Aralia elata (Miq) Seem Extract Decreases O -GlcNAc Transferase Expression and Retinal Cell Death in Diabetic Mice

Abstract

Aralia elata (Miq) Seem (AES) is a medicinal plant used in traditional Chinese and Korean medicine for the treatment of several diseases, including diabetes. This study aimed to investigate the neuroprotective effect of AES extract against high glucose-induced retinal injury in diabetic mice. AES extract (20 and 100 mg/kg body weight) was orally administered to control mice or mice with streptozotocin-induced diabetes. Protein levels of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element-binding protein (SREBP)-1, thioredoxin-interacting protein (TXNIP), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) were analyzed by western blotting. Colocalization of terminal deoxynucleotide transferase-mediated dUTP nicked-end labeling (TUNEL)-positive ganglion cells and OGT, ChREBP, or TXNIP were monitored using double immunofluorescence analysis. Interaction between ChREBP and OGT was assessed using coimmunoprecipitation analysis. AES extract protected the retinas from neuronal injury and decreased levels of OGT, ChREBP, TXNIP, SREBP-1, FAS, and ACC in the diabetic retinas. AES extract reduced colocalization of TUNEL-positive ganglion cells and OGT, ChREBP, or TXNIP in the diabetic retinas. Coimmunoprecipitation analysis indicated that AES extract reduced interaction between ChREBP and OGT and attenuated ganglion cell death in diabetic retinas. Moreover, the ChREBP that colocalized with OGT or the TUNEL signal was significantly decreased in diabetic mice treated with AES extract. These findings show that AES extract can alleviate OGT-, ChREBP-, TXNIP-, or SREBP-1-related retinal injury in diabetic retinopathy.

Introduction

Diabetic retinopathy (DR) is a common complication of diabetes that leads to blindness in the western world.¹ Neurodegenerative changes such as neural apoptosis and loss of ganglion cells occur in the earliest stages of DR.² Aralia elata (Miq) Seem (AES) has been known to be safe and effective for the treatment of several diseases, including diabetes.^{3

–12} Saponins and several glycosides seem to be the main bioactive components of AES.^{5,8,11,13
–15} A previous study found that AES extract affects blood glucose levels and could protect against neuronal death in DR.¹⁶ We examined how AES protects the ganglion cells of the inner retinal layers from cell death in DR. Studies show that lipid synthesis dysregulation is associated with obesity and type 2 diabetes, and that carbohydrate-responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP) are two transcriptional regulators of glucose and lipid metabolism that affect lipogenic gene expression.¹⁷

ChREBP plays a role in various processes, ranging from glucolipotoxicity to apoptosis or proliferation in specific cell types.¹⁸ ChREBP activity is regulated through multiple posttranslational modifications.¹⁹ Hyperglycemia-induced O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins increases glucotoxicity.²⁰ Of note, O-GlcNAc modification increases ChREBP protein levels and transcriptional activity in liver.²¹ O-GlcNAc transferase (OGT) facilitates the interaction and O-GlcNAc modification of key insulin signaling regulators.²² Moreover, thioredoxin-interacting protein (TXNIP), which is transcriptionally regulated by ChREBP,²³ is a crucial mediator of glucotoxicity-induced β-cell apoptosis.²⁴ The mechanism through which glucose activates ChREBP is complex,¹⁸ and its role in DR has not yet been elucidated.

SREBP, a master regulator of fatty acid synthase (FAS), controls lipogenesis²⁵ and is involved in type 2 diabetes.²⁶ SREBPs, especially SREBP-1c, have a critical role in the pathogenesis of the metabolic syndrome, and therefore identification of compounds that inhibit SREBPs may be useful in the treatment of metabolic syndrome or to ameliorate its inherent complications.²⁷ In this study, we assessed whether AES affects the adverse changes in retinal neuronal death of diabetic mice, in part, driven by posttranslational modification of ChREBP and SREBP, with the aim of facilitating the development of novel agents for protecting against retinal damage in DR.

Materials and Methods

Sample preparation for high performance liquid chromatography

AES freeze-dried powder prepared as described previously⁶ was obtained from Medvill Co., Ltd. (Seoul, Korea). The reference standard (quercetin) and AES (1 g) was dissolved in CH₃CN (50 mL) in the concentration of 1 mg/mL and stored at 4°C. The filtrates were concentrated under vacuum and filtered with a 0.45 μm filter before use.

High performance liquid chromatography analysis

Chromatographic separation was performed on Agilent 1200 high performance liquid chromatography (HPLC) system equipped with YMC-C18 column (4.6 × 250 mm, 5-mm) (Agilent Technologies, San Jose, CA, USA). The column temperature was maintained at 35°C, and the flow rate was 1 mL/min. The detector was UV 260 nm. One of the peaks detected by HPLC analysis was identified as the quercetin (Fig. 1A, B).

FIG. 1.

HPLC and MS chromatograms of CH3CN extract of AES (A and C, respectively), and standard Quercetin (B and D, respectively). *Quercetin identified in AES. AES, Aralia elata (Miq) Seem; HPLC, high performance liquid chromatography; MS, mass spectrometry. Color images available online at www.liebertpub.com/jmf

Mass spectrometry

Identification of peaks was performed using a Synapt G2 quadrupole time-of-flight mass spectrometer (Waters), which was equipped with an electrospray ionization (ESI). The instrument was operated using ESI source in positive and negative mode simultaneously. The source voltage was set to 3100 and −2500 V for ESI+ and ESI−, respectively. The cone voltage, desolvation gas (nitrogen) flow rate, and desolvation temperature were 35 V, 800 L/h, and 350°C, respectively. Mass data were obtained in positive ion mode by a full scan of m/z 50–1200. Data acquisition and processing were carried out using a MassLynx software workstation. One of the abundant peaks shown by HPLC analysis was identified as quercetin (Fig. 1C, D), which is likely to be one of the bioactive compounds, which may protect the retinas from neuronal injury.

Animals

Diabetes was induced in male C57BL/6 mice (KOATEC, Pyeongtaek, Korea), as previously described.²⁸ All mice were treated in accordance with the guidelines of the Institutional Animal Care and Use Committee of Gyeongsang National University. Control mice were injected with phosphate-buffered saline (PBS). All mice were sacrificed at 2 months after the final injection of 2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-glucopyranose (STZ) or saline. Blood was obtained by tail puncture, and induction of diabetes was verified weekly after STZ injection by evaluating blood glucose concentrations using a Precision glucometer (Abbott, Alameda, CA, USA). Mice with a blood glucose concentration ≥250 mg/dL were considered diabetic.

Cell culture

The ARPE-19 human retinal pigment epithelial (RPE) cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and grown on cell culture plates at 37°C in Dulbecco's modified Eagle medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 μg/mL streptomycin, and 100 units/mL penicillin (Invitrogen). Cells were grown in low-glucose (5 mM) or high-glucose (25 mM) medium.

Administration of AES

AES freeze-dried powder was orally administered, as described previously.¹⁶ Mice were treated once a day for 8 weeks after the final STZ or saline injections. Mice were divided into four groups; (1) saline-treated control group, (2) saline-treated diabetic group, (3) AES (20 mg/kg)-treated diabetic group, and (4) AES (100 mg/kg)-treated diabetic group. Blood glucose levels and body weights were measured weekly.

Antibodies

The following antibodies were used: OGT (sc-74546, Santa Cruz Biotechnology; ab96718, Abcam, Cambridge, United Kingdom), ChREBP (NB400-135; Novus Biologicals, Littleton, CO, USA), TXNIP (sc-166234; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SREBP-1 (sc-8984; Santa Cruz Biotechnology), FAS (#3180; Cell Signaling Technology, Danvers, MA, USA), acetyl CoA carboxylase (ACC) (#3662; Cell Signaling Technology), γ-Syn (ab55424; Abcam), β-actin (A5441; Sigma, St. Louis, MO, USA), secondary horseradish-peroxidase-conjugated goat anti-mouse IgG (#31430; Pierce Biotechnology, Rockford, IL, USA), and goat anti-rabbit IgG (#31460; Pierce Biotechnology).

Western blotting analysis

Protein extraction and western blotting were performed as described previously.²⁸

Immunoprecipitation

Immunoprecipitation was performed as described previously.²⁹

Immunohistochemistry analysis

Immunohistochemistry was performed on frozen retinal sections (5-μm thick), as described previously.³⁰ In brief, after blocking with serum, the sections were incubated with a primary antibody against OGT, TXNIP, ChREBP, or SHREBP followed by an Alexa 488- or −594-conjugated secondary IgG (Molecular Probes, Carlsbad, CA, USA). The sections were mounted with mounting medium that contained DAPI (ProLong Gold Antifade Reagent; Invitrogen). All retinal images were captured at a distance of ∼0.8–1.0 mm from the optic nerve head using a JP IX2-DSU disk-scanning confocal microscope (Olympus, Hamburg, Germany).

Immunofluorescence analysis

Immunofluorescence analysis was performed as described previously.²⁹ In brief, for double staining of OGT and ChREBP or SREBP, we prepared 5-μm-thick retinal sections. The slices were incubated in blocking solution (2% normal donkey serum, 0.5% Triton X-100, 0.05% sodium azide in 0.05 M PBS, pH 7.4) for 1 h, followed by incubation at 4°C overnight with mixed primary antibodies (anti-OGT and ChREBP or SREBP antibodies). After several washes in 0.1 M PBS, fluorescent secondary antibodies (goat anti-Mouse IgG conjugated to Alexa Fluor 488 [A-11029; Thermo Scientific, Waltham, MA, USA] or donkey anti-Rabbit IgG conjugated to Alexa Fluor 594 [A-21207; Thermo Scientific]) were applied, and sections were mounted with mounting medium, including DAPI (Invitrogen). For double staining of ChREBP, SREBP, or OGT along with TUNEL, ChREBP, SREBP, or OGT was labeled with Alexa 488, and immunofluorescent staining for ChREBP, SREBP, or OGT was followed by TUNEL staining. Digital images were captured using a fluorescence microscope (BX51-DSU; Olympus, Tokyo, Japan), and merged to reveal colocalization of proteins in the retinas.

Statistical analyses

Quantitative analyses were performed using Image J image analysis software (Molecular Devices, Sunnyvale, CA, USA) and GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Data are representative of three independent experiments and presented as the mean ± standard error of the mean. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc analysis or t-test to compare groups. Results were considered statistically significant when P was less than .05.

Results

AES reduces levels of OGT in the diabetic retinas

OGT facilitates O-GlcNAc modification of ChREBP, and O-GlcNAcylation increases ChREBP,²¹ which plays a potential role in glucolipotoxicity and apoptosis.¹⁸ Therefore, we assessed whether AES affects levels of OGT in the diabetic retinas. We found that levels of OGT were significantly elevated in the diabetic retinas compared with that in controls (P < .005; Fig. 2A). However, AES treatment significantly lowered OGT levels in the diabetic retinas, compared with those not treated with the low or high doses of AES (20 or 100 mg/kg, respectively; P < .005; Fig. 2A), consistent with the data in the immunofluorescence analysis (Fig. 2B). We also found that OGT is present mostly in the nuclear region in the ganglion cell layer (GCL) of the diabetic retinas (Fig. 2B).

FIG. 2.

(A) Representative Western blot and quantification of OGT in the retinas of control (CTL, n = 5) and diabetic mice with (DM+AE, n = 5) or without AE (DM, n = 5) at 2 months after induction of diabetes. Band intensity was normalized to β-actin. Data are presented as means ± SEM. **P < 0.005, vs. CTL; ^†† P < 0.001, vs. DM by ANOVA. These experiments were repeated at least three times. (B) Representative images of immunofluorescence staining for OGT plus 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization in the retinas of control (CTL, n = 5) or diabetic mice with or without AES. The arrowheads indicate OGT-positive cells that were stained for DAPI in the retinas of diabetic mice. Scale bar, 50 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OGT, O-GlcNAc transferase; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment. Color images available online at www.liebertpub.com/jmf

AES decreases ChREBP and TXNIP levels and inhibits OGT-ChREBP interaction in the diabetic retinas

ChREBP promotes glucolipotoxicity as well as apoptosis and affects insulin sensitivity.¹⁸ Therefore, we tested whether AES affects ChREBP activation in the diabetic retinas. Western blotting analysis showed that levels of ChREBP were significantly increased in the diabetic retinas compared with that in controls (P < .05; Fig. 3A). Importantly, AES was able to reverse this increase in ChREBP in the diabetic retinas (P < .05; Fig. 3A). In addition, to evaluate the effect of glucose on ChREBP, we monitored ChREBP levels in RPE cells exposed to low or high glucose. Our data show that ChREBP levels were significantly increased in RPE cells exposed to high glucose, compared with the levels in cells exposed to low glucose (P < .05; Fig. 3B). TXNIP is induced by diabetes and transcriptionally regulated by ChREBP,²³ but the potential link between proapoptotic TXNIP and retinal cell death remains elusive. To address the role of TXNIP in retinal cell death in our model, we assessed whether AES affects retinal TXNIP expression levels in the diabetic retinas. Indeed, we found that TXNIP levels were significantly increased in the diabetic retinas, compared with that in the controls; however, AES treatment greatly reversed the diabetes-mediated increase in TXNIP (P < .005 or P < .001, respectively; Fig. 3C). Notably, immunofluorescence analysis showed significant colocalization of TXNIP and the TUNEL signal in the GCL of the diabetic retinas (Fig. 3D), whereas AES treatment attenuated this colocalization (Fig. 3D). Furthermore, we assessed the interaction between OGT and ChREBP, in part, because O-GlcNAcylation activates ChREBP under high glucose conditions, and determined whether ChREBP is O-GlcNAc modified in diabetic retinas. Immunoprecipitation assays showed that the interaction between OGT and ChREBP was significantly increased in the diabetic retinas, compared with the interaction levels in controls (P < .001 or P < .05; Fig. 3E). Strikingly, AES treatment mostly reversed this change in the diabetic retinas (P < .001 or P < .005; Fig. 3E), suggesting that AES lowered the levels of O-GlcNAc-modified ChREBP in the diabetic retinas. Consistent with these data, we found that ChREBP and OGT were mostly colocalized in the GCL of the diabetic retinas (Fig. 3F). However, AES treatment decreased the extent of colocalization, compared with that in diabetic retinas of mice not treated with AES (Fig. 3F). In addition, immunofluorescence analysis showed that ChREBP, OGT, and the TUNEL signal were colocalized in the GCL of the diabetic retinas, but AES treatment greatly reduced the extent of colocalization (Fig. 3G), suggesting that AES protects against GCL death, at least partially through modulation of ChREBP and/or OGT.

FIG. 3.

Representative Western blot and quantification of ChREBP (A) and TXNIP (C) in the retinas of control (CTL, n = 5) and diabetic mice with (DM+AE, n = 5) or without AES (DM, n = 5) at 2 months after induction of diabetes. Band intensity was normalized to β-actin. Data are presented as mean ± SEM. *P < 0.05, **P < 0.005, vs. CTL; ^† P < 0.05, ^††† P < 0.001, vs. DM by ANOVA. (B) Representative Western blot and quantification of ChREBP in retinal pigment epithelial cells incubated in low (LG, 5 mM) or high glucose medium (HG, 25 mM). Band intensity was normalized to β-actin. Data are presented as means ± SEM. **P < 0.01 vs. LG by t-test. (D) Representative images of double immunofluorescence staining for TXNIP and TUNEL in the retinas of control (CTL, n = 5) or diabetic mice with or without AES. The arrowheads indicate TUNEL-positive cells that were stained for TXNIP in the retinas of diabetic mic. Scale bar, 50 μm. (E) Binding of OGT to ChREBP. Representative immunoblots and quantification of co-immunoprecipitated ChREBP to OGT in the retinas of control or diabetic mice with or without AES. Tissue lysates were subjected to immuno-precipitation (IP) with an anti-OGT antibody and immunoblotted with an anti-ChREBP antibody. The same blots were reprobed with the immunoprecipitating antibody to confirm equal protein loading. Densitometry of co-immunoprecipitated ChREBP to OGT was normalized to IgG. Data are presented as means ± SEM. (n = 57 per group). *P < 0.05, ***P < 0.001, vs. CTL; ^†† P < 0.005, ^††† P < 0.001, vs. DM by ANOVA. (F) Representative images of double immunofluorescence staining for ChREBP and OGT plus 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization in the retinas of control (CTL) or diabetic mice with (DM+AE) or without AES (DM). The arrowheads indicate OGT-positive cells that were stained for ChREBP in the retinas of diabetic mice. Scale bar, 50 çm. (G) Representative images of immunofluorescence staining for ChREBP and OGT plus TUNEL in the retinas of control (CTL, n = 5) or diabetic mice with or without AES. To confirm the death of cells in conjunction with ChREBP and OGT expression, immunofluorescent staining of ChREBP using Alexa Fluor 488 goat anti-rabbit IgG (green), TUNEL (red) and OGT (violet) were performed consecutively in the same sections. The arrowheads indicate TUNEL-positive cells that were stained for ChREBP and OGT in the retinas of diabetic mice. Scale bar, 50 μm. ChREBP, carbohydrate-responsive element-binding protein; TXNIP, thioredoxin-interacting protein; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment. Color images available online at www.liebertpub.com/jmf

AES suppresses SREBP-1 and decreases FAS and ACC in the diabetic retina

SREBP-1c expression is transcriptionally stimulated by insulin and is itself, in turn, a major mediator of insulin action.³¹ Therefore, we assessed whether SREBP-1 is involved in retinal cell death in DR and whether AES affects SREBP-1 in the diabetic retinas. SREBP-1 is synthesized as a precursor form, and after proteolytic cleavage, its mature active form migrates into the nucleus where it can bind both sterol regulatory elements and E-boxes.³² Western blotting analysis showed that levels of both the precursor and mature SREBP-1 protein were significantly increased in the diabetic retinas compared with that in controls (P < .05; Fig. 4A). However, AES treatment significantly reversed this increase in precursor and mature SREBP-1 in the diabetic retinas (P < .005 and P < .05, respectively; Fig. 4A), suggesting that AES significantly inhibits activation of SREBP-1. SREBP-1 also activates the transcription of lipogenic genes such as FAS³³ and ACC.³⁴ Next, we evaluated the effect of AES on FAS and ACC. Indeed, we found that levels of FAS and ACC were significantly increased in the diabetic retinas, compared with that in controls (P < .05 and P < .005, respectively; Fig. 4B, C), These data suggest increased activity of SREBP-1 in diabetic mice, which may, in turn, promote insulin action on FAS and ACC. However, AES treatment significantly reversed the elevation in FAS and ACC levels in the diabetic retinas (P < .005 and P < .05, respectively; Fig. 4B, C). Because activated AMP-activated protein kinase (AMPK) may reduce ACC and suppress lipogenic enzymes such as SREBP-1,³⁵ our results suggest that AES activates AMPK in the diabetic retinas, which would eventually lower SREBP-1 levels. Moreover, we found that SREBP-1 colocalizes with OGT and the TUNEL signal (Fig. 4D or E, respectively), suggesting that SREBP-1 may be O-GlcNAc modified or contribute to GCL cell death in the diabetic retinas. Intriguingly, SREBP-1 colocalization with OGT and the TUNEL signal was greatly reduced in the retinas of diabetic mice treated with AES (Fig. 4D or E, respectively).

FIG. 4.

Representative Western blot and quantification of SREBP-1 (A), FAS (B) and ACC (C) in the retinas of control (CTL, n = 5) and diabetic mice with (DM+AE, n = 5) or without AES (DM, n = 7). Band intensity was normalized to β-actin. Data are presented as means ± SEM. *P < 0.05, **P < 0.005, vs. CTL; ^† P < 0.05, ^††† P < 0.005, vs. DM by ANOVA. (D) Representative images of double immunofluorescence staining for SREBP-1 and OGT plus 4′,6-diamidino-2-phenylindole (DAPI) for nuclear localization in the retinas of control (CTL) or diabetic mice with (DM+AE) or without AES (DM). The arrowheads indicate SREBP-1-positive cells that were stained for OGT in the retinas of diabetic mice. Scale bar, 50 μm. (E) Representative images of immunofluorescence staining for SREBP-1 and TUNEL plus DAPI for nuclear localization in the retinas of control (CTL, n = 5) or diabetic mice with (DM+AE) or without AES (DM). To confirm the death of cells in conjunction with SREBP-1 expression, immunofluorescent staining for SREBP using Alexa Fluor 488 goat anti-rabbit IgG (green) and TUNEL (red) were performed consecutively in the same sections. The arrowheads indicate TUNEL-positive cells that were stained for SREBP-1 in the retinas of diabetic mice. Scale bar, 50 μm. ACC, acetyl CoA carboxylase; FAS, fatty acid synthase; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; SREBP, sterol regulatory element-binding protein. Color images available online at www.liebertpub.com/jmf

AES inhibits retinal cell death through OGT

OGT is associated with insulin resistance³⁶ and may trigger the programmed cell death in diabetes mellitus and neurodegeneration.³⁷ Based on this role of OGT in diabetes, we hypothesized that OGT promotes retinal cell death in the inner retinal layers, which is an early characteristic of diabetes that eventually progresses to retinopathy.^38,39 To test whether AES affects OGT-related cell death, we first assessed OGT immunoreactivity in the GCL of the diabetic retinas. We found that OGT immunoreactivity in the GCL of the diabetic retinas was greatly increased compared with that in controls, while AES treatment restored this diabetes-associated increase (Fig. 5). Moreover, TUNEL-positive ganglion cells were also significantly increased in the diabetic retinas, compared with that in controls, and AES treatment decreased the numbers of dying ganglion cells in the diabetic retinas (Fig. 5). Double immunofluorescence analysis showed that OGT colocalized with most TUNEL-positive ganglion cells in the GCL of diabetic retinas, while AES treatment significantly reduced the OGT signal in TUNEL-positive regions (Fig. 5). Sequential double-immunofluorescent staining for γ-Syn, the ganglion cell marker, and OGT was performed. Indeed, most of the TUNEL-positive and OGT signal also colocalized with γ-Syn, confirming that the OGT signal was coincident with ganglion cells undergoing apoptosis.

FIG. 5.

Effect of AES on OGT and ganglion cell death in the retinas of diabetic mice. Color images available online at www.liebertpub.com/jmf

Discussion

O-GlcNAc dysregulation is linked to diabetes,^40,41 while OGT triggers apoptosis in diabetes.³⁷ Hyperglycemia promotes the rapid and reversible accumulation of O-GlcNAc protein, specifically in beta cells in vivo, and animals pretreated with STZ also accumulate O-GlcNAc protein in their beta cells when hyperglycemic.⁴² In this study, we provide new evidence for increased OGT protein expression in the retinas of diabetic mice. Our data also show that AES treatment reversed in the diabetes-associated OGT increase and protected the retinas of diabetic mice from neuronal cell death in the GCL. OGT may play a role in triggering the programmed cell death observed in diseases such as diabetes mellitus and neurodegeneration.³⁷ Our data showed that OGT was related to the cell death in the GCL of the diabetic retinas, as indicated by double immunofluorescence analysis of OGT and the TUNEL assay. Our finding that AES administration reduced retinal cell death related to OGT in diabetic mice is of particular note, as this finding supports further development of AES as a protective agent in DR.

We also found that ChREBP protein and its colocalization with the TUNEL signal in the GCL of the diabetic retinas was increased. Importantly, AES reduced levels of ChREBP, which is known to play a crucial role in apoptosis¹⁸ and is correlated with insulin sensitivity in early type 2 diabetes.⁴³ Because ChREBP is involved in metabolic adaptation to changing glucose levels, and alterations in glucose homeostasis are associated with metabolic diseases such as type 2 diabetes,¹⁸ reduction of ChREBP by AES will likely uncover new therapeutic opportunities in DR. Moreover, treatment with AES downregulated TXNIP, consistent with a recent study where AMPK was found to decrease TXNIP protein levels.⁴⁴ AMPK activation has been suggested to be a good target in treatment of type 2 diabetes.⁴⁵ Therefore, the similarity in the effects of AES and AMPK are intriguing. Also, a previous study showed that increased beta cell TXNIP expression induced by high glucose plays a major role in the pathogenesis of pancreatic beta cell loss and diabetes.²⁴ As such, we found that TXNIP was increased and colocalized with TUNEL-positive ganglion cells in the GCL of the diabetic retinas, and these diabetes-associated changes could be attenuated by treatment of the diabetic mice with AES. Furthermore, ChREBP has been shown to interact with OGT and is O-GlcNAc modified in HEK293T cells and mouse hepatocytes.²¹ Consistent with these data, we found that ChREBP interacts with and is colocalized with OGT as well as with TUNEL-positive GCL of the diabetic retinas. Our data also show that ChREBP contributes to neuronal cell death of the diabetic retinas. Strikingly, AES restored the extent of the ChREBP-OGT interaction, suggesting that AES reduces GCL cell death through its effects on ChREBP and OGT. In response to elevated glucose, ChREBP translocates to the nucleus.⁴⁶ We detected nuclear ChREBP in the diabetic retinas, suggesting that ChREBP is activated by high glucose, whereas AES reverses this activation. Previous studies also showed that ChREBP acts in synergy with SREBP-1 and is necessary for the induction of lipogenic genes such as ACC and FAS in response to glucose and insulin.^47
–49 Notably, SREBP has a critical role in the pathogenesis of the metabolic syndrome and controlling pathways for insulin resistance.^26,50 Intriguingly, AES reduced levels of activated SREBP-1 in the diabetic retinas, and concomitantly lowered levels of FAS, a known target of SREBP, consistent with a previous report.²⁵ Because SREBP-1 has been implicated in type 2 diabetes²⁶ and the pathogenesis of β cell dysfunction,⁵¹ our results suggest that SREBP-1may be a novel therapeutic target in DR, and that AES can inhibit SREBP-1. Moreover, AES-mediated decrease in SREBP-1 levels may indicate activation of AMPK, because activated AMPK suppresses SREBP-1.³⁵ Therefore, AES-mediated reduction in SREBP-1 suggests that AES could be a useful therapeutic agent in DR. Therefore, the combined potent effects of AES on FAS and ACC, an action likely induced synergistically by ChREBP and SREBP-1, warrant further studies of this unique antidiabetic agent.

STZ, a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins, and therefore, STZ toxicity is a result of the sustained accumulation of O-GlcNAc after a hyperglycemic episode.⁴² These data suggest that the diabetogenic effect of STZ may enhance OGT action.

In conclusion, our findings demonstrate the neuroprotective action of AES extract in the retinas of this mouse model of diabetes, and provide support for the role of AES as a novel inhibitor of OGT, an important regulator of retinal cell death in the GCL of diabetic mice. Our data suggest that the neuroprotective function of AES in DR is mediated at least partially through ChREBP. Further studies are needed to elucidate the molecular pathway(s) invoked by AES and to address its utility as a potential therapeutic agent in DR.

Footnotes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT, and Future Planning (2014049413) and NRF-2015R1A5A2008833.

Author Disclosure Statement

No competing financial interests exist.

References

Caldwell

, Bartoli

, Behzadian

, et al.: Vascular endothelial growth factor and diabetic retinopathy: Pathophysiological mechanisms and treatment perspectives. Diabetes Metab Res Rev, 2003; 19:442–455.

van Dijk

, Verbraak

, Kok

, et al.: Early neurodegeneration in the retina of type 2 diabetic patients. Invest Ophthalmol Vis Sci, 2012; 53:2715–2719.

Lee

, Kim

, Choi

, Kang

, Han

, Kim

: Effects of water extract of 1:1 mixture of Phellodendron cortex and Aralia cortex on polyol pathway and oxidative damage in lenses of diabetic rats. Phytother Res, 1999; 13:555–560.

Lee

, Kim

, Hong

, Kang

, Kim

: Water extract of 1:1 mixture of Phellodendron cortex and Aralia cortex has inhibitory effects on oxidative stress in kidney of diabetic rats. J Ethnopharmacol, 2000; 73:429–436.

Yoshikawa

, Murakami

, Harada

, Murakami

, Yamahara

, Matsuda

: Bioactive saponins and glycosides. VII. On the hypoglycemic principles from the root cortex of Aralia elata Seem.: Structure related hypoglycemic activity of oleanolic acid oligoglycoside. Chem Pharm Bull (Tokyo), 1996; 44:1923–1927.

Chung

, Choi

, Lee

, et al.: Water extract of Aralia elata prevents cataractogenesis in vitro and in vivo. J Ethnopharmacol, 2005; 101:49–54.

Luo

, Dong

, Yu

, Sun

: Total aralosides of Aralia elata (Miq) seem (TASAES) ameliorate nonalcoholic steatohepatitis by modulating IRE1α-mediated JNK and NF-κB pathways in ApoE^-/- mice. J Ethnopharmacol, 2015; 163:241–250.

Satoh

, Sakai

, Katsumata

, et al.: Oleanolic acid saponins from root-bark of Aralia elata . Phytochemistry, 1994; 36:147–152.

Wang

, Xu

, et al.: Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. J Ethnopharmacol, 2014; 155:240–247.

10.

, Zhou

, Zhang

, Cai

, Zhao

: Protective effect of total aralosides of Aralia elata (Miq) Seem (TASAES) against diabetic cardiomyopathy in rats during the early stage, and possible mechanisms. Exp Mol Med, 2009; 41:538–547.

11.

Yoshikawa

, Harada

, Matsuda

, Murakami

, Yamahara

, Murakami

: Elatosides A and B, potent inhibitors of ethanol absorption in rats from the bark of Aralia elata Seem: The structure-activity relationships of oleanolic acid oligoglycosides. Chem Pharm Bull (Tokyo), 1993; 41:2069–2071.

12.

Hwang

, Hwang

, Kim

, Choe

: Extracts from Aralia elata (Miq) Seem alleviate hepatosteatosis via improving hepatic insulin sensitivity. BMC Complement Altern Med, 2015; 15:347.

13.

Song

, Nakamura

, Ma

, Hattori

, Xu

: Four new saponins from the root bark of Aralia elata . Chem Pharm Bull (Tokyo), 2000; 48:838–842.

14.

Sakai

, Katsumata

, Satoh

, et al.: Oleanolic acid saponins from root bark of Aralia elata . Phytochemistry, 1994; 35:1319–1324.

15.

Kuang

, Sun

, Zhang

, Okada

, Okuyama

: Two new saponins, congmuyenosides A and B, from the leaves of Aralia elata collected in Heilongjiang, China. Chem Pharm Bull (Tokyo), 1996; 44:2183–2185.

16.

Kim

, Yoo

, Kim

, et al.: Aralia elata prevents neuronal death by downregulating tonicity response element binding protein in diabetic retinopathy. Ophthalmic Res, 2015; 54:85–95.

17.

Postic

, Dentin

, Denechaud

, Girard

: ChREBP, a transcriptional regulator of glucose and lipid metabolism. Annu Rev Nutr, 2007; 27:179–192.

18.

Filhoulaud

, Guilmeau

, Dentin

, Girard

, Postic

: Novel insights into ChREBP regulation and function. Trends Endocrinol Metab, 2013; 24:257–268.

19.

Havula

, Hietakangas

: Glucose sensing by ChREBP/MondoA-Mlx transcription factors. Semin Cell Dev Biol, 2012; 23:640–647.

20.

Hart

, Housley

, Slawson

: Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins. Nature, 2007; 446:1017–1022.

21.

Guinez

, Filhoulaud

, Rayah-Benhamed

, et al.: O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver. Diabetes, 2011; 60:1399–1413.

22.

Hanover

, Krause

, Love

: Bittersweet memories: Linking metabolism to epigenetics through O-GlcNAcylation. Nat Rev Mol Cell Biol, 2012; 13:312–321.

23.

Cha-Molstad

, Saxena

, Chen

, Shalev

: Glucose-stimulated expression of Txnip is mediated by carbohydrate response element-binding protein, p300, and histone H4 acetylation in pancreatic beta cells. J Biol Chem, 2009; 284:16898–16905.

24.

Chen

, Saxena

, Mungrue

, Lusis

, Shalev

: Thioredoxin-interacting protein: A critical link between glucose toxicity and beta-cell apoptosis. Diabetes, 2008; 57:938–944.

25.

Shimano

: SREBP-1c and TFE3, energy transcription factors that regulate hepatic insulin signaling. J Mol Med (Berl), 2007; 85:437–444.

26.

Verges

: New insight into the pathophysiology of lipid abnormalities in type 2 diabetes. Diabetes Metab, 2005; 31:429–439.

27.

Soyal

, Nofziger

, Dossena

, Paulmichl

, Patsch

: Targeting SREBPs for treatment of the metabolic syndrome. Trends Pharmacol Sci, 2015; 36:406–416.

28.

Kim

, Choi

, Kim

, et al.: Protein kinase C delta regulates anti-apoptotic αB-crystallin in the retina of type 2 diabetes. Neurobiol Dis, 2007; 28:293–303.

29.

Kim

, Choi

, Lee

, et al.: Decreased interaction between FoxO3a and Akt correlates with seizure-induced neuronal death. Epilepsy Res, 2014; 108:367–378.

30.

Kim

, Park

, et al.: Reduction of experimental diabetic vascular leakage and pericyte apoptosis in mice by delivery of αA-crystallin with a recombinant adenovirus. Diabetologia, 2012; 55:2835–2844.

31.

Foretz

, Guichard

, Ferre

, Foufelle

: Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc Natl Acad Sci U S A, 1999; 96:12737–12742.

32.

Brown

, Goldstein

: The SREBP pathway: Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell, 1997; 89:331–340.

33.

Magana

, Osborne

: Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter. J Biol Chem, 1996; 271:32689–32694.

34.

Lopez

, Bennett

, Sanchez

, Rosenfeld

, Osborne

: Sterol regulation of acetyl coenzyme A carboxylase: A mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci U S A, 1996; 93:1049–1053.

35.

Zhou

, Myers

, Li

, et al.: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest, 2001; 108:1167–1174.

36.

Yang

, Ongusaha

, Miles

, et al.: Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature, 2008; 451:964–969.

37.

Shin

, Love

, Hanover

: Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis. Amino Acids, 2011; 40:885–893.

38.

Cheung

, Fung

, Lo

, et al.: Aldose reductase deficiency prevents diabetes-induced blood-retinal barrier breakdown, apoptosis, and glial reactivation in the retina of db/db mice. Diabetes, 2005; 54:3119–3125.

39.

Sun

, Oates

, Coutcher

, Gerhardinger

, Lorenzi

: A selective aldose reductase inhibitor of a new structural class prevents or reverses early retinal abnormalities in experimental diabetic retinopathy. Diabetes, 2006; 55:2757–2762.

40.

Hart

, Slawson

, Ramirez-Correa

, Lagerlof

: Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease. Annu Rev Biochem, 2011; 80:825–858.

41.

Slawson

, Copeland

, Hart

: O-GlcNAc signaling: A metabolic link between diabetes and cancer?. Trends Biochem Sci, 2010; 35:547–555.

42.

Liu

, Paterson

, Chin

, Kudlow

: Glucose stimulates protein modification by O-linked GlcNAc in pancreatic β cells: Linkage of O-linked GlcNAc to β cell death. Proc Natl Acad Sci U S A, 2000; 97:2820–2825.

43.

Kursawe

, Caprio

, Giannini

, et al.: Decreased transcription of ChREBP-alpha/beta isoforms in abdominal subcutaneous adipose tissue of obese adolescents with prediabetes or early type 2 diabetes: Associations with insulin resistance and hyperglycemia. Diabetes, 2013; 62:837–844.

44.

Kawaguchi

, Osatomi

, Yamashita

, Kabashima

, Uyeda

: Mechanism for fatty acid “sparing” effect on glucose-induced transcription: Regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase. J Biol Chem, 2002; 277:3829–3835.

45.

Winder

, Hardie

: AMP-activated protein kinase, a metabolic master switch: Possible roles in type 2 diabetes. Am J Physiol, 1999; 277:E1–E10.

46.

Stoltzman

, Peterson

, Breen

, Muoio

, Billin

, Ayer

: Glucose sensing by MondoA:Mlx complexes: A role for hexokinases and direct regulation of thioredoxin-interacting protein expression. Proc Natl Acad Sci U S A, 2008; 105:6912–6917.

47.

Dentin

, Pegorier

, Benhamed

, et al.: Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression. J Biol Chem, 2004; 279:20314–20326.

48.

Ishii

, Iizuka

, Miller

, Uyeda

: Carbohydrate response element binding protein directly promotes lipogenic enzyme gene transcription. Proc Natl Acad Sci U S A, 2004; 101:15597–15602.

49.

, Tsatsos

, Towle

: Direct role of ChREBP.Mlx in regulating hepatic glucose-responsive genes. J Biol Chem, 2005; 280:12019–12027.

50.

Shimomura

, Matsuda

, Hammer

, Bashmakov

, Brown

, Goldstein

: Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell, 2000; 6:77–86.

51.

Wang

, Maechler

, Antinozzi

, et al.: The transcription factor SREBP-1c is instrumental in the development of beta-cell dysfunction. J Biol Chem, 2003; 278:16622–16629.