Contribution of inducible and neuronal nitric oxide synthases to mitochondrial damage and melatonin rescue in LPS-treated mice

NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS^?/?) and neuronal nitric oxide synthase (nNOS^?/?) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.     

Development of Mice with Osteoblast-Specific Connexin43 Gene Deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43^fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43^±; Colα1(I)-Cre or Cx43^±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43^fl/?; Colα1(I)-Cre or Cx43^fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

We hypothesized that NO^• is generated in isolated cardiac mitochondria as the source for ONOO⁻ production during oxidative stress. We monitored generation of ONOO⁻ from guinea pig isolated cardiac mitochondria subjected to excess Ca²⁺ uptake before adding succinate and determined if ONOO⁻ production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl₂ and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O₂^•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO⁻ to form dityrosine (diTyr) in proportion to the ONOO⁻ present. We found that exposing mitochondria to excess CaCl₂ before succinate resulted in an increase in diTyr and amplex red fluorescence (H₂O₂) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa²⁺ and succinate, increased mitochondrial ONOO⁻ production via NO^• and O₂^•−. Changes in mitochondrial ONOO⁻ production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO^• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO⁻ production, PTIO decreased production of ONOO⁻, and TEMPOL decreased ONOO⁻ levels by converting more O₂^•− to H₂O₂. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO⁻ into the buffer is dependent both on O₂^•− released from mitochondria and NO^• derived from a mtCa²⁺-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Expression of nitric oxide synthase isoforms in the dorsal horn of monoarthritic rats: effects of competitive and uncompetitive <Emphasis Type="Italic">N</Emphasis>-methyl-D-aspartate antagonists

Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.     

Hypoxia-induced Activation of Epidermal Growth Factor Receptor (EGFR) Kinase in the Cerebral Cortex of Newborn Piglets: The Role of Nitric Oxide

The present study aims to investigate the mechanism of EGFR kinase activation during hypoxia and tests the hypothesis that hypoxia-induced increased activation of EGFR kinase in the cerebral cortical membrane fraction of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS). Fifteen newborn piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor (Hx-nNOSi, n = 5). Hypoxia was induced by an FiO2 of 0.07 for 60 min. nNOS inhibitor I (selectivity >2,500 vs. endothelial NOS, eNOS, and >500 vs. inducible NOS, iNOS) was administered (0.4 mg/kg, i. v.) 30 min prior to hypoxia. EGFR kinase tyrosine phosphorylation at Tyr1173, an index of activation of EGFR kinase, was determined by Western blot analysis using an anti-phospho (pTyr¹¹⁷³)-EGFR kinase antibody. Protein bands were analyzed by imaging densitometry and expressed as absorbance (OD × mm²). EGFR kinase activity was determined radiochemically using immunopurified enzyme. EGFR kinase activity was expressed as pmols/mg protein/hr. Density of phosphor (pTyr¹¹⁷³)-EGFR kinase (OD × mm²) was 60.2 ± 9.8 in Nx, 177.0 ± 26.9 in Hx (P < 0.05 vs. Nx) and 79.9 ± 15.7 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Activity of EGFR kinase (pmoles/mg protein/hr) was 4,603 ± 155 in Nx, 8,493 ± 427 in Hx (P < 0.05 vs. Nx) and 4,516 ± 104 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Pretreatment with nNOS inhibitor prevented the hypoxia-induced increased phosphorylation and increased activity of EGFR kinase. We conclude that the mechanism of hypoxia-induced increased activation of EGFR kinase is mediated by nNOS-derived NO.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.     

Expression profiles of NOS isoforms in gingiva of nNOS knockout mice

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from l-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20–25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.     

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury

Background

The factors contributing to chronic Chagas'' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 ^−/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.

Methodology

Rhesus monkeys and C57BL/6 and Nos2 ^−/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2⁺ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.

Results

Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2⁺ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 ^−/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.

Conclusion

T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas'' heart disease.     

Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21 d of chronic social isolation stress, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold (4 °C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic–pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.     

ER stress is implicated in mitochondrial dysfunction-induced apoptosis of pancreatic beta cells

Mitochondrial dysfunction induces apoptosis of pancreatic β-cells and leads to type 2 diabetes, but the mechanism involved in this process remains unclear. Chronic endoplasmic reticulum (ER) stress plays a role in the apoptosis of pancreatic β-cells; therefore, in current study, we investigated the implication of ER stress in mitochondrial dysfunction-induced β-cells apoptosis. Metabolic stress induced by antimycin or oligomycin was used to impair mitochondrial function in MIN6N8 cells, which are mouse pancreatic β-cells. Impaired mitochondria dysfunction increased ER stress proteins such as p-eIF2α, GRP78 and GRP 94, as well as ER stress-associated apoptotic factor, CHOP, and activated JNK. AMP-activated protein kinase (AMPK) was also activated under mitochondria dysfunction by metabolic stress. However, the inhibition of AMPK by treatment with compound C, inhibitor of AMPK, and overexpression of mutant dominant negative AMPK (AMPKK45R) blocked the induction of ER stress, which was consist-ent with the decreased β-cell apoptosis and increase of insulin content. Furthermore, mitochondrial dysfunction increased the expression of the inducible nitric oxide synthase (iNOS) gene and the production of nitric oxide (NO), but NO production was prevented by compound C and mutant dominant negative AMPK (AMPK-K45R). Moreover, treatment with 1400W, which is an inhibitor of iNOS, prevented ER stress and apoptosis induced by mitochondrial dysfunction. Treatment of MIN6N8 cells with lipid mixture, physiological conditions of impaired mitochondria function, activated AMPK, increased NO production and induced ER stress. Collectively, these data demonstrate that mitochondrial dysfunction activates AMPK, which induces ER stress via NO production, resulting in pancreatic β-cells apoptosis.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

Thisstudy examined mRNA and protein expressions of neuronal (nNOS),inducible (iNOS), and endothelial nitric oxide synthases (eNOS) inperipheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. Onesciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve werequantitatively measured by competitive PCR, and protein was determinedby Western blotting and immunohistochemical staining. The resultsshowed that, after ischemia (2 h), both nNOS and eNOS proteinexpressions decreased. After I/R (2 h of ischemia followed by3 h of reperfusion), expression of both nNOS and eNOS mRNA andprotein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal thedynamic expression of individual NOS isoforms during the course of I/Rinjury. An understanding of this modulation on a cellular and molecularlevel may lead to understanding the mechanisms of I/R injury and tomethods of ameliorating peripheral nerve injury.

     

Diet‐induced obesity impairs AKT signalling in the retina and causes retinal degeneration

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high‐fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT₁, eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4‐hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin‐resistance‐induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes. Copyright © 2012 John Wiley & Sons, Ltd.