Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin.
BACKGROUND
Heparan sulfate proteoglycans (HSPGs) contain glycosaminoglycan (GAG) chains made primarily of heparan sulfate (HS). Hyperglycemia in diabetes leads to endothelial injury and nephropathy, retinopathy and atherosclerosis. Decreased HSPG may contribute to diabetic endothelial injury. Decreased tissue HS in diabetes has been reported, however, endothelial HS changes are poorly studied.


OBJECTIVE
To determine total GAGs, including HS, in endothelium under hyperglycemic conditions and the protective effect of insulin and heparin.


METHODS
Confluent primary porcine aortic endothelial cells (PAECs) were divided into control, glucose (30 mM), insulin (0.01 unit/ml) and glucose plus insulin treatment groups for 24, 48 and 72 hours. Additionally, PAECs were treated with glucose, heparin (0.5 microg/ml) and glucose plus heparin for 72 hours. GAGs were isolated from cells and medium. GAG concentrations were determined by the carbazole assay and agarose gel electrophoresis.


RESULTS
GAGs were significantly increased only in control and glucose plus insulin groups at 72 versus 24 hours. Glucose decreased cell GAGs and increased medium GAGs, and insulin alone decreased cell GAGs at all times compared to control. In the glucose plus insulin group, cell GAGs were less than control at 24 hours, and greater than glucose or insulin alone at 48 and 72 hours while GAGs in medium were greater than control at all times and glucose at 72 hours. Heparin increased GAGs in glucose treated cells and medium.


CONCLUSIONS
High glucose and insulin alone reduces endothelial GAGs. In hyperglycemic conditions, heparin or insulin preserves GAGs which may protect cells from injury. Insulin is an effective diabetic therapy since it not only lowers blood glucose, but also protects endothelium.
Han
Juying
J
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada. juh884@mail.usask.ca
Zhang
Fuming
F
Xie
Jin
J
Linhardt
Robert J
RJ
Hiebert
Linda M
LM
D000818:Animals; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004347:Drug Interactions; D004587:Electrophoresis, Agar Gel; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000032:analysis / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000032:analysis / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000032:analysis / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D034421:Sus scrofa; D013552:Swine; D053667:Syndecans / Q000032:analysis / Q000378:metabolism; D013997:Time Factors
D005947:Glucose; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007328:Insulin; D053667:Syndecans; C071927:perlecan
Kidney Int. 2000 Jun;57(6):2492-501
10844618
Exp Nephrol. 1997 May-Jun;5(3):180-8
9208276
J Vasc Res. 2001 Sep-Oct;38(5):415-22
11561143
J Biol Chem. 1987 Apr 5;262(10):4708-16
2951371
Annu Rev Cell Biol. 1992;8:365-93
1335744
Annu Rev Biochem. 1991;60:443-75
1883201
Diabetologia. 1989 Apr;32(4):219-26
2668076
J Surg Res. 1980 Jun;28(6):556-62
6448319
Biochim Biophys Acta. 2004 Apr 7;1672(1):36-45
15056491
Diabetologia. 1992 Feb;35(2):183-6
1547925
Kidney Int. 1995 Nov;48(5):1508-16
8544408
J Biol Chem. 2000 Jan 14;275(2):1463-70
10625699
Diabetes. 1983 Apr;32(4):337-42
6219905
Kidney Int. 2006 Dec;70(12):2100-8
17051139
Experientia. 1993 May 15;49(5):447-55
8500599
Biochem Soc Trans. 1997 Feb;25(1):220-4
9056874
Am J Pathol. 1981 May;103(2):271-82
7195151
Cardiovasc Diabetol. 2005 Aug 09;4:12
16086844
Arch Biochem Biophys. 1991 Mar;285(2):205-10
1897927
Experientia. 1995 Sep 29;51(9-10):863-72
7556568
Life Sci. 1984 Dec 24;35(26):2613-20
6239964
Arch Biochem Biophys. 1995 Apr 20;318(2):286-94
7733656
J Cell Physiol. 1989 Aug;140(2):305-10
2745565
J Lab Clin Med. 1990 Apr;115(4):422-32
1691256
Isr Med Assoc J. 2002 Nov;4(11):996-1002
12489489
N Engl J Med. 1983 Oct 27;309(17):1001-9
6225948
J Am Soc Nephrol. 1994 Mar;4(9):1683-9
8011978
Int J Biochem Cell Biol. 2000 Feb;32(2):115-20
10687947
Diabetes. 1982 May;31(5 Pt 1):418-25
7152134
Diabetologia. 1994 Mar;37(3):313-20
8174847
Exp Biol Med (Maywood). 2007 Jul;232(7):927-34
17609509
Semin Thromb Hemost. 1991;17 Suppl 1:47-56
2068571
Am J Cardiol. 1999 Jul 8;84(1A):21J-24J
10418854
Circulation. 2000 Apr 4;101(13):1539-45
10747347
J Biol Chem. 1983 Oct 10;258(19):11672-7
6225778
Arterioscler Thromb Vasc Biol. 2004 May;24(5):858-63
15031130
Diabetologia. 1993 Apr;36(4):316-22
8477876
Kidney Int. 1992 Aug;42(2):285-91
1328749
Eur J Nutr. 2006 Oct;45(7):369-75
16810465
Biochemistry. 2006 May 9;45(18):5703-11
16669614
J Clin Invest. 1997 Dec 1;100(11 Suppl):S67-75
9413405
Am J Med. 1988 Nov 28;85(5A):113-8
3057888