ABSTRACT
Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% +/- 5 (mean +/- S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.
Subject(s)
Adipocytes/metabolism , Insulin/pharmacology , Phosphoproteins/genetics , Signal Transduction , Transfection/methods , 3-Phosphoinositide-Dependent Protein Kinases , Adipocytes/drug effects , DNA-Binding Proteins/metabolism , Electroporation/methods , Enzyme Activation/drug effects , Enzyme Activation/physiology , Gene Expression , Glucose Transporter Type 4 , Humans , Insulin/metabolism , Insulin Receptor Substrate Proteins , Monosaccharide Transport Proteins/metabolism , Muscle Proteins/metabolism , Mutation/genetics , Phosphoproteins/biosynthesis , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , Protein Transport/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction/drug effects , Transcription Factors/metabolism , ets-Domain Protein Elk-1ABSTRACT
BACKGROUND/AIMS: Epidermis forms the protective barrier of the skin by its outermost layer, stratum corneum. The purpose of this study was to investigate the epidermal barrier in view of epidermal calcium release (ECR), phosphate release, transepidermal water loss (TEWL) and skin surface pH. Calcium is mainly an intracellular ion. Calcium was sampled introducing a new and simple washout chamber technique for the study of epidermal release in vivo. METHODS: Test sites on forearms of 13 healthy subjects were pre-treated with 24 h water occlusion, 24 h 2% sodium lauryl sulphate (SLS) or tape stripped. Both untreated and pre-treated test sites were exposed to a water washout chamber with 200 micro deionized water as a solvent. Water washout chambers were removed after two hours and calcium and phosphate in the water was analyzed. Transepidermal water loss and pH were measured before and after the trial. RESULTS: pH increased after tape stripping and after exposure to SLS. Transepidermal water loss increased significantly at all test sites. Calcium was significantly released from SLS-treated sites but not from tape stripped sites. There was generally a correlation between ECR, phosphate release, TEWL and pH. In this study ECR is showed to be a barrier marker of high reproducibility. CONCLUSIONS: Epidermal calcium release or ECR is found useful as an indicator of skin barrier function. Calcium release and increase of pH appear mainly to illustrate direct and corrosive damage to epidermal cells and functions contrasting TEWL, in this experiment probably reflecting intercellular damage of fracturing as exemplified by mechanical damage resulting from surface stripping. This new distinction of skin barrier damage into cellular damage resulting from a corrosive chemical trauma and intercellular damage and fracturing resulting from a mechanical trauma is exemplified in SLS provocative testing and tape stripping, the former characterized by increased ECR. The washout chamber technique was deemed technically reliable and reproducible, and has a major potential in experimental dermatology and skin pharmacology for the study of in vivo epidermal release of a range of endogenous and exogenous substances.
Subject(s)
Calcium/metabolism , Epidermis/metabolism , Adult , Dermatology/methods , Epidermis/drug effects , Female , Forearm , Humans , Hydrogen-Ion Concentration , Male , Middle Aged , Phosphates/metabolism , Reproducibility of Results , Sodium Dodecyl Sulfate/pharmacology , Water Loss, InsensibleABSTRACT
Apresentaçäo de um caso de odontoma combinado e seu tratamento cirúrgico: biópsia excisional