear structures in liver biopsy samples.

Norihiro Imai, Yuki Ohsaki, Jinglei Cheng, Hanna Kawecka, Jingjing Zhang, Fumitaka Mizuno, Taku Tanaka, Shinya Yokoyama, Kenta Yamamoto, Takanori Ito, Yoji Ishizu, Takashi Honda, Tetsuya Ishikawa, Michał Woźniak, Hiroaki Wake, Hiroki Kawashima

Hepatology research : the official journal of the Japan Society of Hepatology 55 ( 7 ) 1065 - 1074 2025.07 [International journal]

AIM: Glycogenated nuclei (GN) are glycogen deposits within the nuclei and are a frequent pathological finding in metabolic dysfunction-associated steatotic liver disease. This study aimed to investigate the relationship between GN and two morphologically distinct types of intranuclear lipid droplets in liver biopsy specimens and to explore their respective pathological significance. METHODS: We analyzed 135 liver biopsy specimens. A portion of the liver biopsy specimen was examined using transmission electron microscopy (TEM) to investigate intranuclear lipid droplets in hepatocytes. Nuclear inclusion bodies with clear boundaries and unstained areas on hematoxylin and eosin staining were identified as nuclear glycogen. RESULTS: TEM revealed nucleoplasmic lipid droplets (nLD) in 65% of liver biopsy specimens and invagination of cytoplasmic lipid droplets into the nucleus in 30% of specimens. In contrast, light microscopy detected GN in 82% of specimens. No significant correlations were observed between the frequencies of the two types of intranuclear lipid droplets and nuclear glycogen levels. A significant positive correlation was observed between the frequency of nLD and transaminase levels. Glycogenated nuclei were frequently observed in liver biopsy specimens from patients with MASLD; however, their frequency did not significantly correlate with the degree of hepatic steatosis. Instead, a significant positive correlation was observed between nuclear glycogen and blood HbA1c levels. CONCLUSIONS: The two types of intranuclear lipid droplets and nuclear glycogen observed in liver biopsy specimens showed no significant correlation in their formation frequencies, suggesting that they possess distinct pathological significance.

Region-specific assessment of the mechanical properties of each hamstring muscle in human cadavers using shear wave elastography.

Gakuto Nakao, Taiki Kodesho, Kazuma Yamagata, Risa Adachi, Koki Ishiyama, Kazuyoshi Kozawa, Kota Watanabe, Yuki Ohsaki, Masaki Katayose, Keigo Taniguchi

Clinical biomechanics (Bristol, Avon) 127 106586 - 106586 2025.07 [International journal]

BACKGROUND: Understanding regional mechanical properties of individual hamstring muscles is essential for accurately interpreting their functional behavior during elongation. However, how mechanical stress varies within muscles during elongation remains unclear. This study aimed to examine whether mechanical stresses differ among the hamstring muscles and at various regions within each muscle. METHODS: Fifteen cadavers were dissected to study the biceps femoris long head, semitendinosus, and semimembranosus muscles. Proximal and distal tendons were attached to a mechanical testing machine, and muscles were stretched from slack length to 8 % strain. Muscle length was measured with a tape measure, and anatomical cross-sectional areas at proximal (33 %) and distal (67 %) regions were determined using B-mode ultrasonography. Strain and stress were calculated to assess mechanical properties, and shear modulus was measured using shear wave elastography at the same regions. FINDINGS: A linear correlation between shear modulus and stress was found for all hamstring muscles (P < 0.01). Significant interactions among muscle, region, and strain were observed, with post-hoc tests revealing that the biceps femoris long head and semimembranosus had higher shear modulus than the semitendinosus after 0.5 % strain. The proximal biceps femoris long head showed increased shear modulus after 5 % strain, and proximal semimembranosus showed higher values after 0.5 % strain compared with the distal region. INTERPRETATION: The study findings reveal region-specific variations in the mechanical properties both among and within the hamstring muscles. Combining shear wave elastography with mechanical testing offers a non-destructive approach for characterizing these variations in passive muscle behavior.

Ultrasound-guided serratus posterior superior muscle block: an anatomical study investigating the extent of injected dye and the mechanism of action of a simulated injection in Thiel soft-embalmed cadavers.

Atsushi Sawada, Tatsuya Kunigo, Yuki Ohsaki, Kanna Nagaishi, Michiaki Yamakage

Journal of anesthesia 2025.04 [Domestic journal]

PURPOSE: The extent of local anesthetic spread and mechanism of action of serratus posterior superior muscle (SPSM) block is still debatable. This cadaveric study aimed to evaluate the anatomical spread of 30 or 20 mL of dye following a simulated SPSM block in Thiel soft-embalmed cadavers. METHODS: Simulated SPSM block injections were administered bilaterally in four cadavers (left side: 30 mL, right side: 20 mL). Anatomical dissection was performed to evaluate the extent of spread of the injected dye over the ribcage, and to document staining of the musculature of the back following a simulated SPSM block. RESULTS: The extents of spread (mean ± SD) of 30 mL and 20 mL of dye over the ribcage were 8.5 ± 3.9 cm and 5.5 ± 1.6 cm, respectively. Dye spread to the peri-scapular fascia was observed in all four of the simulated SPSM blocks with 30 mL of dye, but not with 20 mL. Dye spread into the rhomboid major muscle was observed in two of the simulated SPSM blocks with 30 mL of dye. CONCLUSION: Our findings suggest that the SPSM block using 30 mL of dye has a tendency to spread wider compared to the SPSM block using 20 mL of injectate, and that the block might serve as an interfascial block rather than a segmental nerve block. Further studies in a large sample of human participants are required to confirm the optimal volume of local anesthetic in the SPSM block.

Ultrasound-assisted middle thoracic epidural catheter placement utilizing the most dorsal sites of bilateral transverse process roots as anatomical landmarks: A cadaveric observational study and a clinical randomized controlled trial.

Tatsuya Kunigo, Yusuke Yoshikawa, Shunichi Niki, Masahiro Ohtani, Mami Muraki, Asako Nitta, Yuki Ohsaki, Kanna Nagaishi, Michiaki Yamakage

Journal of clinical anesthesia 101 111740 - 111740 2025.02 [International journal]

STUDY OBJECTIVE: We developed an innovative method for ultrasound-assisted thoracic epidural catheter placement and assessed its potential to reduce procedural duration for trainees. DESIGN: A cadaveric observational study and a clinical randomized controlled trial. SETTING: Sapporo Medical University Hospital. PATIENTS: A total of 52 adult patients scheduled for thoracic or abdominal surgery and four cadavers. INTERVENTIONS: Patients were randomly assigned to either group receiving conventional palpation (conventional group) or combination of the ultrasound examination and conventional palpation (ultrasound group). MEASUREMENTS: The primary outcome was total procedure time (sum of skin marking time and needling time) by trainees. The secondary outcomes were (1) skin marking time, (2) needling time, (3) multiple skin punctures, (4) needle redirection, (5) complications, and (6) failed cases. MAIN RESULTS: Through dissection of four cadavers, the most dorsal site of the transverse process root was identifiable by ultrasound and the reliable indicator of the interlaminar space. We devised ultrasound-assisted middle thoracic epidural catheter placement utilizing the most dorsal sites of bilateral transverse process roots as anatomical landmarks. Trainees in the ultrasound group had significantly longer skin marking time and significantly shorter needling time than those in the conventional group (107 [87-158] vs 46 s [34-54] s, p < 0.001 and 197 [156-328] vs 341 [303-488] s, p = 0.003). Consequently, there was no significant difference between the two groups in total procedure time (326 [263-467] s vs 391 [354-533] s, p = 0.167). Moreover, the probability of trainee failure in epidural anesthesia was significantly lower in the ultrasound group (2/26 [17.7 %] vs 10/26 [38.5 %], p = 0.019). CONCLUSIONS: Our novel technique for thoracic epidural catheter placement resulted in expedited needling and enhanced success rates among trainees, although there was no significant difference between total procedure time when using ultrasound guidance and that when using conventional palpation.

Passive muscle tension changes in the biceps femoris long head after biceps femoris short head detachment: A human cadaver study.

Gakuto Nakao, Kazuma Yamagata, Risa Adachi, Koki Ishiyama, Kazuyoshi Kozawa, Kota Watanabe, Yuki Ohsaki, Kousuke Shiwaku, Norio Hayashi, Masaki Katayose, Keigo Taniguchi

Journal of biomechanics 179 112480 - 112480 2025.01 [International journal]

The functional role of the biceps femoris short head (BFsh) remains unclear. Clarifying the functional role of each biceps femoris head may provide useful insights into the reduction of biceps femoris long head (BFlh) injuries. This study aimed to clarify whether the passive tension in the BFlh would change with BFsh detachment using cadavers. The shear modulus of the BFlh and BFsh was measured using ultrasonic shear wave elastography as an index of passive tension in three tissue processing conditions (intact, removal of all tissues from skin to deep fascia, and BFsh detachment) in four limb positions, with hip [H0°, H90°] and knee [K0°, K90°] joint angles under each tissue processing condition. The measurement site was the distal 30 % of the line connecting the sciatic tuberosity (100 %) and fibular head (0 %). Three-way analysis of variance was conducted with muscles, tissue processing, and positions as factors, which revealed a significant interaction (P < 0.01). The post-test results indicated that the BFlh was significantly higher than the BFsh at H90° and K0° before tissue processing; however, no difference was observed between the muscles after skin and deep fascia removal. After BFsh detachment, the shear modulus of the BFsh decreased, whereas that of the BFlh significantly increased (P < 0.01), suggesting that the BFsh might be involved in the passive tension reduction of the BFlh in a lengthened position, as the shear modulus of the BFlh increased after detachment.

Effects of superficial tissue and intermuscular connections on rectus femoris muscle shear modulus heterogeneity.

Taiki Kodesho, Takuya Kato, Gakuto Nakao, Yu Yokoyama, Yuhei Saito, Kota Watanabe, Yuki Ohsaki, Masaki Katayose, Keigo Taniguchi

Journal of ultrasound 27 ( 3 ) 449 - 455 2024.09 [International journal]

INTRODUCTION: Intramuscular heterogeneity exists in the shear modulus of the rectus femoris (RF) muscle. However, the underlying heterogeneity mechanisms are not entirely understood. Previous research has reported that detachment of superficial tissues reduces the shear modulus by 50%. The aim of this study was to examine the effects of the skin, deep fascia, and intermuscular connections on the shear modulus of the RF at multiple sites. MATERIALS AND METHODS: Eleven donors were fixed using the Thiel method. Measurements were performed at 0°, 60°, and 120° knee flexion in a neutral hip position. Tissue processing was performed under four conditions: superficial tissue (CONT), skin off (SKIN), deep fascia detachment (FASC), and intermuscular connections detachment (ALL). The shear modulus at the proximal, central, and distal regions were measured using ultrasound shear wave elastography. The study was approved by the Sapporo Medical University Ethical Committee. RESULTS: Three-way ANOVA revealed no significant interaction between treatment, site, and angle (P = 0.156), treatment and angle (P = 0.067), or site and angle (P = 0.441). There was a significant effect of treatment (P < 0.001), site (P = 0.010), and angle (P < 0.001) and interaction between treatment and site (P < 0.001). The proximal shear modulus was greater than the central for CONT. There were no significant differences between the measurement sites for SKIN. The distal shear modulus was greater than the proximal for FASC. The distal shear modulus was also greater than the proximal and central for ALL. CONCLUSIONS: Intramuscular regional differences that influence superficial tissue and intermuscular connections of RF elasticity heterogeneity were observed.

Validity of Elastic Imaging Evaluation of Hamstring Muscles With Knee Contracture Using Ultrasound Shear Wave Elastography.

Gakuto Nakao, Taiki Kodesho, Kazuma Yamagata, Kota Watanabe, Yuki Ohsaki, Masaki Katayose, Keigo Taniguchi

Cureus 16 ( 8 ) e68343 2024.08 [International journal]

PURPOSE: This study used ultrasound shear wave elastography (SWE) to evaluate the mechanical properties of hamstring muscles from cadaveric specimens with knee flexion contractures. METHODS: Hamstring muscles for tensile testing were harvested from Thiel soft-embalmed cadavers with and without knee flexion contracture. Muscle specimens were mounted on a testing machine. The initial load detected when a tensile load was applied to the distal end was used as the slack length. The cross-sectional areas of the muscle at slack length were measured at the proximal and distal sites using B-mode ultrasonography. Subsequently, the muscle specimen was elongated from the slack length to 8% strain, with the shear modulus measured using SWE. Young's modulus (stress/strain) was calculated based on the displacement and tensile force obtained from the tensile test. RESULTS: Regression analysis showed a significant positive linear relationship between the Young's and shear moduli for all specimens at all the sites (P < 0.01 and coefficient of determination: 0.95-0.99). The Young's and average shear moduli at the proximal and distal sites were higher in all hamstring muscles with contractures than in those without contractures. CONCLUSIONS: SWE can be used to estimate Young's moduli of hamstring muscles with contractures. Muscle specimens with contractures exhibited higher resistance to elongation, thereby indicating that their mechanical properties differed from those of muscles without contractures.

Correction: Relationship between shear elastic modulus and passive muscle force in human hamstring muscles using a Thiel soft-embalmed cadaver.

Gakuto Nakao, Taiki Kodesho, Takuya Kato, Yu Yokoyama, Yuhei Saito, Yuki Ohsaki, Kota Watanabe, Masaki Katayose, Keigo Taniguchi

Journal of medical ultrasonics (2001) 51 ( 3 ) 541 - 541 2024.07 [Domestic journal]

Stress-strain relationship of individual hamstring muscles: A human cadaver study.

Gakuto Nakao, Taiki Kodesho, Kazuma Yamagata, Kota Watanabe, Yuki Ohsaki, Masaki Katayose, Keigo Taniguchi

Journal of the mechanical behavior of biomedical materials 153 106473 - 106473 2024.05 [International journal]

The incidence of hamstring muscle strain varies among muscles, suggesting that the mechanical stresses associated with elongation may differ among muscles. However, the passive mechanical properties of whole human muscles have rarely been directly measured and clarified. This study aimed to clarify the stress-strain relationship of the hamstring muscles using a soft-embalmed Thiel cadaver. The long heads of the biceps femoris (BFlh), semimembranosus (SM), and semitendinosus (ST) muscles were dissected from eight cadavers. The proximal and distal hamstring tendons were affixed to the mechanical testing machine. Slack length was defined as the muscle length at the initial loading point detected upon the application of a tensile load. Muscle length was measured using a tape measure, and the anatomical cross-sectional area (ACSA) of the muscle was measured at the proximal and distal sites using B-mode ultrasonography. In the loading protocol, the muscle was elongated from its slack length to a maximum of 8% strain at an average rate of 0.83 L0/s, and the amount of displacement and tensile load were measured for each muscle. Further, the strain (%, displacement/slack muscle length) and stress (kPa, tensile load/ACSA) were calculated to evaluate the mechanical properties. Two-way repeated-measures analysis of variance (ANOVA) was used to compare stress changes with increasing muscle strain. A significant interaction between the muscle and strain factors was observed with respect to stress. Post-hoc tests revealed higher stresses in the BFlh and SM than in ST after 3% strain (P < 0.01). However, no significant differences were observed between the BFlh and SM groups. At 8% strain, the BFlh, SM, and ST exhibited stresses of 63.7 ± 12.1, 53.7 ± 23.2, and 21.0 ± 11.9 kPa, respectively. The results indicate that the stress changes associated with muscle strain differed among muscles. In particular, the stress applied to the three muscles at the same strain was found to be higher in the BFlh and SM. Thus, these findings suggest that increased mechanical stress during elongation may contribute to the frequent occurrence of muscle strain in BFlh and SM.

Fish oil-derived n-3 polyunsaturated fatty acids downregulate aquaporin 9 protein expression of liver and white adipose tissues in diabetic KK mice and 3T3-L1 adipocytes.

Yuzuru Iizuka, Satoshi Hirako, Hyounju Kim, Nobuhiro Wada, Yuki Ohsaki, Naoko Yanagisawa

The Journal of nutritional biochemistry 124 109514 - 109514 2024.02 [International journal]

Aquaporin 9 (AQP9) is an integral membrane protein that facilitates glycerol transport in hepatocytes and adipocytes. Glycerol is necessary as a substrate for gluconeogenesis in the physiological fasted state, suggesting that inhibiting AQP9 function may be beneficial for treating type 2 diabetes associated with fasting hyperglycemia. The n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are rich in fish oil and lower the risk of metabolic syndrome; however, the effects of EPA and DHA on AQP9 expression in obese and type 2 diabetes are unclear. The KK mouse is an animal model of obesity and type 2 diabetes because of the polymorphisms on leptin receptor gene, which results in a part of cause for obese and diabetic conditions. In this study, we determined the effect of fish oil-derived n-3 PUFA on AQP9 protein expression in the liver and white adipose tissue (WAT) of KK mice and mouse 3T3-L1 adipocytes. The expression of AQP9 protein in the liver, epididymal WAT, and inguinal WAT were markedly decreased following fish oil administration. We also demonstrated that n-3 PUFAs, such as DHA, and to a lesser extent EPA, downregulated AQP9 protein expression in 3T3-L1 adipocytes. Our results suggest that fish oil-derived n-3 PUFAs may regulate the protein expressions of AQP9 in glycerol metabolism-related organs in KK mice and 3T3-L1 adipocytes.

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line.

Takumi Konno, Takayuki Kohno, Shin Kikuchi, Arisa Kura, Kimihito Saito, Tadahi Okada, Hiroshi Shimada, Yuya Yamazaki, Tomoki Sugiyama, Motoki Matsuura, Yuki Ohsaki, Tsuyoshi Saito, Takashi Kojima

Tissue barriers 2304443 - 2304443 2024.01 [International journal]

It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-β and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-β receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-β and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-β and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

I-gel Plus acts as a superior conduit for fiberoptic intubation than standard i-gel.

Tomohiro Chaki, Shunsuke Tachibana, Sho Kumita, Satoshi Sato, Tomoki Hirahata, Yuta Ikeshima, Yuki Ohsaki, Michiaki Yamakage

Scientific reports 13 ( 1 ) 18381 - 18381 2023.10 [International journal]

The supraglottic airway (SGA) is widely used. I-gel Plus is a next-generation i-gel with some improvements, including facilitation of fiberoptic tracheal intubation (FOI). To compare the performance of i-gel Plus and standard i-gel as conduits for FOI, a Thiel-embalmed cadaveric study was conducted. Twenty-two anesthesiologists were enrolled as operators in Experiment 1. The i-gel Plus and standard i-gel were inserted into one cadaver, and the FOI was performed through each SGA. The primary outcome was time required for FOI. The secondary outcomes were the number of attempts and visual analog scale (VAS) score for difficulty in FOI. Moreover, fiberoptic views of the vocal cords in each SGA were assessed by an attending anesthesiologist using nine cadavers in Experiment 2. The percentage of glottic opening (POGO) score without fiberscope tip upward flexion and upward angle of the fiberscope tip to obtain a 100% POGO score were evaluated as secondary outcomes. The time for FOI through i-gel Plus was significantly shorter than that through standard i-gel (median (IQR), i-gel Plus: 30.3 (25.4-39.0) s, vs standard i-gel: 54.7 (29.6-135.0) s; median of differences, 24.4 s; adjusted 95% confidence interval, 3.0-105.7; adjusted P = 0.040). Although the number of attempts for successful FOI was not significantly different, the VAS score for difficulty in the i-gel Plus group was significantly lower (easier) than that in the standard i-gel group. Moreover, i-gel Plus required a significantly smaller upward angle of the fiberscope tip to obtain a 100% POGO score. FOI can be performed more easily using i-gel Plus than using standard i-gel because of the improved fiberoptic visibility of vocal cords.

Modified thoracoabdominal nerve block through perichondrial approach (M-TAPA): an anatomical study to evaluate the spread of dye after a simulated injection in soft embalmed Thiel cadavers.

Atsushi Sawada, Sho Kumita, Asako Nitta, Yuki Ohsaki, Michiaki Yamakage

Regional anesthesia and pain medicine 48 ( 8 ) 403 - 407 2023.08 [International journal]

BACKGROUND AND OBJECTIVES: There is still no consensus on the analgesic range and mechanisms of action of modified thoracoabdominal nerve block through perichondrial approach (M-TAPA). This cadaveric study aimed to determine the spread of an injectate following simulated M-TAPA. METHODS: Simulated M-TAPA injections (n=8) were administered on both sides of soft embalmed Thiel cadavers with 25 mL of a saline-soluble dye. Anatomic dissection was performed to document staining (deeply, faintly, or not stained) of the anterior cutaneous branches of the thoracoabdominal nerves and determine the extent of the injectate spread of the dye to the intercostal space in the thoracic cage following a simulated M-TAPA. RESULTS: The median (IQR) dermatome of the stained segmental nerve was T10 (T8-T11) and the median (IQR) number of stained segmental nerves was 3 (4-2). The T9, T10 and T11 segmental nerves were stained in 75%, 100% and 62.5% of simulated M-TAPA, respectively. Conversely, the T8 segmental nerve was stained in only 25% of simulated M-TAPA. No injectate spread of dye to the intercostal space in the thoracic cage was observed in eight simulated injections of M-TAPA. CONCLUSION: Our findings suggest that M-TAPA most likely involves the T9, T10 and T11 segmental nerves and that the local anesthetic may not spread to the intercostal space in the thoracic cage in M-TAPA. Further studies are required to confirm the precise mechanism of action and efficacy of M-TAPA in a large sample of human participants.

Relationship between shear elastic modulus and passive muscle force in human hamstring muscles using a Thiel soft-embalmed cadaver.

Gakuto Nakao, Taiki Kodesho, Takuya Kato, Yu Yokoyama, Yuhei Saito, Yuki Ohsaki, Kota Watanabe, Masaki Katayose, Keigo Taniguchi

Journal of medical ultrasonics (2001) 50 ( 3 ) 275 - 283 2023.07 [Domestic journal]

PURPOSE: Assessing muscle flexibility and architecture is important for hamstring strain injury (HSI) prevention. We investigated the relationship between shear modulus and passive force in hamstring muscles at different sites and the effect of muscle architecture on the slope of the shear modulus-passive force using shear wave elastography (SWE). METHODS: The biceps femoris long head (BFlh), semitendinosus (ST), and semimembranosus (SM) muscles were dissected from nine Thiel-embalmed cadavers and fixed to a custom-made mechanical testing machine. Calibrated weights (0-1800 g) were applied gradually in 150-g increments. The shear modulus and anatomical cross-sectional area (ACSA) were measured at proximal, central, and distal points using SWE. The muscle mass and length were measured before the loading test. The shear modulus-passive load relationship of each tested muscle region was analyzed by fitting a least-squares regression line. The increase in shear modulus slope per unit load was calculated and compared between the muscles before and after normalization by the muscle mass, length, and ACSA. RESULTS: The shear modulus and passive force for all hamstring muscles in each region showed a statistically significant linear correlation. Furthermore, the increase in shear modulus slope was greater for BFlh and ST than for SM (P < 0.05), but after normalization by the muscle length and ACSA, there were no significant differences among the muscles. CONCLUSION: The local mechanical properties of individual hamstring muscles can be indirectly estimated using SWE, and the slope of increase in shear modulus reflects characteristics of the muscle architecture.

Distinct features of two lipid droplets types in cell nuclei from patients with liver diseases

Norihiro Imai, Yuki Ohsaki, Jinglei Cheng, Jingjing Zhang, Fumitaka Mizuno, Taku Tanaka, Shinya Yokoyama, Kenta Yamamoto, Takanori Ito, Yoji Ishizu, Takashi Honda, Masatoshi Ishigami, Hiroaki Wake, Hiroki Kawashima

Scientific Reports ( Springer Science and Business Media LLC ) 13 ( 1 ) 2023.04

Authorship： Lead author , Corresponding author

Abstract Lipid droplets (LDs) have been observed in the nuclei of hepatocytes; however, their significance in liver disease remains unresolved. Our purpose was to explore the pathophysiological features of intranuclear LDs in liver diseases. We included 80 patients who underwent liver biopsies; the specimens were dissected and fixed for electron microscopy analysis. Depending on the presence of adjacent cytoplasmic invagination of the nuclear membrane, LDs in the nuclei were classified into two types: nucleoplasmic LDs (nLDs) and cytoplasmic LD invagination with nucleoplasmic reticulum (cLDs in NR). nLDs were found in 69% liver samples and cLDs in NR were found in 32%; no correlation was observed between the frequencies of the two LD types. nLDs were frequently found in hepatocytes of patients with nonalcoholic steatohepatitis, whereas cLDs in NR were absent from the livers of such patients. Further, cLDs in NR were often found in hepatocytes of patients with lower plasma cholesterol level. This indicates that nLDs do not directly reflect cytoplasmic lipid accumulation and that formation of cLDs in NR is inversely correlated to the secretion of very low-density lipoproteins. Positive correlations were found between the frequencies of nLDs and endoplasmic reticulum (ER) luminal expansion, suggesting that nLDs are formed in the nucleus upon ER stress. This study unveiled the presence of two distinct nuclear LDs in various liver diseases.

Oleic acid‐bound <scp>FABP7</scp> drives glioma cell proliferation through regulation of nuclear lipid droplet formation

Banlanjo Abdulaziz Umaru, Yoshiteru Kagawa, Yuki Ohsaki, Yijun Pan, Chuck T. Chen, Daniel K. Chen, Toshiaki Abe, Subrata Kumar Shil, Hirofumi Miyazaki, Shuhei Kobayashi, Motoko Maekawa, Yui Yamamoto, Tunyanat Wannakul, Shuhan Yang, Richard P. Bazinet, Yuji Owada

The FEBS Journal ( Wiley ) 290 ( 7 ) 1798 - 1821 2022.11

Oxygen-Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats.

Shin Kikuchi, Takayuki Kohno, Takashi Kojima, Haruyuki Tatsumi, Yuki Ohsaki, Takafumi Ninomiya

Cellular and molecular neurobiology 2022.06 [International journal]

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

Nuclear lipid droplets form in the inner nuclear membrane in a seipin-independent manner.

Kamil Sołtysik, Yuki Ohsaki, Tsuyako Tatematsu, Jinglei Cheng, Asami Maeda, Shin-Ya Morita, Toyoshi Fujimoto

The Journal of cell biology 220 ( 1 ) 2021.01 [Refereed] [International journal]

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1β expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.

Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets.

Yuta Ogasawara, Jinglei Cheng, Tsuyako Tatematsu, Misaki Uchida, Omi Murase, Shogo Yoshikawa, Yuki Ohsaki, Toyoshi Fujimoto

Nature communications 11 ( 1 ) 4480 - 4480 2020.09 [Refereed] [International journal]

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.

Intracellular Organelles Driven by Liquid-Liquid Phase Separation

Shunsuke F. SHIMOBAYASHI, Yuki OHSAKI

Seibutsu Butsuri ( Biophysical Society of Japan ) 60 ( 5 ) 267 - 271 2020

Universal phase behaviors of intracellular lipid droplets.

Shunsuke F Shimobayashi, Yuki Ohsaki

Proceedings of the National Academy of Sciences of the United States of America 116 ( 51 ) 25440 - 25445 2019.12 [Refereed] [International journal]

Lipid droplets are cytoplasmic microscale organelles involved in energy homeostasis and handling of cellular lipids and proteins. The core structure is mainly composed of two kinds of neutral lipids, triglycerides and cholesteryl esters, which are coated by a phospholipid monolayer and proteins. Despite the liquid crystalline nature of cholesteryl esters, the connection between the lipid composition and physical states is poorly understood. Here, we present a universal intracellular phase diagram of lipid droplets, semiquantitatively consistent with the in vitro phase diagram, and reveal that cholesterol esters cause the liquid-liquid crystal phase transition under near-physiological conditions. We moreover combine in vivo and in vitro studies, together with the theory of confined liquid crystals, to suggest that the radial molecular alignments in the liquid crystallized lipid droplets are caused by an anchoring force at the droplet surface. Our findings on the phase transition of lipid droplets and resulting molecular organization contribute to a better understanding of their biological functions and diseases.

Nuclear lipid droplets derive from a lipoprotein precursor and regulate phosphatidylcholine synthesis

Kamil So{\l}tysik, Yuki Ohsaki, Tsuyako Tatematsu, Jinglei Cheng, Toyoshi Fujimoto

Nature Communications ( Springer Nature ) 10 ( 1 ) 1230 - 1230 2019.12 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

Nuclear lipid droplets derive from a lipoprotein precursor and regulate phosphatidylcholine synthesis.

Kamil Sołtysik, Yuki Ohsaki, Tsuyako Tatematsu, Jinglei Cheng, Toyoshi Fujimoto

Nature communications 10 ( 1 ) 473 - 473 2019.01 [International journal]

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CDP-choline diacylglycerol phosphotransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.

Duo in a Mystical Realm—Nuclear Lipid Droplets and the Inner Nuclear Membrane

Kamil Sołtysik, Yuki Ohsaki, Toyoshi Fujimoto

Contact ( SAGE Publications ) 2 251525641989696 - 251525641989696 2019.01 [Refereed] [Invited]

The lipid droplet (LD) is a cytoplasmic organelle, but it also exists in the nucleus under some conditions or in some cell types. New studies have revealed that nuclear LDs do not occur by haphazard entry of cytoplasmic LDs. Instead, they are generated by specific mechanisms that are increasingly understood. The inner nuclear membrane (INM) plays a critical role in nuclear LD formation in both mammalian hepatocytes and budding yeast, although in significantly different ways. Hepatocyte nuclear LDs derive from precursors of very low-density lipoprotein lacking apolipoprotein B-100, which form in the endoplasmic reticulum lumen and accumulate in intranuclear extensions of the perinuclear space called type I nucleoplasmic reticulum. In contrast, nuclear LDs in yeast are generated by triglyceride synthesized in the INM. Nuclear LDs in hepatocytes and budding yeast are both instrumental in the regulation of phospholipid synthesis; however, again they function in different ways. As the full functional importance is as yet unknown, the close relationship of nuclear LDs and the INM is an attractive target of research from both physiological and pathological perspectives.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells

Takir Gizem Gulevin, Ohsaki Yuki, Morotomi-Yano Keiko, Yano Ken-ichi, Saitoh Hisato

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 504 ( 2 ) 485-490 - 490 2018.10 [Refereed] [International journal]

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3

Hana Kimura, Kohei Arasaki, Yuki Ohsaki, Toyoshi Fujimoto, Takayuki Ohtomo, Junji Yamada, Mitsuo Tagaya

Journal of Lipid Research ( American Society for Biochemistry and Molecular Biology Inc. ) 59 ( 5 ) 805 - 819 2018 [Refereed] [International journal]

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

A New Electron Microscopic Method to Observe the Distribution of Phosphatidylinositol 3,4-bisphosphate

Sharmin Aktar, Sho Takatori, Takuma Tsuji, Minami Orii, Yuki Ohsaki, Jinglei Cheng, Toyoshi Fujimoto

ACTA HISTOCHEMICA ET CYTOCHEMICA ( JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY ) 50 ( 5 ) 141 - 147 2017 [Refereed] [Domestic journal]

Phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4) P-2] is a phosphoinositide that plays important roles in signal transduction, endocytosis, and cell migration among others. The intracellular distribution of PtdIns(3,4) P-2 has mainly been studied by observing the distribution of GFP-tagged PtdIns(3,4) P-2-binding protein domains in live cells and by labeling with anti-PtdIns(3,4) P-2 antibody in fixed cell samples, but these methods only offer low spatial resolution results and may have pitfalls. In the present study, we developed an electron microscopic method to observe the PtdIns(3,4) P-2 distribution using the SDS-treated freeze-fracture replica labeling method. The recombinant GST-tagged pleckstrin homology (PH) domain of TAPP1 was used as the binding probe, and its binding to PtdIns(3,4) P-2 in the freeze-fracture replica was confirmed by using liposomes containing different phosphoinositides and by the lack of labeling by a mutant probe, in which one amino acid in the PH domain was substituted. The method was applied to NIH3T3 cell samples and showed that the increase of PtdIns(3,4) P-2 in cells treated with hydrogen peroxide occurs in the cytoplasmic leaflet of the plasma membrane, except in the caveolar membrane. The present method can define the distribution of PtdIns(3,4) P-2 at a high spatial resolution and will facilitate our understanding of the physiological function of this less studied phosphoinositide.

The lipid droplet and the endoplasmic reticulum

Yuki Ohsaki, Kamil Sołtysik, Toyoshi Fujimoto

Advances in Experimental Medicine and Biology ( Springer New York LLC ) 997 111 - 120 2017 [Invited] [International journal]

Authorship： Lead author

Lipid droplets (LDs) are often found adjacent to the endoplasmic reticulum (ER). The ER–LD association may appear morphologically similar to the prototypical membrane contact sites found between the ER and other organelles, but the functional relationship between the ER and LDs is unique in that highly hydrophobic lipid esters are transported between them. This transportation is thought to occur through some form of membrane continuity, but its details are yet to be defined. Lipin, seipin, and FIT proteins, which are located at the ER–LD interface, may be involved in the lipid ester transport and probably play important roles for functional connectivity of the two organelles. More recently, LDs in the nucleus were found to be closely adhered to the inner nuclear membrane, representing a specialized form of the ER–LD association. In this article, we will give an overview of the ER–LD association, which is still filled with many unanswered questions.

PML isoform II plays a critical role in nuclear lipid droplet formation

Yuki Ohsaki, Takeshi Kawai, Yukichika Yoshikawa, Jinglei Cheng, Eija Jokitalo, Toyoshi Fujimoto

JOURNAL OF CELL BIOLOGY ( ROCKEFELLER UNIV PRESS ) 212 ( 1 ) 29 - 38 2016.01 [Refereed] [International journal]

Authorship： Lead author

Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP : phosphocholine cytidylyltransferase a and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types.

電子顕微鏡による脂肪滴と膜脂質の探索

藤本豊士, 大崎雄樹, 辻琢磨

Proceedings of Clinical Electron Microscopy 35 1‐3 2016

Inhibition of Niemann-Pick-Type C1-Like1 by Ezetimibe Activates Autophagy in Human Hepatocytes and Reduces Mutant alpha 1-Antitrypsin Z Deposition

Takeshi Yamamura, Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Tsuyako Tatematsu, Jinglei Cheng, Masato Okada, Naoki Ohmiya, Yoshiki Hirooka, Hidemi Goto, Toyoshi Fujimoto

HEPATOLOGY ( WILEY-BLACKWELL ) 59 ( 4 ) 1591 - 1599 2014.04 [Refereed] [International journal]

Autophagy can degrade aggregate-prone proteins, but excessive autophagy can have adverse effects. It would be beneficial if autophagy could be enhanced in a cell type-specific manner, but this has been difficult because the basic mechanism of autophagy is common. In the present study we found that inhibition of Niemann-Pick-type C1-like 1 (NPC1L1) by ezetimibe activates autophagy only in hepatocytes and small intestinal epithelia, but not in other cells. Ezetimibe induced accumulation of free cholesterol in the late endosome/lysosome and increased partitioning of a Ragulator component, LAMTOR1, in rafts. The latter change led to down-regulation of mammalian target of rapamycin (mTOR)C1 activity by decreasing mTOR recruitment to the late endosome/lysosome and activated autophagy. A primary effect of ezetimibe was found to be a decrease of free cholesterol in the plasma membrane, because all the results caused by ezetimibe were suppressed by supplementation of cholesterol as a methyl-beta-cyclodextrin complex. By enhancing autophagy in human primary hepatocytes with ezetimibe, insoluble mutant alpha 1-antitrypsin Z was reduced significantly. Conclusion: Inhibition of NPC1L1 by ezetimibe activates autophagy in human hepatocytes by modulating cholesterol homeostasis. Ezetimibe may be used to ameliorate liver degeneration in alpha 1-antitrypsin deficiency. (Hepatology 2014;59:1591-1599)

Open Questions in Lipid Droplet Biology

Yuki Ohsaki, Michitaka Suzuki, Toyoshi Fujimoto

CHEMISTRY & BIOLOGY ( CELL PRESS ) 21 ( 1 ) 86 - 96 2014.01 [Refereed] [Invited] [International journal]

Authorship： Lead author

Lipid droplets (LDs) have been the focus of intense research for the past decade because of their active engagement in lipid metabolism and relationship with diseases. In contrast to other intracellular organelles, LDs are composed of a mass of hydrophobic lipid esters that is covered with a phospholipid monolayer. The unique architecture makes the LD a formidable object to study by the methods available today, and many fundamental questions remain unanswered. This review focuses on some of those questions, such as how LDs form and grow, how proteins move to and from LDs, and how LDs are related to protein degradation; we will also discuss what is not known about LDs. We think that small LDs that have thus far eluded analysis are the key to resolving many of the above-mentioned questions.

Inhibition of ADP-ribosylation suppresses aberrant accumulation of lipidated apolipoprotein B in the endoplasmic reticulum

Yuki Ohsaki, Jinglei Cheng, Kazushi Yamairi, Xiaoyue Pan, M. Mahmood Hussain, Toyoshi Fujimoto

FEBS LETTERS ( ELSEVIER SCIENCE BV ) 587 ( 22 ) 3696 - 3702 2013.11 [Refereed] [International journal]

Authorship： Lead author

ApoB-crescent, an endoplasmic reticulum (ER)-lipid droplet amalgamation structure, is a useful marker to indicate aberrant lipidated apolipoprotein B accumulation in the hepatocyte ER. Blockade of the ER-to-Golgi transport by either vesicle transport inhibitors or dominant-negative Arf1 caused a significant increase in ApoB-crescents. However, a low concentration of Brefeldin A induced the same result without affecting protein secretion, suggesting ADP-ribosylation as an additional mechanism. ADP-ribosylation inhibitors not only suppressed the increase of ApoB-crescents, but also rapidly dissolved existing ApoB-crescents. These results implicate the involvement of ADP-ribosylation in the ApoB-crescent formation and maintenance process at the ER. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Alleviation of seipinopathy-related ER stress by triglyceride storage.

Maarit Hölttä-Vuori, Veijo T Salo, Yuki Ohsaki, Maximiliano L Suster, Elina Ikonen

Human molecular genetics 22 ( 6 ) 1157 - 66 2013.03 [International journal]

Mutations affecting the N-glycosylation site in Berardinelli-Seip lipodystrophy (BSCL)-associated gene BSCL2/seipin lead to a dominantly inherited spastic paraplegia termed seipinopathy. While the loss of function of seipin leads to severe congenital lipodystrophy, the effects of seipin N-glycosylation mutations on lipid balance in the nervous system are unknown. In this study, we show that expression of seipin N-glycosylation mutant N88S led to decreased triglyceride (TG) content in astrocytoma and motor neuron cell lines. This was corrected by supplementation with exogenous oleic acid. Upon oleic acid loading, seipin N88S protein was relocated from the endoplasmic reticulum (ER) to the surface of lipid droplets and this was paralleled by alleviation of ER stress induced by the mutant protein. This effect was not limited to seipin N88S, as oleic acid loading also reduced tunicamycin-induced ER stress in motor neuron cells. Furthermore, both seipin N88S and tunicamycin-induced ER stress were decreased by inhibiting lipolysis, suggesting that lipid droplets protected neuronal cells from ER stress. In developing zebrafish larvae, seipin N88S expression led to TG imbalance and reduced spontaneous free swimming. Importantly, supplementation with exogenous oleic acid reduced ER stress in the zebrafish head and increased fish motility. We propose that the decreased TG content contributes to the pathology induced by seipin N88S, and that rescuing TG levels may provide a novel therapeutic strategy in seipinopathy.

Imaging Lipid Droplets by Electron Microscopy

Toyoshi Fujimoto, Yuki Ohsaki, Michitaka Suzuki, Jinglei Cheng

LIPID DROPLETS ( ELSEVIER ACADEMIC PRESS INC ) 116 227 - 251 2013 [Refereed] [International journal]

The lipid droplet (LD) is different from other cellular organelles in that most of its volume is made of lipid esters and its surface is lined by a phospholipid monolayer. This uniquely lipid-dominant structure poses a problem for electron microscopy (EM) because the aldehydes commonly used as a fixative do not react with most lipids. To circumvent this difficulty and utilize the high resolving power of EM, many methods have been developed. In this chapter, we discuss methods that have been used and/or are potentially useful to study LDs. The methods include conventional EM to observe the LD core, cryoelectron microscopy to observe the LD surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-embedding, and cryosectioning methods), and freeze-fracture. Each method has strong and weak points and therefore some caution is necessary in interpreting the obtained results. In combination with methods of other disciplines, the electron microscopic techniques should contribute significantly to solving the remaining questions on LDs.

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion

Eija Nissila, Yuki Ohsaki, Marion Weber-Boyvat, Julia Perttila, Elina Ikonen, Vesa M. Olkkonen

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1821 ( 12 ) 1472 - 1484 2012.12 [Refereed] [International journal]

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [S-35]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in beta-lipoprotein secretion by human hepatocytes. (C) 2012 Elsevier B.V. All rights reserved.

Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets

Michitaka Suzuki, Toshihiko Otsuka, Yuki Ohsaki, Jinglei Cheng, Takako Taniguchi, Hisashi Hashimoto, Hisaaki Taniguchi, Toyoshi Fujimoto

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY ) 23 ( 5 ) 800 - 810 2012.03 [Refereed] [International journal]

Apolipoprotein B-100 (ApoB) is the principal component of very low density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the endoplasmic reticulum (ER) lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remain unknown. In the present study, we demonstrate that ApoB after lipidation is dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulates as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which is almost confined to LDs in this cell type, decreases recruitment of p97 to LDs and causes an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induces an accumulation of lipidated ApoB in the ER lumen but does not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bind with each other and with lipidated ApoB and show colocalization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the predislocation and postdislocation steps, respectively.

Translation inhibitors induce formation of cholesterol ester-rich lipid droplets.

Michitaka Suzuki, Yuki Ohsaki, Tsuyako Tatematsu, Yuki Shinohara, Takashi Maeda, Jinglei Cheng, Toyoshi Fujimoto

PloS one 7 ( 8 ) e42379 2012 [International journal]

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.

Lipid droplet biogenesis: when the endoplasmic reticulum starts to fatten up

Veijo T. Salo, Yuki Ohsaki, Elina Ikonen

CURRENT OPINION IN LIPIDOLOGY ( LIPPINCOTT WILLIAMS & WILKINS ) 22 ( 6 ) 505 - 506 2011.12 [Refereed] [International journal]

Cytoplasmic oxysterol-binding proteins: sterol sensors or transporters?

Terhi Vihervaara, Maurice Jansen, Riikka-Liisa Uronen, Yuki Ohsaki, Elina Ikonen, Vesa M. Olkkonen

CHEMISTRY AND PHYSICS OF LIPIDS ( ELSEVIER IRELAND LTD ) 164 ( 6 ) 443 - 450 2011.09 [Refereed] [International journal]

Families of oxysterol-binding protein (ORP) homologues are present in eukaryotes from yeast to man. Their hallmark feature is a characteristic ligand binding domain that, for several family members, has been shown to accommodate different oxysterols and/or cholesterol. ORPs of the "long" subtype contain targeting determinants for the endoplasmic reticulum and to other organelle membranes, the most prominent of which are phosphoinositide-binding pleckstrin homology domains, while "short" ORPs comprise a ligand binding domain with little additional sequences. There is increasing evidence that both long and short ORPs can be enriched at membrane contact sites, junctions of the endoplasmic reticulum with other organelles, where they are suggested to execute regulatory or sterol transfer functions. In this review we discuss the current evidence for putative roles of ORPs as sterol sensors or transporters. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Lipid droplets: size matters

Michitaka Suzuki, Yuki Shinohara, Yuki Ohsaki, Toyoshi Fujimoto

JOURNAL OF ELECTRON MICROSCOPY ( OXFORD UNIV PRESS ) 60 ( 1 ) S101 - S116 2011.08 [Refereed] [Domestic journal]

The lipid droplet (LD), an organelle that exists ubiquitously in various organisms, from bacteria to mammals, has attracted much attention from both medical and cell biology fields. The LD in white adipocytes is often treated as the prototype LD, but is rather a special example, considering that its size, intracellular localization and molecular composition are vastly different from those of non-adipocyte LDs. These differences confer distinct properties on adipocyte and non-adipocyte LDs. In this article, we address the current understanding of LDs by discussing the differences between adipocyte and non-adipocyte LDs.

Role of ORPs in Sterol Transport from Plasma Membrane to ER and Lipid Droplets in Mammalian Cells

Maurice Jansen, Yuki Ohsaki, Laura Rita Rega, Robert Bittman, Vesa M. Olkkonen, Elina Ikonen

TRAFFIC ( WILEY-BLACKWELL PUBLISHING, INC ) 12 ( 2 ) 218 - 231 2011.02 [Refereed] [International journal]

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.

Lysosomal accumulation of mTOR is enhanced by rapamycin

Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 134 ( 6 ) 537 - 544 2010.12 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth that integrates signals from growth factors and nutrients. Recent studies have shown that an mTOR-containing complex, mTORC1, is targeted to lysosomes in the presence of amino acids and activated by Rheb GTPase resident in that compartment. In this study, we found that treatment with the mTOR inhibitors rapamycin and Torin1 significantly enhanced lysosomal accumulation of mTOR and Raptor. This phenomenon was not observed in the absence of amino acids but was restored upon addition of l-leucine or protein synthesis inhibitors. mTOR was not concentrated in autophagosomes that were induced by rapamycin. These results suggest that the lysosome harbors both active and inactive forms of mTOR in the presence of amino acids.

Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins

Yuko Shimizu, Takayuki Hishiki, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Atsushi Kato, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Kunitada Shimotohno

VIROLOGY ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 407 ( 1 ) 152 - 159 2010.11 [Refereed] [International journal]

The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity. (C) 2010 Elsevier Inc. All rights reserved.

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Takayuki Hishiki, Yuko Shimizu, Reiri Tobita, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Takaji Wakita, Thomas F. Baumert, Yusuke Miyanari, Kunitada Shimotohno

JOURNAL OF VIROLOGY ( AMER SOC MICROBIOLOGY ) 84 ( 22 ) 12048 - 12057 2010.11 [Refereed] [International journal]

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy

Yuki Ohsaki, Yuki Shinohara, Michitaka Suzuki, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 133 ( 4 ) 477 - 480 2010.04 [Refereed] [International journal]

Authorship： Lead author

The lipid droplet (LD) has become a focus of intense research. Fluorescence labeling is indispensable for the cell biological analysis of the LD, and a lipophilic fluorescence dye, BODIPY 493/503, which emits bright green fluorescence has been used extensively for LD labeling. The dye is convenient for double fluorescence labeling, but we noticed that it emits red fluorescence under certain conditions, which could lead to erroneous interpretations. We propose a protocol to preclude such a possibility.

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

Jinglei Cheng, Akikazu Fujita, Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 132 ( 3 ) 281 - 291 2009.09 [Refereed] [International journal]

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.

Biogenesis of cytoplasmic lipid droplets: From the lipid ester globule in the membrane to the visible structure

Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara, Akikazu Fujita, Toyoshi Fujimoto

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1791 ( 6 ) 399 - 407 2009.06 [Refereed] [International journal]

Authorship： Lead author

The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaflets is supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that these processes may give rise to the structural and functional diversities of the CLD is discussed. (C) 2008 Elsevier B.V. All rights reserved.

脂質を観察する方法

藤田秋一, 大崎雄樹, 藤本豊士

日本組織細胞化学会編組織細胞化学2009 35-43 2009

Lipid Droplet-Associated Proteins Protect Renal Tubular Cells from Fatty Acid-Induced Apoptosis

Yoshimichi Urahama, Yuki Ohsaki, Yutaka Fujita, Shoichi Maruyama, Yukio Yuzawa, Seiichi Matsuo, Toyoshi Fujimoto

AMERICAN JOURNAL OF PATHOLOGY ( ELSEVIER SCIENCE INC ) 173 ( 5 ) 1286 - 1294 2008.11 [Refereed] [International journal]

Proteinuria is a major cause of tubulointerstitial kidney damage, and free fatty acids bound to albumin are thought to play an important role in its pathogenesis. However, the mechanism whereby proteinuria causes tubulointerstitial damage to the kidney is unclear. Using primary human renal proximal tubular cells, we observed that albumin replete with fatty acids (rBSA) and defatted albumin (dBSA) complexed with linoleic acid (LA) induced significantly more apoptosis than did defatted albumin alone. Oxidative stress was partially involved in apoptotic induction by LA/dBSA but not by rBSA. Administration of fatty acid-bound BSA increased the number of lipid droplets (LDs) and the LD-associated proteins, adipocyte differentiation-related protein and TIP47. LDs are organelles that store esterified fatty acids, and the LD-associated proteins are presumed to facilitate LD formation. Knockdown of adipocyte differentiation-related protein or TIP47 by RNA interference enhanced induction of apoptosis by both rBSA and LA/dBSA. Apoptotic induction was observed similarly when either rBSA or LA/dBSA was applied to only the apical surfaces of polarized LLC-PK1 cells. The present results suggest that LDs and LD-associated proteins have protective effects against apoptosis induced by fatty acid-bound albumin by sequestering free fatty acids. Therapeutic manipulation of these LD-associated proteins could aid in the amelioration of nephritic diseases. (Anti J Pathol 2008, 173:1286-1294; DOI: 10.2353/ajpath.2008.080137)

Lipid droplets: a classic organelle with new outfits

Toyoshi Fujimoto, Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 130 ( 2 ) 263 - 279 2008.08 [Refereed] [International journal]

Lipid droplets are depots of neutral lipids that exist virtually in any kind of cell. Recent studies have revealed that the lipid droplet is not a mere lipid blob, but a major contributor not only to lipid homeostasis but also to diverse cellular functions. Because of the unique structure as well as the functional importance in relation to obesity, steatosis, and other prevailing diseases, the lipid droplet is now reborn as a brand new organelle, attracting interests from researchers of many disciplines.

*Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100

Ohsaki, Y. Cheng, J. Suzuki, M. Fujita, A. Fujimoto, T

J Cell Sci 121 ( 14 ) 2415-22 - 22 2008.06 [Refereed] [International journal]

Authorship： Lead author

Apolipoprotein B-100 (ApoB) is a major component of very-low-density lipoproteins, and is deposited in a region around lipid droplets (LDs) called the ;ApoB-crescent'. The ApoB-crescent is thought to be related to ApoB degradation because it drastically increases when proteasome or autophagy is inhibited. In the present study, we found that ApoB-crescents were significantly reduced when ApoB lipidation was suppressed by either the inhibition or knockdown of the microsomal triglyceride-transfer protein. By contrast, ApoB-crescents increased under conditions that are presumed to cause lipidated ApoB abnormalities in secretory compartments. By electron microscopic analyses, we identified the ApoB-crescent as a thin cholesterol-rich ER cistern fused to an LD, and - topologically - this structure is equivalent to a lipid-ester globule between the two leaflets of the ER membrane. ApoB localized in the thin cisternal lumen, and its binding to LDs was resistant to alkaline treatment. Overexpression of ADRP or TIP47 suppressed the increase in the number of ApoB-crescents, whereas knockdown of these proteins had the opposite effect. From these results, we inferred that the ApoB-crescent is f

Platelet-derived growth factor signals play critical roles in differentiation of longitudinal smooth muscle cells in mouse embryonic gut.

Kurahashi, M, Niwa, Y, Cheng, J, Ohsaki, Y, Fujita, A, Goto, H, Fujimoto, Y, Torihashi, S

Neurogastroenterol Motil. 20 ( 5 ) 521-531 2008.01 [Refereed]

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor alpha gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor alpha and beta. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut.

脂肪滴形成のメカニズム

藤本豊士, 大崎雄樹, 鈴木倫毅

膜 MEMBRANE 33 ( 1 ) 17-23 2008.01 [Invited]

All-trans-retinol generated by rhodopsin photobleaching induces rapid recruitment of TIP47 to lipid droplets in the retinal pigment epithelium

Eiko Tsuiki, Akikazu Fujita, Yuki Ohsaki, Jinglei Cheng, Toshiaki Irie, Kiwamu Yoshikawa, Haruki Senoo, Kazuaki Mishima, Takashi Kitaoka, Toyoshi Fujimoto

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE ( ASSOC RESEARCH VISION OPHTHALMOLOGY INC ) 48 ( 6 ) 2858 - 2867 2007.06 [Refereed] [International journal]

Purpose. To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE). Methods. Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC. Results. Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly. Conclusions. All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: A potential basis for glial cell activation in the NPC brain

Michitaka Suzuki, Yuko Sugimoto, Yuki Ohsaki, Makoto Ueno, Shinsuke Kato, Yukisato Kitamura, Hiroshi Hosokawa, Joanna P. Davies, Yiannis A. Ioannou, Marie T. Vanier, Kousaku Ohno, Haruaki Ninomiya

JOURNAL OF NEUROSCIENCE ( SOC NEUROSCIENCE ) 27 ( 8 ) 1879 - 1891 2007.02 [Refereed] [International journal]

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin- 6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1(-/-) mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1(-/-) mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1(-/-) mouse brain.

Cholesterol depletion induces autophagy.

Jinglei Cheng, Yuki Ohsaki, Kumi Tauchi-Sato, Akikazu Fujita, Toyoshi Fujimoto

Biochemical and biophysical research communications 351 ( 1 ) 246 - 52 2006.12 [International journal]

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

Cheng J, Ohsaki Y, Tauchi-Sato K, Fujita A, Fujimoto T.

Cheng J, Ohsaki Y, Tauchi-Sato K, Fujita A, Fujimoto T

Biochem. Biophys. Res. Commun. 351 ( 1 ) 246-252 2006.12 [Refereed]

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of ra

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Yuki Ohsaki, Takashi Maeda, Marl Maeda, Kumi Tauchi-Sato, Toyoshi Fujimoto

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 347 ( 1 ) 279 - 287 2006.08 [Refereed] [International journal]

Authorship： Lead author

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47. (c) 2006 Elsevier Inc. All rights reserved.

Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

Y Ohsaki, Y Sugimoto, M Suzuki, H Hosokawa, T Yoshimori, JP Davies, YA Ioannou, MT Vanier, K Ohno, H Ninomiya

JOURNAL OF CELL SCIENCE ( COMPANY OF BIOLOGISTS LTD ) 119 ( 13 ) 2643 - 2653 2006.07 [Refereed] [International journal]

Authorship： Lead author

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1( P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(delta LLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1( E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.

Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B

Yuki Ohsaki, Jinglei Cheng, Akikazu Fujita, Toshinobu Tokumoto, Toyoshi Fujimoto

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY ) 17 ( 6 ) 2674 - 2683 2006.06 [Refereed] [International journal]

Authorship： Lead author , Corresponding author

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in < 10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal. inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

Proteasomal and autophagic pathways converge on lipid droplets

Toyoshi Fujimoto, Yuki Ohsaki

Autophagy ( Taylor and Francis Inc. ) 2 ( 4 ) 299 - 301 2006 [Refereed] [Invited]

Apolipoprotein B (apoB) is the primary protein of very low-density lipoproteins (VLDL). We found that apoB accumulated on the surface of cytoplasmic lipid droplets (LDs) of hepatocytes when the proteasomal or autophagic processes were suppressed. ApoB associated with LDs was poly-ubiquitinated and surrounded by autophagic vacuoles. Moreover, proteasomal subunits were concentrated around LDs. Our data suggest that apoB that is destined to be degraded remains adhered to LDs until it is broken down by the proteasomal and autophagic pathways. We speculate that the LD surface serves as a platform to prevent hydrophobic apoB from forming aggregates, and that LDs may play a similar role for other aggregation-prone hydrophobic proteins. ©2006 Landes Bioscience.

Cytoplasmic lipid droplets - Rediscovery of an old structure as a unique platform

Toyoshi Fujimoto, Yuki Ohsaki

INTEGRATED MOLECULAR MEDICINE FOR NEURONAL AND NEOPLASTIC DISORDERS ( BLACKWELL PUBLISHING ) 1086 104 - 115 2006 [Refereed] [International journal]

Cytoplasmic lipid droplets (LDs) exist in virtually any kind of cell. They have a core of lipid esters covered by the surface phospholipid monolayer. A number of proteins related to various cell functions are present in LDs and/or LD-rich subcellular fractions, suggesting that LDs are an independent organelle that is engaged in various cellular activities. Furthermore, a recent study suggested that LDs are a platform where the proteasomal and autophagic pathways converge. The molecular composition and architecture of LDs are discussed here, with special reference to the technical difficulties encountered when analyzing this unique organelle.

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

Y Ohsaki, T Maeda, T Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 124 ( 5 ) 445 - 452 2005.11 [Refereed] [International journal]

Authorship： Lead author

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

カベオラ，ラフトと血管

藤本豊士, 藤田豊, 大崎雄樹

血管医学 5 455-461 2004.10 [Invited]

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of niemann-pick C1-deficient cells

Yuko Sugimoto, Haruaki Ninomiya, Yuki Ohsaki, Katsumi Higaki, Joanna P. Davies, Yiannis A. Ioannou, Kousaku Ohno

Proceedings of the National Academy of Sciences of the United States of America 98 ( 22 ) 12391 - 12396 2001.10

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/ endoplasmic reticulum does not.

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