[Regulation effect of d-alpha-tocopherol on the expression of hexokinase induced by high glucose in cultured human peritoneal mesothelium cells].
OBJECTIVE
To investigate the effect of d-alpha-tocopherol on the expression of hexokinase (HK) induced by high glucose in cultured human peritoneal mesothelium cell (HPMC).


METHODS
Specimens of human omentum were obtained from consenting patient undergoing elective abdominal surgery. HPMC were isolated and subcultured by enzymatic disaggregation. Morphology and immunocytochemical method were used for identification. HPMC were divided into normal glucose group (0.1% glucose, equal to 5.5 mmol/L), high glucose group (0.5%, 1.0%, 1.5%, 2.5%, 4.25% glucose) and d-alpha-tocopherol group. After 24 h, standard G6PDH-coupled assay, temperature sensitive essay were used to detect the activity of total HK and its isozyme. Immunocytochemical staining was used for observation the intracellular location of HKII. Western blotting was used to analyze the protein expression of HKII and RT-PCR was used to detect the mRNA expression of HKII. The net glucose utilization was assayed by glucose disappearance from medium by hexokinase method.


RESULTS
Primary cultured HPMC reacted positively for cytokeratin and vimentin and had numerous surface microvilli under electron microscope. High glucose induced HK activity in a dose-dependent manner and increased HKII isoform selectively. At concentration of 1.5%, 2.5% and 4.25% glucose for 24 h, the relative activity of total HK were 115.4%, 129.1% and 155.2%, respectively comparing with normal control (P < 0.05), and selectively increased HKII isoform expression. D-alpha-tocopherol blocked the activity of total HK and HKII induced by glucose. The relative activity of total HK inhibited by d-alpha-tocopherol was 82.1% (P = 0.001). After incubated with d-alpha-tocopherol, the net glucose utilization were decreased from (25.3 +/- 3.9) mmol/L to (17.3 +/- 2.1) mmol/L (P = 0.018). HKII stained light brown in cytoplasm of HPMC in normal group, accompanying with the increased concentration of glucose, the staining of HKII became strong and accumulated to nuclear. D-alpha-tocopherol made it thinning. The protein and mRNA expression of HKII were accorded with its activity.


CONCLUSIONS
D-alpha-tocopherol inhibited the expression of HKII in activity, protein and mRNA induced by high glucose and decreased the net glucose utilization, which might become a method to improve ultrafiltration in peritoneal dialysis.
Fan
Yi
Y
Department of Nephrology, First Clinical College, China Medical University, Shenyang 110001, China.
Ma
Jian-fei
JF
Ding
Hong
H
Zhang
Jing-hua
JH
Han
Jie
J
Jia
Hai-yan
HY
Wang
Li-ning
LN
D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000096:biosynthesis*; D006801:Humans; D010537:Peritoneum / Q000166:cytology; D012333:RNA, Messenger / Q000096:biosynthesis; D024502:alpha-Tocopherol / Q000494:pharmacology*
D012333:RNA, Messenger; D006593:Hexokinase; D024502:alpha-Tocopherol; D005947:Glucose