Introduction
Diabetic retinopathy is considered a neurodegenerative disease in which visual dysfunction is initiated in early diabetes [
1]. As recent studies reveal, many of the diabetic complications are associated with oxidative stress [
2‐
4] as well as inflammation [
4,
5]. However, the underlying mechanism in diabetic retinal degeneration remains to be elucidated. Moreover, a definitive therapy for its prevention is not available at this time.
Several intracellular signalling pathways downstream of inflammation are associated with oxidative stress [
4‐
7]. One such pathway, angiotensin II type 1 receptor (AT1R) signalling, is pathogenic in the development of diabetic complications [
3,
8]. In fact, the streptozotocin (STZ)-induced mouse model of diabetes has a decrease in responses of the oscillatory potentials (OPs) in electroretinograms (ERGs) through retinal AT1R signalling, as we have previously reported [
8]. Another report showed that an angiotensin II converting enzyme inhibitor prevented the OP changes, supporting the idea that angiotensin II signal is important in diabetic retinopathy [
9]. OPs reflect the functioning of the inner retina [
10], and are already abnormal in early diabetes, in both human patients and experimental animals [
8,
11‐
13]. This is at least in part because of the decrease in the level of synaptophysin caused by AT1R signalling in the retina [
8]. Synaptophysin is a synaptic membrane protein that is abundant in the inner plexiform layer (IPL), where AT1R is also produced [
14], and plays a critical role in OPs. In neurons, AT1R signalling activates extracellular signal-regulated kinase (ERK) to induce excessive degradation of synaptophysin, through the ubiquitin–proteasome system [
8]. Therefore, AT1R signalling is one of the key modulators of diabetic retinopathy. However, whether or not these diabetic neurodegenerative changes can be prevented by suppressing reactive oxygen species (ROS) in the retina remains to be elucidated. On the other hand, retinal ganglion cells [
15‐
18] and a subset of amacrine cells in the inner nuclear layer (INL) [
19] are lost to apoptosis in diabetes, as shown by caspase-3 activation and TUNEL staining, and can be attenuated by administration of the soluble factor, brain-derived neurotrophic factor (BDNF) [
19]. However, the relationship between BDNF and oxidative stress in diabetes is still obscure. Thus, evaluating the contribution of ROS in diabetic retinopathy may help establish a new therapy.
Here, we focus on lutein, a xanthophyll carotenoid and an antioxidant, which is spread throughout the retina. Lutein is not synthesised in vivo and needs to be obtained through the diet, and is then delivered to the retina. It corresponds to the macular pigment in the retina with its optical isomer zeaxanthin. Long-term oral intake of lutein is reported to elevate serum lutein levels [
20,
21], which correlate with the macular pigment density [
20,
22], indicating that lutein constantly taken from the diet accumulates in the retina. Our previous data confirmed that lutein administration increases lutein levels in the choroid and retinal pigment epithelial cells in the eye, and suppresses inflammatory signalling in a model of laser-induced choroidal neovascularisation [
7]. We previously reported in an endotoxin-induced uveitis model that lutein administration suppresses ROS and inflammatory signalling in the retina, and prevents the visual dysfunction [
6] caused by rhodopsin degradation [
23]. Therefore, growing evidence shows the role of lutein as a suppressor of ROS induced by inflammation [
6,
7,
24,
25]. However, the effect of this antioxidant, lutein, in diabetic neurodegeneration is not fully understood.
In this study, we analysed whether constant lutein intake suppresses the neurodegenerative changes in the retina of STZ-induced diabetic mice. We first checked body weight or blood glucose level changes and the level of ROS generated in the retina of diabetic mice, with or without constant intake of lutein-supplemented diet. Then, visual function was measured, and biochemical and histological changes in the retina were analysed. We showed the neurodegenerative influence of oxidative stress in the diabetic retina that provides us with a possible mechanism involved in the pathogenesis of diabetic retinopathy.
Discussion
We have demonstrated that diabetes-induced oxidative stress in the retina is reduced by a constant intake of lutein (Fig.
1) without changing body weight or blood glucose levels (Table
1). Importantly, impairment of visual function measured by OPs of the ERGs (Fig.
2), as well as ERK activation, the subsequent synaptophysin reduction and BDNF depletion after 1 month of diabetes, were all suppressed by lutein (Fig.
3). Later, at 4 months of diabetes, histological changes (Fig.
4) caused by apoptosis (Fig.
5) in the retina of diabetic mice were inhibited by feeding the lutein-supplemented diet all the time after onset of diabetes.
Superoxide anion can be generated in the mitochondria under high glucose [
26]. However, lutein prevented oxidative stress in the retina of diabetic mice without body weight or blood glucose level changes (Table
1), suggesting that lutein may have scavenged ROS, at least in the retinal cells. This is consistent with the fact that lutein is delivered into the IPL, which consists of the neurites of inner retinal cells, as well as photoreceptor cells [
27,
28].
In accord with clinical and experimental data [
8,
11,
13,
29], OPs in ERGs were changed in early diabetes. Pharmacological studies using tetrodotoxin and glycine suggested the cellular origin of OPs as neurons with synapse formation in the inner retina [
30‐
32]. Lack of synaptophysin induces a decrease in synaptic vesicles, which disturbs neurotransmitter release and synaptic network activity [
33]. Thus, the present data showing that OPs were preserved when synaptophysin levels were rescued by ROS reduction are realistic.
In addition, it should be also noted that synaptic activity promotes cell survival [
34,
35] and vision protection in the following phase (discussed below), through increasing the levels of intracellular calcium ion in neurons, which are triggered by synaptic activity, i.e. the neuronal electric stimuli [
34,
35].
Our results showed that ERK activation was suppressed and the synaptophysin level was preserved when the ROS level was decreased by lutein (Figs
1 and
3), indicating that ROS activated ERK to reduce synaptophysin levels in diabetes, as AT1R signalling did in our previous study [
8]. In cardiac fibroblasts, angiotensin II increases the intracellular ROS and ERK activation through AT1R, to induce cardiac hypertrophy. The increase is cancelled by the NADH/NADPH oxidase inhibitor that reduces intracellular ROS, indicating that angiotensin II stimulates ROS production via AT1R and NADH/NADPH oxidase, to activate ERK [
36]. Thus, ROS generation in the diabetic retina may have promoted ERK activation downstream of angiotensin II, and lutein inhibited this AT1R-mediated diabetic change in the retina.
We also found that reduction of BDNF was attenuated by lutein, indicating that this change was partly caused by excessive oxidative stress. Since BDNF is regulated by neuronal synaptic activity [
37,
38], the positive effect of lutein on BDNF may have been through preservation of the synaptophysin level and the subsequent neuronal synaptic activity in the retina of diabetic mice.
As regards the roles of BDNF, it regulates neurotransmitter release and neuronal activity [
38,
39]. Thus, BDNF reduction may also have been involved in visual impairment shown by OPs in ERGs. Another role of BDNF is to promote survival of inner retinal cells [
19,
40]. Thus, this oxidative stress-mediated BDNF reduction may, to some extent, have contributed to the obvious histological changes subsequently appearing in the inner retina.
Histological changes in the inner retina were clearly observed in 4-month-diabetic mice (Figs
4 and
5). Consistent with previous reports showing caspase-3 activation and TUNEL staining [
16,
18,
41,
42], cell loss in the STZ-induced diabetic retina of this study was also through apoptosis pathway. This long-term effect may be caused by multiple signals activated by diabetes. However, lutein’s ability to maintain synaptophysin levels and neuronal network activity (discussed above), together with the resulting maintenance of BDNF production, may be involved in promoting the survival of neurons in the inner retina, interrupting the positive feedback loop for progressive neurodegeneration. Interestingly, in another progressive neurodegenerative disease, Alzheimer’s disease, reductions in synaptophysin [
43] and BDNF [
44] are also observed, and oxidative stress is involved in the pathogenesis. However, whether or not lutein will have any effect on the progression of Alzheimer’s disease is still unknown.
Taken as a whole, the current evidence clearly indicates that the visual impairment occurring in early diabetes is regulated by oxidative stress, which was prevented by constant lutein intake.
The relationship between a lutein diet and eye disease has been mainly analysed in human age-related macular degeneration (AMD) and cataract [
45]. AMD may partly be exacerbated by blue light (the blue-light hazard), and lutein may act as a blue-light filter to prevent subsequent photochemical injury to the retina that is mediated by oxidative stress. Several previous human studies have suggested, and a large-scale clinical study (the Age-Related Eye Disease Study 2) is now ongoing, to show the effect of lutein in reducing risk of progression of AMD, which is, at least in part, accelerated by light exposure [
46]. But, the present animal study showed that lutein does have the effect, in vivo, of suppressing oxidative stress independently of blue-light exposure. This antioxidative effect is also reported in another field: increase in dietary intake of lutein is protective against the development of early atherosclerosis from the results of epidemiological human data, and in vitro and in vivo mouse model experiments [
21]. Future clinical studies aimed at analysing the effect of a daily lutein administration in human diabetic retinopathy are anticipated. Other epidemiological data, showing that a higher plasma concentration of lutein/zeaxanthin and lycopene is associated with significantly lower odds of diabetic retinopathy [
47], may also support this idea.
In summary, we have shown that lutein prevents diabetes-induced visual dysfunction caused by damage to the inner neural retina that ultimately leads to retinal cell death. Lutein inhibited oxidative stress, thereby preserving the neuroprotective pathways in the early diabetic retina.
Acknowledgements
We thank Y. Oike (Kumamoto University, Japan) for advice on preparing the manuscript, and I. Kawamori and H. Koizumi for technical assistance. This study was supported by a grant from Wakasa Seikatsu Co., and partly by a grant-in-aid from the Ministry of Education, Science and Culture of Japan to Y. Ozawa.