Skeletal muscle insulin resistance is induced by 4-hydroxy-2-hexenal, a by-product of n-3 fatty acid peroxidation

Diabetologia. 2018 Mar;61(3):688-699. doi: 10.1007/s00125-017-4528-4. Epub 2018 Jan 3.


Aims/hypothesis: Oxidative stress is involved in the pathophysiology of insulin resistance and its progression towards type 2 diabetes. The peroxidation of n-3 polyunsaturated fatty acids produces 4-hydroxy-2-hexenal (4-HHE), a lipid aldehyde with potent electrophilic properties able to interfere with many pathophysiological processes. The aim of the present study was to investigate the role of 4-HHE in the development of insulin resistance.

Methods: 4-HHE concentration was measured in plasma from humans and rats by GC-MS. Insulin resistance was estimated in healthy rats after administration of 4-HHE using hyperinsulinaemic-euglycaemic clamps. In muscle cells, glucose uptake was measured using 2-deoxy-D-glucose and signalling pathways were investigated by western blotting. Intracellular glutathione was measured using a fluorimetric assay kit and boosted using 1,2-dithiole-3-thione (D3T).

Results: Circulating levels of 4-HHE in type 2 diabetic humans and a rat model of diabetes (obese Zucker diabetic fatty rats), were twice those in their non-diabetic counterparts (33 vs 14 nmol/l, p < 0.001), and positively correlated with blood glucose levels. During hyperinsulinaemic-euglycaemic clamps in rats, acute intravenous injection of 4-HHE significantly altered whole-body insulin sensitivity and decreased glucose infusion rate (24.2 vs 9.9 mg kg-1 min-1, p < 0.001). In vitro, 4-HHE impaired insulin-stimulated glucose uptake and signalling (protein kinase B/Akt and IRS1) in L6 muscle cells. Insulin-induced glucose uptake was reduced from 186 to 141.9 pmol mg-1 min-1 (p < 0.05). 4-HHE induced carbonylation of cell proteins and reduced glutathione concentration from 6.3 to 4.5 nmol/mg protein. Increasing intracellular glutathione pools using D3T prevented 4-HHE-induced carbonyl stress and insulin resistance.

Conclusions/interpretation: 4-HHE is produced in type 2 diabetic humans and Zucker diabetic fatty rats and blunts insulin action in skeletal muscle. 4-HHE therefore plays a causal role in the pathophysiology of type 2 diabetes and might constitute a potential therapeutic target to taper oxidative stress-induced insulin resistance.

Keywords: 4-HHE; Insulin resistance; Lipid aldehyde; Peroxidation; Skeletal muscle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aldehydes / pharmacology*
  • Animals
  • Blood Glucose / drug effects
  • Blotting, Western
  • Diabetes Mellitus, Type 2 / blood
  • Diabetes Mellitus, Type 2 / metabolism
  • Fatty Acids, Omega-3 / blood
  • Female
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Insulin / blood
  • Insulin / pharmacology
  • Insulin Resistance / physiology*
  • Lipid Peroxidation / drug effects*
  • Male
  • Middle Aged
  • Muscle, Skeletal / drug effects*
  • Muscle, Skeletal / metabolism*
  • Oxidative Stress / drug effects
  • Rats
  • Rats, Zucker
  • Thiones / pharmacology
  • Thiophenes / pharmacology


  • Aldehydes
  • Blood Glucose
  • Fatty Acids, Omega-3
  • Insulin
  • Thiones
  • Thiophenes
  • 4-hydroxy-2-hexenal
  • 1,2-dithiol-3-thione