Izumi Iseda

Vaspin Is an Adipokine Ameliorating ER Stress in Obesity as a Ligand for Cell-Surface GRP78/MTJ-1 Complex

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.

The Role for HNF-1β-Targeted Collectrin in Maintenance of Primary Cilia and Cell Polarity in Collecting Duct Cells

Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1α, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1β and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to γ-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1β and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1β regulated genes.

Serum Vaspin Concentrations Are Closely Related to Insulin Resistance, and rs77060950 at SERPINA12 Genetically Defines Distinct Group with Higher Serum Levels in Japanese Population

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 × 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.

Visceral Adipose Tissue-derived Serine Proteinase Inhibitor Inhibits Apoptosis of Endothelial Cells as a Ligand for the Cell-Surface GRP78/Voltage-dependent Anion Channel Complex

Rationale: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. Objective: The role of vaspin in the diabetic vascular complications remains elusive and we investigated the effects of vaspin on the vascular function under diabetic milieu. Methods and Results: Adenovirus carrying full-length of vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb and Pdgfrb genes were significantly reduced by the treatment of Vaspin-Ad. In cuff injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells (HAEC). Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to HAEC, subjected to tandem tag purification and LC-MS/MS, and we identified GRP78 (78 kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78 and VDAC (voltage-dependent anion channel) on plasma membrane was confirmed by the immunoprecipitation studies using aortae of Vaspin Tg mice. The binding assay using 125 I-vaspin in HAEC revealed high affinity binding (Kd=0.565×10 -9 M) by the treatment of 5 υM thapsigargin, which recruited GRP78 from ER to plasma membrane by inducing ER stress. In HAEC, vaspin induced phosphorylation of Akt, inhibited the kringle 5-induced Ca 2+ influx, and subsequent apoptosis. Conclusions: Vaspin is a novel ligand for cell-surface GRP78/VDAC complex in endothelial cells, promotes the proliferation, inhibits the apoptosis, and protects the vascular injuries in diabetes.Rationale: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. Objective: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. Methods and Results: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using 125I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10–9 m) by the treatment of 5 &mgr;M thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca2+ influx and subsequent apoptosis. Conclusions: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.

Vaspin Inhibits Apoptosis of Endothelial Cells as a Ligand for Cell-Surface GRP78/VDAC Complex

Rationale: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. Objective: The role of vaspin in the diabetic vascular complications remains elusive and we investigated the effects of vaspin on the vascular function under diabetic milieu. Methods and Results: Adenovirus carrying full-length of vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb and Pdgfrb genes were significantly reduced by the treatment of Vaspin-Ad. In cuff injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells (HAEC). Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to HAEC, subjected to tandem tag purification and LC-MS/MS, and we identified GRP78 (78 kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78 and VDAC (voltage-dependent anion channel) on plasma membrane was confirmed by the immunoprecipitation studies using aortae of Vaspin Tg mice. The binding assay using 125 I-vaspin in HAEC revealed high affinity binding (Kd=0.565×10 -9 M) by the treatment of 5 υM thapsigargin, which recruited GRP78 from ER to plasma membrane by inducing ER stress. In HAEC, vaspin induced phosphorylation of Akt, inhibited the kringle 5-induced Ca 2+ influx, and subsequent apoptosis. Conclusions: Vaspin is a novel ligand for cell-surface GRP78/VDAC complex in endothelial cells, promotes the proliferation, inhibits the apoptosis, and protects the vascular injuries in diabetes.Rationale: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. Objective: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. Methods and Results: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using 125I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10–9 m) by the treatment of 5 &mgr;M thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca2+ influx and subsequent apoptosis. Conclusions: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

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 Siaα2-6Gal/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 = 7.29×10−8) 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 = 3.89×10−4). 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.

Comparison of insulin detemir and insulin glargine on glycemic variability in patients with type 1 and type 2 diabetes.

AIMS To compare the glycemic variability of insulin detemir and insulin glargine in type 1 and type 2 diabetic patients. METHODS 15 type 1 and 14 type 2 diabetic patients receiving intensive insulin therapy with insulin glargine were enrolled. Before and after switching insulin glargine to insulin detemir, we assessed fasting glucose variability using the standard deviation (SD) and the coefficient of variance (CV) of self-monitored fasting blood sugar (FBS) levels. RESULTS The SD and CV values were significantly decreased in type 1 diabetes after switching the therapy, though there was no significant difference in type 2 diabetes. The frequency of hypoglycemia was decreased in type 1 diabetes and there was no change in type 2 diabetes. The changes of the CV value also showed significant positive correlation with fasting serum CPR levels in all patients and total insulin dose in type 1 diabetes. The changes of frequency of hypoglycemia showed significant positive correlation with total and basal insulin dose adjusted for body weight in type 1 diabetes. CONCLUSION The present study demonstrated lower within-subject variability of insulin detemir compared to insulin glargine, suggesting that the basal insulin replacement with insulin detemir may provide a useful therapeutic strategy for uncontrolled type 1 diabetes with high glucose variability.

A Pregnant Patient with Brittle Type 1 Diabetes Successfully Managed by CSII Therapy with Insulin Aspart

Continuous subcutaneous insulin infusion (CSII) therapy is widely accepted for brittle type 1 diabetes, since it has the benefit of less frequency of hypoglycemia and better management of the dawn phenomenon compared to multiple daily insulin injection (MDI) therapy. On the other hand, insulin aspart, one of the rapid-acting insulin analogs, was approved for Pregnancy Category B rating by the United States Food and Drug Administration (FDA) in 2007, whereas the FDA created the rating system and approved a Pregnancy Category B rating for human regular insulin in 1979.

A Case of Proinsulin-Secreting Malignant Insulinoma in an Elderly Patient with Cerebral Infarction

The diagnosis of insulinoma is sometimes challenging and the symptoms of hypoglycemia may be overlooked in elderly patients with cerebrovascular diseases and higher cerebral dysfunction. Recently, there have been an increasing number of case reports of insulinoma secreting proinsulin without hyperinsulinemia. We report a case of proinsulinsecreting malignant insulinoma in a 72-year-old man with aphasia due to cerebral infarction. He manifested no apparent symptoms, although he was incidentally found to have recurrent hypoglycemia by plasma glucose measurements. The Fajans, Grunt and Turner indexes were all within normal range and the arterial stimulation and venous sampling (ASVS) test revealed no increase in insulin concentration. However, proinsulin concentration was markedly elevated (2470 pmol/l) and well-differentiated endocrine adenocarcinoma, malignant insulinoma, with 2 cm in size in the tail of the pancreas was surgically removed. The diagnosis of hypoglycemia requires careful attention especially in elderly persons and/or patients with cerebrovascular diseases. Measurement of proinsulin is recommended in the diagnosis of insulinoma without hyperinsulinemia.