Kazuyuki Hida

Thiazolidinediones Ameliorate Diabetic Nephropathy via Cell Cycle–Dependent Mechanisms

Thiazolidinediones are ligands for peroxisome proliferator–activated receptor (PPAR)-γ, widely used as insulin sensitizer in type 2 diabetic patients and implicated in apoptosis, cell proliferation, and cell cycle regulation. Here, the effect of thiazolidinediones on G1-phase cell cycle arrest, the hallmark in diabetic nephropathy, was investigated. Eight-week-old male Otsuka Long-Evans Tokushima fatty rats were treated with pioglitazone (1 mg · kg body wt−1 · day−1) until 50 weeks of age and compared with insulin treatment. Although similar HbA1c levels were observed in both groups, pioglitazone significantly inhibited glomerular hypertrophy and mesangial matrix expansion and reduced urinary albumin excretion compared with the insulin-treated group. In addition, pioglitazone significantly reduced the number of glomerular p27Kip1-positive cells. Because prominent expression of PPAR-γ was observed in podocytes in glomeruli and cultured cells, conditionally immortalized mouse podocyte cells were cultured under 5.5 and 25 mmol/l d-glucose supplemented with pioglitazone. Pioglitazone inhibited cell hypertrophy revealed by [3H]thymidine and [3H]proline incorporation, and pioglitazone reversed high glucose–induced G1-phase cell cycle arrest, i.e., an increase in G0/G1 phase and decrease in S and G2 phases. Pioglitazone suppressed high glucose–induced phosphorylation of p44/42 mitogen-activated protein kinase and reduced Bcl-2 and p27Kip1 protein levels. Besides glucose-lowering action, pioglitazone ameliorates diabetic nephropathy via cell cycle–dependent mechanisms.

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.

Relationship between reduced serum IGF-I levels and accumulation of visceral fat in Japanese men.

OBJECTIVE: To investigate whether the changes in IGF-I concentrations after weight reduction in Japanese overweight men are associated with changes in visceral and subcutaneous fat.DESIGN: Cross-sectional and longitudinal clinical intervention study with exercise education.SUBJECTS: One-hundred and twelve Japanese overweight men aged 30–59 y (body mass index (BMI) 28.4±2.5 kg/m2) and 33 normal-weight men aged 30–39 y (BMI 22.1±1.5 kg/m2) at baseline. From the participants, 56 randomly selected overweight men (BMI 28.8±2.8) were further enrolled into a 1 y exercise program.MEASUREMENTS: Fat distribution was evaluated by visceral fat (V) and subcutaneous fat (S) areas measured with computed tomography scanning at umbilical levels, metabolic parameters and hormones including insulin, leptin and IGF-I at baseline and after 1 y.RESULTS: In 112 overweight subjects at baseline, insulin (10.5±5.0 µU/ml) and leptin (6.4±3.7 ng/ml) significantly correlated with both V (r=0.260, P=0.0073; r=0.410, P<0.0001) and S areas (r=0.377, P<0.0001; r=0.613, P<0.0001), respectively. IGF-I (156.8±48.7 µU/ml) significantly and negatively correlated with V area (r=−0.242, P=0.0125) and age (r=−0.192, P=0.0480). In normal-weight men aged 30–39 y (n=33) and age-matched subjects (n=30) selected from the 112 overweight men, the serum IGF-I further tightly correlated with V area (r=−0.467, P<0.0001). Visceral fat area and age were independently related to serum IGF-I levels by multiple regression analysis. By intervention with exercise education, 56 overweight subjects showed an increase in daily steps (6224±2781 to 7898±4141 steps/day) and reduction of BMI (28.8±2.8 to 27.7±2.9). ΔIGF-I significantly correlated with ΔV area (r=−0.432, P=0.0009) but not with ΔS area or ΔBMI.CONCLUSION: The present study indicated a negative correlation between IGF-I levels and visceral fat at baseline as well as an association between the reduction in visceral fat and increase in IGF-I levels after an exercise intervention.

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.

Identification of adipocyte adhesion molecule (ACAM), a novel CTX gene family, implicated in adipocyte maturation and development of obesity

Few cell adhesion molecules have been reported to be expressed in mature adipocytes, and the significance of cell adhesion process in adipocyte biology is also unknown. In the present study, we identified ACAM (adipocyte adhesion molecule), a novel homologue of the CTX (cortical thymocyte marker in Xenopus) gene family. ACAM cDNA was isolated during PCR-based cDNA subtraction, and its mRNA was shown to be up-regulated in WATs (white adipose tissues) of OLETF (Otsuka Long-Evans Tokushima fatty) rats, an animal model for Type II diabetes and obesity. ACAM, 372 amino acids in total, has a signal peptide, V-type (variable) and C2-type (constant) Ig domains, a single transmembrane segment and a cytoplasmic tail. The amino acid sequence in rat is highly homologous to mouse (94%) and human (87%). ACAM mRNA was predominantly expressed in WATs in OLETF rats, and increased with the development of obesity until 30 weeks of age, which is when the peak of body mass is reached. Western blot analysis revealed that ACAM protein, approx. 45 kDa, was associated with plasma membrane fractions of mature adipocytes isolated from mesenteric and subdermal adipose deposits of OLETF rats. Up-regulation of ACAM mRNAs in obesity was also shown in WATs of genetically obese db/db mice, diet-induced obese ICR mice and human obese subjects. In primary cultured mouse and human adipocytes, ACAM mRNA expression was progressively up-regulated during differentiation. Several stably transfected Chinese-hamster ovary K1 cell lines were established, and the quantification of ACAM mRNA and cell aggregation assay revealed that the degree of homophilic aggregation correlated well with ACAM mRNA expression. In summary, ACAM may be the critical adhesion molecule in adipocyte differentiation and development of obesity.

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.

RXR antagonism induces G0/G1 cell cycle arrest and ameliorates obesity by up‐regulating the p53–p21

The peroxisome proliferator activated receptor‐γ (PPARγ) agonist, pioglitazone (PIO), exerts anti‐diabetic properties associated with increased fat mass, whereas the retinoid X receptor (RXR) antagonist HX531 demonstrates anti‐obesity and anti‐diabetic effects with reduced body weight and fat pad mass. The cell cycle abnormality in adipocytes has not been well‐investigated in obesity or during treatment with modulators of nuclear receptors. We therefore investigated cell size and cell cycle distributions of adipocytes in vivo and examined the expression of cell cycle regulators in cultured human visceral preadipocytes. The cell size distribution and cell cycle analyses of in vivo adipocytes derived from OLETF rats demonstrated that HX531 brought about G0/G1 cell cycle arrest associated with the inhibition of cellular hypertrophy, which resulted in the reduction of fat pad mass. In contrast, PIO promoted proliferation activities associated with the increase in M + late M:G0 + G1 ratio and the appearance of both small and hypertrophied adipocytes. In cultured human visceral preadipocytes HX531 up‐regulated cell cycle regulators, p53, p21

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