Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

Abstract

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

Fig. 1.

Free radical production in murine liver after 1 h of acute acetone treatment. C57BL/B6 mice were administered a single intragastric injection of acetone (2,500 mg/kg) to mimic ketosis, and spin trapping was performed by an ip injection of α-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN, 20 mg/mouse). Representative electron paramagnetic resonance (EPR) spectra of POBN radical adducts detected in the lipid extracts of livers from acetone-treated (A) or control (B) mice 1 h after POBN injection. Spectra are representatives of at least 5 independent experiments.

Fig. 2.

Lipid radical production in the liver of mice containing the disrupted gp91phox gene (gp91^phox−/−) and inducible nitric oxide synthase (iNOS) gene (iNOS^−/−) or mice pretreated with the inhibitor 1400W. Lipid radical adduct production was measured by EPR spectroscopy. Knockout mice were injected intragastrically with acetone (2,500 mg/kg) and with the spin trap POBN (20 mg/mouse ip) for 1 h. Lipid radical adducts were determined in the lipid extracts of liver samples. Each experiment was made in quadruplicate. Representative EPR spectra of lipid radicals detected in the liver of acetone-treated C57BL mice (A), acetone-treated NADPH oxidase knockout mice (B), acetone-treated iNOS knockout mice (C), and acetone-treated C57Bl mice pretreated with the iNOS inhibitor 1400W (15 mg/kg) (D).

Fig. 3.

iNOS overexpression in murine liver tissues upon chronic acetone treatment. Mice received 2% acetone for 5 days in their drinking water. A: immunofluorescence detection of iNOS using confocal microscopy. Incubation of liver slices with primary anti-iNOS and Alexafluor 488 secondary antibody shows significant increase of iNOS expression in acetone-treated animals. The enzyme expression was more pronounced around the centralobular vein. Omitting the primary antibody demonstrated only minor background fluorescence (data not shown). B: RT-PCR and Western blot analysis of iNOS protein expression in liver tissues of control mice and acetone-treated mice 1, 3, and 5 days after treatment. Control and acetone-treated liver homogenate samples containing equal amounts of protein (40 μg/lane) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-iNOS monoclonal antibody. Total RNA was extracted from liver samples, and quantitative RT-PCR was performed. Data are representative of 3 independent experiments. C: statistical evaluation of Western blot band intensities and quantitative real-time PCR analysis of iNOS mRNA after a time course of acetone treatment. *P < .05 vs. control group.

Fig. 4.

Protein oxidation and immunofluorescence detection of protein radical formation after 5 days of acetone treatment. Mice were receiving 2% acetone in their drinking water for 5 days together with the ip administration of 20 μl 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) 3 times/day. A: incubation of liver slices with primary anti-DMPO and Alexafluor 488 secondary rabbit antibody shows enhanced protein oxidation in acetone-treated liver (intense green staining). Omitting the primary antibody or applying the anti-DMPO antibody only without DMPO treatment demonstrated only minor background fluorescence (data not shown). B: compared with wild-type mice treated with acetone, iNOS knockout mice show a significant decrease in protein radical formation, whereas no changes were detected in the liver of NADPH oxidase knockout mice. C: quantitative comparison of the levels of protein oxidation. Confocal images were compared using the Scion Image program, and the degree of protein oxidation was expressed as mean fluorescence intensity in each group. P < 0.05 vs. control group (*) and vs. acetone-treated group (#).

Fig. 5.

Immunohistochemical detection of 4-hydroxynonenal in liver as a marker of lipid peroxidation and lipid-protein conjugation. Positive staining showing a centrolobular pattern was observed in acetone-treated livers after 21 days while no significant staining was present in control liver samples (top). Only minor background staining was detected in control or acetone-treated iNOS knockout animals (bottom).