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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
3-Phosphoinositide-Dependent Protein Kinases
-
Adipocytes / drug effects
-
Adipocytes / metabolism*
-
DNA-Binding Proteins / metabolism
-
Electroporation / methods
-
Enzyme Activation / drug effects
-
Enzyme Activation / physiology
-
Gene Expression
-
Glucose Transporter Type 4
-
Humans
-
Insulin / metabolism
-
Insulin / pharmacology*
-
Insulin Receptor Substrate Proteins
-
Monosaccharide Transport Proteins / metabolism
-
Muscle Proteins / metabolism
-
Mutation / genetics
-
Phosphoproteins / biosynthesis
-
Phosphoproteins / genetics*
-
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
-
Transfection / methods*
-
ets-Domain Protein Elk-1
Substances
-
DNA-Binding Proteins
-
ELK1 protein, human
-
Glucose Transporter Type 4
-
IRS3P protein, human
-
Insulin
-
Insulin Receptor Substrate Proteins
-
Monosaccharide Transport Proteins
-
Muscle Proteins
-
Phosphoproteins
-
Proto-Oncogene Proteins
-
SLC2A4 protein, human
-
Transcription Factors
-
ets-Domain Protein Elk-1
-
3-Phosphoinositide-Dependent Protein Kinases
-
AKT1 protein, human
-
Protein Serine-Threonine Kinases
-
Proto-Oncogene Proteins c-akt