ABSTRACT
Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10(-8)âmol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10(-8)âmol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10(-8)âmol/l and glucose accumulation at 10(-7)âmol/l in HMVEC-Cs. TNF-α dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.
Subject(s)
Endothelial Cells/metabolism , Myocardium/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/physiology , Receptor, Insulin/genetics , Receptor, Insulin/physiology , Anti-Inflammatory Agents/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Coronary Vessels/physiology , Endothelial Cells/drug effects , Endothelial Cells/physiology , Gene Expression , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Inflammation/prevention & control , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Microvessels/drug effects , Microvessels/metabolism , Microvessels/physiology , Receptor, IGF Type 1/agonists , Receptor, IGF Type 1/metabolism , Receptor, Insulin/agonists , Receptor, Insulin/metabolismABSTRACT
We compared insulin and IGF effects in adipocytes expressing IR (insulin receptors), and preadipocytes expressing IR and IGF-IR (IGF-I receptors). Treatment of adipocytes with insulin, IGF-II or IGF-I resulted in phosphorylation of IR. Order of potency was insulin>IGF-II>IGF-I. In preadipocytes IR, IGF-IR and insulin/IGF-I hybrid receptors (HR) were detected. Treatment of preadipocytes with IGF-I and IGF-II 10(-8)M resulted in activation of IGF-IR and IR whereas insulin was more potent in activating IR, with no effect on IGF-IR. In adipocytes glucose transport was 100-fold more sensitive to insulin than to IGFs and the maximal effect was higher with insulin. In preadipocytes glucose accumulation and DNA synthesis was equally sensitive to insulin and IGFs but the maximal effect was higher with IGF-I. In conclusion, insulin and IGF-I activate their cognate receptors and IGF-I also HR. IGF-II activates IR, IGF-IR and HR. Insulin and IGF-I are partial agonists to each other's receptors.
Subject(s)
Adipocytes/drug effects , Insulin-Like Growth Factor II/pharmacology , Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolism , Adipocytes/metabolism , Cells, Cultured , DNA Replication , Glucose/metabolism , Humans , Immunoprecipitation , Insulin/physiology , Phosphorylation , Protein Binding , Thymidine/metabolismABSTRACT
Mature adipocytes originate from fibroblast-like precursor cells, preadipocytes, which differentiate to obtain the characteristics of adipocytes. Our aim was to investigate how differentiation of human preadipocytes affects the distribution of insulin receptors (IR) and IGF-I receptors (IGF-IR) and other cell characteristics. Preadipocytes were differentiated using indomethacine, dexamethasone, isobutyl-methylxantine (IBMX) and high concentration of insulin. Gene expression was quantified by real-time RT-PCR in preadipocytes (PA), differentiated preadipocytes (dPA) and mature adipocytes (mAD). The amount of expressed receptor protein was analyzed using receptor specific ELISAs and Western blot. We also studied DNA synthesis with radiolabeled thymidine incorporation and glucose accumulation with radiolabeled glucose. Differentiation of PA increased gene expression of IR but not IGF-IR. GLUT4, growth hormone receptor (GHR) and adiponectin appeared or increased. In PA and dPA only IR-A was expressed whereas also IR-B was detected in mAD. By Western blot and ELISA, IR and IGF-IR were detected in PA, dPA and mAD. During differentiation the ratio of IR to IGF-IR increased severalfold. In PA both the IR and the IGF-IR was phosphorylated by their own ligand at 1 nM and in dPA the activation of both receptors was stimulated by IGF-I, but not insulin, at 1 nM. Accumulation of glucose in PA was increased by insulin at 10nM and by IGF-I at 1 nM and 10nM. DNA synthesis was increased by insulin and IGF-I at 10nM. In conclusion, both IR and IGF-IR are present in human preadipocytes and adipocytes. Differentiation is characterized by an increased IR/IGF-IR ratio.
