Diabetic Retinopathy: The Role of Mitochondria in the Neural Retina and Microvascular Disease

Abstract

Diabetic retinopathy (DR), a common chronic complication of diabetes mellitus and the leading cause of vision loss in the working-age population, is clinically defined as a microvascular disease that involves damage of the retinal capillaries with secondary visual impairment. While its clinical diagnosis is based on vascular pathology, DR is associated with early abnormalities in the electroretinogram, indicating alterations of the neural retina and impaired visual signaling. The pathogenesis of DR is complex and likely involves the simultaneous dysregulation of multiple metabolic and signaling pathways through the retinal neurovascular unit. There is evidence that microvascular disease in DR is caused in part by altered energetic metabolism in the neural retina and specifically from signals originating in the photoreceptors. In this review, we discuss the main pathogenic mechanisms that link alterations in neural retina bioenergetics with vascular regression in DR. We focus specifically on the recent developments related to alterations in mitochondrial metabolism including energetic substrate selection, mitochondrial function, oxidation-reduction (redox) imbalance, and oxidative stress, and critically discuss the mechanisms of these changes and their consequences on retinal function. We also acknowledge implications for emerging therapeutic approaches and future research directions to find novel mitochondria-targeted therapeutic strategies to correct bioenergetics in diabetes. We conclude that retinal bioenergetics is affected in the early stages of diabetes with consequences beyond changes in ATP content, and that maintaining mitochondrial integrity may alleviate retinal disease.

Full text

antioxidants
Review
Diabetic Retinopathy: The Role of Mitochondria in
the Neural Retina and Microvascular Disease
David J. Miller , M. Ariel Cascio and Mariana G. Rosca *
Department of Foundational Sciences, Central Michigan University College of Medicine,
Mount Pleasant, MI 48858, USA; mille15d@cmich.edu (D.J.M.); ariel.cascio@cmich.edu (M.A.C.)
*Correspondence: rosca1g@cmich.edu; Tel.: +1-989-774-6556
Received: 19 August 2020; Accepted: 18 September 2020; Published: 23 September 2020


Abstract:
Diabetic retinopathy (DR), a common chronic complication of diabetes mellitus and the
leading cause of vision loss in the working-age population, is clinically defined as a microvascular
disease that involves damage of the retinal capillaries with secondary visual impairment. While its
clinical diagnosis is based on vascular pathology, DR is associated with early abnormalities in
the electroretinogram, indicating alterations of the neural retina and impaired visual signaling.
The pathogenesis of DR is complex and likely involves the simultaneous dysregulation of multiple
metabolic and signaling pathways through the retinal neurovascular unit. There is evidence that
microvascular disease in DR is caused in part by altered energetic metabolism in the neural retina
and specifically from signals originating in the photoreceptors. In this review, we discuss the main
pathogenic mechanisms that link alterations in neural retina bioenergetics with vascular regression
in DR. We focus specifically on the recent developments related to alterations in mitochondrial
metabolism including energetic substrate selection, mitochondrial function, oxidation-reduction
(redox) imbalance, and oxidative stress, and critically discuss the mechanisms of these changes and
their consequences on retinal function. We also acknowledge implications for emerging therapeutic
approaches and future research directions to find novel mitochondria-targeted therapeutic strategies
to correct bioenergetics in diabetes. We conclude that retinal bioenergetics is affected in the early stages
of diabetes with consequences beyond changes in ATP content, and that maintaining mitochondrial
integrity may alleviate retinal disease.
Keywords: diabetic retinopathy; mitochondria; oxidative stress; redox; photoreceptor
1. Introduction
Diabetes mellitus is a growing public health problem, reaching pandemic proportions in the
United States and worldwide [
1
]. Diabetic retinopathy (DR) is the leading cause of irreversible visual
impairment and blindness in the working-age population [
2
]. The Diabetes Control and Complications
Trial concluded that tight metabolic control can delay the development and slow the progression of
DR. However, good metabolic control is often difficult to achieve and does not guarantee complete
protection against DR, suggesting that there are additional contributing factors that remain to be
discovered [
3
,
4
]. While targeted therapies are effective in mitigating the sight-threatening complications
of proliferative diabetic retinopathy (PDR) [
5
], new therapeutic approaches are needed to manage the
milder non-proliferative disease. Thus, there is an urgent need to better understand the early stages of
DR in order to develop new strategies to halt its progression.
DR is clinically defined as a microvascular disease [
6
], and can be broadly classified into two distinct
stages on the basis of the presence of neovascularization. While non-proliferative diabetic retinopathy
(NPDR) is characterized by blood flow alterations, pericyte loss, downregulation of endothelial cell
tight junctions [
7
], and thickening of the basement membrane [
8
], PDR presents with sight-threatening
Antioxidants 2020,9, 905; doi:10.3390/antiox9100905 www.mdpi.com/journal/antioxidants

Antioxidants 2020,9, 905 2 of 29
neovascularization that may precipitate retinal detachment and blindness. Recent work has shown that
retinal neurodegeneration precedes clinically detectable microvascular damage [
9
–
13
]. Since Wolter’s
first observation of neuronal cell death in the diabetic retina [
14
], numerous studies have described
early neuronal apoptosis and alterations in visual signaling. Retinal ganglion cells (RGCs) of the optic
nerve undergo apoptosis at a rate higher than any other retinal cell [
15
]. These changes are associated
with a subjective decline in the quality of vision including impaired contrast sensitivity and color
vision [
16
–
18
], and altered visual signaling as assessed by the electroretinogram (ERG). In addition to
changes in the a- and b-waves on the ERG, alterations in the amplitude of the oscillatory potential
(photopic and scotopic oscillatory potentials, which are initiated in the inner retina [
19
]) have been
suggested to predict the progression of DR [20,21].
In light of these findings, new discoveries into retinal physiology have emphasized the role of
the neurovascular unit in DR [
6
], which refers to the physical and biochemical interaction between
neurons (RGCs, amacrine cells, bipolar cells, and horizontal cells), glia (Müller cells and astrocytes),
and the microvascular network (endothelial cells and pericytes) [
22
,
23
]. The key role of this interaction
in neurodevelopment [
24
] and normal neurovascular signaling [
25
] has led to the hypothesis that DR
may result from the uncoupling of the neurovascular unit [
26
,
27
]. Nevertheless, the effect and timing
of cellular dysfunction throughout the neurovascular unit in DR has yet to be determined.
One of the classical and prevailing theories explaining the pathogenesis of DR is that diabetes
enhances oxidative stress, which in turn damages the retinal microvasculature [
28
]. The term oxidative
stress refers to an imbalance between reactive oxygen species (ROS) production and antioxidant
defenses. Because of their role in oxidative metabolism, mitochondria are key sources of increased
ROS in diabetes [
29
–
31
]. Oxidative stress originating in mitochondria of endothelial cells has been
reported to enhance multiple seemingly independent pathways, each contributing to the development
of microvascular complications [
32
,
33
]. Most current knowledge is derived from this “unifying theory”
that was developed on cultured aortic endothelial cells and has since been extrapolated to the
retinal microvasculature. However, recent work by Du et al. [
34
] determined that diabetes-induced
oxidative stress originates from the photoreceptors rather than endothelial cells. In this model,
photoreceptor-induced oxidative stress was associated with increased inflammation, which is widely
regarded as an important pathogenic mechanism of DR, and contributes to vascular regression in the
diabetic retina [
35
]. The critical role of the neural retina in the development of microvascular disease is
further supported by studies of patients with retinitis pigmentosa who exhibit both photoreceptor
degeneration and protection against DR [36,37].
While performing the core metabolic function of energy production, mitochondria are critical gears
in a currently expanding number of cellular functions including redox homeostasis [
38
] and programmed
cell death [
39
]. An increased mitochondrial oxidative stress reveals a change in mitochondrial function.
The importance of understanding the role of bioenergetics in the diabetic neural retina is supported by
the knowledge that inherited mitochondrial diseases cause retinal disease and visual impairment [
40
],
and is further highlighted by the heterogeneity of the neural retinal cells regarding the contribution
of their mitochondria to cellular ATP and oxidative stress [
41
,
42
]. This review will summarize the
recent developments related to alterations in mitochondrial bioenergetics in the neural retina, as well
as the consequences of these alterations on retinal function. We will conclude by acknowledging
emerging therapeutic approaches to correct mitochondrial bioenergetic-related functions and maintain
the mitochondrial integrity in diabetes.
2. Normal Retinal Structure
The retina is a highly organized tissue consisting of at least 10 distinct layers, which can be broadly
divided into an inner and outer retina (Figure 1).

Antioxidants 2020,9, 905 3 of 29
Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 30
Figure 1. The structure of the retina. (A) Electron microscopy images of the mouse outer retina.
Mitochondria are shown in the figure inset and are indicated by white arrows. (B) Confocal image of
Figure 1.
The structure of the retina. (
A
) Electron microscopy images of the mouse outer retina.
Mitochondria are shown in the figure inset and are indicated by white arrows. (
B
) Confocal image
of the mouse retina depicting rhodopsin (green) and cell nuclei (blue). (
C
) Distribution of cell nuclei
(blue) in the mouse retina. The numbers represent the retinal layers: 1—retinal pigment epithelium
(RPE, detached); 2—outer nuclear layer; 3—inner nuclear layer; 4—ganglion cell layer. Rhodopsin
(green fluorescence) is present in stacks of membranous disks of the photoreceptor outer segments
(OS). 40,6-diamidino-2-phenylindole (DAPI, blue) stains the nuclei in all nuclear layers and the RPE.

Antioxidants 2020,9, 905 4 of 29
The inner retina includes the RGCs as well as two nuclear layers with the photoreceptor soma.
Photoreceptors are the light-sensitive cells responsible for phototransduction and present as either rods
and cones expressing the visual pigments rhodopsin and opsin, respectively. The outer retina includes
the photoreceptor outer segments (OSs) and the underlying retinal pigment epithelium (RPE). The RPE
rests on Bruch’s membrane, a multi-layered structure that separates the outer retina from the choroid
choriocapillaris. The inner retina receives blood from three local vascular plexuses, while photoreceptors
are primarily supplied by the choriocapillaris. Therefore, although the photoreceptors are physically
distant from the inner retina where DR manifests as a microvascular disease, both structures contribute
to the pathophysiology of DR but their cooperative signals are yet to be identified.
3. Pathophysiology of DR
The pathogenesis of the early stages of DR remains poorly understood. Pericyte death has been
considered the central mechanism for the loss of retinal vascular integrity in diabetes [
43
]. However,
the seminal work of Mizutani et al. [
44
] revealed early and accelerated death of both retinal pericyte and
endothelial cells in diabetic rodents and humans. While endothelial cells are replaced by proliferation,
migration, or neighbor cell redeployment, pericytes do not regenerate, and their absence is evidenced by
the presence of “pericyte ghosts” in the capillary wall. Dynamic high resolution microscopy determined
that the decrease in blood flow favors the process of vasoregression [
45
,
46
]. As an indisputable event in the
diabetic retina, pericyte loss has been observed in all rodent models of both type 1 (T1D) and type 2 (T2D)
diabetes [
47
–
49
]. Moreover, genetic pericyte elimination recapitulates the early features of experimental
DR, including acellular capillaries, microaneurysms, and blood–retinal barrier abnormalities, all of which
underline the seminal role of pericytes to maintain retinal capillary integrity [
50
]. While pericytes likely
play a similar role in humans [
51
], progress in this area is limited by the scarcity of human retinal tissue
and the inherent difficulties of translational research [52].
Previous studies have focused primarily on the retinal microvasculature. However, a recent
growing body of literature indicates that diabetes causes cellular dysfunction and loss of virtually all
retinal cell populations [
13
,
53
–
58
], as measured qualitatively by ERG and quantitatively by optical
coherence tomography, revealing a decrease in retinal thickness [
10
,
59
]. Diabetes-induced alterations
of neuronal cells and photoreceptors are particularly important as the death of these cells is not
matched by similar rates of regeneration [
60
]. Due to the large surface area of outer segments (OS),
photoreceptors are highly sensitive to incident photons and have a high capacity for ion exchange
that must be supported by ATP. Abnormalities in photoreceptors have been reported in multiple
models of insulin-dependent diabetes in both rodents [
61
,
62
] and rabbits [
63
]. Similar observations
have been reported in zebrafish exposed to hyperglycemia [
64
]. In diabetic patients, photoreceptor
integrity is altered and the OS length shortened, changes that have been associated with decreased
visual acuity [
65
–
67
]. While altered photoreceptor morphology appears modest at 3–6 months of
hyperglycemia [
62
,
68
], the functional abnormalities are more severe and include impaired function of
the Na
+
/K
+
ATPase pump [
69
,
70
]. In photoreceptors, the Na
+
/K
+
ATPase pump is critical not only for
normal ion homeostasis, but also for the “dark current”, a physiologic event that can be assessed by
the a-wave on the ERG. Subsequent studies have expanded upon this work and observed changes in
the amplitude and latency of the a-wave as early events in streptozotocin (STZ)-induced diabetes [
71
].
Similar abnormalities in the ERG have also been noted in diabetic patients, and suggested to precede
and predict the microvascular histopathology [72]. These findings are consistent with the hypothesis
that early visual dysfunction precedes morphologic neurodegeneration and vascular regression in DR
(Figure 2).

Antioxidants 2020,9, 905 5 of 29
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 30
Figure 2. Proposed pathophysiology of diabetic retinopathy (DR). The early stages of diabetes
mellitus are characterized by alterations in bioenergetics and substrate selection in a variety of cell
types. In the retina, these changes cause oxidative stress and are associated with early visual deficits
such as impaired contrast sensitivity. Mitochondrial oxidative stress alters mitochondrial metabolism
and upregulates multiple seemingly independent pathways leading to retinal disease. Mitochondrial
dysfunction also changes the redox state that further enhances oxidative stress. Therefore,
mitochondrial-generated oxidative stress may precede overt neurodegeneration and microvascular
disease. Abbreviations: AGEs, advanced glycation end products; DR, diabetic retinopathy; mt,
mitochondrial. : increased; : decreased.
4. Retinal Bioenergetics and Mitochondrial Substrate Selection
4.1. ATP-Consuming Processes in the Retina
While all retinal cells rely on ATP as a fuel source, the photoreceptors are the largest consumers.
Photoreceptors use more than 75% of oxygen of the retina and contain more than 75% of retinal
mitochondria to produce large amounts of ATP by oxidative phosphorylation (Oxphos), which is
necessary for phototransduction [73]. Phototransduction, the process by which photons are converted
into electrical signals in photoreceptors, relies on the cycling of 11-cis retinal, a vitamin A derivative
bound to an opsin G-protein-coupled receptor (GPCR). In the presence of light, 11-cis retinal is
isomerized to all-trans retinal. This photoisomerization results in a conformational change of the
opsin GPCR, leading to a signaling cascade that causes the closure of sodium ion channels,
hyperpolarization of the cell, and decreased glutamate release with depolarization of bipolar cells
initiating phototransduction. In the dark, 11-cis retinal holds the opsin GPCR in an inactive
conformation allowing the entry of sodium ions with glutamate release, thus inhibiting bipolar cells.
This latter process is referred to as the “dark current”, a high ATP consuming process needed to
maintain a steady influx of sodium ions and keep a constant membrane potential.
In order to provide a constant supply of 11-cis retinal, all-trans retinal must be converted back to
11-cis retinal through a series of redox reactions collectively referred to as the visual cycle [74]. The
visual cycle involves proteolysis of the visual pigment (opsin or rhodopsin) and release of all-trans
retinal into the RPE, where it is converted to 11-cis retinal. The rate of 11-cis retinal regeneration is
determined by the availability of ATP and nicotinamide adenine dinucleotide phosphate (NADPH)
[75], further supporting the proposition that the visual cycle is highly dependent on bioenergetic
support. Photoreceptors undergo daily shedding, losing approximately 10% of their OS to
phagocytosis by the RPE [76]. Continuous shedding of “used” OS discs and replacement with newly
assembled discs, a critical process to maintain normal photoreceptor function, also consumes a large
amount of ATP and NADPH. Photoreceptors are supported by adjacent Müller cells [77]; studies
have shown that disruption of Müller cell metabolism results in impaired assembly of nascent
Figure 2.
Proposed pathophysiology of diabetic retinopathy (DR). The early stages of diabetes mellitus
are characterized by alterations in bioenergetics and substrate selection in a variety of cell types. In the
retina, these changes cause oxidative stress and are associated with early visual deficits such as impaired
contrast sensitivity. Mitochondrial oxidative stress alters mitochondrial metabolism and upregulates
multiple seemingly independent pathways leading to retinal disease. Mitochondrial dysfunction also
changes the redox state that further enhances oxidative stress. Therefore, mitochondrial-generated
oxidative stress may precede overt neurodegeneration and microvascular disease. Abbreviations:
AGEs, advanced glycation end products; DR, diabetic retinopathy; mt, mitochondrial.
↑
: increased;
↓: decreased.
4. Retinal Bioenergetics and Mitochondrial Substrate Selection
4.1. ATP-Consuming Processes in the Retina
While all retinal cells rely on ATP as a fuel source, the photoreceptors are the largest consumers.
Photoreceptors use more than 75% of oxygen of the retina and contain more than 75% of retinal
mitochondria to produce large amounts of ATP by oxidative phosphorylation (Oxphos), which is
necessary for phototransduction [
73
]. Phototransduction, the process by which photons are converted
into electrical signals in photoreceptors, relies on the cycling of 11-cis retinal, a vitamin A derivative
bound to an opsin G-protein-coupled receptor (GPCR). In the presence of light, 11-cis retinal is
isomerized to all-trans retinal. This photoisomerization results in a conformational change of
the opsin GPCR, leading to a signaling cascade that causes the closure of sodium ion channels,
hyperpolarization of the cell, and decreased glutamate release with depolarization of bipolar cells
initiating phototransduction. In the dark, 11-cis retinal holds the opsin GPCR in an inactive conformation
allowing the entry of sodium ions with glutamate release, thus inhibiting bipolar cells. This latter
process is referred to as the “dark current”, a high ATP consuming process needed to maintain a steady
influx of sodium ions and keep a constant membrane potential.
In order to provide a constant supply of 11-cis retinal, all-trans retinal must be converted back
to 11-cis retinal through a series of redox reactions collectively referred to as the visual cycle [
74
].
The visual cycle involves proteolysis of the visual pigment (opsin or rhodopsin) and release of all-trans
retinal into the RPE, where it is converted to 11-cis retinal. The rate of 11-cis retinal regeneration is
determined by the availability of ATP and nicotinamide adenine dinucleotide phosphate (NADPH) [
75
],
further supporting the proposition that the visual cycle is highly dependent on bioenergetic support.
Photoreceptors undergo daily shedding, losing approximately 10% of their OS to phagocytosis by the
RPE [
76
]. Continuous shedding of “used” OS discs and replacement with newly assembled discs,
a critical process to maintain normal photoreceptor function, also consumes a large amount of ATP
and NADPH. Photoreceptors are supported by adjacent Müller cells [
77
]; studies have shown that
disruption of Müller cell metabolism results in impaired assembly of nascent photoreceptor OS [
78
].

Antioxidants 2020,9, 905 6 of 29
Thus, the retina is a highly active tissue and requires a remarkable amount of oxygen and ATP to
sustain its normal functions.
4.2. ATP-Generating Processes in the Retina and the Heterogeneity of Retinal Bioenergetics
The major sources of ATP in the retina are extramitochondrial glycolysis and mitochondrial Oxphos
(Figure 3). In the 1920s, Warburg and Krebs reported that the mammalian retina, as a whole, has a
metabolism largely based on aerobic glycolysis, converting 80–96% of glucose to lactic acid [
79
]. However,
more recent research has demonstrated that the distribution of glycolysis and oxidative metabolism
varies throughout the retina [
80
]. While neurotransmission in the inner retina is supported almost
entirely by glycolysis, phototransduction in the outer retina is supported by mitochondrial Oxphos [
80
].
Mitochondrial Oxphos occurs in the inner mitochondrial membrane in which invaginations called cristae
greatly increase the surface area for electron transport and ATP production. The electron transport chain
(ETC) consists of four complexes (I-IV) that oxidize nicotinamide adenine dinucleotide (NADH) and flavin
adenine dinucleotide (FADH
2
) to NAD
+
and FAD
+
, respectively. Through a series of redox reactions,
the ETC transfers electrons towards molecular oxygen and H
+
into the intermembrane space. This process
creates a transmembrane electrochemical gradient, which is used by ATP synthase (complex V) for the
phosphorylation of adenosine diphosphate (ADP) to ATP. In addition to the ETC complexes, mitochondrial
Oxphos also relies on ubiquinone (coenzyme Q) and cytochrome c (cyt c), two mobile electron carriers that
shuttle electrons between ETC complexes [81].
A comprehensive investigation into oxidative metabolism revealed that retinal mitochondrial
Oxphos operates in basal conditions at maximal capacity without a significant reserve capacity [
82
],
suggesting that mitochondrial defects have a significant impact on retinal energy homeostasis. In most
tissues, Oxphos is a tightly coupled process in which substrate oxidation is paired by ATP synthesis [
81
].
In the retina, mitochondria are reportedly less coupled, allowing proton leakage through the inner
membrane without ATP synthesis [
82
]. Weak coupling between electron transport and ATP synthesis
suggests that mitochondrial oxidative metabolism in the retina supports other functions in addition to
ATP production, such as maintaining the NADH/NAD
+
and FADH
2
/FAD
+
redox ratios. This concept
is one of the core focus of our review, and will be further detailed in the following sections.
Despite the high demand for ATP and NADPH described previously, photoreceptor OSs have
limited glycolytic capacity and are devoid of mitochondria (Figure 1), relying on the inner segments
(IS) for their energetic needs. Accordingly, photoreceptor ISs have the highest capacity for glycolysis,
tricarboxylic acid (TCA) cycle, mitochondrial Oxphos, and creatine phosphate-mediated shuttling
of ATP into the cytosol [
80
]. Photoreceptor ISs contain high amounts of hexokinase 2 (HK2) on
the mitochondrial outer membrane, which catalyzes the rate-limiting step of glycolysis, namely,
the conversion of glucose into glucose-6-phosphate. From here, a portion of glucose proceeds
through glycolysis and the TCA cycle, which provides the GTP needed for phototransduction.
Glucose-6-phosphate is also utilized in the pentose phosphate pathway, which is the primary source
of NADPH used in anabolic reactions and the regeneration of cytosolic reduced glutathione (GSH),
a major antioxidant defense mechanism. Between the two photoreceptor populations, cones contain
10-fold more mitochondria and thus have a much greater ATP-generating capacity than rods. Cone ISs
also contain greater amounts of creatine phosphate, suggesting that cones provide the cytosolic ATP
more efficiently than rods in the setting of high energetic demand [80].
The outer retina exhibits light-induced changes in oxygen consumption and ATP production [
82
].
As mentioned previously, phototransduction requires a steady influx of ATP and NAPDH both
for the regeneration of 11-cis retinal and photoreceptor OS. Light has been shown to stimulate the
accumulation of ribose-5-phosphate, an intermediate in the pentose phosphate pathway, which likely
reflects increased NADPH production and anabolic metabolism. Oxygen consumption also correlates
with the rod dark current, which imposes a high energetic demand in mammals [
83
], accounting for
41% of total retinal oxygen consumption [
84
]. When oxygen supply is inadequate, the dark current

Antioxidants 2020,9, 905 7 of 29
may be partially supported by glycolysis, indicating that more ATP is needed and extracted in the dark
from energetic fuel substrates through their oxidation.
In contrast to the outer retina, the inner retina does not exhibit significant light-induced changes
in oxidative metabolism. Nevertheless, neurons and glia of the inner retina also require a steady state
[ATP] for neurotransmission. Müller cells, the most abundant glial cell in the retina, are critical to the
maintenance of the neurovascular unit and perform important functions such as synaptic transmission
regulation, handling of nutrients and waste products, maintenance of the “tightness” of the blood-retinal
barrier, and survival of neurons and endothelial cells. Müller cells rely primarily on glycolysis and are
rich in glycogen reserves [
85
,
86
]. Although glucose is their preferred substrate, Müller cells also utilize
extracellular glutamate [
87
], and for this reason are believed to play a role in preventing glutamate
excitotoxicity. Cell culture experiments have confirmed that Müller cells exhibit aerobic glycolysis and
provide lactate that can be transferred to retinal neurons for metabolic support [
88
]. Their Oxphos
capacity, however, is limited. It is suggested that this metabolic heterogeneity of the retina likely plays
an important role in the cell-specific vulnerability to diabetes.
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 30
may be partially supported by glycolysis, indicating that more ATP is needed and extracted in the
dark from energetic fuel substrates through their oxidation.
In contrast to the outer retina, the inner retina does not exhibit significant light-induced changes
in oxidative metabolism. Nevertheless, neurons and glia of the inner retina also require a steady state
[ATP] for neurotransmission. Müller cells, the most abundant glial cell in the retina, are critical to the
maintenance of the neurovascular unit and perform important functions such as synaptic
transmission regulation, handling of nutrients and waste products, maintenance of the “tightness” of
the blood-retinal barrier, and survival of neurons and endothelial cells. Müller cells rely primarily on
glycolysis and are rich in glycogen reserves [85,86]. Although glucose is their preferred substrate,
Müller cells also utilize extracellular glutamate [87], and for this reason are believed to play a role in
preventing glutamate excitotoxicity. Cell culture experiments have confirmed that Müller cells
exhibit aerobic glycolysis and provide lactate that can be transferred to retinal neurons for metabolic
support [88]. Their Oxphos capacity, however, is limited. It is suggested that this metabolic
heterogeneity of the retina likely plays an important role in the cell-specific vulnerability to diabetes.
Figure 3. Glycolysis and oxidative phosphorylation in the retina. The retina relies on glycolysis and
mitochondrial oxidative phosphorylation (Oxphos) as sources of ATP. Glucose uptake into retinal
cells occurs via insulin-dependent glucose transporter 4 (GLUT4) and insulin-independent glucose
transporter 1 (GLUT1). Fatty acid (FA) uptake is not hormonally regulated, but rather potentially
driven by circulating availability (FAcirc) [89,90]. In the retina, FA uptake is regulated by a lipid sensor,
the free fatty acid lipid receptor 1 (Ffar1), and mediated by the very low density lipoprotein receptor
(VLDLR). In retinal cells, glucose follows multiple metabolic pathways, including glycolysis and the
polyol pathway, the latter of which leads to the production of sorbitol and ultimately fructose.
Pyruvate is either converted to lactate or transported into mitochondria where it is converted by
pyruvate dehydrogenase (PDH) to acetyl coenzyme A (AcCoA), which enters the tricarboxylic acid
(TCA) cycle. PDH is inhibited by pyruvate dehydrogenase kinases that are activated by excessive
acetyl-CoA and nicotinamide adenine dinucleotide (NADH). For simplicity, other glucose metabolic
pathways are not shown. FAs, which are released from triglycerides and imported into retinal cells
via the VLDLR, are converted to fatty acyl-CoA, shuttled into the mitochondria via carnitine
palmitoyltransferases 1 and 2 (CPT1 and 2), and oxidized via FA β-oxidation. FA β-oxidation yields
NADH, flavin adenine dinucleotide (FAHD2), and AcCoA, which are further oxidized by the electron
transport chain (ETC) complexes in the process of Oxphos with ATP synthesis. FA β-oxidation is
Figure 3.
Glycolysis and oxidative phosphorylation in the retina. The retina relies on glycolysis and
mitochondrial oxidative phosphorylation (Oxphos) as sources of ATP. Glucose uptake into retinal
cells occurs via insulin-dependent glucose transporter 4 (GLUT4) and insulin-independent glucose
transporter 1 (GLUT1). Fatty acid (FA) uptake is not hormonally regulated, but rather potentially driven
by circulating availability (FA
circ
) [
89
,
90
]. In the retina, FA uptake is regulated by a lipid sensor, the free
fatty acid lipid receptor 1 (Ffar1), and mediated by the very low density lipoprotein receptor (VLDLR).
In retinal cells, glucose follows multiple metabolic pathways, including glycolysis and the polyol
pathway, the latter of which leads to the production of sorbitol and ultimately fructose.
Pyruvate is
either
converted to lactate or transported into mitochondria where it is converted by pyruvate dehydrogenase
(PDH) to acetyl coenzyme A (AcCoA), which enters the tricarboxylic acid (TCA) cycle. PDH is inhibited
by pyruvate dehydrogenase kinases that are activated by excessive acetyl-CoA and nicotinamide adenine
dinucleotide (NADH). For simplicity, other glucose metabolic pathways are not shown. FAs, which are
released from triglycerides and imported into retinal cells via the VLDLR, are converted to fatty acyl-CoA,
shuttled into the mitochondria via carnitine palmitoyltransferases 1 and 2 (CPT1 and 2), and oxidized
via FA
β
-oxidation. FA
β
-oxidation yields NADH, flavin adenine dinucleotide (FAHD
2
), and AcCoA,
which are further oxidized by the electron transport chain (ETC) complexes in the process of Oxphos

Antioxidants 2020,9, 905 8 of 29
with ATP synthesis. FA
β
-oxidation is inhibited by malonyl-CoA (an intermediate of FA synthesis),
FADH
2
/FAD
+
ratios, and NADH/NAD
+
ratios. Malonyl-CoA is degraded by malonyl-CoA decarboxylase
(MCD), thus decreasing its inhibitory effect on CPT1. Although described in other organs, these regulatory
steps are yet to be identified in the retina. Mitochondrial Oxphos provides the bulk of retinal ATP.
As electrons transfer NADH and FADH
2
to molecular oxygen by ETC complexes, an electrochemical
gradient is built across the mitochondrial inner membrane (IM). This gradient is used by the complex V to
produce ATP. Mitochondria-generated ATP is transferred to the cytosol by the creatine kinase (CK) shuttle
to sustain the normal functions of the retinal cells. The inset included here is an electron micrograph of a
mouse rod photoreceptor and shows the inner segment mitochondria. For simplicity, the nicotinamide
nucleotide transhydrogenase, a mitochondrial inner membrane enzymethat reduces nicotinamide adenine
dinucleotide phosphate (NADPH
+
) by oxidizing NADH and using the mitochondrial proton-motive
force, is not shown in this figure. The oxidized NAD+and NADP+are shown in red.
4.3. Substrate Selection and Energy Production
Tissues with a high metabolic rate, such as the heart, often utilize multiple energy sources (glucose,
amino acids, fatty acids), which confers some degree of metabolic flexibility during periods of scarcity
and surplus [
91
]. The retina also uses multiple fuel sources to generate the electron carriers NADH and
FADH
2
, which donate their electrons directly to complex I and coenzyme Q-complex III, respectively,
and ultimately establish the electrochemical gradient that drives ATP synthesis [81].
In addition to glucose, palmitate (C16:0), one of the most abundant fatty acids (FAs) in the human
body, can also be used as a fuel substrate for retinal mitochondrial energy production [
92
]. In the retina,
cellular FA uptake is mediated by the very low density lipoprotein receptor (VLDLR) that is expressed on
both photoreceptor and RPE cells. Moreover, the expression of proteins involved in FA
β
-oxidation has
been reported throughout the retina, including RGCs, photoreceptors, and Müller cells, albeit in varying
amounts [
93
,
94
], indicating that retinal cells possess the machinery to oxidize lipids as fuel sources.
FA oxidation is essential for retinal metabolism and function. In support of this concept, genetic mutations
in specific enzymes involved in
β
-oxidation cause mitochondrial dysfunction, pigmentary retinopathy,
and ultimately vision loss [
95
]. In mice, knockout of the peroxisome proliferative-activated receptor-
α
(PPAR
α
), a nuclear receptor that modulates lipoprotein lipase expression and triglyceride metabolism,
causes decreased lipid metabolism and retinal neurodegeneration [
96
]. Despite recognizing FA as a fuel
source, the involvement of potential changes in FA
β
-oxidation remain largely unexplored in the retina in
diabetes, a disease associated with increased FA availability. Nevertheless, two large-scale clinical trials
known as the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) and Action to Control
Cardiovascular Risk in Diabetes (ACCORD) studies have shown that the PPAR
α
-agonist fenofibrate
slows the progression of DR [
97
,
98
]. These findings raise the possibility that diabetes-induced alterations
in mitochondrial FA β-oxidation may contribute to retinal dysfunction.
Further research has investigated the role of FA
β
-oxidation in the retinal microvasculature.
Surprisingly, impaired FA oxidation in retinal endothelial cells neither results in energy depletion
nor does it disturb redox homeostasis [
99
], suggesting that FA oxidation in these cells likely supports
cellular functions in addition to ATP production. Using isotope labeling experiments, Schoors et al. [
99
]
demonstrated that FA-derived carbon units are incorporated into aspartate (a nucleotide precursor)
and eventually DNA. The same group also showed that blockade of carnitine palmitoyl transferase 1
(CPT1), the rate-limiting enzyme in FA
β
-oxidation, inhibits pathological neovascularization in mice.
These data suggest a novel role of FA oxidation in endothelial cell proliferation and maintenance of the
neurovascular unit. These findings raise the important question of whether, in diabetes, the retina
exhibits a “metabolic switch” towards increased FA oxidation, similar to other high-energy consuming
organs (e.g., heart, kidney). While such data are scarce, at least one study has indicated that the retina
can increase the expression of FA oxidation enzymes in diabetic rats [
100
]. Subsequent studies will
be needed, however, to characterize and quantify the relative contribution of individual energetic
substrates to energy production in the diabetic retina.

Antioxidants 2020,9, 905 9 of 29
Diabetes is characterized by decreased insulin action (a decrease in either secretion or sensitivity)
and increased availability of energetic substrates (i.e., glucose and FAs). In the heart, also a highly
energy consuming organ, increased availability of FAs is associated with a rapid decrease in glucose
oxidation [
101
], thus reducing metabolic flexibility and increasing reliance on FAs for ATP production.
In hepatocytes and adipocytes, increased availability of glucose leads to increased flux through the TCA
cycle and increased production malonyl coenzyme A (malonyl-CoA), which inhibits CPT1 and spares
lipids from
β
-oxidation. Similar to the heart, these changes indicate a decrease in metabolic flexibility.
In the retina, glucose uptake occurs via both insulin-independent glucose transporter 1 (GLUT1) and
insulin-dependent glucose transporter 4 (GLUT4) [
102
,
103
]. The retina also expresses a lipid sensor
known as free fatty acid receptor 1 (Ffar1) (Figure 3) [
92
]. Ffar1 regulates insulin secretion in the islets
of Langerhans [
104
] and neuronal function in the brain [
105
]. Interestingly, Ffar1 has been shown
to downregulate GLUT1 expression in VLDLR-deficient photoreceptors, resulting in a dual glucose
and lipid substrate uptake [
92
], which predictably leads to low levels of TCA cycle intermediates.
Several TCA cycle intermediates including
α
-ketoglutarate and succinate have been shown to
modulate the stabilization of hypoxia-inducible factor 1
α
[
106
]. In VLDLR-deficient photoreceptors,
low
α
-ketoglutarate stabilizes hypoxia-inducible factor 1
α
and promotes neovascularization [
92
].
While similar studies have yet to be performed in the context of DR, these findings suggest multiple
mechanisms by which the selection of energetic substrates, all plentiful in diabetes, may change retinal
disease progression.
5. Alterations in Mitochondrial Oxidative Metabolism in the Neural Retina
5.1. Localization of Mitochondria in the Retina
An increasingly popular view is that diabetic milieu leads to the uncoupling of the retinal
neurovascular unit [
6
], a concept that implies impaired crosstalk between neurons, glia, and the
microvascular network [
23
]. While Müller cells rely primarily on glycolysis for ATP, those located in
highly vascularized portions of the retina are rich in mitochondria [
107
], raising the possibility that
mitochondrial dysfunction in Müller cells may have neurovascular consequences. Mitochondria also
concentrate in the photoreceptor IS and the most external ends of the RPE (Figure 1), which likely reflects
their migration toward the oxygen-rich choriocapillaris during neurodevelopment [
108
]. The specific
distribution of mitochondria in the retina may explain, at least in part, the regional susceptibility to
neurodegeneration and microvascular lesions in DR.
5.2. Diabetes Alters Mitochondrial Function in the Retina
The function of healthy and diseased mitochondria can be evaluated by measuring oxygen
consumption rates in isolated mitochondria, retinal explants, or cultured retinal cells. However,
due to limitations imposed by the small sample volume, many studies have resorted to studying
mitochondrial function in retinal homogenate rather than individual cells or specific retinal layers.
Retinal mitochondria exhibit a biphasic response to diabetes, characterized by an early and transient
activation followed by a later decline. Masser et al. [
100
] showed that basal and ATP-linked oxygen
consumption rates were significantly elevated in the retina of 3-month-old diabetic rats. In the same
study, proteomic analysis revealed elevated levels of several FA
β
-oxidation enzymes and antioxidant
proteins, suggesting a positive adaptive response of the retina to the diabetic milieu. This response
mirrors the diabetic heart in that it indicates an energetic shift toward reliance on FA
β
-oxidation and
metabolic inflexibility [
101
]. Another study reported increased mitochondrial oxygen consumption in
diabetic rats at 3 weeks of hyperglycemia [
109
], which was associated with increased specific activities
of complexes I, II, and III. Despite an increase in oxygen consumption and ETC complex activity,
ATP generation was unchanged due to mitochondrial uncoupling, suggesting mitochondrial activation
and inefficiency rather than mitochondrial defects. Similarly, in a model of spontaneous T2D in the
cone-rich diurnal Nile rat, a short-term (2 month) hyperglycemia increased complex I-dependent

Antioxidants 2020,9, 905 10 of 29
mitochondrial respiration and was associated with increased cytochrome c access to cytochrome c
oxidase, suggesting a change in composition or organization of the mitochondrial inner membrane [
110
].
Increased mitochondrial membrane permeability was confirmed in isolated mitochondria from retinas
of Zucker diabetic fatty rats with 6 weeks of persistent hyperglycemia and was associated with a
concomitant decrease in mitochondrial complex III-specific activity [111].
Osorio-Paz et al. [
109
] reported that longer (approximately 6 weeks) durations of STZ-induced
diabetes in rats caused a decreased cytochrome c-reducing activity of complex III, while complexes
II and IV were hyperactive when measured in isolated retinal mitochondria. These changes in ETC
complex-specific activities reflect a decrease in oxygen consumption of retinal mitochondria energized
with a combination of energetic substrates (glutamate and malate) that are generating NADH to be
oxidized by complex I. Intriguingly, the generated ATP was unchanged. Mitochondrial respiration
in the presence of substrates feeding electrons into complex I (NADH pathway) and II (succinate
pathway) involves complexes III and IV, as well as mobile electron carriers such as co-enzyme Q and
cytochrome c. Therefore, it is expected that a change in individual components will affect the whole
Oxphos pathway. However, the effect of individual components on the whole integrative function
depends upon the control of that component on the Oxphos. In comparison with heart, where the
impact of individual components on the Oxphos and ATP synthesis has been determined, this control
has yet to be investigated in the normal and diabetic retina. While it is unclear if complex III defect
is limiting for the Oxphos in the diabetic retina, a decreased complex III activity led to increased
superoxide in a mouse model of STZ-induced diabetes; both were normalized by overexpressing the
mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD) [112].
In contrast with short-term diabetes, a long-term (18 months) sustained hyperglycemia in T2D Nil
diurnal rats caused a decrease in NADH-supported mitochondrial respiration accompanied by an
increase in succinate contribution to the maximal Oxphos capacity in the whole retinas, thus confirming
a partial decline in mitochondrial bioenergetics [
110
]. As NADH-induced mitochondrial respiration is
supported by complex I, co-enzyme Q, complex III, cytochrome c, and complexes IV and V, these results
suggest a potential defect in any of these Oxphos subunits.
Mitochondrial DNA (mtDNA) follows a similar response in the diabetic retina. Alterations in the
morphology and function in the neural retina occurring within the first 3 months of diabetes in rats
are not associated with changes in mtDNA in isolated retinal synaptosomes. These findings suggest
that alterations of mtDNA in synaptosomes are not causative for the early neural retina dysfunction
in diabetes [
100
]. In addition, in a model of T1D in rats, while an elevated oxidative stress was
detected as early as 15 days of diabetes, mtDNA damage was observed much later at 6 months due
to inactivation of the DNA repair/replication enzymes [
113
]. A similar temporal relationship was
observed in endothelial cells exposed to high glucose [
114
]. These data suggest that the oxidative
damage of mtDNA is fully compensated in early stages of diabetes while the altered mtDNA in later
stages leads to a decline in mitochondrial transcription and secondary ETC defects. A summary of the
mitochondrial alterations in DR is presented in Tables 1and 2.

Antioxidants 2020,9, 905 11 of 29
Table 1. Summary of investigations into altered mitochondrial homeostasis in the diabetic retina by cell type *.
Cell Type Oxidative Stress Mitochondrial Morphology Mitochondrial Turnover References
RGCs 4 4 Unknown [115–117]
Bipolar cells 4 4 Unknown [117]
Müller cells 4 4 4 [118–124]
Photoreceptors 4 4 4 [121]
Endothelial cells 4 4 4 [113,114,125–131]
* Not an exhaustive list. Abbreviations: RGC, retinal ganglion cell. 4: this feature was confirmed in the respective cells, as supported by references.
Table 2. Summary of electron transport chain protein expression and activity in the diabetic retina by cell type *.
Cell Type Complex I Complex II †Complex III Complex IV Complex V
(ATP Synthase) References
RGCs ↓ ↓ Unknown ↓Unknown [132,133]
Bipolar cells Unknown Unknown Unknown Unknown Unknown
Müller cells Unknown ↔Unknown ↔Unknown [119]
Photoreceptors Unknown Unknown Unknown Varies Unknown [121]
Retinal homogenate Biphasic ‡Biphasic Biphasic Biphasic Varies [100,109–112,121,134]
Pericytes Unknown ↓Unknown Unknown Unknown [135]
Endothelial cells ↓ ↓ ↓ ↓ ↓ [129,136–139]
* Not an exhaustive list.
†
The activity of complex II was inferred in this study from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which measures the
ability of succinate dehydrogenase in complex II to reduce tetrazolium salts [
140
].
‡
Biphasic changes in electron transport complex activity are characterized by early activation followed
by a later decline. The exact timing varies by model. Abbreviations: RGC, retinal ganglion cell. ↓: decreased; ↔: unchanged.

Antioxidants 2020,9, 905 12 of 29
6. Mitochondria-Derived Oxidative Stress in the Diabetic Retina
Oxidative stress is a critical component of altered homeostasis across multiple cell types involved
in DR [
141
]. ROS are defined by the presence of a highly reactive oxygen molecule, and are
generated as normal byproducts of cellular redox reactions. Mitochondria generate superoxide during
oxidation-reduction reactions as some electrons may leak to univalently reduce molecular oxygen.
A number of mitochondrial antioxidant defense mechanisms are in place to prevent the increase in
oxidative stress, including MnSOD, catalase, reduced glutathione, and thioredoxin. Mitochondria are
both the producers and targets of oxidative stress, with the latter altering mtDNA and mitochondrial
proteins leading to mitochondrial dysfunction. Defects in the ETC further amplify the risk of increased
oxidative stress. In the retina, this self-perpetuating cycle has been referred to as the “metabolic memory”
phenomenon, a hypothesis that is supported by the persistence of altered mtDNA, decreased activity
of ETC complexes, and increased oxidative stress throughout the retina despite the reinstitution of
good glycemic control [142].
The proposition that increased oxidative stress is a key pathogenic factor in the development of DR is
supported by finding of insufficient antioxidant defenses in diabetic patients [
143
]. Antioxidant approaches
alleviate diabetes-induced vascular lesions in the retina [
112
,
144
,
145
]. Moreover, the causal link between
increased mitochondrial-generated oxidative stress and retinal microvascular disease in diabetes is
supported by the effect of overexpressing the mitochondrial antioxidant enzyme, MnSOD, to decrease the
number of acellular capillaries in diabetic mice [
112
]. Therefore, mitochondria are directly implicated in
the development of diabetic microvascular lesions.
Mitochondrial dysfunction in retinal endothelial cells has been identified as the upstream
contributor to diabetic vascular disease in diabetes [
146
,
147
]. Recent evidence indicates that the
neurovascular unit is functionally affected before the onset of retinal microvascular disease, and that
the neural retina is affected by oxidative stress originating from the photoreceptors [
148
]. The role of
photoreceptors as oxidative stress generators is supported by the observation that human patients with
photoreceptor degeneration and retinitis pigmentosa have less severe DR than diabetics with intact
photoreceptors as well as diabetic mice lacking photoreceptors due to opsin deficiency [
34
]. The work
of Du et al. [
34
] unequivocally identified photoreceptors as the major source of superoxide generated
by retinas of diabetic mice, and showed that mitochondria contributes to at least 50% of oxidative
stress, thus complementing the NADPH oxidase. Their deletion inhibited the expected increase in
superoxide and inflammatory proteins in the remaining retina in diabetic mice. Of note, Müller cells
cultured in high glucose also exhibit increased oxidative stress [
149
], but their contribution to DR
in vivo is unknown.
There are two potential mechanisms explaining the increased superoxide production by the
diabetic photoreceptors. The first hypothesis is that defects of the mitochondrial ETC interrupt the
normal electron flow to fully reduce oxygen, thus leading to accumulation of electrons at sites within
the ETC, which are accepted by oxygen to generate superoxide [
150
]. The ETC sites prone to leak
electrons to oxygen are complexes I and III [
151
,
152
]. In support of this hypothesis, we recently
reported that correcting the electron flow within the complex I-deficient ETC decreased oxidative stress
and photoreceptor damage [
153
]. The second hypothesis is that an early increase in mitochondrial
oxidative phosphorylation fed by an increased FA
β
-oxidation brings additional sites of electron leak to
oxygen, as was shown for the heart [
154
,
155
] and kidney tubules in diabetes [
156
]. These possibilities
are not mutually exclusive, but they are yet to be investigated in the retina.
Superoxide is dismutated to hydrogen peroxide, a highly permeable compound that may support
the crosstalk between retinal cells and affect neighbor cells, thus providing a link between neural retina
and microvasculature. However, this crosstalk has not yet been investigated in the retina. Oxidative stress
increases the expression of pro-inflammatory proteins [
157
] and enhance retinal inflammation [
158
]
that contributes to early DR [
159
]. In addition, in the microvasculature, increased oxidative stress
is also associated with increased apoptosis [
114
,
160
,
161
], further compromising the integrity of the
neurovascular unit.

Antioxidants 2020,9, 905 13 of 29
7. NAD Pool and the NADH/NAD+Redox Ratio in the Diabetic Retina
7.1. NAD Pool
Nicotinamide adenine dinucleotide (NAD) is a coenzyme for redox enzymes, shuttling electrons
from glycolysis and the TCA cycle to complex I in the ETC. The oxidized form, NAD
+
, is also a
co-substrate for non-redox reactions such as those catalyzed by sirtuin (SIRT) and poly (ADP-ribose)
polymerase (PARP) families of proteins [
162
,
163
]. This link represents a highly conserved mechanism by
which redox status influences a wide range of cellular and metabolic functions, including cell signaling,
DNA transcription, and programmed cell death. Recent work by Lin et al. [
164
] suggest that NAD
is essential for vision. In this study, specific deficiency of NAD in rod photoreceptor for 6 weeks led
to massive atrophy of the entire neurosensory retina, affecting the microvasculature, RPE, and optic
nerve. The complete absence of the outer nuclear layer (photoreceptor nuclei) indicates that cone
photoreceptors are also secondarily affected by rod NAD deficiency. These results strongly suggest that
retinal photoreceptors are essential for the integrity of the whole retina. Although similar studies have
not yet been conducted in the context of DR, these findings suggest that alterations of the photoreceptors
precede the vascular regression in diabetes. During a short (3-week) period of NAD deficiency,
mitochondrial morphology was maintained as normal, whereas at 4 weeks, mitochondrial cristae
were lost, and photoreceptor OS were disrupted. Metabolomic analysis showed that NAD deficiency
causes dysregulation of multiple metabolic pathways including the TCA cycle, mitochondrial protein
biosynthesis, and propionate metabolism with accumulation of acylcarnitines, and also decreased ATP
production. Both glycolysis and mitochondrial Oxphos were affected. These results highlight the critical
role of metabolism and bioenergetics to maintain the photoreceptor integrity. The same research group
identified the decreased retinal NAD pool as an early feature in retinal disease caused by STZ-induced
diabetes at 3 weeks of sustained hyperglycemia [
164
]. NAD deficiency caused photoreceptor death and
diminished rod ERG recordings. These data support the concept that mitochondrial dysfunction in
photoreceptors in the neural retina proceed the vascular regression in the diabetic retina.
7.2. NADH/NAD+Redox Ratio
The “hyperglycemic pseudohypoxia” hypothesis [
165
,
166
] suggests that diabetes is associated
with an increased cellular NADH/NAD
+
redox ratio attributed to an increased flux through the polyol
pathway and resulting in altered metabolism and neurovascular dysfunction. The polyol pathway is a
two-step reaction involving the reduction of glucose to sorbitol and the subsequent oxidation of sorbitol
to fructose [
167
]. The rate-limiting step in this pathway is catalyzed by aldose reductase, which is
expressed in all cells and utilizes NADPH as an electron donor. Importantly, aldose reductase is activated
by hyperglycemia. The second step in the polyol pathway is catalyzed by sorbitol dehydrogenase,
which uses NAD
+
as an oxidizing agent to produce fructose and thus increases the NADH/NAD
+
redox
ratio. Studies have shown that both sorbitol and fructose accumulate in diabetic tissues, including the
retina [
168
,
169
], suggesting that the polyol pathway may contribute to oxidative stress in DR. This finding
is supported by the observation that genetic knockout of aldose reductase protects retinal endothelial
cells from oxidative stress [170].
The redox state is compartmentalized between cellular organelles. The cytosolic NADPH/NADP
+
is maintained in a reduced state necessary for drive biosynthetic and antioxidant processes. In energized
mitochondria NADH exceeds NAD
+
to provide electrons for the ETC while the cytosol has a higher
NAD
+
, reflecting a relatively oxidized redox state [
171
]. Mitochondrial NADH/NAD
+
redox ratio
is closely related with mitochondrial function. We recently reported that a mitochondrial complex
I defect directly results in an increased NADH/NAD
+
ratio in cultured photoreceptor cells [
153
].
Diederen et al. [
172
] found no significant differences in NADH/NAD
+
redox ratios in the whole retina
of 6-month-old STZ-induced diabetic mice. The redox status in specific cellular organelles including
mitochondria was not assessed in this study. It may be predicted that mitochondrial redox state is
unchanged in early stages of DR when considering the biphasic response of retinal mitochondria

Antioxidants 2020,9, 905 14 of 29
to the diabetic milieu. An early enhanced mitochondrial function would be expected to maintain
a normal NADH/NAD
+
redox ratios until ETC defects begin to manifest. While complex I and IV
defects are associated with the accumulation of NADH and an increased NADH/NAD
+
ratio [
173
,
174
],
measures taken to correct ETC defects can be used to decrease NADH and restore redox balance [
175
].
These data are consistent with the hypothesis that ETC defects cause an increase in NADH and a
reduced redox microenvironment in retinal mitochondria.
A potential mechanism that may increase cellular NADH/NAD
+
redox ratio is the activation of
poly (ADP-ribose) polymerases (PARPs), a family of proteins best known for their role in DNA repair.
PARPs are activated in response to DNA damage and catalyze the transfer and polymerization of
ADP-ribose to DNA repair enzymes. This reaction requires NAD
+
, leading some to hypothesize that
PARP activation could lead to NAD
+
depletion and altered redox homeostasis. PARP-deficient mice are
protected against diabetes [
176
], and exhibit preserved redox homeostasis and mitochondrial function [
177
].
The mitochondrial protective effect is mediated by activating the NAD
+
-dependent deacetylases, sirtuins
(SIRTs). Among the large family of SIRT proteins, SIRT1 is an extramitochondrial protein with a wide
range of functions in both metabolism and aging. Recent work by Mishra et al. [
178
] have shown that the
SIRT1 promoter is hypermethylated in STZ-induced diabetic mice. SIRT1 overexpression protected the
mice against mitochondrial damage, neural dysfunction, RGC degeneration, and blood–retinal barrier
breakdown. A role of mitochondrial sirtuins in retinal disease is supported by the finding that genetic
knockout of SIRT3, a mitochondrial SIRT, mirrors NAD
+
deficiency and leads to early and rapid retinal
degeneration [164].
The immediate consequence of an increased mitochondrial NADH/NAD
+
redox ratio is reductive
stress (increased NADH) and NAD
+
deficiency, which is detrimental to photoreceptor integrity [
153
].
Mitochondrial production of ROS is largely governed by the NADH/NAD
+
ratio, as an increased
[NADH] slows the ETC flux. The antioxidant defense is supported by the NADPH/NADP
+
redox
ratio. NADPH is a potent reducing agent involved the regeneration of antioxidant compounds such
as reduced glutathione. Importantly, the mitochondrial NADH/NAD
+
and NADPH/NADP
+
redox
couples are linked by nicotinamide nucleotide transhydrogenase (NNT), an enzyme that leverages the
proton-motive force in the oxidation of NADH and the simultaneous reduction of NADP
+
(Figure 4A).
NNT maintains a NADPH/NADP
+
ratio several-fold higher than the NADH/NAD
+
ratio, and thus is
a physiologically relevant source of NADPH that drives the reduction of H
2
O
2
[
179
]. While NNT is
reported to be expressed exclusively in cardiac tissue [
180
], we provide evidence here that the NNT
protein is also expressed in the retina (Figure 4B). The role of NNT to maintain the mitochondrial redox
state and antioxidant defense is yet to be determined.
Antioxidants 2020, 9, x FOR PEER REVIEW 14 of 30
expected to maintain a normal NADH/NAD+ redox ratios until ETC defects begin to manifest. While
complex I and IV defects are associated with the accumulation of NADH and an increased
NADH/NAD+ ratio [173,174], measures taken to correct ETC defects can be used to decrease NADH
and restore redox balance [175]. These data are consistent with the hypothesis that ETC defects cause
an increase in NADH and a reduced redox microenvironment in retinal mitochondria.
A potential mechanism that may increase cellular NADH/NAD+ redox ratio is the activation of
poly (ADP-ribose) polymerases (PARPs), a family of proteins best known for their role in DNA repair.
PARPs are activated in response to DNA damage and catalyze the transfer and polymerization of
ADP-ribose to DNA repair enzymes. This reaction requires NAD+, leading some to hypothesize that
PARP activation could lead to NAD+ depletion and altered redox homeostasis. PARP-deficient mice
are protected against diabetes [176], and exhibit preserved redox homeostasis and mitochondrial
function [177]. The mitochondrial protective effect is mediated by activating the NAD+-dependent
deacetylases, sirtuins (SIRTs). Among the large family of SIRT proteins, SIRT1 is an
extramitochondrial protein with a wide range of functions in both metabolism and aging. Recent
work by Mishra et al. [178] have shown that the SIRT1 promoter is hypermethylated in STZ-induced
diabetic mice. SIRT1 overexpression protected the mice against mitochondrial damage, neural
dysfunction, RGC degeneration, and blood–retinal barrier breakdown. A role of mitochondrial
sirtuins in retinal disease is supported by the finding that genetic knockout of SIRT3, a mitochondrial
SIRT, mirrors NAD+ deficiency and leads to early and rapid retinal degeneration [164].
The immediate consequence of an increased mitochondrial NADH/NAD+ redox ratio is
reductive stress (increased NADH) and NAD+ deficiency, which is detrimental to photoreceptor
integrity [153]. Mitochondrial production of ROS is largely governed by the NADH/NAD+ ratio, as
an increased [NADH] slows the ETC flux. The antioxidant defense is supported by the
NADPH/NADP+ redox ratio. NADPH is a potent reducing agent involved the regeneration of
antioxidant compounds such as reduced glutathione. Importantly, the mitochondrial NADH/NAD+
and NADPH/NADP+ redox couples are linked by nicotinamide nucleotide transhydrogenase (NNT),
an enzyme that leverages the proton-motive force in the oxidation of NADH and the simultaneous
reduction of NADP+ (Figure 4A). NNT maintains a NADPH/NADP+ ratio several-fold higher than
the NADH/NAD+ ratio, and thus is a physiologically relevant source of NADPH that drives the
reduction of H2O2 [179]. While NNT is reported to be expressed exclusively in cardiac tissue [180],
we provide evidence here that the NNT protein is also expressed in the retina (Figure 4B). The role
of NNT to maintain the mitochondrial redox state and antioxidant defense is yet to be determined.
Figure 4. Nicotinamide nucleotide transhydrogenase (NNT). (A) NNT is a mitochondrial enzyme

Antioxidants 2020,9, 905 15 of 29
that oxidizes NADH, and therefore supplements complex I in the process of regenerating NAD
+
.
In addition, the enzyme forms NADPH that is critical to maintain the antioxidant potency of
mitochondria by maintaining the reduced glutathione (GSH) [
181
,
182
]. (
B
) Western blot analysis of
the NNT protein expression in a variety of tissues in both male in female mice. Abbreviations: UCP,
uncoupling protein; H, heart; K, kidney; Br, brain; R, retina; BAT, brown adipose tissue; WAT, white
adipose tissue; L, liver.
8. Alterations in Mitochondrial Turnover
8.1. Mitochondrial Biogenesis and Mitophagy
The balance between mitochondrial formation and destruction regulates the cellular mitochondrial
mass. Mitochondrial biogenesis is the cellular process to increase total mitochondrial content. This process
relies on the coordinated action of cell signaling molecules, molecular chaperones, and transcription
factors, all of which working in tandem to replicate the mitochondrial genome and proteome. One of the
most upstream factors is the peroxisome proliferator-activated receptor gamma coactivator 1-
α
(PGC-1
α
),
which has been referred to as the “master regulator” of mitochondrial biogenesis [
183
]. Among its
many downstream targets is mitochondrial transcription factor A (TFAM), which is translocated to the
mitochondrial matrix and initiates mitochondrial genome replication. In the past decade, a series of
studies conducted by Santos et al. [
127
,
129
,
184
] have shown that mitochondrial biogenesis is altered
in both experimental and human DR. Specifically, Santos et al. [
129
] found that nuclear-mitochondrial
translocation of TFAM is impaired within 12 months of STZ-induced diabetes. Subsequent experiments
by the same group determined that TFAM is ubiquitinated and targeted for proteasomal degradation,
and its translocation to the matrix is impaired [
128
]. Overexpression of MnSOD or administration of the
exogenous antioxidant lipoid acid had a positive impact on mitochondrial localization of TFAM and
mtDNA copy number [
127
,
129
], further supporting a role of oxidative stress as an upstream regulator
of mitochondrial biogenesis. An important limitation, however, is that most of these studies were
performed in retinal homogenate or endothelial cells. Thus, whether these specific findings translate to
specific cell populations within the neural retina is an important question that remains to be explored.
Mitophagy is a specialized form of macro-autophagy by which damaged or excessive mitochondria
are selectively targeted for lysosomal degradation. Several groups have reported that Müller cells
grown in high glucose exhibit enhanced mitophagy [
119
–
121
,
124
]. This phenomenon is thought
to occur in part as a consequence of hyperglycemia-induced expression of thioredoxin-interacting
protein (TXNIP), which binds to and inhibits the antioxidants thioredoxin 1 and thioredoxin 2.
Hyperglycemia-induced expression of TXNIP is observed in the vasculature, pericytes, and the
RPE [
130
,
185
], suggesting a conserved mechanism across cell types. Knockdown of TXNIP reduces
oxidative stress, improves ATP synthesis, and restores mitophagic flux [
119
]. Some of these findings
have since been validated in the db/db mouse model by Zhou et al. [
124
]. When considered together
with the work of Santos et al. [
114
,
127
–
129
,
186
], these findings suggest that DR is characterized by a
gradual decrease in mitochondrial content, both due to impaired biogenesis and enhanced mitophagy.
Recent work by Hombrebueno et al. [121] suggests that these processes have a temporal relationship.
Using the spontaneous Ins2Akita diabetic mouse model, Hombrebueno et al. [121] observed enhanced
PTEN-induced kinase (PINK1)-dependent mitophagy in both Müller cells and photoreceptors within
the first 2 months of diabetes. While increased mitochondrial biogenesis can compensate for enhanced
mitophagy, compensatory mechanisms begin to fail at 8 months of diabetes, resulting in decreased
mitochondrial mass.
8.2. Fusion–Fission Dynamics in the Retina
Mitochondria exist in a constant flux of fusion and fission, which is necessary to respond to the
energy requirements of the cell (for a review, see [
187
]). This process is regulated by mitofusin-2
(Mfn2) and dynamin-related protein 1 (Drp1), two antagonistic GTPases that regulate fusion and fission,

Antioxidants 2020,9, 905 16 of 29
respectively. In human DR, Mfn2 protein levels are reduced, while Drp1 is increased, suggesting an
imbalance between these two GTPases that favors mitochondrial fission, reduced mtDNA, and possibly
decreased ATP synthesis [
188
]. These findings have also been observed in Müller cells and photoreceptors
grown in high-glucose conditions [
119
]. Although the exact mechanism is largely unexplored, a shift
toward mitochondrial fission may also be due to oxidative damage. In support of this hypothesis,
administration of melatonin, which is well-known for its antioxidant properties, preserved mitochondrial
fusion in photoreceptors both
in vitro
and
in vivo
[
118
]. Alternatively, enhanced mitochondrial fission
may be due to increased methylation at the MFN2 promoter site, as shown in endothelial cells [
136
].
However, this finding alone does not explain the reported increase in Drp1, which independently favors
mitochondrial fission. This area of research is still in its preliminary stages, and subsequent studies will be
necessary to confirm its significance in DR.
9. Consequences of Increased Oxidative Stress in the Neural Retina
9.1. Diabetic Milieu Alters Ion Channel Homeostasis in the Retina
Early in its course, diabetes causes a paradoxical closure of the L-type calcium ion channels
(LTCCs) in the dark, as suggested by manganese-enhanced magnetic resonance imaging (MEMRI)
studies that have shown that photoreceptor uptake of manganese (a calcium surrogate) is significantly
reduced in the dark-adapted rodents [
189
]. Because these ion channels are essential for the regulated
release of neurotransmitters at the photoreceptor synapses, paradoxically closed photoreceptor LTCCs
in the dark have significant functional consequences. Alterations in ion channel homeostasis have also
been reported at the level of the mitochondria. The expression of the mitochondrial calcium uniporter
(MCU), which plays important roles in calcium buffering and ion channel homeostasis, is decreased in
photoreceptors grown in high glucose conditions [
190
]. Retinal neurons cultured in high glucose exhibit
increased mitochondrial calcium load, associated with depolarization of mitochondrial membrane and
ROS generation. Similar observations were made in retinas from 9-week-old diabetic rats [191].
Long-term diabetes causes mitochondrial ETC defects and decreases mitochondrial respiration
efficiency in the retina, with both being canonical causes of energy deficit. However, available data
do not support the hypothesis that ATP deficit is responsible for the dysfunction in ion channels as
retinal [ATP] is unchanged in diabetes [
192
]. Decreasing oxidative stress in diabetic rodents with
copper/zinc superoxide dismutase (Cu/Zn SOD) overexpression or lipoic acid administration corrected
the diabetes-induced ion flux abnormalities in photoreceptors in the dark [
145
,
193
], suggesting that
abnormalities in ion homeostasis are induced by increased oxidative stress. Studies showing that
diabetes elevates oxidative stress in the retina by both promoting ROS production and suppressing the
antioxidant defense [117] also provide strong support for this hypothesis.
9.2. Apoptosis
The intrinsic (mitochondrial) pathway of apoptosis is initiated by increased permeabilization of the
mitochondrial outer membrane and activation of the apoptotic signaling cascade.
Notably, the intrinsic
pathway is induced by increased oxidative stress [
194
], making this pathway highly relevant in DR,
as virtually all cell types in the retina experience hyperglycemia-induced oxidative stress [
116
,
123
,
164
,
195
].
RGCs [
133
] and the retinal microvasculature in particular have been shown to undergo oxidative
stress-induced apoptosis [
114
,
160
]. Several groups have reported that administration of exogenous
antioxidants preserved mitochondrial integrity and prevent cell death in the diabetic retina [
116
,
133
].
These findings
reiterate the concept that oxidative stress is an early event in the pathogenesis of DR,
whereas cell death generally occurs as a later and secondary event.

Antioxidants 2020,9, 905 17 of 29
10. Therapeutic Implications
10.1. Therapies Focused on Maintaining the Integrity of Retinal Mitochondria
The Diabetes Control and Complications Trial showed that intensive insulin replacement therapy
reduces the incidence and slows the progression of DR [
5
]. However, the incidence of DR remains high,
and many patients still progress to PDR despite advances in diabetes care. Accordingly, second-line
treatments are frequently necessary to manage the later complications of DR. These therapies are
highly effective in managing the sight-threatening complications of DR. Thus, it is critical to better
understand the early stages of DR and develop new therapies to prevent its progression. The need for
new therapies in early stages of DR is further highlighted by the “metabolic memory” phenomenon
(for a review, see [
2
]), which may be a consequence of persistent mitochondrial damage and oxidative
stress [
142
]. This hypothesis is consistent with the benefit of mitochondrial targeted therapy to preserve
mitochondrial integrity and vision in experimental DR.
An important antioxidant therapy is the Szeto–Schiller (SS) tetra-peptide, SS-31 (elamipretide),
which is concentrated in the inner mitochondrial membrane and selectively stabilizes cardiolipin [
196
],
a phospholipid critical for mitochondrial integrity and function, which is prone to oxidative damage in
diabetes [
197
]. SS-31 enhances the interaction between cytochrome c and cardiolipin to facilitate better
electron transfer from complex III to complex IV, and reduces mitochondrial oxidative stress [
198
,
199
].
Evidence from animal studies suggests that SS-31 could reduce the risk of vascular disease in diabetes,
as administration of SS-31 alleviated the microvascular retinal disease in rodent models of DR [
200
,
201
],
and increased SIRT1 while ameliorating retinal inflammation in rodent and human subjects with
T2D [
202
]. However, human studies have shown limited efficacy to date, and no studies have been
performed in patients with DR.
Numerous studies have suggested that oxidative stress may be modulated by uncoupling proteins
(UCPs), a family of proteins named for their ability to uncouple electron transport from ATP synthesis.
These proteins decrease mitochondrial ROS production by decreasing the electrochemical gradient [
203
],
a process called “mild uncoupling”. The theory is based on the observation of Korshunov et al. [
204
] that
ROS generation increases in an exponential manner when mitochondrial membrane potential exceeds a
threshold that is higher than that corresponding to the mitochondrial energetic state in
in vivo
settings.
The hypothesis is also based on the assumption that the diabetic milieu increases the availability
of energetic substrates (glucose, FAs) to the retina, which are oxidized to increase mitochondrial
membrane potential (“hyperpolarization”). This hypothesis is supported by the observation that
UCP2-deficient mice exhibit increased ROS generation [
205
], whereas overexpression of the UCP2 gene
preserves mitochondrial function in human umbilical endothelial cells [
206
]. In the retina, UCP2 protein
levels and activity are increased in retinal endothelial cells grown in high-glucose conditions [
207
],
which may indicate a compensatory response to oxidative stress. However, administration of the
uncoupling agent niclosamide ethanolamine has shown no benefit in the treatment of diabetes or
its complications in db/db mice [
208
] indicating the lack of benefit of uncouplers in
in vivo
settings.
A potential explanation is that Oxphos is a process regulated by energy demand rather than substrate
availability. In diabetes, increased substrate availability does not increase oxidative metabolism that
exceeds ATP synthesis [
109
], a mechanism that would lead to “hyperpolarization”. While UCPs may
be an important endogenous defense mechanism in the setting of oxidative stress, their therapeutic
utility is unclear.
Improving the efficiency of electron flow within the defective mitochondrial ETC may be an
optimal approach to relieve the increased electron density at specific ETC sites and eliminate the
risk of oxygen univalent reduction and superoxide formation. Methylene blue, a redox compound
that provides an alternative electron route between mitochondrial complex I and cytochrome c [
209
],
preserves mitochondrial and photoreceptor integrity by preventing oxidative stress in a model of
complex I defect [153] and experimental diabetes [34].

Antioxidants 2020,9, 905 18 of 29
Idebenone, a synthetic benzoquinone that mediates electron transfer to complex III by bypassing
complex I [
210
], is a free radical scavenger [
211
], reduces intracellular ROS and increases ATP production
in complex I-defective cells [
212
–
214
], and promotes an increase in mitochondrial mass by regulating
mitophagy [
215
]. Its mitochondrial protective effects have recently been investigated as a drug therapy
for Leber’s hereditary optic neuropathy, a rare genetic mitochondrial disease that causes rapid and
progressive bilateral vision loss in young adults. A 24-week multi-center double-blind, randomized,
placebo-controlled trial in patients with Leber’s hereditary optic neuropathy show a mild benefit
in visual acuity [
216
] that was confirmed when treatment started 5 years after the diagnosis [
217
].
The benefic outcomes are considered a result of idebenone to restore the bioenergetics in the remaining
dysfunctional RGC. Evidence for efficacy of idebenone in human patients with primary or acquired
mitochondrial defects are still limited, and restrict its use in DR.
10.2. Clinical Trials and the Role of Antioxidants in the Management of DR
Despite the evidence that antioxidants can slow the progression of DR, clinical trials have had
mixed results. This topic was very recently reviewed by Garcia-Medina et al. [
218
], but is summarized
here for completeness. The most successful interventions have been those that use combined antioxidant
therapy (CAT). The Diabetes Visual Function Supplement Study showed that CAT may improve visual
acuity and contrast sensitivity among participants with T1D and T2D without clinically detectable
retinopathy or with mild non-proliferative diabetic retinopathy (NPDR) [
219
]. This finding is supported
by the prior work of Hu et al. [
220
], who showed that patients with NPDR have lower levels of lutein
and zeaxanthin, and further demonstrated that supplementation with these antioxidants reduces
oxidative stress and may improve visual function. Although the previous two studies suggest a role of
CAT in the management of DR, the conclusions are limited by the short study duration (6 and 3 months,
respectively) relative to the chronicity of DR. To date, the longest trial that has been conducted was a
5-year follow-up of patients taking a commercially available multi-vitamin showing that antioxidants
may slow the progression of DR as detected by ophthalmic examination [
221
]. However, in contrast
to the above studies, the investigators did not observe a significant improvement in visual acuity.
These discrepancies highlight the need for additional studies to find better therapeutic strategies
that decrease the mitochondrial ROS generation by preserving mitochondrial function rather than
scavenging the already generated ROS.
11. Conclusions
The pathogenesis of DR is complex and likely involves the simultaneous dysregulation of
multiple metabolic and signaling pathways throughout the retinal neurovascular unit. Alterations in
mitochondrial function has broader consequences than changes in ATP content. Increased oxidative
stress and alterations in the redox balance are interrelated mechanisms that are altered by diabetes,
and their effect on retinal structure and function in diabetes is yet to be explored. The benefit
of maintaining retinal energetic flexibility and optimal fuel selection between plentiful competing
substrates (glucose versus FA) in diabetes remains largely unexplored, and may represent a promising
area of research. Although the present review focuses on the roles of mitochondrial dysfunction and
oxidative stress, cellular dysfunction in the retina can take many forms, including neuroinflammation
and blood–retinal barrier breakdown. These processes likely occur in parallel and thus future studies
should adopt a comprehensive approach that appreciates the interconnectedness of the retina.
Author Contributions:
Conceptualization, D.J.M., M.A.C., and M.G.R.; resources, D.J.M. and M.G.R.;
writing—original draft preparation, D.J.M.; writing—review and editing, M.A.C. and M.G.R.; visualization, D.J.M.
and M.G.R.; supervision, M.A.C. and M.G.R.; project administration, M.A.C. and M.G.R.; funding acquisition,
M.G.R. All authors reviewed and edited the references. All authors have read and agreed to the published version
of the manuscript.
Funding:
This research was funded by the American Heart Association, grant number 18AIREA33990023.
MGR and DJM were also funded by startup funds and a student research publication grant, respectively,
provided by the Central Michigan University College of Medicine.

Antioxidants 2020,9, 905 19 of 29
Acknowledgments:
We thank Timothy Kern for his feedback during writing the manuscript. We apologize to
authors of other important studies that were not included in this review because of space limitations.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Saeedi, P.; Petersohn, I.; Salpea, P.; Malanda, B.; Karuranga, S.; Unwin, N.; Colagiuri, S.; Guariguata, L.;
Motala, A.A.; Ogurtsova, K.; et al. Global and regional diabetes prevalence estimates for 2019 and projections
for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9th edition.
Diabetes Res
.
Clin. Pract. 2019,157, 107843. [CrossRef] [PubMed]
2.
Yau, J.W.Y.; Rogers, S.L.; Kawasaki, R.; Lamoureux, E.L.; Kowalski, J.W.; Bek, T.; Chen, S.J.; Dekker, J.M.;
Fletcher, A.; Grauslund, J.; et al. Global Prevalence and Major Risk Factors of Diabetic Retinopathy.
Diabetes Care 2012,35, 556–564. [CrossRef] [PubMed]
3.
Klein, R.; Knudtson, M.D.; Lee, K.E.; Gangnon, R.; Klein, B.E.K. The Wisconsin Epidemiologic Study of
Diabetic Retinopathy: XXII the twenty-five-year progression of retinopathy in persons with type 1 diabetes.
Ophthalmology 2008,115, 1859–1868. [CrossRef] [PubMed]
4.
Hirsch, I.B.; Brownlee, M. Beyond Hemoglobin A1c—Need for Additional Markers of Risk for Diabetic
Microvascular Complications. JAMA 2010,303, 2291–2292. [CrossRef] [PubMed]
5.
Royle, P.; Mistry, H.; Auguste, P.; Shyangdan, D.; Freeman, K.; Lois, N.; Waugh, N. Pan-retinal
photocoagulation and other forms of laser treatment and drug therapies for non-proliferative diabetic
retinopathy: Systematic review and economic evaluation. Health Technol. Assess.
2015
,19, 1–247. [CrossRef]
6.
Antonetti, D.A.; Klein, R.; Gardner, T.W. Diabetic retinopathy. N. Engl. J. Med.
2012
,366, 1227–1239.
[CrossRef]
7.
Antonetti, D.A.; Barber, A.J.; Khin, S.; Lieth, E.; Tarbell, J.M.; Gardner, T.W. Vascular Permeability in
Experimental Diabetes Is Associated With Reduced Endothelial Occludin Content: Vascular endothelial
growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group. Diabetes
1998
,
47, 1953–1959. [CrossRef]
8.
Roy, S.; Maiello, M.; Lorenzi, M. Increased expression of basement membrane collagen in human diabetic
retinopathy. J. Clin. Investig. 1994,93, 438–442. [CrossRef]
9.
Biallosterski, C.; Van Velthoven, M.E.J.; Michels, R.P.J.; Schlingemann, R.O.; Devries, J.H.; Verbraak, F.D.
Decreased optical coherence tomography-measured pericentral retinal thickness in patients with diabetes
mellitus type 1 with minimal diabetic retinopathy. Br. J. Ophthalmol. 2007,91, 1135–1138. [CrossRef]
10.
Van Dijk, H.W.; Kok, P.H.B.; Garvin, M.; Sonka, M.; Devries, J.H.; Michels, R.P.J.; Van Velthoven, M.E.J.;
Schlingemann, R.O.; Verbraak, F.D.; AbraMoff, M.D. Selective Loss of Inner Retinal Layer Thickness in Type
1 Diabetic Patients with Minimal Diabetic Retinopathy. Investig. Opthalmol. Vis. Sci.
2009
,50, 340–3409.
[CrossRef]
11.
Simo, R.; Hernandez, C.; European Consortium for the Early Treatment of Diabetic Retinopathy
(EUROCONDOR). Neurodegeneration is an early event in diabetic retinopathy: Therapeutic implications.
Br. J. Ophthalmol. 2012,96, 1285–1290. [CrossRef] [PubMed]
12.
Vujosevic, S.; Midena, E. Retinal Layers Changes in Human Preclinical and Early Clinical Diabetic Retinopathy
Support Early Retinal Neuronal and Müller Cells Alterations. J. Diabetes Res. 2013, 905058. [CrossRef]
13.
Yang, J.H.; Kwak, H.W.; Kim, T.G.; Han, J.; Moon, S.W.; Yu, S.Y. Retinal Neurodegeneration in Type II Diabetic
Otsuka Long-Evans Tokushima Fatty Rats. Investig. Opthalmol. Vis. Sci.
2013
,54, 3844–3851. [CrossRef]
[PubMed]
14. Wolter, J.R. Diabetic Retinopathy. Am. J. Ophthalmol. 1961,51, 1123/1251–1141/1269. [CrossRef]
15.
Barber, A.J.; Lieth, E.; Khin, S.A.; Antonetti, D.A.; Buchanan, A.G.; Gardner, T.W. Neural apoptosis in the
retina during experimental and human diabetes. Early onset and effect of insulin. J. Clin. Investig.
1998
,102,
783–791. [CrossRef]
16.
Sokol, S.; Moskowitz, A.; Skarf, B.; Evans, R.; Molitch, M.; Senior, B. Contrast Sensitivity in Diabetes with
and Without Background Retinopathy. Arch. Ophthalmol. 1985,103, 51–54. [CrossRef] [PubMed]
17.
Roy, M.S.; Gunkel, R.D.; Podgor, M.J. Color Vision Defects in Early Diabetic Retinopathy. Arch. Ophthalmol.
1986,104, 225–228. [CrossRef] [PubMed]

Antioxidants 2020,9, 905 20 of 29
18.
Cho, N.-C. Selective Loss of S-Cones in Diabetic Retinopathy. Arch. Ophthalmol.
2000
,118, 1393–1400.
[CrossRef] [PubMed]
19.
Wachtmeister, L. Oscillatory potentials in the retina: What do they reveal. Prog. Retin Eye Res.
1998
,17,
485–521. [CrossRef]
20.
Bresnick, G.H.; Korth, K.; Groo, A.; Palta, M. Electroretinographic oscillatory potentials predict progression
of diabetic retinopathy. Preliminary report. Arch. Ophthalmol. 1984,102, 1307–1311. [CrossRef] [PubMed]
21.
Coupland, S.G. A comparison of oscillatory potential and pattern electroretinogram measures in diabetic
retinopathy. Doc. Ophthalmol. 1987,66, 207–218. [CrossRef] [PubMed]
22.
Iadecola, C. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat. Rev. Neurosci.
2004,5, 347–360. [CrossRef] [PubMed]
23.
Metea, M.R.; Newman, E.A. Signalling within the neurovascular unit in the mammalian retina. Exp. Physiol.
2007,92, 635–640. [CrossRef]
24.
Bilimoria, P.M.; Stevens, B. Microglia function during brain development: New insights from animal models.
Brain Res. 2015,1617, 7–17. [CrossRef]
25.
Koehler, R.C.; Gebremedhin, D.; Harder, D.R. Role of astrocytes in cerebrovascular regulation. J. Appl. Physiol.
2006,100, 307–317. [CrossRef]
26.
Kurihara, T. Development and pathological changes of neurovascular unit regulated by hypoxia response in
the retina. Prog. Brain Res. 2016,225, 201–211. [CrossRef]
27.
Gardner, T.W.; Davila, J.R. The neurovascular unit and the pathophysiologic basis of diabetic retinopathy.
Graefes Arch. Clin. Exp. Ophthalmol. 2017,255, 1–6. [CrossRef]
28.
Kowluru, R.A.; Chan, P.S. Oxidative stress and diabetic retinopathy. Exp. Diabetes Res.
2007
,2007, 43603.
[CrossRef]
29.
Hammes, H.P. Diabetic retinopathy: Hyperglycaemia, oxidative stress and beyond. Diabetologia
2018
,61,
29–38. [CrossRef]
30.
Kim, J.A.; Wei, Y.; Sowers, J.R. Role of mitochondrial dysfunction in insulin resistance. Circ. Res.
2008
,102,
401–414. [CrossRef] [PubMed]
31.
Sivitz, W.I.; Yorek, M.A. Mitochondrial dysfunction in diabetes: From molecular mechanisms to functional
significance and therapeutic opportunities. Antioxid. Redox Signal. 2010,12, 537–577. [CrossRef]
32.
Du, X.L.; Edelstein, D.; Rossetti, L.; Fantus, I.G.; Goldberg, H.; Ziyadeh, F.; Wu, J.; Brownlee, M.
Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway
and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc. Natl. Acad.
Sci. USA 2000,97, 12222–12226. [CrossRef] [PubMed]
33.
Nishikawa, T.; Edelstein, D.; Du, X.L.; Yamagishi, S.-I.; Matsumura, T.; Kaneda, Y.; Yorek, M.A.; Beebe, D.;
Oates, P.J.; Hammes, H.-P.; et al. Normalizing mitochondrial superoxide production blocks three pathways
of hyperglycaemic damage. Nature 2000,404, 787–790. [CrossRef] [PubMed]
34.
Du, Y.; Veenstra, A.; Palczewski, K.; Kern, T.S. Photoreceptor cells are major contributors to diabetes-induced
oxidative stress and local inflammation in the retina. Proc. Natl. Acad. Sci. USA
2013
,110, 16586–16591.
[CrossRef] [PubMed]
35.
Tonade, D.; Liu, H.; Kern, T.S. Photoreceptor Cells Produce Inflammatory Mediators That Contribute to
Endothelial Cell Death in Diabetes. Investig. Ophthalmol. Vis. Sci. 2016,57, 4264–4271. [CrossRef]
36.
Arden, G.B. The absence of diabetic retinopathy in patients with retinitis pigmentosa: Implications for
pathophysiology and possible treatment. Br. J. Ophthalmol. 2001,85, 366–370. [CrossRef] [PubMed]
37.
De Gooyer, T.E.; Stevenson, K.A.; Humphries, P.; Simpson, D.A.C.; Gardiner, T.A.; Stitt, A.W. Retinopathy Is
Reduced during Experimental Diabetes in a Mouse Model of Outer Retinal Degeneration. Investig. Opthalmol.
Vis. Sci. 2006,47, 5561–5568. [CrossRef] [PubMed]
38.
Jezek, P.; Holendova, B.; Plecita-Hlavata, L. Redox Signaling from Mitochondria: Signal Propagation and Its
Targets. Biomolecules 2020,10, 93. [CrossRef] [PubMed]
39.
Bock, F.J.; Tait, S.W.G. Mitochondria as multifaceted regulators of cell death. Nat. Rev. Mol. Cell Biol.
2020
,
21, 85–100. [CrossRef]
40.
Yu-Wai-Man, P.; Newman, N.J. Inherited eye-related disorders due to mitochondrial dysfunction. Hum. Mol.
Genet. 2017,26, R12–R20. [CrossRef]
41.
Hoegger, M.J.; Lieven, C.J.; Levin, L.A. Differential production of superoxide by neuronal mitochondria.
BMC Neurosci. 2008,9, 4. [CrossRef] [PubMed]

Antioxidants 2020,9, 905 21 of 29
42.
Remor, A.P.; de Matos, F.J.; Ghisoni, K.; da Silva, T.L.; Eidt, G.; Burigo, M.; de Bem, A.F.; Silveira, P.C.;
de Leon, A.; Sanchez, M.C.; et al. Differential effects of insulin on peripheral diabetes-related changes in
mitochondrial bioenergetics: Involvement of advanced glycosylated end products. Biochim. Biophys. Acta
2011,1812, 1460–1471. [CrossRef]
43.
Cogan, D.G.; Kuwabara, T. Capillary Shunts in the Pathogenesis of Diabetic Retinopathy. Diabetes
1963
,12,
293–300. [CrossRef] [PubMed]
44.
Mizutani, M.; Kern, T.S.; Lorenzi, M. Accelerated death of retinal microvascular cells in human and
experimental diabetic retinopathy. J. Clin. Investig. 1996,97, 2883–2890. [CrossRef] [PubMed]
45.
Franco, C.A.; Jones, M.L.; Bernabeu, M.O.; Geudens, I.; Mathivet, T.; Rosa, A.; Lopes, F.M.; Lima, A.P.;
Ragab, A.; Collins, R.T.; et al. Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel
Regression. PLoS Biol. 2015,13, e1002125. [CrossRef]
46.
Lenard, A.; Daetwyler, S.; Betz, C.; Ellertsdottir, E.; Belting, H.-G.; Huisken, J.; Affolter, M. Endothelial Cell
Self-fusion during Vascular Pruning. PLoS Biol. 2015,13, e1002126. [CrossRef]
47. Cogan, D.G.; Kuwabara, T. The Mural Cell in Perspective. Arch. Ophthalmol. 1967,78, 133–139. [CrossRef]
48.
Pfister, F.; Feng, Y.; vom Hagen, F.; Hoffmann, S.; Molema, G.; Hillebrands, J.L.; Shani, M.; Deutsch, U.;
Hammes, H.P. Pericyte migration: A novel mechanism of pericyte loss in experimental diabetic retinopathy.
Diabetes 2008,57, 2495–2502. [CrossRef]
49.
Warmke, N.; Griffin, K.J.; Cubbon, R.M. Pericytes in diabetes-associated vascular disease. J. Diabetes Complicat.
2016,30, 1643–1650. [CrossRef]
50.
Valdez, C.N.; Arboleda-Velasquez, J.F.; Amarnani, D.S.; Kim, L.A.; D’Amore, P.A. Retinal Microangiopathy
in a Mouse Model of Inducible Mural Cell Loss. Am. J. Pathol. 2014,184, 2618–2626. [CrossRef]
51.
Hammes, H.-P.; Feng, Y.; Pfister, F.; Brownlee, M. Diabetic Retinopathy: Targeting Vasoregression. Diabetes
2011,60, 9–16. [CrossRef] [PubMed]
52.
Santos, G.S.P.; Prazeres, P.; Mintz, A.; Birbrair, A. Role of pericytes in the retina. Eye
2018
,32, 483–486.
[CrossRef] [PubMed]
53.
Nilsson, M.; von Wendt, G.; Wanger, P.; Martin, L. Early detection of macular changes in patients with
diabetes using Rarebit Fovea Test and optical coherence tomography. Br. J. Ophthalmol.
2007
,91, 1596–1598.
[CrossRef] [PubMed]
54.
Valverde, A.M.; Miranda, S.; Garc
í
a-Ram
í
rez, M.; Gonz
á
lez-Rodriguez,
Á
.; Hern
á
ndez, C.; Sim
ó
, R.
Proapoptotic and survival signaling in the neuroretina at early stages of diabetic retinopathy. Mol. Vis.
2013
,
19, 47–53.
55.
Trudeau, K.; Molina, A.J.A.; Guo, W.; Roy, S. High Glucose Disrupts Mitochondrial Morphology in Retinal
Endothelial Cells. Am. J. Pathol. 2010,177, 447–455. [CrossRef]
56.
Kern, T.S.; Barber, A.J. Retinal ganglion cells in diabetes. J. Physiol.
2008
,586, 4401–4408. [CrossRef] [PubMed]
57.
Gastinger, M.J.; Singh, R.S.J.; Barber, A.J. Loss of Cholinergic and Dopaminergic Amacrine Cells in
Streptozotocin-Diabetic Rat and Ins2Akita-Diabetic Mouse Retinas. Investig. Opthalmol. Vis. Sci.
2006
,47,
3143–3150. [CrossRef] [PubMed]
58.
Szabo, K.; Enzsoly, A.; Dekany, B.; Szabo, A.; Hajdu, R.I.; Radovits, T.; Matyas, C.; Olah, A.; Laurik, L.K.;
Somfai, G.M.; et al. Histological Evaluation of Diabetic Neurodegeneration in the Retina of Zucker Diabetic
Fatty (ZDF) Rats. Sci. Rep. 2017,7, 8891. [CrossRef]
59.
Rodrigues, E.B.; Urias, M.G.; Penha, F.M.; Badaro, E.; Novais, E.; Meirelles, R.; Farah, M.E. Diabetes induces
changes in neuroretina before retinal vessels: A spectral-domain optical coherence tomography study. Int. J.
Retina Vitreous 2015,1, 4. [CrossRef]
60.
Barber, A.J.; Gardner, T.W.; Abcouwer, S.F. The Significance of Vascular and Neural Apoptosis to the Pathology
of Diabetic Retinopathy. Investig. Opthalmol. Vis. Sci. 2011,52, 1156–1163. [CrossRef]
61.
Enzsoly, A.; Szabo, A.; Kantor, O.; David, C.; Szalay, P.; Szabo, K.; Szel, A.; Nemeth, J.; Lukats, A. Pathologic
alterations of the outer retina in streptozotocin-induced diabetes. Investig. Ophthalmol. Vis. Sci.
2014
,55,
3686–3699. [CrossRef] [PubMed]
62.
Hombrebueno, J.R.; Chen, M.; Penalva, R.G.; Xu, H. Loss of synaptic connectivity, particularly in second order
neurons is a key feature of diabetic retinal neuropathy in the Ins2Akita mouse. PLoS ONE
2014
,9, e97970.
[CrossRef] [PubMed]

Antioxidants 2020,9, 905 22 of 29
63.
Zarebska, A.; Czerny, K.; Bakiera, K.; Cichacz-Kwiatkowska, B.; Lis-Sochocka, M.; Kis, G.; Wojtowicz, Z.
Histological changes in the retina in experimental alloxan-induced diabetes in rabbits. Ann. Univ. Mariae
Curie Sklodowska Med. 2001,56, 81–84. [PubMed]
64.
Alvarez, Y.; Chen, K.; Reynolds, A.L.; Waghorne, N.; O’Connor, J.J.; Kennedy, B.N. Predominant cone
photoreceptor dysfunction in a hyperglycaemic model of non-proliferative diabetic retinopathy. Dis. Model.
Mech. 2010,3, 236–245. [CrossRef]
65.
Shin, H.J.; Chung, H.; Kim, H.C. Association between integrity of foveal photoreceptor layer and visual
outcome in retinal vein occlusion. Acta Ophthalmol. 2011,89, e35–e40. [CrossRef]
66.
Forooghian, F.; Stetson, P.F.; Meyer, S.A.; Chew, E.Y.; Wong, W.T.; Cukras, C.; Meyerle, C.B.; Ferris, F.L., 3rd.
Relationship between photoreceptor outer segment length and visual acuity in diabetic macular edema.
Retina 2010,30, 63–70. [CrossRef]
67.
Ito, S.; Miyamoto, N.; Ishida, K.; Kurimoto, Y. Association between external limiting membrane status and
visual acuity in diabetic macular oedema. Br. J. Ophthalmol. 2013,97, 228–232. [CrossRef]
68.
Park, S.H.; Park, J.W.; Park, S.J.; Kim, K.Y.; Chung, J.W.; Chun, M.H.; Oh, S.J. Apoptotic death of photoreceptors
in the streptozotocin-induced diabetic rat retina. Diabetologia 2003,46, 1260–1268. [CrossRef]
69.
Ottlecz, A.; Bensaoula, T. Captopril ameliorates the decreased Na+,K(+)-ATPase activity in the retina of
streptozotocin-induced diabetic rats. Investig. Ophthalmol. Vis. Sci. 1996,37, 1633–1641.
70.
Ottlecz, A.; Garcia, C.A.; Eichberg, J.; Fox, D.A. Alterations in retinal Na+, K(+)-ATPase in diabetes:
Streptozotocin-induced and Zucker diabetic fatty rats. Curr. Eye Res. 1993,12, 1111–1121. [CrossRef]
71.
Phipps, J.A.; Fletcher, E.L.; Vingrys, A.J. Paired-flash identification of rod and cone dysfunction in the diabetic
rat. Investig. Ophthalmol. Vis. Sci. 2004,45, 4592–4600. [CrossRef]
72.
Kern, T.S.; Berkowitz, B.A. Photoreceptors in diabetic retinopathy. J. Diabetes Investig.
2015
,6, 371–380.
[CrossRef] [PubMed]
73.
Country, M.W. Retinal metabolism: A comparative look at energetics in the retina. Brain Res.
2017
,1672,
50–57. [CrossRef]
74.
Tang, P.H.; Kono, M.; Koutalos, Y.; Ablonczy, Z.; Crouch, R.K. New insights into retinoid metabolism and
cycling within the retina. Prog. Retin Eye Res. 2013,32, 48–63. [CrossRef]
75.
Kolesnikov, A.V.; Ala-Laurila, P.; Shukolyukov, S.A.; Crouch, R.K.; Wiggert, B.; Estevez, M.E.;
Govardovskii, V.I.; Cornwall, M.C. Visual cycle and its metabolic support in gecko photoreceptors. Vision
Res. 2007,47, 363–374. [CrossRef] [PubMed]
76.
LaVail, M.M. Rod outer segment disk shedding in rat retina: Relationship to cyclic lighting. Science
1976
,194,
1071–1074. [CrossRef] [PubMed]
77.
Wang, X.; Iannaccone, A.; Jablonski, M.M. Contribution of Muller cells toward the regulation of photoreceptor
outer segment assembly. Neuron Glia Biol. 2005,1, 1–6. [CrossRef] [PubMed]
78.
Jablonski, M.M.; Iannaccone, A. Targeted disruption of Muller cell metabolism induces photoreceptor
dysmorphogenesis. Glia 2000,32, 192–204. [CrossRef]
79. Hurley, J.B. Warburg’s vision. Elife 2017,6, e29217. [CrossRef]
80.
Rueda, E.M.; Johnson, J.E., Jr.; Giddabasappa, A.; Swaroop, A.; Brooks, M.J.; Sigel, I.; Chaney, S.Y.; Fox, D.A.
The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative
phosphorylation, and ~P transferring kinases. Mol. Vis. 2016,22, 847–885.
81.
Mitchell, P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. 1966. Biochim. Biophys.
Acta 2011,1807, 1507–1538. [CrossRef] [PubMed]
82.
Du, J.; Rountree, A.; Cleghorn, W.M.; Contreras, L.; Lindsay, K.J.; Sadilek, M.; Gu, H.; Djukovic, D.; Raftery, D.;
Satr
ú
stegui, J.; et al. Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse
Retina. J. Biol. Chem. 2016,291, 4698–4710. [CrossRef] [PubMed]
83.
Linsenmeier, R.A. Effects of light and darkness on oxygen distribution and consumption in the cat retina.
J. Gen. Physiol. 1986,88, 521–542. [CrossRef] [PubMed]
84.
Ames, A., 3rd; Li, Y.Y.; Heher, E.C.; Kimble, C.R. Energy metabolism of rabbit retina as related to function:
High cost of Na+transport. J. Neurosci. 1992,12, 840–853. [CrossRef]
85.
Perezleon, J.A.; Osorio-Paz, I.; Francois, L.; Salceda, R. Immunohistochemical localization of glycogen
synthase and GSK3beta: Control of glycogen content in retina. Neurochem. Res.
2013
,38, 1063–1069.
[CrossRef]

Antioxidants 2020,9, 905 23 of 29
86.
Winkler, B.S.; Arnold, M.J.; Brassell, M.A.; Puro, D.G. Energy metabolism in human retinal Muller cells.
Investig. Ophthalmol. Vis. Sci. 2000,41, 3183–3190.
87.
Toft-Kehler, A.K.; Skytt, D.M.; Svare, A.; Lefevere, E.; Van Hove, I.; Moons, L.; Waagepetersen, H.S.; Kolko, M.
Mitochondrial function in Müller cells—Does it matter? Mitochondrion 2017,36, 43–51. [CrossRef]
88.
Tsacopoulos, M.; Poitry-Yamate, C.L.; MacLeish, P.R.; Poitry, S. Trafficking of molecules and metabolic signals
in the retina. Prog. Retin Eye Res. 1998,17, 429–442. [CrossRef]
89.
Puchalska, P.; Crawford, P.A. Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and
Therapeutics. Cell Metab. 2017,25, 262–284. [CrossRef]
90.
Bayeva, M.; Sawicki, K.T.; Ardehali, H. Taking diabetes to heart–deregulation of myocardial lipid metabolism
in diabetic cardiomyopathy. J. Am. Heart Assoc. 2013,2, e000433. [CrossRef]
91.
Muoio, D.M. Metabolic inflexibility: When mitochondrial indecision leads to metabolic gridlock. Cell
2014
,
159, 1253–1262. [CrossRef] [PubMed]
92.
Joyal, J.S.; Sun, Y.; Gantner, M.L.; Shao, Z.; Evans, L.P.; Saba, N.; Fredrick, T.; Burnim, S.; Kim, J.S.; Patel, G.;
et al. Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1. Nat. Med.
2016,22, 439–445. [CrossRef] [PubMed]
93.
Tyni, T.; Johnson, M.; Eaton, S.; Pourfarzam, M.; Andrews, R.; Turnbull, D.M. Mitochondrial fatty acid
beta-oxidation in the retinal pigment epithelium. Pediatr. Res. 2002,52, 595–600. [CrossRef] [PubMed]
94.
Atsuzawa, K.; Nakazawa, A.; Mizutani, K.; Fukasawa, M.; Yamamoto, N.; Hashimoto, T.; Usuda, N.
Immunohistochemical localization of mitochondrial fatty acid beta-oxidation enzymes in Muller cells of
the retina. Histochem. Cell. Biol. 2010,134, 565–579. [CrossRef] [PubMed]
95.
Fletcher, A.L.; Pennesi, M.E.; Harding, C.O.; Weleber, R.G.; Gillingham, M.B. Observations regarding
retinopathy in mitochondrial trifunctional protein deficiencies. Mol. Genet. Metab.
2012
,106, 18–24. [CrossRef]
96.
Pearsall, E.A.; Cheng, R.; Matsuzaki, S.; Zhou, K.; Ding, L.; Ahn, B.; Kinter, M.; Humphries, K.M.;
Quiambao, A.B.; Farjo, R.A.; et al. Neuroprotective effects of PPARalpha in retinopathy of type 1 diabetes.
PLoS ONE 2019,14, e0208399. [CrossRef]
97.
Group, A.S.; Group, A.E.S.; Chew, E.Y.; Ambrosius, W.T.; Davis, M.D.; Danis, R.P.; Gangaputra, S.;
Greven, C.M.; Hubbard, L.; Esser, B.A.; et al. Effects of medical therapies on retinopathy progression in type
2 diabetes. N. Engl. J. Med. 2010,363, 233–244. [CrossRef]
98.
Keech, A.C.; Mitchell, P.; Summanen, P.A.; O’Day, J.; Davis, T.M.; Moffitt, M.S.; Taskinen, M.R.; Simes, R.J.;
Tse, D.; Williamson, E.; et al. Effect of fenofibrate on the need for laser treatment for diabetic retinopathy
(FIELD study): A randomised controlled trial. Lancet 2007,370, 1687–1697. [CrossRef]
99.
Schoors, S.; Bruning, U.; Missiaen, R.; Queiroz, K.C.; Borgers, G.; Elia, I.; Zecchin, A.; Cantelmo, A.R.;
Christen, S.; Goveia, J.; et al. Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature
2015,520, 192–197. [CrossRef]
100.
Masser, D.R.; Otalora, L.; Clark, N.W.; Kinter, M.T.; Elliott, M.H.; Freeman, W.M. Functional changes in the
neural retina occur in the absence of mitochondrial dysfunction in a rodent model of diabetic retinopathy.
J. Neurochem. 2017,143, 595–608. [CrossRef]
101.
Lopaschuk, G.D. Fatty Acid Oxidation and Its Relation with Insulin Resistance and Associated Disorders.
Ann. Nutr. Metab. 2016,68 (Suppl. 3), 15–20. [CrossRef]
102.
Swarup, A.; Samuels, I.S.; Bell, B.A.; Han, J.Y.S.; Du, J.; Massenzio, E.; Abel, E.D.; Boesze-Battaglia, K.;
Peachey, N.S.; Philp, N.J. Modulating GLUT1 expression in retinal pigment epithelium decreases glucose
levels in the retina: Impact on photoreceptors and Muller glial cells. Am. J. Physiol. Cell Physiol.
2019
,316,
C121–C133. [CrossRef]
103.
Sanchez-Chavez, G.; Pena-Rangel, M.T.; Riesgo-Escovar, J.R.; Martinez-Martinez, A.; Salceda, R. Insulin
stimulated-glucose transporter Glut 4 is expressed in the retina. PLoS ONE
2012
,7, e52959. [CrossRef]
[PubMed]
104.
Itoh, Y.; Kawamata, Y.; Harada, M.; Kobayashi, M.; Fujii, R.; Fukusumi, S.; Ogi, K.; Hosoya, M.; Tanaka, Y.;
Uejima, H.; et al. Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature
2003,422, 173–176. [CrossRef] [PubMed]
105.
Falomir-Lockhart, L.J.; Cavazzutti, G.F.; Gimenez, E.; Toscani, A.M. Fatty Acid Signaling Mechanisms in
Neural Cells: Fatty Acid Receptors. Front. Cell Neurosci. 2019,13, 162. [CrossRef] [PubMed]

Antioxidants 2020,9, 905 24 of 29
106.
Pan, Y.; Mansfield, K.D.; Bertozzi, C.C.; Rudenko, V.; Chan, D.A.; Giaccia, A.J.; Simon, M.C. Multiple factors
affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase
activity in vivo and in vitro. Mol. Cell Biol. 2007,27, 912–925. [CrossRef] [PubMed]
107.
Germer, A.; Biedermann, B.; Wolburg, H.; Schuck, J.; Grosche, J.; Kuhrt, H.; Reichelt, W.; Schousboe, A.;
Paasche, G.; Mack, A.F.; et al. Distribution of mitochondria within Muller cells–I. Correlation with retinal
vascularization in different mammalian species. J. Neurocytol. 1998,27, 329–345. [CrossRef]
108.
Stone, J.; van Driel, D.; Valter, K.; Rees, S.; Provis, J. The locations of mitochondria in mammalian
photoreceptors: Relation to retinal vasculature. Brain Res. 2008,1189, 58–69. [CrossRef]
109.
Osorio-Paz, I.; Uribe-Carvajal, S.; Salceda, R. In the Early Stages of Diabetes, Rat Retinal Mitochondria
Undergo Mild Uncoupling due to UCP2 Activity. PLoS ONE 2015,10, e0122727. [CrossRef]
110.
Han, W.H.; Gotzmann, J.; Kuny, S.; Huang, H.; Chan, C.B.; Lemieux, H.; Sauve, Y. Modifications in
Retinal Mitochondrial Respiration Precede Type 2 Diabetes and Protracted Microvascular Retinopathy.
Investig. Ophthalmol. Vis. Sci. 2017,58, 3826–3839. [CrossRef]
111.
Kowluru, R.A.; Mishra, M.; Kowluru, A.; Kumar, B. Hyperlipidemia and the development of diabetic
retinopathy: Comparison between type 1 and type 2 animal models. Metabolism
2016
,65, 1570–1581.
[CrossRef] [PubMed]
112.
Kanwar, M.; Chan, P.S.; Kern, T.S.; Kowluru, R.A. Oxidative damage in the retinal mitochondria of diabetic
mice: Possible protection by superoxide dismutase. Investig. Ophthalmol. Vis. Sci.
2007
,48, 3805–3811.
[CrossRef] [PubMed]
113.
Madsen-Bouterse, S.A.; Mohammad, G.; Kanwar, M.; Kowluru, R.A. Role of mitochondrial DNA damage
in the development of diabetic retinopathy, and the metabolic memory phenomenon associated with its
progression. Antioxid. Redox Signal. 2010,13, 797–805. [CrossRef] [PubMed]
114.
Santos, J.M.; Tewari, S.; Kowluru, R.A. A compensatory mechanism protects retinal mitochondria from initial
insult in diabetic retinopathy. Free Radic. Biol. Med. 2012,53, 1729–1737. [CrossRef] [PubMed]
115.
Wang, H.; Zheng, Z.; Gong, Y.; Zhu, B.; Xu, X. U83836E inhibits retinal neurodegeneration in early-stage
streptozotocin-induced diabetic rats. Ophthalmic Res. 2011,46, 19–24. [CrossRef] [PubMed]
116.
Fan, Y.; Lai, J.; Yuan, Y.; Wang, L.; Wang, Q.; Yuan, F. Taurine Protects Retinal Cells and Improves Synaptic
Connections in Early Diabetic Rats. Curr. Eye Res. 2020,45, 52–63. [CrossRef]
117.
Li, X.; Zhang, M.; Zhou, H. The morphological features and mitochondrial oxidative stress mechanism of the
retinal neurons apoptosis in early diabetic rats. J. Diabetes Res. 2014,2014, 678123. [CrossRef]
118.
Chang, J.Y.; Yu, F.; Shi, L.; Ko, M.L.; Ko, G.Y. Melatonin Affects Mitochondrial Fission/Fusion Dynamics in
the Diabetic Retina. J. Diabetes Res. 2019,2019, 8463125. [CrossRef]
119.
Devi, T.S.; Lee, I.; Huttemann, M.; Kumar, A.; Nantwi, K.D.; Singh, L.P. TXNIP links innate host defense
mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia:
Implications for diabetic retinopathy. Exp. Diabetes Res. 2012,2012, 438238. [CrossRef]
120.
Devi, T.S.; Somayajulu, M.; Kowluru, R.A.; Singh, L.P. TXNIP regulates mitophagy in retinal Muller cells under
high-glucose conditions: Implications for diabetic retinopathy. Cell Death Dis. 2017,8, e2777. [CrossRef]
121.
Hombrebueno, J.R.; Cairns, L.; Dutton, L.R.; Lyons, T.J.; Brazil, D.P.; Moynagh, P.; Curtis, T.M.; Xu, H. Uncoupled
turnover disrupts mitochondrial quality control in diabetic retinopathy. JCI Insight
2019
,4, e129760. [CrossRef]
[PubMed]
122.
Krugel, K.; Wurm, A.; Pannicke, T.; Hollborn, M.; Karl, A.; Wiedemann, P.; Reichenbach, A.; Kohen, L.;
Bringmann, A. Involvement of oxidative stress and mitochondrial dysfunction in the osmotic swelling of
retinal glial cells from diabetic rats. Exp. Eye Res. 2011,92, 87–93. [CrossRef] [PubMed]
123. Tien, T.; Zhang, J.; Muto, T.; Kim, D.; Sarthy, V.P.; Roy, S. High Glucose Induces Mitochondrial Dysfunction
in Retinal Muller Cells: Implications for Diabetic Retinopathy. Investig. Ophthalmol. Vis. Sci.
2017
,58,
2915–2921. [CrossRef] [PubMed]
124.
Zhou, P.; Xie, W.; Meng, X.; Zhai, Y.; Dong, X.; Zhang, X.; Sun, G.; Sun, X. Notoginsenoside R1 Ameliorates
Diabetic Retinopathy through PINK1-Dependent Activation of Mitophagy. Cells
2019
,8, 213. [CrossRef]
[PubMed]
125.
Mishra, M.; Kowluru, R.A. DNA Methylation-a Potential Source of Mitochondria DNA Base Mismatch in
the Development of Diabetic Retinopathy. Mol. Neurobiol. 2019,56, 88–101. [CrossRef] [PubMed]
126.
Mohammad, G.; Duraisamy, A.J.; Kowluru, A.; Kowluru, R.A. Functional Regulation of an Oxidative Stress
Mediator, Rac1, in Diabetic Retinopathy. Mol. Neurobiol. 2019,56, 8643–8655. [CrossRef]

Antioxidants 2020,9, 905 25 of 29
127.
Santos, J.M.; Kowluru, R.A. Role of mitochondria biogenesis in the metabolic memory associated with the
continued progression of diabetic retinopathy and its regulation by lipoic acid. Investig. Ophthalmol. Vis. Sci.
2011,52, 8791–8798. [CrossRef]
128.
Santos, J.M.; Kowluru, R.A. Impaired transport of mitochondrial transcription factor A (TFAM) and the metabolic
memory phenomenon associated with the progression of diabetic retinopathy. Diabetes Metab.Res. Rev.
2013
,29,
204–213. [CrossRef]
129.
Santos, J.M.; Tewari, S.; Goldberg, A.F.; Kowluru, R.A. Mitochondrial biogenesis and the development of
diabetic retinopathy. Free Radic. Biol. Med. 2011,51, 1849–1860. [CrossRef]
130.
Schulze, P.C.; Yoshioka, J.; Takahashi, T.; He, Z.; King, G.L.; Lee, R.T. Hyperglycemia promotes oxidative
stress through inhibition of thioredoxin function by thioredoxin-interacting protein. J. Biol. Chem.
2004
,279,
30369–30374. [CrossRef]
131.
Zou, Y.L.; Luo, W.B.; Xie, L.; Mao, X.B.; Wu, C.; You, Z.P. Targeting human 8-oxoguanine DNA glycosylase to
mitochondria protects cells from high glucose-induced apoptosis. Endocrine
2018
,60, 445–457. [CrossRef]
[PubMed]
132.
Zhu, H.; Zhang, W.; Zhao, Y.; Shu, X.; Wang, W.; Wang, D.; Yang, Y.; He, Z.; Wang, X.; Ying, Y.
GSK3beta-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting
synaptic and mitochondrial functions. Mol. Neurodegener. 2018,13, 62. [CrossRef] [PubMed]
133.
Liu, W.Y.; Liou, S.S.; Hong, T.Y.; Liu, I.M. Protective Effects of Hesperidin (Citrus Flavonone) on High Glucose
Induced Oxidative Stress and Apoptosis in a Cellular Model for Diabetic Retinopathy. Nutrients
2017
,9, 1312.
[CrossRef] [PubMed]
134.
Kowluru, R.A.; Mishra, M.; Kumar, B. Diabetic retinopathy and transcriptional regulation of a small molecular
weight G-Protein, Rac1. Exp. Eye Res. 2016,147, 72–77. [CrossRef] [PubMed]
135.
Devi, T.S.; Hosoya, K.; Terasaki, T.; Singh, L.P. Critical role of TXNIP in oxidative stress, DNA damage and
retinal pericyte apoptosis under high glucose: Implications for diabetic retinopathy. Exp. Cell Res.
2013
,319,
1001–1012. [CrossRef]
136.
Duraisamy, A.J.; Mohammad, G.; Kowluru, R.A. Mitochondrial fusion and maintenance of mitochondrial
homeostasis in diabetic retinopathy. Biochim. Biophys. Acta Mol. Basis Dis.
2019
,1865, 1617–1626. [CrossRef]
137.
Leal, E.C.; Aveleira, C.A.; Castilho, A.F.; Serra, A.M.; Baptista, F.I.; Hosoya, K.; Forrester, J.V.; Ambrosio, A.F.
High glucose and oxidative/nitrosative stress conditions induce apoptosis in retinal endothelial cells by a
caspase-independent pathway. Exp. Eye Res. 2009,88, 983–991. [CrossRef]
138.
Madsen-Bouterse, S.A.; Zhong, Q.; Mohammad, G.; Ho, Y.S.; Kowluru, R.A. Oxidative damage of
mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase. Free Radic.
Res. 2010,44, 313–321. [CrossRef]
139.
Xie, L.; Zhu, X.; Hu, Y.; Li, T.; Gao, Y.; Shi, Y.; Tang, S. Mitochondrial DNA oxidative damage triggering
mitochondrial dysfunction and apoptosis in high glucose-induced HRECs. Investig. Ophthalmol. Vis. Sci.
2008,49, 4203–4209. [CrossRef]
140.
Van Meerloo, J.; Kaspers, G.J.; Cloos, J. Cell sensitivity assays: The MTT assay. Methods Mol. Biol.
2011
,731,
237–245. [CrossRef]
141. Betteridge, D.J. What is oxidative stress? Metabolism 2000,49, 3–8. [CrossRef]
142.
Kowluru, R.A. Diabetic retinopathy, metabolic memory and epigenetic modifications. Vision Res.
2017
,139,
30–38. [CrossRef] [PubMed]
143.
Kowluru, R.A.; Tang, J.; Kern, T.S. Abnormalities of retinal metabolism in diabetes and experimental
galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy.
Diabetes 2001,50, 1938–1942. [CrossRef]
144.
Kowluru, R.; Kern, T.S.; Engerman, R.L. Abnormalities of retinal metabolism in diabetes or galactosemia.
II. Comparison of gamma-glutamyl transpeptidase in retina and cerebral cortex, and effects of antioxidant
therapy. Curr. Eye Res. 1994,13, 891–896. [CrossRef] [PubMed]
145.
Berkowitz, B.A.; Gradianu, M.; Bissig, D.; Kern, T.S.; Roberts, R. Retinal ion regulation in a mouse model
of diabetic retinopathy: Natural history and the effect of Cu/Zn superoxide dismutase overexpression.
Investig. Ophthalmol. Vis. Sci. 2009,50, 2351–2358. [CrossRef] [PubMed]
146.
Kowluru, R.A.; Abbas, S.N. Diabetes-induced mitochondrial dysfunction in the retina. Investig. Ophthalmol.
Vis. Sci. 2003,44, 5327–5334. [CrossRef]

Antioxidants 2020,9, 905 26 of 29
147.
Madsen-Bouterse, S.; Mohammad, G.; Kowluru, R.A. Glyceraldehyde-3-phosphate dehydrogenase in
retinal microvasculature: Implications for the development and progression of diabetic retinopathy.
Investig. Ophthalmol. Vis. Sci. 2010,51, 1765–1772. [CrossRef]
148.
Lott, M.E.; Slocomb, J.E.; Shivkumar, V.; Smith, B.; Gabbay, R.A.; Quillen, D.; Gardner, T.W.; Bettermann, K.
Comparison of retinal vasodilator and constrictor responses in type 2 diabetes. Acta Ophthalmol.
2012
,90,
e434–e441. [CrossRef]
149.
Du, Y.; Miller, C.M.; Kern, T.S. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells.
Free Radic. Biol. Med. 2003,35, 1491–1499. [CrossRef]
150.
Adam-Vizi, V. Production of reactive oxygen species in brain mitochondria: Contribution by electron transport
chain and non-electron transport chain sources. Antioxid. Redox. Signal. 2005,7, 1140–1149. [CrossRef]
151.
Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J.
2009
,417, 1–13. [CrossRef]
[PubMed]
152. Bek, T. Mitochondrial dysfunction and diabetic retinopathy. Mitochondrion 2017,36, 4–6. [CrossRef]
153.
Mekala, N.K.; Kurdys, J.; Depuydt, M.M.; Vazquez, E.J.; Rosca, M.G. Apoptosis inducing factor deficiency
causes retinal photoreceptor degeneration. The protective role of the redox compound methylene blue.
Redox. Biol. 2019,20, 107–117. [CrossRef] [PubMed]
154.
Austin, S.; Klimcakova, E.; St-Pierre, J. Impact of PGC-1alpha on the topology and rate of superoxide
production by the mitochondrial electron transport chain. Free Radic. Biol. Med.
2011
,51, 2243–2248.
[CrossRef] [PubMed]
155.
St-Pierre, J.; Buckingham, J.A.; Roebuck, S.J.; Brand, M.D. Topology of superoxide production from different
sites in the mitochondrial electron transport chain. J. Biol. Chem.
2002
,277, 44784–44790. [CrossRef]
[PubMed]
156.
Rosca, M.G.; Vazquez, E.J.; Chen, Q.; Kerner, J.; Kern, T.S.; Hoppel, C.L. Oxidation of fatty acids is the source
of increased mitochondrial reactive oxygen species production in kidney cortical tubules in early diabetes.
Diabetes 2012,61, 2074–2083. [CrossRef]
157.
Ji, L.L.; Gomez-Cabrera, M.C.; Vina, J. Exercise and hormesis: Activation of cellular antioxidant signaling
pathway. Ann. N. Y. Acad. Sci. 2006,1067, 425–435. [CrossRef]
158.
Roy, S.; Kern, T.S.; Song, B.; Stuebe, C. Mechanistic Insights into Pathological Changes in the Diabetic Retina:
Implications for Targeting Diabetic Retinopathy. Am. J. Pathol. 2017,187, 9–19. [CrossRef]
159. Tang, J.; Kern, T.S. Inflammation in diabetic retinopathy. Prog. Retin Eye Res. 2011,30, 343–358. [CrossRef]
160.
Tewari, S.; Santos, J.M.; Kowluru, R.A. Damaged mitochondrial DNA replication system and the development
of diabetic retinopathy. Antioxid. Redox. Signal. 2012,17, 492–504. [CrossRef]
161.
Tewari, S.; Zhong, Q.; Santos, J.M.; Kowluru, R.A. Mitochondria DNA replication and DNA methylation in
the metabolic memory associated with continued progression of diabetic retinopathy. Investig. Ophthalmol.
Vis. Sci. 2012,53, 4881–4888. [CrossRef] [PubMed]
162.
Smith, J.S.; Brachmann, C.B.; Celic, I.; Kenna, M.A.; Muhammad, S.; Starai, V.J.; Avalos, J.L.;
Escalante-Semerena, J.C.; Grubmeyer, C.; Wolberger, C.; et al. A phylogenetically conserved NAD+-dependent
protein deacetylase activity in the Sir2 protein family. Proc. Natl. Acad. Sci. USA
2000
,97, 6658–6663. [CrossRef]
[PubMed]
163.
Canto, C.; Sauve, A.A.; Bai, P. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes.
Mol. Aspects Med. 2013,34, 1168–1201. [CrossRef] [PubMed]
164.
Lin, J.B.; Kubota, S.; Ban, N.; Yoshida, M.; Santeford, A.; Sene, A.; Nakamura, R.; Zapata, N.; Kubota, M.;
Tsubota, K.; et al. NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice. Cell Rep.
2016
,17,
69–85. [CrossRef]
165.
Ido, Y.; Nyengaard, J.R.; Chang, K.; Tilton, R.G.; Kilo, C.; Mylari, B.L.; Oates, P.J.; Williamson, J.R. Early
neural and vascular dysfunctions in diabetic rats are largely sequelae of increased sorbitol oxidation.
Antioxid. Redox. Signal. 2010,12, 39–51. [CrossRef]
166.
Williamson, J.R.; Chang, K.; Frangos, M.; Hasan, K.S.; Ido, Y.; Kawamura, T.; Nyengaard, J.R.; van den Enden, M.;
Kilo, C.; Tilton, R.G. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes
1993
,42, 801–813.
[CrossRef]
167.
Lorenzi, M. The polyol pathway as a mechanism for diabetic retinopathy: Attractive, elusive, and resilient.
Exp. Diabetes Res. 2007,2007, 61038. [CrossRef]

Antioxidants 2020,9, 905 27 of 29
168.
Li, Q.; Hwang, Y.C.; Ananthakrishnan, R.; Oates, P.J.; Guberski, D.; Ramasamy, R. Polyol pathway and
modulation of ischemia-reperfusion injury in Type 2 diabetic BBZ rat hearts. Cardiovasc. Diabetol.
2008
,7, 33.
[CrossRef]
169.
Obrosova, I.G.; Drel, V.R.; Kumagai, A.K.; Szabo, C.; Pacher, P.; Stevens, M.J. Early diabetes-induced
biochemical changes in the retina: Comparison of rat and mouse models. Diabetologia
2006
,49, 2525–2533.
[CrossRef]
170.
Tang, J.; Du, Y.; Petrash, J.M.; Sheibani, N.; Kern, T.S. Deletion of aldose reductase from mice inhibits
diabetes-induced retinal capillary degeneration and superoxide generation. PLoS ONE
2013
,8, e62081.
[CrossRef]
171.
Berthiaume, J.M.; Kurdys, J.G.; Muntean, D.M.; Rosca, M.G. Mitochondrial NAD(+)/NADH Redox State and
Diabetic Cardiomyopathy. Antioxid. Redox Signal. 2019,30, 375–398. [CrossRef]
172.
Diederen, R.M.H.; Starnes, C.A.; Berkowitz, B.A.; Winkler, B.S. Reexamining the Hyperglycemic
Pseudohypoxia Hypothesis of Diabetic Oculopathy. Investig. Ophtal. Vis. Sci.
2006
,47, 2726. [CrossRef]
[PubMed]
173.
Karamanlidis, G.; Lee, C.F.; Garcia-Menendez, L.; Kolwicz, S.C., Jr.; Suthammarak, W.; Gong, G.;
Sedensky, M.M.; Morgan, P.G.; Wang, W.; Tian, R. Mitochondrial complex I deficiency increases protein
acetylation and accelerates heart failure. Cell Metab. 2013,18, 239–250. [CrossRef] [PubMed]
174.
Sung, H.J.; Ma, W.; Wang, P.Y.; Hynes, J.; O’Riordan, T.C.; Combs, C.A.; McCoy, J.P., Jr.; Bunz, F.; Kang, J.G.;
Hwang, P.M. Mitochondrial respiration protects against oxygen-associated DNA damage. Nat. Commun.
2010,1, 5. [CrossRef] [PubMed]
175.
Akie, T.E.; Liu, L.; Nam, M.; Lei, S.; Cooper, M.P. OXPHOS-Mediated Induction of NAD+Promotes Complete
Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease. PLoS ONE
2015
,10, e0125617.
[CrossRef]
176.
Masutani, M.; Suzuki, H.; Kamada, N.; Watanabe, M.; Ueda, O.; Nozaki, T.; Jishage, K.; Watanabe, T.;
Sugimoto, T.; Nakagama, H.; et al. Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to
streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. USA 1999,96, 2301–2304. [CrossRef]
177.
Bai, P.; Canto, C.; Oudart, H.; Brunyanszki, A.; Cen, Y.; Thomas, C.; Yamamoto, H.; Huber, A.; Kiss, B.;
Houtkooper, R.H.; et al. PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation.
Cell Metab. 2011,13, 461–468. [CrossRef]
178.
Mishra, M.; Duraisamy, A.J.; Kowluru, R.A. Sirt1- A Guardian of the Development of Diabetic Retinopathy.
Diabetes 2018,67, 745–754. [CrossRef]
179.
Rydstrom, J. Mitochondrial NADPH, transhydrogenase and disease. Biochim. Biophys. Acta
2006
,1757,
721–726. [CrossRef]
180.
Nickel, A.G.; von Hardenberg, A.; Hohl, M.; Loffler, J.R.; Kohlhaas, M.; Becker, J.; Reil, J.C.; Kazakov, A.;
Bonnekoh, J.; Stadelmaier, M.; et al. Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in
Heart Failure. Cell Metab. 2015,22, 472–484. [CrossRef]
181.
Fisher-Wellman, K.H.; Lin, C.T.; Ryan, T.E.; Reese, L.R.; Gilliam, L.A.; Cathey, B.L.; Lark, D.S.; Smith, C.D.;
Muoio, D.M.; Neufer, P.D. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase
constitute an energy-consuming redox circuit. Biochem. J. 2015,467, 271–280. [CrossRef] [PubMed]
182.
Ronchi, J.A.; Francisco, A.; Passos, L.A.; Figueira, T.R.; Castilho, R.F. The Contribution of Nicotinamide
Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and
Counterbalanced by Other Sources of NADPH in Liver Mitochondria. J. Biol. Chem.
2016
,291, 20173–20187.
[CrossRef]
183.
Ventura-Clapier, R.; Garnier, A.; Veksler, V. Transcriptional control of mitochondrial biogenesis: The central
role of PGC-1alpha. Cardiovasc. Res. 2008,79, 208–217. [CrossRef] [PubMed]
184.
Santos, J.M.; Mishra, M.; Kowluru, R.A. Posttranslational modification of mitochondrial transcription
factor A in impaired mitochondria biogenesis: Implications in diabetic retinopathy and metabolic memory
phenomenon. Exp. Eye Res. 2014,121, 168–177. [CrossRef] [PubMed]
185.
Devi, T.S.; Yumnamcha, T.; Yao, F.; Somayajulu, M.; Kowluru, R.A.; Singh, L.P. TXNIP mediates high
glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells.
Biol. Open 2019,8, bio038521. [CrossRef]
186.
Santos, J.M.; Mohammad, G.; Zhong, Q.; Kowluru, R.A. Diabetic retinopathy, superoxide damage and
antioxidants. Curr. Pharm. Biotechnol. 2011,12, 352–361. [CrossRef]

Antioxidants 2020,9, 905 28 of 29
187.
Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol.
2010
,11,
872–884. [CrossRef]
188.
Zhong, Q.; Kowluru, R.A. Diabetic retinopathy and damage to mitochondrial structure and transport
machinery. Investig. Ophthalmol. Vis. Sci. 2011,52, 8739–8746. [CrossRef]
189.
Berkowitz, B.A.; Roberts, R.; Luan, H.; Bissig, D.; Bui, B.V.; Gradianu, M.; Calkins, D.J.; Vingrys, A.J.
Manganese-enhanced MRI studies of alterations of intraretinal ion demand in models of ocular injury.
Investig. Ophthalmol. Vis. Sci. 2007,48, 3796–3804. [CrossRef]
190.
Bangi, B.B.; Ginjupally, U.; Nadendla, L.K.; Mekala, M.R.; Lakshmi B, J.; Kakumani, A. Evaluation of
Gustatory Function in Oral Submucous Fibrosis Patients and Gutka Chewers. Asian Pac. J. Cancer Prev.
2019
,
20, 569–573. [CrossRef]
191.
Haider, S.Z.; Sadanandan, N.P.; Joshi, P.G.; Mehta, B. Early Diabetes Induces Changes in Mitochondrial
Physiology of Inner Retinal Neurons. Neuroscience 2019,406, 140–149. [CrossRef]
192.
Kern, T.S.; Kowluru, R.A.; Engerman, R.L. Abnormalities of retinal metabolism in diabetes or galactosemia:
ATPases and glutathione. Investig. Ophthalmol. Vis. Sci. 1994,35, 2962–2967.
193.
Berkowitz, B.A.; Roberts, R.; Stemmler, A.; Luan, H.; Gradianu, M. Impaired apparent ion demand in
experimental diabetic retinopathy: Correction by lipoic Acid. Investig. Ophthalmol. Vis. Sci.
2007
,48,
4753–4758. [CrossRef]
194. Kannan, K.; Jain, S.K. Oxidative stress and apoptosis. Pathophysiology 2000,7, 153–163. [CrossRef]
195.
Chen, C.; Peng, S.; Chen, F.; Liu, L.; Li, Z.; Zeng, G.; Huang, Q. Protective effects of pioglitazone on vascular
endothelial cell dysfunction induced by high glucose via inhibition of IKKalpha/beta-NFkappaB signaling
mediated by PPARgamma in vitro. Can. J. Physiol. Pharmacol. 2017,95, 1480–1487. [CrossRef] [PubMed]
196.
Birk, A.V.; Liu, S.; Soong, Y.; Mills, W.; Singh, P.; Warren, J.D.; Seshan, S.V.; Pardee, J.D.; Szeto, H.H.
The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with
cardiolipin. J. Am. Soc. Nephrol. 2013,24, 1250–1261. [CrossRef] [PubMed]
197.
Dudek, J. Role of Cardiolipin in Mitochondrial Signaling Pathways. Front. Cell Dev. Biol.
2017
,5, 90.
[CrossRef]
198.
Birk, A.V.; Chao, W.M.; Bracken, C.; Warren, J.D.; Szeto, H.H. Targeting mitochondrial cardiolipin and the
cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis.
Br. J. Pharmacol. 2014,171, 2017–2028. [CrossRef]
199.
Szeto, H.H.; Liu, S.; Soong, Y.; Birk, A.V. Improving mitochondrial bioenergetics under ischemic conditions
increases warm ischemia tolerance in the kidney. Am. J. Physiol. Renal Physiol.
2015
,308, F11–F21. [CrossRef]
200.
Alam, N.M.; Mills, W.C.t.; Wong, A.A.; Douglas, R.M.; Szeto, H.H.; Prusky, G.T. A mitochondrial therapeutic
reverses visual decline in mouse models of diabetes. Dis. Model. Mech. 2015,8, 701–710. [CrossRef]
201.
Huang, J.; Li, X.; Li, M.; Li, J.; Xiao, W.; Ma, W.; Chen, X.; Liang, X.; Tang, S.; Luo, Y. Mitochondria-targeted
antioxidant peptide SS31 protects the retinas of diabetic rats. Curr. Mol. Med.
2013
,13, 935–945. [CrossRef]
[PubMed]
202.
Escribano-Lopez, I.; Diaz-Morales, N.; Iannantuoni, F.; Lopez-Domenech, S.; de Maranon, A.M.;
Abad-Jimenez, Z.; Banuls, C.; Rovira-Llopis, S.; Herance, J.R.; Rocha, M.; et al. The mitochondrial antioxidant
SS-31 increases SIRT1 levels and ameliorates inflammation, oxidative stress and leukocyte-endothelium
interactions in type 2 diabetes. Sci. Rep. 2018,8, 15862. [CrossRef] [PubMed]
203.
Brand, M.D.; Esteves, T.C. Physiological functions of the mitochondrial uncoupling proteins UCP2 and
UCP3. Cell Metab. 2005,2, 85–93. [CrossRef] [PubMed]
204.
Korshunov, S.S.; Skulachev, V.P.; Starkov, A.A. High protonic potential actuates a mechanism of production
of reactive oxygen species in mitochondria. FEBS Lett. 1997,416, 15–18. [CrossRef]
205.
Arsenijevic, D.; Onuma, H.; Pecqueur, C.; Raimbault, S.; Manning, B.S.; Miroux, B.; Couplan, E.;
Alves-Guerra, M.C.; Goubern, M.; Surwit, R.; et al. Disruption of the uncoupling protein-2 gene in
mice reveals a role in immunity and reactive oxygen species production. Nat. Genet.
2000
,26, 435–439.
[CrossRef]
206.
He, Y.; Luan, Z.; Fu, X.; Xu, X. Overexpression of uncoupling protein 2 inhibits the high glucose-induced
apoptosis of human umbilical vein endothelial cells. Int. J. Mol. Med. 2016,37, 631–638. [CrossRef]
207.
Cui, Y.; Xu, X.; Bi, H.; Zhu, Q.; Wu, J.; Xia, X.; Qiushi, R.; Ho, P.C. Expression modification of uncoupling
proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: The role of reactive
oxygen species in diabetic retinopathy. Exp. Eye Res. 2006,83, 807–816. [CrossRef]

Antioxidants 2020,9, 905 29 of 29
208.
Hinder, L.M.; Sas, K.M.; O’Brien, P.D.; Backus, C.; Kayampilly, P.; Hayes, J.M.; Lin, C.M.; Zhang, H.;
Shanmugam, S.; Rumora, A.E.; et al. Mitochondrial uncoupling has no effect on microvascular complications
in type 2 diabetes. Sci. Rep. 2019,9, 881. [CrossRef]
209.
Wen, Y.; Li, W.; Poteet, E.C.; Xie, L.; Tan, C.; Yan, L.J.; Ju, X.; Liu, R.; Qian, H.; Marvin, M.A.; et al. Alternative
mitochondrial electron transfer as a novel strategy for neuroprotection. J. Biol. Chem.
2011
,286, 16504–16515.
[CrossRef]
210.
Haefeli, R.H.; Erb, M.; Gemperli, A.C.; Robay, D.; Courdier Fruh, I.; Anklin, C.; Dallmann, R.; Gueven, N.
NQO1-dependent redox cycling of idebenone: Effects on cellular redox potential and energy levels. PLoS
ONE 2011,6, e17963. [CrossRef]
211.
Mordente, A.; Martorana, G.E.; Minotti, G.; Giardina, B. Antioxidant properties of 2,3-dimethoxy-5-methyl-6-
(10-hydroxydecyl)-1,4-benzoquinone (idebenone). Chem. Res. Toxicol.
1998
,11, 54–63. [CrossRef] [PubMed]
212.
Heitz, F.D.; Erb, M.; Anklin, C.; Robay, D.; Pernet, V.; Gueven, N. Idebenone protects against retinal damage
and loss of vision in a mouse model of Leber’s hereditary optic neuropathy. PLoS ONE
2012
,7, e45182.
[CrossRef] [PubMed]
213.
Erb, M.; Hoffmann-Enger, B.; Deppe, H.; Soeberdt, M.; Haefeli, R.H.; Rummey, C.; Feurer, A.; Gueven, N.
Features of idebenone and related short-chain quinones that rescue ATP levels under conditions of impaired
mitochondrial complex I. PLoS ONE 2012,7, e36153. [CrossRef]
214.
Yu-Wai-Man, P.; Soiferman, D.; Moore, D.G.; Burte, F.; Saada, A. Evaluating the therapeutic potential of
idebenone and related quinone analogues in Leber hereditary optic neuropathy. Mitochondrion
2017
,36,
36–42. [CrossRef] [PubMed]
215.
Dombi, E.; Diot, A.; Morten, K.; Carver, J.; Lodge, T.; Fratter, C.; Ng, Y.S.; Liao, C.; Muir, R.; Blakely, E.L.;
et al. The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy.
Neurology 2016,86, 1921–1923. [CrossRef]
216.
Klopstock, T.; Yu-Wai-Man, P.; Dimitriadis, K.; Rouleau, J.; Heck, S.; Bailie, M.; Atawan, A.; Chattopadhyay, S.;
Schubert, M.; Garip, A.; et al. A randomized placebo-controlled trial of idebenone in Leber’s hereditary
optic neuropathy. Brain 2011,134, 2677–2686. [CrossRef]
217.
Pemp, B.; Kircher, K.; Reitner, A. Visual function in chronic Leber’s hereditary optic neuropathy during
idebenone treatment initiated 5 to 50 years after onset. Graefes Arch. Clin. Exp. Ophthalmol.
2019
,257,
2751–2757. [CrossRef]
218. Garcia-Medina, J.J.; Rubio-Velazquez, E.; Foulquie-Moreno, E.; Casaroli-Marano, R.P.; Pinazo-Duran, M.D.;
Zanon-Moreno, V.; Del-Rio-Vellosillo, M. Update on the Effects of Antioxidants on Diabetic Retinopathy:
In Vitro Experiments, Animal Studies and Clinical Trials. Antioxidants 2020,9, 561. [CrossRef]
219.
Chous, A.P.; Richer, S.P.; Gerson, J.D.; Kowluru, R.A. The Diabetes Visual Function Supplement Study
(DiVFuSS). Br. J. Ophthalmol. 2016,100, 227–234. [CrossRef]
220.
Hu, B.J.; Hu, Y.N.; Lin, S.; Ma, W.J.; Li, X.R. Application of Lutein and Zeaxanthin in nonproliferative diabetic
retinopathy. Int. J. Ophthalmol. 2011,4, 303–306. [CrossRef]
221.
Garcia-Medina, J.J.; Pinazo-Duran, M.D.; Garcia-Medina, M.; Zanon-Moreno, V.; Pons-Vazquez, S. A 5-year
follow-up of antioxidant supplementation in type 2 diabetic retinopathy. Eur. J. Ophthalmol.
2011
,21, 637–643.
[CrossRef] [PubMed]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).