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Articles by T Nakano
Total Records ( 7 ) for T Nakano
  T Wada , S Hori , M Sugiyama , E Fujisawa , T Nakano , H Tsuneki , K Nagira , S Saito and T. Sasaoka
 

Maternal insulin resistance is essential for efficient provision of glucose to the fetus. Although elevation of placental hormones is known to relate to the development of insulin resistance, the precise underlying mechanism of maternal insulin resistance is unknown. Therefore, we examined the molecular mechanisms of progesterone causing insulin resistance in 3T3-L1 adipocytes. Progesterone at 10–4 M, but not 10–5 M, reduced the amount of IRS-1. As a result, insulin-induced phosphorylation of IRS-1, the association of IRS-1 with p85, and subsequent phosphorylation of Akt1 and -2 was decreased moderately by 10–4 M progesterone. Subsequently, insulin-induced translocation of GLUT4 to the plasma membrane evaluated by immunostaining on the plasma membrane sheet by confocal laser microscope was also decreased by 10–4 M progesterone. In contrast, 2-[3H]deoxyglucose (2DG) uptake was markedly inhibited by both 10–5 and 10–4 M progesterone in a dose-dependent manner. Surprisingly, 2DG uptake elicited by adenovirus-mediated expression of constitutive-active mutant of PI 3-kinase (myr-p110) and Akt (myr-Akt) was suppressed by progesterone. Interestingly, insulin-induced tyrosine phosphorylation of Cbl and activation of TC10 were inhibited by progesterone at 10–5 M. These results indicate that progesterone is implicated in insulin resistance during pregnancy by inhibiting the PI 3-kinase pathway at the step of 1) IRS-1 expression and 2) distal to Akt and 3) by suppressing the PI 3-kinase-independent pathway of TC10 activation by affecting Cbl phosphorylation.

  E Hokazono , S Osawa , T Nakano , Y Kawamoto , Y Oguchi , T Hotta , Y Kayamori , D Kang , Y Cho , K Shiba and K. Sato
  Background

Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method.

Methods

Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg2+, Fe2+, Cu2+ and Zn2+. The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS).

Results

The within-run and between-run variations (coefficient of variation [CV]) were 0.92–1.01% and 0.75–1.43%, respectively. The linearity was 0–7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) – 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4°C without daily calibration.

Conclusion

The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.

  M Ai , A Tanaka , K Shimokado , R Ohtani , A Inazu , J Kobayashi , H Mabuchi , T Nakano and K. Nakajima
  Background

We found a unique cholesteryl ester transfer protein (CETP) deficient case with markedly elevated serum triglyceride (TG) as well as high-density lipoprotein cholesterol (HDL-C) levels. Most of the CETP deficiency cases were reported to have normal or reduced serum TG with elevated HDL-C.

Methods

The case subject was a 40-year-old male with a compound heterozygous CETP deficiency. Two heterozygous CETP deficient cases and 10 normal volunteers were also recruited as controls. They underwent an oral fat tolerance test (OFTT) and their blood was taken at fasting and during the OFTT to be used for laboratory tests.

Results

The case subject had apolipoprotein E (apo-E) phenotype 4/2 with fatty liver but without any cardiovascular disease. His serum TG, HDL-C, apo-AI and apo-B48 levels were significantly higher, but the low-density lipoprotein cholesterol level was lower than controls. Although post-heparin plasma lipoprotein lipase and hepatic lipase (both mass and activity) were nearly normal, the serum level of angiopoietin-like-protein-3 was extremely elevated. While his serum remnant-like particles-TG (RLP-TG) and total TG levels significantly increased after a fat load, the RLP-cholesterol (RLP-C) level did not increase during OFTT.

Conclusions

The case subject was different from the common CETP deficient cases reported previously. Also, the results indicated that the metabolic pathways of RLP-C and RLP-TG formation in the postprandial state are controlled independently in CETP deficient cases. CETP deficiency itself may not be atherogenic, while one with elevated RLPs may be atherogenic. These cases may have raised the controversy of whether CETP deficiency is atherogenic or not.

  K Nakajima , J Kobayashi , H Mabuchi , T Nakano , Y Tokita , T Nagamine , S Imamura , M Ai , S Otokozawa and E. F. Schaefer
  Background

The relationship between plasma angiopoietin-like protein 3 (ANGPTL3), and lipoprotein lipase (LPL) activity and hepatic triglyceride lipase (HTGL) activity has not been investigated in the metabolism of remnant lipoproteins (RLPs) and high-density lipoprotein (HDL) in human plasma.

Methods

ANGPTL3, LPL activity, HTGL activity, RLP-C and RLP-TG and small, dense LDL-cholesterol (sd LDL-C) were measured in 20 overweight and obese subjects in the fasting and postprandial states.

Results

Plasma TG, RLP-C, RLP-TG and sd LDL-C were inversely correlated with LPL activity both in the fasting and postprandial states, but not correlated with HTGL activity and ANGPTL3. However, plasma HDL-C was positively correlated with LPL activity both in the fasting and postprandial states, while inversely correlated with HTGL activity. ANGPTL3 was inversely correlated with HTGL activity both in the fasting and postprandial states, but not correlated with LPL activity.

Conclusion

HTGL plays a major role in HDL metabolism, but not RLP metabolism. These findings suggest that ANGPTL3 is strongly associated with the inhibition of HTGL activity and regulates HDL metabolism, but not associated with the inhibition of LPL activity for the metabolism of RLPs in human plasma.

  T Nakano , S Sekine , K Ito and T. Horie
 

The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 ± 2.1 nmol/cm to 18.1 ± 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.

  T Nakano , S Sekine , K Ito and T. Horie
 

The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 ± 2.1 nmol/cm to 18.1 ± 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.

  J Seino , K Ishii , T Nakano , N Ishida , M Tsujimoto , Y Hashimoto and S. Takashima
 

Using the basic local alignment search tool (BLAST) algorithm to search the Oryza sativa (Japanese rice) nucleotide sequence databases with the Arabidopsis thaliana UDP-galactose transporter sequences as queries, we found a number of sequences encoding putative O. sativa UDP-galactose transporters. From these, we cloned four putative UDP-galactose transporters, designated OsUGT1, 2, 3 and 4, which exhibited high sequence similarity with Arabidopsis thaliana UDP-galactose transporters. OsUGT1, 2, 3 and 4 consisted of 350, 337, 345 and 358 amino acids, respectively, and all of these proteins were predicted to have multiple transmembrane domains. To examine the UDP-galactose transporter activity of the OsUGTs, we introduced the OsUGTs’ expression vectors into UDP-galactose transporter activity-deficient Lec8 cells. Our results showed that transfection with OsUGT1, 2 and 3 resulted in recovery of the deficit phenotype of Lec8 cells, but transfection with OsUGT4 did not. The results of an in vitro nucleotide sugar transport assay of OsUGTs, carried out with a yeast expression system, suggested that OsUGT4 is a UDP-glucose transporter rather than a UDP-galactose transporter. Although plants have multiple UDP-galactose transporter genes, phylogenic analysis indicates that plant UDP-galactose transporter genes are not necessarily evolutionary related to each other.

 
 
 
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