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

Eur J Endocrinol. 2003 Jan;148(1):157-67. doi: 10.1530/eje.0.1480157.

Abstract

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.

Design and 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes / cytology
  • Adipocytes / drug effects
  • Adipocytes / metabolism
  • Animals
  • Cells, Cultured
  • Diabetes Mellitus, Type 2 / drug therapy
  • Diabetes Mellitus, Type 2 / metabolism
  • Diabetes Mellitus, Type 2 / physiopathology
  • Glucose / pharmacokinetics*
  • Glucose Transporter Type 4
  • Hypoglycemic Agents / pharmacology*
  • Insulin / pharmacology*
  • Insulin Receptor Substrate Proteins
  • Insulin Resistance / physiology*
  • Intracellular Signaling Peptides and Proteins
  • Male
  • Monosaccharide Transport Proteins / metabolism
  • Muscle Proteins*
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction / drug effects

Substances

  • Glucose Transporter Type 4
  • Hypoglycemic Agents
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Intracellular Signaling Peptides and Proteins
  • Irs1 protein, rat
  • Irs2 protein, rat
  • Monosaccharide Transport Proteins
  • Muscle Proteins
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • Slc2a4 protein, rat
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Glucose