Dietary effects on the retina of hamsters

Abstract

The retina is a sensory tissue in the back of the eye, which captures visual information and relays it to the brain. The retinal pigment epithelium separates the neural retina from the choroidal (systemic) circulation and is thereby exposed to circulating lipoprotein particles. Herein, we used hamsters and conducted various retinal evaluations of animals fed either a normal diet or a Western-type diet (WTD). Prior to evaluations, hamsters were injected with indocyanine green (ICG), a fluorescent dye that binds to various proteins and lipids in the systemic circulation. The WTD increased plasma levels of total and HDL cholesterol 1.8- and 2.1-fold, respectively, and led to additional HDL₂ and HDL₃ subpopulations. The diet also increased the ICG fluorescence in the retinal pigment epithelium and the underlying choroidal circulation on histological tracking and altered retinal protein abundance as assessed by proteomics. Functional enrichments were found in the retinal gene expression, energy production, intracellular transport, cytoskeleton- and synapse-related processes, and protein ubiquitination. The biochemical basis linking the WTD, retinal energy production, and retinal neurotransmission was suggested as well. The data obtained were then compared with those from our previous investigations of hamsters and different mouse genotypes. We identified common retinal processes that can be affected by circulating lipoprotein particles regardless of the mechanism by which their levels and subpopulations were altered (through diet or genetic modification). Thus, we obtained novel mechanistic insights into how lipids in the systemic circulation can affect the retina.

Keywords: HDL; cholesterol; diet; hamster; indocyanine green; lipoprotein; retina.

FIGURE 1

The neural retina, retinal pigment epithelium (RPE), and vascular networks. (A) Schematic representation of the retina comprised of the neural retina and RPE and served by two vascular networks, intraretinal and choroidal. The endothelial cells of the intraretinal vascular network are linked by tight junctions, which along with Muller cells, astrocytes, and pericytes establish the inner blood–retinal barrier that prevents passage of plasma proteins and other macromolecules in or out of the blood vessels. The outer blood‐retinal barrier is formed by the endothelial cells of the choroidal vascular network, by Bruch's membrane, and by the RPE cells. The endothelial cells of the choroidal vasculature are fenestrated and supply the retina with nutrients, water, and ions, while removing waste material. Bruch's membrane allows passive diffusion of various molecules, and the RPE cells form tight junctions but have different receptors on its basal side to take up certain molecules. Thus, the outer blood‐retinal barrier plays an essential role in retinal physiology by mediating a bi‐directional molecules' exchange between the retina and choroidal circulation (ChC). (B) Schematic representation of an RPE cell showing the location of the receptors for different LPP particles., , , The ABCA1 and ABCG1 transporters that efflux cholesterol to HDL are also shown., , , , , , , , BrM‐LPP, Bruch's membrane lipoprotein particles, , , ; oxLDl, oxidized LDL. Panel A for this figure was licensed from Carlson Stock Art and is used with permission; panel B was taken from and modified.

FIGURE 2

In vivo imaging of hamster retina by ICGA. Representative images (n = 5) of an early, intermediate and late‐stage fundus ICG fluorescence with the laser beam being focused either on the choroidal or retinal vascular networks. White numbers in the upper right corner indicate the post‐ICG injection time in minutes.

FIGURE 3

Plasma lipid profiles in hamsters (A) and mice (B) on different diets. Data were analyzed by a two‐tailed unpaired Student's t‐test (n = 5–7 animals per group); dots represent the measurements in individual animals. *p ≤ .05; **p ≤ .01.

FIGURE 4

Plasma LPP isolation by three KBr density ultracentrifugations. After each ultracentrifugation, 1‐mL fractions were collected from the tube top to the tube bottom and their fluorescence and light scattering were measured. Fraction pooling for hamsters on normal diet (ND) and western‐type diet (WTD) is indicated by the cyan and lavender highlights, respectively.

FIGURE 5

SDS‐PAGE and Western blot analyses of the HDL subpopulations isolated by density ultracentrifugations from hamster plasma. The same amount of protein (1 μg) from each HDL subpopulation was applied per each lane for SDS‐PAGE (A). For Western blots (B–D), the protein amount per lane was 0.3 μg for detection of serum albumin and 1 μg for detection of the isoforms of apolipoprotein C (APOC). The molecular weight standards were run in the middle gel lane. ND, normal diet; WTD, Western‐type diet.

FIGURE 6

Histological ICG tracking. Representative retinal cross‐sections from the eyes of hamsters on normal diet (ND) and Western‐type diet (WTD) (n = 3 animals per diet) 17 min post‐intraperitoneal ICG injection. Control hamster (Cntl) was injected with sterile water. Color boxes indicate enlarged chorioretinal regions shown at the bottom. The quantification of ICG fluorescence in relative fluorescence units (RFU) is also shown (B). Scale bars 25 μm. GCL, the ganglion cell layer; IPL, the inner plexiform layer; INL, the inner nuclear layer; OPL, the outer plexiform layer; ONL, the outer nuclear layer; OS, the photoreceptor outer segments; RPE, retinal pigment epithelium. **p ≤ .01.

FIGURE 7

Retinal proteomics of hamsters on western‐type diet (WTD) versus normal diet (ND). (A) The identification of differentially expressed proteins in different databases and changes in the protein abundance. (B) Biological processes enriched with differentially expressed proteins. ha, hu, and mo indicate the identification of differentially expressed proteins in hamster, human, and mouse databases, respectively. See main text for details.

FIGURE 8

Proteomics analyses. (A) Some of the enzymatic reactions catalyzed by differentially expressed proteins. x, a fold change in protein abundance in hamsters on WTD versus ND. PC catalyzes the irreversible conversion of pyruvate into oxaloacetate, OAA. SLC25A11 exports α‐KG from mitochondria into the cytosol in exchange for malate or other dicarboxylic acids. SLC25A22 imports glutamate and H⁺ from cytosol to mitochondria in exchange for OH⁻. SDHB is a dual function enzyme, catalyzing the oxidation of succinate to fumarate in the TCA cycle and also transferring electrons from succinate to ubiquinone (Q) in the mitochondrial electron transport chain. SLC25A4 imports ADP into the mitochondrial matrix for ATP synthesis, and exports ATP to fuel the cell., ATP 5A1, 5B, and 5D are subunits of ATP synthase, which produces ATP from ADP and P_i (inorganic phosphate) using the energy generated by the electron transport chain (ETC). Dashed lines indicate multiple reactions. (B) Overlapping processes and protein families suggested by a comparison of differentially expressed proteins in hamsters on WTD versus ND with those in mice, APOB100 versus WT, on ND. The ATP family is represented by the subunits of ATP synthase in the ETC, which generates 90% of cellular ATP. The NDUF family members are the NADH–ubiquinone oxidoreductase/dehydrogenases from different electron transport chain complexes., , , The BAN family is represented by BANF1 (barrier‐to‐autointegration factor 1), which plays fundamental roles in nuclear assembly, chromatin organization, and gene expression. The HIST family is various histones, that is, proteins that bind DNA and form chromatin. The SMARC family is the SWI/SNF‐related matrix‐associated Actin‐dependent regulators of chromatin. The FAM members are the deubiquinating enzymes. Conversely the UBE2 members are different ubiquitin‐conjugating enzymes E2. The PPP family are different subunits of phosphoprotein phosphatases with PPP1R9B, which overlapped in hamsters and mice, being a protein phosphatase 1 and Actin filament binding protein localized to dendritic spines. The SLC family is represented by different mitochondrial and vesicular amine or amino acid transporters. Finally, the members of the CCDC (coiled‐coil domain‐containing) protein family are involved in a wide variety of biological functions.,