Increased MAPK activation and impaired insulin signaling in subcutaneous microvascular endothelial cells in type 2 diabetes: the role of endothelin-1.
OBJECTIVE
To establish a method for isolation and culture of subcutaneous microvascular endothelial cells (MVEC) from small human tissue biopsies to compare gene and protein expression of insulin signaling molecules in MVEC from insulin-resistant and healthy control subjects.


METHODS
Stromavascular cells from subcutaneous needle biopsies of type 2 diabetic and control subjects were expanded in culture and the endothelial cells selected with magnetic immune separation. Western blots and RT-PCR were used for protein and gene expression assays.


RESULTS
At least 99% of the expanded primary MVEC could be characterized as endothelial cells. The expression of insulin receptors was low, but insulin increased tyrosine phosphorylation of both the insulin receptor and insulin receptor substrate (IRS)-1 and activated protein kinase B (PKB). The IRS-1 protein expression was reduced and the serine phosphorylation of PKB in response to insulin attenuated whereas basal and insulin-stimulated phosphorylation of extracellular signal-related kinase (ERK)1/2 was increased in type 2 diabetes MVEC. Endothelin (ET)-1 mRNA levels were significantly higher in type 2 diabetes cells. The addition of ET-1 increased the phosphorylation of mitogen-activated protein kinase (MAPK), an effect antagonized by the MEK-1 inhibitor PD98059. Furthermore, the endothelin ET(A) and ET(B) receptor antagonists BQ123 and BQ788 decreased basal MAPK activity in type 2 diabetes MVEC and prevented the ET-1-induced activation.


CONCLUSIONS
We developed a system for isolation and culture of human MVEC from small needle biopsies. Our observations support the concept of "selective" insulin resistance, involving IRS-1 and the PI3kinase pathway, as an underlying factor for a dysregulated microvascular endothelium in type 2 diabetes. Our data also support a role of ET-1 for the increased MAPK activity seen in nonstimulated type 2 diabetes MVEC.
Gogg
Silvia
S
Lundberg Laboratory for Diabetes Research, Center of Excellence for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. silvia.gogg@medic.gu.se
Smith
Ulf
U
Jansson
Per-Anders
PA
D000273:Adipose Tissue / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D001707:Biopsy, Needle; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*; D019332:Endothelin-1 / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics; D008833:Microcirculation; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics; D012016:Reference Values
D019332:Endothelin-1; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010768:Phosphoserine; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases