Frontiers Media SA Acta Medica Okayama 2297-055X 8 2021 Novel Urinary Glycan Biomarkers Predict Cardiovascular Events in Patients With Type 2 Diabetes: A Multicenter Prospective Study With 5-Year Follow Up (U-CARE Study 2) 668059 EN Koki Mise Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mariko Imamura Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Yamaguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mayu Watanabe Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chigusa Higuchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Katayama Diabetes Center, Okayama University Hospital Satoshi Miyamoto Center for Innovative Clinical Medicine, Okayama University Hospital Haruhito A. Uchida Department of Chronic Kidney Disease and Cardiovascular Disease, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuko Nakatsuka Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jun Eguchi Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyuki Hida Department of Diabetology and Metabolism, National Hospital Organization Okayama Medical Center Tatsuaki Nakato Okayama Saiseikai General Hospital Atsuhito Tone Okayama Saiseikai General Hospital Sanae Teshigawara Okayama Saiseikai General Hospital Takashi Matsuoka Kurashiki Central Hospital Shinji Kamei Kurashiki Central Hospital Kazutoshi Murakami Kurashiki Central Hospital Ikki Shimizu The Sakakibara Heart Institute of Okayama Katsuhiro Miyashita Japanese Red Cross Okayama Hospital Shinichiro Ando Okayama City General Medical Center Tomokazu Nunoue Nunoue Clinic Michihiro Yoshida Center for Innovative Clinical Medicine, Okayama University Hospital Masao Yamada GlycoTechnica Ltd. Kenichi Shikata Center for Innovative Clinical Medicine, Okayama University Hospital Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Although various biomarkers predict cardiovascular event (CVE) in patients with diabetes, the relationship of urinary glycan profile with CVE in patients with diabetes remains unclear. Methods: Among 680 patients with type 2 diabetes, we examined the baseline urinary glycan signals binding to 45 lectins with different specificities. Primary outcome was defined as CVE including cardiovascular disease, stroke, and peripheral arterial disease. Results: During approximately a 5-year follow-up period, 62 patients reached the endpoint. Cox proportional hazards analysis revealed that urinary glycan signals binding to two lectins were significantly associated with the outcome after adjustment for known indicators of CVE and for false discovery rate, as well as increased model fitness. Hazard ratios for these lectins (+1 SD for the glycan index) were UDA (recognizing glycan: mixture of Man5 to Man9): 1.78 (95% CI: 1.24-2.55, P = 0.002) and Calsepa [High-Man (Man2-6)]: 1.56 (1.19-2.04, P = 0.001). Common glycan binding to these lectins was high-mannose type of N-glycans. Moreover, adding glycan index for UDA to a model including known confounders improved the outcome prediction [Difference of Harrel's C-index: 0.028 (95% CI: 0.001-0.055, P = 0.044), net reclassification improvement at 5-year risk increased by 0.368 (0.045-0.692, P = 0.026), and the Akaike information criterion and Bayesian information criterion decreased from 725.7 to 716.5, and 761.8 to 757.2, respectively]. Conclusion: The urinary excretion of high-mannose glycan may be a valuable biomarker for improving prediction of CVE in patients with type 2 diabetes, and provides the rationale to explore the mechanism underlying abnormal N-glycosylation occurring in patients with diabetes at higher risk of CVE. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 9 3 2014 Pemt deficiency ameliorates endoplasmic reticulum stress in diabetic nephropathy e92647 EN Mayu Watanabe Atsuko Nakatsuka Kazutoshi Murakami Kentaro Inoue Takahiro Terami Chigusa Higuchi Akihiro Katayama Sanae Teshigawara Jun Eguchi Daisuke Ogawa Eijiro Watanabe Jun Wada Hirofumi Makino Phosphatidylethanolamine N-methyltransferase (Pemt) catalyzes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) mainly in the liver. Under an obese state, the upregulation of Pemt induces endoplasmic reticulum (ER) stress by increasing the PC/PE ratio in the liver. We targeted the Pemt gene in mice to explore the therapeutic impact of Pemt on the progression of diabetic nephropathy and diabetes, which was induced by the injection of streptozotocin (STZ). Although the blood glucose levels were similar in STZ-induced diabetic Pemt+/+ and Pemt−/−mice, the glomerular hypertrophy and albuminuria in Pemt−/− mice were significantly reduced. Pemt deficiency reduced the intraglomerular F4/80-positive macrophages, hydroethidine fluorescence, tubulointerstitial fibrosis and tubular atrophy. The expression of glucose-regulated protein-78 (GRP78) was enriched in the renal tubular cells in STZ-induced diabetic mice, and this was ameliorated by Pemt deficiency. In mProx24 renal proximal tubular cells, the treatment with ER-stress inducers, tunicamycin and thapsigargin, increased the expression of GRP78, which was reduced by transfection of a shRNA lentivirus for Pemt (shRNA-Pemt). The number of apoptotic cells in the renal tubules was significantly reduced in Pemt−/− diabetic mice, and shRNA-Pemt upregulated the phosphorylation of Akt and decreased the cleavage of caspase 3 and 7 in mProx24 cells. Taken together, these findings indicate that the inhibition of Pemt activity ameliorates the ER stress associated with diabetic nephropathy in a model of type 1 diabetes and corrects the functions of the three major pathways downstream of ER stress, i.e. oxidative stress, inflammation and apoptosis. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 6 2016 Insufficiency of phosphatidylethanolamine N-methyltransferase is risk for lean non-alcoholic steatohepatitis 21721 EN Atsuko Nakatsuka Makoto Matsuyama Satoshi Yamaguchi Akihiro Katayama Jun Eguchi Kazutoshi Murakami Sanae Teshigawara Daisuke Ogawa Nozomu Wada Tetsuya Yasunaka Fusao Ikeda Akinobu Takaki Eijiro Watanabe Jun Wada 　Although obesity is undoubtedly major risk for non-alcoholic steatohepatitis (NASH), the presence of lean NASH patients with normal body mass index has been recognized. Here, we report that the insufficiency of phosphatidylethanolamine N-methyltransferase (PEMT) is a risk for the lean NASH. The Pemt−/− mice fed high fat-high sucrose (HFHS) diet were protected from diet-induced obesity and diabetes, while they demonstrated prominent steatohepatitis and developed multiple liver tumors. Pemt exerted inhibitory effects on p53-driven transcription by forming the complex with clathrin heavy chain and p53, and Pemt−/− mice fed HFHS diet demonstrated prominent apoptosis of hepatocytes. Furthermore, hypermethylation and suppressed mRNA expression of F-box protein 31 and hepatocyte nuclear factor 4α resulted in the prominent activation of cyclin D1. PEMT mRNA expression in liver tissues of NASH patients was significantly lower than those with simple steatosis and we postulated the distinct clinical entity of lean NASH with insufficiency of PEMT activities. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 2045-2322 5 2015 Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis 16920 EN Akihiro Katayama Atsuko Nakatsuka Jun Eguchi Kazutoshi Murakami Sanae Teshigawara Motoko Kanzaki Tomokazu Nunoue Kazuyuki Hida Nozomu Wada Tetsuya Yasunaka Fusao Ikeda Akinobu Takaki Kazuhide Yamamoto Hiroshi Kiyonari Hirofumi Makino Jun Wada Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Gpnmb is classified as a type 1 membrane protein and its soluble form is secreted by ADAM10-mediated cleavage. Gpnmb mRNA was found in the Kupffer cells and white adipose tissues (WATs) and its upregulation in obesity was recently found. Here, we generated aP2 promoter-driven Gpnmb transgenic (Tg) mice and the overexpression of Gpnmb ameliorated the fat accumulation and fibrosis of the liver in diet-induced obesity model. Soluble form of Gpnmb in sera was elevated in Gpnmb Tg mice and Gpnmb concentrated in hepatic macrophages and stellate cells interacted with calnexin, which resulted in the reduction of oxidative stress. In the patients with non-alcoholic steatohepatitis, serum soluble GPNMB concentrations were higher compared with the patients with simple steatosis. The GPNMB is a promising biomarker and therapeutic target for the development and progression of NAFLD in obesity. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 69 2 2015 Complication of Chronic Eosinophilic Pneumonia in an Elderly Patient with Sjögren Syndrome 123 127 EN Koichi Waseda Hideharu Hagiya Yoshihisa Hanayama Tomohiro Terasaka Kosuke Kimura Takao Tsuzuki Kou Hasegawa Takahiro Nada Eri Nakamura Kazutoshi Murakami Eisei Kondo Fumio Otsuka Case Report 10.18926/AMO/53342 An 81-year-old Japanese male with primary Sjögren syndrome (pSS) developed a low-grade fever and productive cough which were refractory to antibiotic therapy. Based on the high level of eosinophils observed in his bronchial alveolar lavage, he was diagnosed with chronic eosinophilic pneumonia (CEP) and successfully treated by oral prednisolone. Interstitial lung diseases associated with pSS (pSS-ILDs) usually present as nonspecific interstitial pneumonia or usual interstitial pneumonia; therefore, the present case is extremely unique in that the patientʼs condition was complicated with CEP. A diagnosis of advanced gallbladder cancer was made in the patientʼs clinical course, suggesting the advisability of a whole-body workup in cases of pSS, especially in elderly patients. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 6 2013 Urinary angiotensinogen is a marker for tubular injuries in patients with type 2 diabetes 233 240 EN Takahiro Terami Jun Wada Kentaro Inoue Atsuko Nakatsuka Daisuke Ogawa Sanae Teshigawara Kazutoshi Murakami Akihiro Katayama Jun Eguchi Hirofumi Makino Purpose: Urinary angiotensinogen has been reported as a marker for the activation of intrarenal renin–angiotensin system (RAS) in various kidney diseases. To investigate the importance of urinary angiotensinogen in diabetic nephropathy, we compared the urinary levels of angiotensinogen, albumin, and α1-microglobulin. Materials and methods: Japanese patients with type 2 diabetes at various stages of nephropathy (n=85) were enrolled, and we measured albumin/creatinine ratio (ACR) and urinary excretion of angiotensinogen and α1-microglobulin. We also compared the clinical data of the patients treated with or without angiotensin II receptor blockers or angiotensin-converting enzyme inhibitors (RAS inhibitors [+], n=51; RAS inhibitors [−], n=34). Results: Urinary angiotensinogen levels positively correlated with ACR (r =0.367, P=3.84×10-4) and urinary α1-microglobulin (r=0.734, P=1.32 × 10-15), while they negatively correlated with estimated glomerular filtration ratio (eGFR) (r=−0.350, P=1.02 × 10-3) and high-density lipoprotein cholesterol (r=−0.216, P=0.049). Multiple regression analysis was carried out to predict urinary angiotensinogen levels by employing eGFR, ACR, and urinary α1-microglobulin as independent variables; only urinary α1-microglobulin entered the regression equation at a significant level. Although ACR was higher in the RAS inhibitors (+) group, urinary α1-microglobulin and angiotensinogen did not show significant increase in the RAS inhibitors (+) group. Conclusion: Urinary angiotensinogen is well correlated with urinary α1-microglobulin and reflected the tubular injuries which may be associated with the intrarenal RAS activation in patients with type 2 diabetes. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 8 10 2013 Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray EN Kentaro Inoue Jun Wada Jun Eguchi Atsuko Nakatsuka Sanae Teshigawara Kazutoshi Murakami Daisuke Ogawa Takahiro Terami Akihiro Katayama Atsuhito Tone Izumi Iseda Kazuyuki Hida Masao Yamada Tomohisa Ogawa Hirofumi Makino We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Sia alpha 2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography-tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40 +/- 0.43; A2, 0.60 +/- 0.53; A3 1.57 +/- 1.13 ng/gCr; p = 7.29x10(-8)) and of GFR stages (G1, 0.39 +/- 0.39; G2, 0.49 +/- 0.45; G3, 1.25 +/- 1.18; G4, 1.34 +/- 0.80 ng/gCr; p = 3.89x10(-4)). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881-11.844) and 3.739 (1.785-7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy. No potential conflict of interest relevant to this article was reported.

Biomed Central Ltd Acta Medica Okayama 1471-2369 14 2013 Serum galectin-9 levels are elevated in the patients with type 2 diabetes and chronic kidney disease EN Yuko Kurose Jun Wada Motoko Kanzaki Sanae Teshigawara Atsuko Nakatsuka Kazutoshi Murakami Kentaro Inoue Takahiro Terami Akihiro Katayama Mayu Watanabe Chigusa Higuchi Jun Eguchi Nobuyuki Miyatake Hirofumi Makino Background: Galectin-9 (Gal-9) induces apoptosis in activated T helper 1 (T(H)1) cells as a ligand for T cell immunoglobulin mucin-3 (Tim-3). Gal-9 also inhibits the G1 phase cell cycle arrest and hypertrophy in db/db mice, the hallmark of early diabetic nephropathy, by reversing the high glucose-induced up-regulation of cyclin dependent kinase inhibitors such as p27(Kip1) and p21(Cip1). Methods: We investigated the serum levels of Gal-9 in the patients with type 2 diabetes and various stages of chronic kidney disease (CKD) (n = 182). Results: Serum Gal-9 levels in the patients with type 2 diabetes were 131.9 +/- 105.4 pg/ml and Log(10)Gal-9 levels significantly and positively correlated with age (r = 0.227, p = 0.002), creatinine (r = 0.175, p = 0.018), urea nitrogen (r = 0.162, p = 0.028) and osmotic pressure (r = 0.187, p = 0.014) and negatively correlated with estimated glomerular filtration rate (eGFR) (r = -0.188, p = 0.011). Log(10)Gal-9 levels increased along with the progression of GFR categories of G1 to G4, and they were statistically significant by Jonckheere-Terpstra test (p = 0.012). Log(10)Gal-9 levels remained similar levels in albuminuria stages of A1 to A3. Conclusion: The elevation of serum Gal-9 in the patients with type 2 diabetes is closely linked to GFR and they may be related to the alteration of the immune response and inflammation of the patients with type 2 diabetes and CKD. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0012-1797 61 11 2012 Vaspin Is an Adipokine Ameliorating ER Stress in Obesity as a Ligand for Cell-Surface GRP78/MTJ-1 Complex 2823 2832 EN Atsuko Nakatsuka Jun Wada Izumi Iseda Sanae Teshigawara Kanji Higashio Kazutoshi Murakami Motoko Kanzaki Kentaro Inoue Takahiro Terami Akihiro Katayama Kazuyuki Hida Jun Eguchi Chikage Sato Horiguchi Daisuke Ogawa Yasushi Matsuki Ryuji Hiramatsu Hideo Yagita Shigeru Kakuta Yoichiro Iwakura Hirofumi Makino It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue-derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress-induced metabolic dysfunctions. Diabetes 61:2823-2832, 2012 No potential conflict of interest relevant to this article was reported.

Endocrine Soc Acta Medica Okayama 0021-972X 97 7 2012 Serum Vaspin Concentrations Are Closely Related to Insulin Resistance, and rs77060950 at SERPINA12 Genetically Defines Distinct Group with Higher Serum Levels in Japanese Population E1202 E1207 EN Sanae Teshigawara Jun Wada Kazuyuki Hida Atsuko Nakatsuka Jun Eguchi Kazutoshi Murakami Motoko Kanzaki Kentaro Inoue Takahiro Terami Akihiro Katayama Izumi Iseda Yuichi Matsushita Nobuyuki Miyatake John F. McDonald Kikuko Hotta Hirofumi Makino Context: Vaspin is an adipokine with insulin-sensitizing effects identified from visceral adipose tissues of genetically obese rats. Objective: We investigated genetic and nongenetic factors that define serum concentrations of vaspin. Design, Setting and Participants: Vaspin levels were measured with RIA in Japanese subjects with normal fasting plasma glucose (NFG; n = 259) and type 2 diabetes patients (T2D; n = 275). Single nucleotide polymorphisms (SNP) at SERPINA12 (vaspin) gene locus were discovered, and five SNP were genotyped in the subjects with varied body mass index (n = 1138). Results: The level of serum vaspin in 93% of the samples was found to vary from 0.2 to nearly 2 ng/ml in NFG subjects (n = 259) and from 0.2 to nearly 3 ng/ml in T2D patients (n = 275) (Vaspin(Low) group), whereas a significant subpopulation (7%) in both groups displayed much higher levels of 10-40 ng/ml (Vaspin(High) group). In the Vaspin(Low) group, serum vaspin levels in T2D were significantly higher than healthy subjects (0.99 +/- 0.04 vs. 0.86 +/- 0.02 ng/ml; P < 0.01). Both in T2D and genotyped Japanese population, serum vaspin levels closely correlated with homeostasis model of assessment for insulin resistance rather than anthropometric parameters. By genotyping, rs77060950 tightly linked to serum vaspin levels, i.e. CC (0.6 +/- 0.4 ng/ml), CA (18.4 +/- 9.6 ng/ml), and AA (30.5 +/- 5.1 ng/ml) (P < 2 x 10(-16)). Putative GATA-2 and GATA-3 binding consensus site was found at rs77060950. Conclusions: Serum vaspin levels were related to insulin resistance, and higher levels of serum vaspin in 7% of the Japanese population are closely linked to minor allele sequence (A) of rs77060950. (J Clin Endocrinol Metab 97: E1202-E1207, 2012) No potential conflict of interest relevant to this article was reported.