Under the stress of hyperglycemia, the nerve cells, pigment epithelial cells, and capillary endothelial cells in the retina can undergo glycometabolic reprogramming. Dysfunction of the electron transport chain (ETC)ETC, as the core change, results in energy generation disturbance ^[1][24], oxidative stress, abnormal glucose metabolite production, retinal microcirculation disorders, and other pathophysiological changes, inducing the occurrence of retinopathy ^[2][25].

In the case of continuous hyperglycemia, the overload of NADH and FADH2 produced by TCA can induce high mitochondrial membrane potential, which makes the ETC stagnate in complex III, and the electrons and protons carried by coenzyme Q are difficult to transfer to the downstream of the respiratory chain. At this time, oxygen molecules as electron acceptors can generate superoxide, mediating changes in cell metabolism ^[3][26].

Excess accumulation of intracellular superoxide activates PARP with the depletion of nicotinamide adenine dinucleotide (NAD⁺) ^[4][27]. As a result, the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is significantly reduced ^[5][28], and the glycolysis process is inhibited. At this time, the polyol pathway of glucose metabolism is over-activated; in other words, glucose is converted to sorbitol under the action of aldose reductase, and then sorbitol can be oxidized to fructose ^[6][29]. Due to the decrease of the NAD⁺/NADH ratio, sorbitol, the intermediate product of polyol pathway, accumulates, and its high hydrophilicity can lead to cell hypertonicity, damage retinal capillary endothelial cells, and cause micro-circulation disorders ^[7][30]. The activation of the polyol pathway is also accompanied by further depletion of NAD⁺, which has a positive feedback effect on oxidative stress ^[8][31]. The C106T polymorphism in the aldose reductase gene is associated with the severity of retinopathy in type 2 diabetes ^[9][32]. Inhibition of aldose reductase reduces neuronal apoptosis, glial response, and complement deposition and retinal ne-ovascularization in DR ^[10][33].

The inhibition of glycolysis also promotes the metabolism of fructose-6-phosphate via the hexosamine pathway to UDP-N-acetyl glucosamine (UDP-GlcNAc), which promotes the synthesis of proteoglycans and O-linked glycoproteins. O-GlcNAc can covalently modify the transcription factor Sp1, which activates the expression of glucose-responsive gene plasminogen activator inhibitor-1 (Pal-1) in vascular smooth muscle cells ^[11][34], thus promoting DR ^[12][35]. It has been found that the level of hexosamine in the retinal tissue of diabetic patients is increased ^[7][30], which promotes the downstream O-GlcNAc to play a role in signal transduction, transcription regulation, regulation of cytoskeletal dynamics, and cell division ^[13][36]. Glucosamine, a product of the hexosamine metabolic pathway, causes retinal pericyte loss and the formation of acellular capillaries in non-diabetic animals by inhibiting VEGFR2 and Ang2 in the normal retina ^[14][37]. High hexosamine levels can also enhance cellular oxidative stress by positive feedback, further damaging the mitochondrial respiratory chain by promoting ROS generation ^[15][38]. However, oral administration of glucosamine protected retinal neurons in a mouse model of DR. The mechanism of this bidirectional regulation remains to be clarified ^[14][37].

Attenuation of GAPDH activity results in increased concentration of intracellular triose phosphate, which can be decomposed and acylated to diacyl glycerol(DAG) ^[16][39], and also participates in the formation of various advanced glycation end products (AGEs) ^[17][40], all of which play an important role in the pathogenesis of DR. On the one hand, DAG can directly activate protein kinase C (PKC) in the retina, mainly β- and δ-type isozymes, while the activation of PKC-α and -ɛ is also found in the retina of diabetic rats ^[16][39]. On the other hand, AGE-mediated signaling pathways as well as metabolic products of the polyol pathway are also associated with PKC activation ^[18][19][41,42]. Activated PKC increases the activity of cytosolic phospholipase A2 and promotes the production of arachidonic acid and prostaglandin E2 (PGE2); the latter inhibits the activity of Na+/K+ ATPase ^[20][43], leading to cell edema. PKC-α is related to the increased permeability of vascular endothelial cells under the condition of high glucose ^[21][44], and the activation of PKC-β can mediate retinal vasoconstriction and blood flow reduction by inhibiting the production of nitric oxide and increasing the activity of endothelin-1 ^[22][45]. These mechanisms may result in the injury and necrosis of retinal cells and affect the function of the retina. In addition, PKC can induce the expression of vascular endothelial growth factor (VEGF) in retinal tissue, thus increasing vascular permeability and promoting angiogenesis, which is closely related to the non-proliferative and proliferative changes in DR, respectively ^[23][24][46,47].

AGEs refer to a series of proteins, of which amino-groups are modified by intracellular dicarbonyl products (including glyoxal, methylglyoxal, and 3-deoxyglucosone), can be produced massively and secreted to extracellular medium when glycolysis is blocked. AGEs can change the function of intracellular and extracellular proteins, and are associated with intracellular signal transduction, metabolic regulation and cell adhesion ^[3][26]. Stitt et al. found that the content of AGEs in retinal blood vessels of diabetic mice was increased ^[25][48], and AGEs could interact with the receptor for advanced glycation end products (RAGE) on the plasma membrane of adjacent cells, exerting pathological effects, including promoting the activation of nuclear factor Kappa B (NF-κB) in retinal pericytes to induce their apoptosis, up-regulating the expression of VEGF in retinal capillary endothelial cells to increase vascular permeability, and activating the RhoA/ROCK signaling pathway as well as inducing moesin phosphorylation to promote retinal neovascularization ^[26][27][28][49,50,51]. AGEs can also lead to retinal vascular hyperpermeability by disrupting intercellular adhesion and tight junctions ^[29][52]. AGEs can up-regulate the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of endothelial cells, resulting in leukocyte stasis in the microcirculation and causing microcirculation disorders ^[30][53]. Ying et al. proposed that the severity of DR can be predicted by measuring AGEs ^[31][54]. As a result, researchers have begun to explore the possibility of delaying the progression of DR by inhibiting AGEs. Hammes et al. found that AGE-inhibitor amino-guanidine can inhibit the proliferation of abnormal retinal endothelial cells and significantly reduce pericyte shedding, thereby inhibiting the progression of DR ^[32][55]. Endogenous Glyoxalase I can inhibit the production of AGEs, and Maisonpierr et al. have found that its overexpression could inhibit the increase of Angiopoietin-2 (Ang-2) expression in Müller cells induced by hyperglycemia, thereby reducing damage to pericytes and capillary endothelial cells ^[33][56].

Abnormal lipid metabolism occurs in more than 75% of individuals with type 2 diabetes ^[34][57]. Excessive lipid accumulation and abnormal lipid metabolites have been proved to be important mechanisms for the progression of DR. Mitochondria, as the site of lipid metabolism, incur damage related to abnormal lipid metabolism ^[35][58]. High concentration of palmitate culture and high glucose induction can play a synergistic role in retinal endothelial cells, aggravating mtDNA damage ^[36][59]. Excessive production of ceramide, acrolein and incomplete β-oxidation products of fatty acids in retinal cells under high glucose environment are also associated with mitochondrial damage.

Under physiological conditions, activated fatty acids generate acyl-CoA, which enters mitochondria for β-oxidation, followed by terminal metabolism through the Krebs cycle ^[37][60]. In mice with DR, the β-oxidation of fatty acids in the mitochondria of retinal cells is incomplete, and excessive intermediate products accumulate to produce toxic lipid peroxidation products, which in turn cause mitochondrial damage ^[38][39][61,62].

There are many kinds of sphingolipids in mitochondria, including sphingomyelin and ceramide ^[40][41][63,64], as well as metabolism-related enzymes, such as ceramide synthetase, acidic and neutral sphingomyelinase, and neutral ceramidase ^[35][58]. In normal cells, ceramide is minimally expressed in the cytoplasmic membrane, while a high glucose level induces ceramide accumulation in a concentration- and time-dependent manner ^[42][43][44][65,66,67]. Levitsky et al. found that ceramide induced by acid sphingomyelinase increased in mitochondria of retinal pigment epithelial cells in streptozotocin-induced diabetic rats, resulting in respiratory chain dysfunction. Inhibition of acid sphingomyelinase restores the function of the respiratory chain ^[45][68]. In terms of mechanism, excessive ceramide can inhibit the electron transfer function of complex III ^[46][69], promote the production of Sphingosine-1-Phosphate (S1P) and hexadecenal and the activation of BAX/BAK, increasing the permeability of the mitochondrial outer membrane and the release of cytochrome C. Finally, apoptosis is induced ^[47][48][70,71].

Acrolein is overproduced in the retinal cells of diabetic individuals, and the production of its protein-bound products is associated with the progression of DR ^[49][72]. As a biomarker, FDP-lysine can reflect the content of acrolein, which is increased in hemoglobin and vitreous humor in patients with proliferative diabetic retinopathy ^[50][51][73,74], and its level in retinal Müller cells of diabetic animal models also parallels the progress of the disease ^[52][75]. Polyamine oxidation and lipid peroxidation are the main intrinsic pathways of acrolein generation during DR pathology ^[49][53][72,76]. By depleting antioxidants such as glutathione ^[54][77] and promoting oxidative stress by forming protein carbonyls ^[55][78], excessive acrolein reduces the membrane potential of mitochondria and the activity of complexes I, II, and IV, resulting in mitochondrial damage in retinal pigment epithelial cells ^[56][57][79,80]. The corresponding metabolic and biochemical changes also occur in pathological processes such as retinal inflammatory response, ganglion cell degeneration, blood-retinal barrier damage, and Müller cell dysfunction ^[49][72].

Extracellular accumulation of abnormal lipid metabolites can also promote DR. Highly oxidized and glycosylated low-density lipoprotein accumulation in the lumen and extravasation of retinal capillaries is an early feature of DR ^[58][81]. Abnormally modified lipoproteins can induce apoptosis by increasing mitochondrial outer membrane permeability through the Bax pathway ^[59][82].

High glucose can not only reprogramme the metabolism of cells, but also regulate epigenetics by changing the activity of corresponding modifying enzymes, resulting in changes in gene expression, including a number of nuclear-encoded mitochondrial function-related genes and mitochondrial genome genes. The reprogramming of the expression of these genes is closely related to mitochondrial damage.

The most prominent epigenetic change is increased methylation levels ^[60][9]. Among patients with type II diabetes, DNA methylation of key genes in islets increases, which inhibits their expression ^{[61][62][63][64]}[83,84,85,86], and DNA methylation levels are altered in adipose tissue, liver, and skeletal muscle ^{[65][66][67][68][69][70]}[87,88,89,90,91,92]. Methylation of these genes may be directly induced by high glucose and HbA1C ^[63][64][71][85,86,93]. Kowluru et al. found that in the rat model of type 2 diabetes induced by high-fat diet, DNA Methyltransferase 1 (DNMT1) in retinal capillaries was highly expressed in the early stage of diabetes, which could affect the methylation of a series of genes related to retinal damage ^[72][94].

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a component of the NADPH oxidase 2 (Nox2) holoenzyme ^[73][95], and the latter is able to induce ROS production in mitochondria ^[74][96], mediating oxidative stress. In the rat model of type 2 diabetes mellitus induced by a high-fat diet, the Rac1 promoter was methylated in retinal microvascular endothelial cells, and a large number of Nox2 produced promoted the accumulation of intracellular ROS, resulting in the damage to mtDNA and the respiratory chain ^[72][94]. In retinal cells of DR patients, methylation of H3K4 in the Keap1 promoter was significantly increased, which activated Keap1 transcription, blocking the nuclear transport of nuclear factor erythroid-2 related factor 2 (Nrf2), thereby inhibiting its antioxidant effects and exacerbating mitochondrial damage ^[75][76][77][97,98,99]. Histone hypermethylation occurred in Sod2 promoter under high glucose conditions (H3K4 monomethylation and dimethylation decreased, while trimethylation increased), accompanied by an increase in acetylation level (H3K9ac). As a result, the down-regulation of Sod2 expression and the reduction of intracellular antioxidant MnSOD content also promote oxidative stress damage ^[78][79][80][100,101,102]. Mitofusin 2 (Mfn2), which mediates mitochondrial outer membrane fusion, is down-regulated in retinal endothelial cells cultured in high glucose due to hypermethylation of the promoter, interfering with mitochondrial homeostasis ^[81][103].

Mitochondrial DNA (mtDNA), which does not bind to histones, encodes a total of 13 proteins, all of which are important components of the respiratory chain. DNA polymerase γ, encoded by the nuclear gene POLG, is responsible for mtDNA replication. In DR, the CpG island of POLG regulatory region is highly methylated, which inhibits the transcription of POLG and affects the replication of mtDNA ^[82][104]. MutL homolog 1 (MLH1) is involved in mismatch repair during mtDNA replication. In human retinal endothelial cells cultured with high glucose, the methylation level of MLH1 promoter is increased, down-regulating its transcription and affecting the replication accuracy and function of mtDNA ^[83][105], which may reduce the activity of respiratory chain complex I and the antioxidant capacity of mitochondria ^[84][106].

The mtDNA itself is also susceptible to methylation under high glucose conditions. This mostly occurs in the Displacement loop (D-Loop) of mtDNA, which, as a non-coding region, contains transcription elements and also controls DNA polymerase γ-dependent mtDNA replication. D-Loop has a loose structure and is susceptible to various modifying enzymes ^[85][107]. Mitochondrial DNA methyltransferase (DNMT) is also highly expressed in retinal cells under high glucose condition, which can highly methylate the D-Loop region of mtDNA and cause mitochondrial damage in retinal cells ^[86][87][108,109]. Inhibition of DNMT can reduce mtDNA damage, improve transcriptional repression of mtDNA induced by high glucose, and restore ETC function ^[88][110]. At the same time, inhibition of promoter methylation may restore the activity of Sod2 (see above), which can inhibit the methylation of D-Loop in mouse retinal microvascular endothelial cells and ensure the operation of base mismatch repair mechanism ^[89][111].

Interestingly, Zhong et al. found that the promoter methylation level (H3K9me2) of Matrix Metalloproteinase-9 (MMP-9) in retinal microvascular endothelial cells of diabetes mice decreased, while the acetylation level (H3K9Ac) increased ^{[77][90][91][92]}[99,112,113,114], resulting in high expression of MMP-9, damaging mitochondria and inducing apoptosis of retinal microvascular cells ^[90][93][112,115]. In view of this, the influence of a high glucose environment on epigenetic modification of the genome may be diverse and complex. More studies are needed to clarify the expression patterns of mitochondria-related nuclear coding genes and mitochondrial genome in retinal cells under high glucose environment, and the exploration of omics can also provide valuable reference.

Autophagy is a highly conserved biological process, which can be induced by growth factor deficiency, hypoxia, cell starvation, oxidative stress, and other conditions, and maintains cell survival and homeostasis by degrading and reusing some intracellular proteins and organelles.

Mitophagy is a process in which cells selectively degrade damaged mitochondria by acting on themselves to control the number and quality of mitochondria, mediate cell differentiation and metabolic reprogramming ^[94][116], and the most typical pathway is PINK1-Parkin-mediated ubiquitin-dependent mitophagy ^[95][117]. Under stress conditions, when the mitochondrial outer membrane is damaged, the transmembrane transport of PINK1 is blocked and retained on the outer membrane, which recruits and activates the ubiquitin ligase Parkin1; the latter constructs a polyubiquitinated modification chain on the mitochondrial outer membrane protein, recruits the adaptor protein, and then the adaptor protein binds to LC3 to form a double-membrane-wrapped autophagosome. Autophagosomes subsequently bind to lysosomes and damaged mitochondria are engulfed ^[96][118].

Retinal cells can also protect themselves through mitophagy in the environment of high glucose.

Many studies have shown that mitophagy is upregulated during DR ^[97][119]. Devi et al. found that Parkin accumulated in mitochondria of retinal Müller cell line rMC1 under high glucose induction, and then induced autophagy ^[98][120]. Mitophagy plays a protective role in DR, and notoginsenoside R1 (NGR1) can alleviate the damage of retinal Müller cells by further enhancing PINK1-Parkin-dependent mitophagy ^[99][121]. Mitophagy is also critical for the function and survival of cone cells under hyperglycemic conditions ^[100][122]. In retinal vascular endothelial cells, activating bile acid G-protein-coupled membrane receptor (TGR5) enhances mitophagy, thereby reducing endothelial dysfunction and slowing the progression of DR ^[101][123]. In retinal pigment epithelial cells, Sirt3, a deacetylase in mitochondria, can activate mitophagy through Foxo3a/PINK1-Parkin pathway and protect the retina in a high glucose environment ^[102][124]. Normal mitophagy is essential for the maintenance of mitochondrial oxidative phosphorylation and ATP synthesis, which may be one of the mechanisms for mitophagy to exert cytoprotective effects in DR ^[103][125].

Thioredoxin-interacting protein (TXNIP) plays an important role in the maintenance of cellular homeostasis in the pathological process of DR. Under high glucose stress, the expression of TXNIP increases, which can antagonize the oxidative stress ^[104][126]. On the other hand, TXNIP can also play a cytoprotective role in promoting mitochondrial division and autophagy ^[105][127]. Up-regulated TXNIP in high glucose induces nitroso-modification of dynamin related protein 1 (Drp1) to promote mitochondrial fission ^[106][128], while modified Drp1 promotes TXNIP translocation to mitochondria, mediating mitophagy through multiple pathways ^[107][129]. For example, TXNIP can promote LC3BII-mediated mitophagy in retinal Müller cells of diabetic rats ^[98][120]. TXNIP causes nitrosylation, nuclear export, and cytoplasm localization of high mobility group box 1 protein (HMGB1), which competes with the cytoplasm-localized anti-apoptotic protein B-cell lymphoma 2 (BCL-2) for binding to the autophagy-related protein Beclin 1 ^[108][130], thereby inducing autophagy.

As mentioned previously, oxidative stress is prone to occur in cells under high glucose conditions. Antioxidant substances in retinal cells of diabetic individuals are reduced (e.g., MnSOD is reduced ^[109][110][133,134]), while the oxidative system is hyperactive (e.g., Nox2 is increased ^[111][135]), and a large amount of ROS is generated to cause mitochondrial damage. Mitochondrial damage can induce apoptosis of retinal capillary endothelial cells, pericytes, and neurons through content extravasation and activation of apoptosis-related signaling pathways ^{[112][113][114][115]}[136,137,138,139]. Among them, pericytes are more sensitive to high glucose and more prone to apoptosis than endothelial cells ^[116][140].

The opening of the PT pore in the outer mitochondrial membrane and the extravasation of proapoptotic substances are considered to be the characteristic events of mitochondrial pathway apoptosis. In retinal endothelial cells, high glucose can induce the release of mitochondrial cytochrome C, which may be related to the decrease of Cx43 channel activity in the outer mitochondrial membrane ^[82][104]. The release of cytochrome C triggers a caspase-mediated cascade that ultimately induces apoptosis. Santiago et al. found that high glucose-induced apoptosis in retinal neurons was related to the release of apoptosis-inducing factor (AIF) by mitochondria, but not dependent on the activation of caspases ^[117][141]. It is worth mentioning that RNA in mitochondria can also be released into the cytosol during mitochondrial damage induced by high glucose and exist as double-stranded RNA ^[118][142], which can interact with RNA-dependent protein kinase (PKR), mediating apoptosis of retinal neurons ^[119][143]. High glucose activates the NF-κB signaling pathway, enhances oxidative stress in Müller cells, and is also involved in inducing apoptosis in the mitochondrial pathway ^[120][144]. As a transcription factor, NF-kB can promote the expression of Matrix Metalloproteinase-2 (MMP-2) and MMP-9 ^[121][145], both of which are involved in inducing apoptosis of retinal microvascular endothelial cells ^{[90][93][122]}[112,115,146]. In the diabetic state, MMP-2 is activated, which promotes the production of superoxide, accelerating mitochondrial membrane damage and cytochrome C leakage ^[122][146]. MMP-9 is also upregulated in high glucose and accumulates in mitochondria, increasing mitochondrial membrane permeability, which in turn promotes the entry of the pro-apoptotic protein BCL2-associated X protein (Bax) into mitochondria ^[90][93][112,115], mediating the occurrence of apoptosis.

High glucose can also induce apoptosis through the mitochondrial pathway by triggering disequilibrium of calcium homeostasis. Liu et al. found that sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) is inactivated by irreversible oxidative modification of Cys674 residue, which disrupts intracellular calcium homeostasis and promotes apoptosis ^[123][124][147,148].