High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes.
OBJECTIVE
The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins.


METHODS
Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed.


RESULTS
High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration.


CONCLUSIONS
Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.
Burén
Jonas
J
Department of Medicine, Umea University Hospital, S-901 85 Umea, Sweden.
Liu
Hui-Xia
HX
Lauritz
Johan
J
Eriksson
Jan W
JW
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D005947:Glucose / Q000493:pharmacokinetics*; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects
D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526296:Irs1 protein, rat; C526299:Irs2 protein, rat; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; C495694:Slc2a4 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D005947:Glucose