Identification of ATP synthase as a lipid peroxide protein adduct in pancreatic islets from humans with and without type 2 diabetes mellitus

Abstract

Context: Most current knowledge of pancreatic islet pathophysiology in diabetes mellitus has come from animal models. Even though islets from humans are readily available, only a few come from diabetic donors. We had the uncommon opportunity to acquire islets from humans with type 2 diabetes and used it to perform a study not previously done with human or animal islets.

Objectives: Oxidative stress has been proposed as a mechanism for impaired β-cell function in type 2 diabetes. Lipid peroxides caused by reactive oxygen species are damaging to body tissues. The objective was to determine whether lipid peroxide-protein adducts occur in pancreatic islets of humans with type 2 diabetes.

Design: Immunoblots with two antibodies to hydroxynonenal and 2 other antibodies we generated against reactive small aliphatic compounds were used to detect lipid peroxide-protein adducts in islets of patients with type 2 diabetes and controls.

Results: The antibodies reacted strongly to ≥5 islet proteins. The major hydroxynonenal adduct in the islets of type 2 diabetes patients was a 52-kDa protein seen with all 4 antibodies that was also seen in islets of nondiabetic humans, rat islets, and insulinoma cells and in mitochondria of various rat tissues. Nano-LC-MS/MS (liquid chromatography-tandem mass spectrometry) and MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) analysis identified the protein as the β-chain of the mitochondrial F-ATP synthase, an enzyme responsible for 95% of ATP formed in tissues.

Conclusions: Lipid peroxide-protein adducts occur in β-cells in the nondiabetic state and in diabetes. Lipid peroxidation is thought to be damaging to tissues. Analogous to various other unhealthy characteristics, the presence in nondiabetic individuals of lipid peroxide-protein adducts does not necessarily indicate they are not detrimental.

Figure 1.

Hydroxynonenal-protein adducts and other adducts in pancreatic islets from humans with type 2 diabetes and without diabetes and in rat islets and INS-1 832/13 cells. A, Immunoblots of proteins (10 μg protein per lane) from islets of humans with type 2 diabetes (Diab) from Sweden or from rats or INS-1 832/13 cells were probed with antibodies raised against 4-hydroxynonenal (HNE) by L.I.S. or 3-hydroxyoctanoic acid plus nanoic acid (627) and 4-hydroxynonenal plus nanoic acid lactone (628) raised by M.J.M. B, A different antihydroxynonenal antibody (from Chemicon) was used to analyze islets from human donors with type 2 diabetes (T2D) and nondiabetic donors (Non) from the United States. This blot (15 μg protein per lane) is representative of 4 similar blots in which islets from the same human donors with type 2 diabetes and different human donors without diabetes were analyzed. The relative density of the HNE bands are expressed as the average density of the protein bands of the islets from 2 nondiabetic donors set at 100%. The density of the 52-kDa band was not consistently darker in islets from either type of donor as judged from densitometry of the HNE band (as shown in B; also see Results). A blot of β-actin is shown as a control for loading of protein. C, Two-dimensional gel electrophoresis of the 52-kDa HNE-bonded protein from rat kidney mitochondria. The protein was partially purified from rat kidney mitochondrial proteins and then applied to 2 companion 2-dimensional electrophoresis gels that were run in parallel. The gel shown on the right was analyzed by immunoblotting with the anti-HNE antibody; the gel shown on the left was stained for protein, and the protein spot in this gel that corresponded to the anti-HNE–stained protein in the immunoblot was excised and subjected to nano-LC-MS/MS and MALDI-TOF fingerprint analysis to identify the HNE protein adduct.