Dietary Ganglioside and Long-Chain Polyunsaturated Fatty Acids Increase Ganglioside GD3 Content and Alter the Phospholipid Profile in Neonatal Rat Retina

Abstract

purpose. During early development, the ganglioside composition of the retina changes significantly, in that GD3 becomes the primary ganglioside in the mammalian retina. Because gangliosides play an important role in neuronal cell differentiation and proliferation, this change in ganglioside profile may indicate retinal maturation. Dietary long-chain polyunsaturated fatty acids (LCPs) such as 20:4n-6 and 22:6n-3 improve visual acuity in infants. Dietary LCPs stimulate neonatal retinal development by altering membrane phospholipids, which in turn affect cell signaling pathways. It is unknown whether dietary ganglioside and LCPs affect the metabolism of phospholipids and gangliosides during retinal development.

methods. Male Sprague-Dawley rats (18 days old) were fed semipurified diets consisting of 20% fat (control diet) for 2 weeks containing either 0.1% ganglioside enriched in GD3 (GG diet) or 1% 20:4n-6 and 0.5% 22:6n-3 (LCP diet) in the control diet. The profile of ganglioside and phospholipid was measured.

results. The GG diet increased the ganglioside content by 39% in the retina, with a relative increase in GD3 (by 13%). Dietary LCPs significantly increased the relative levels of GD3 (by 19%, P < 0.01). Total phospholipid was decreased by the LCP-supplemented diet (by 28%). Phosphatidylcholine and phosphatidylserine increased with concomitant decreases in phosphatidylinositol and lyso-phosphatidylethanolamine when animals were fed either the LCP or the GG diet.

conclusions. Animals fed dietary ganglioside increased in total retinal ganglioside and GD3 content during retinal development, with a concomitant alteration of phospholipid metabolism. Feeding animals dietary LCPs also affected ganglioside metabolism in the developing retina, suggesting a new mechanism by which these dietary lipids may promote maturation of photoreceptor cells.

An unusual simplified ganglioside composition is observed in rat adult retinal photoreceptor cells, compared with that in other central nervous system (CNS)–derived neurons. ¹ Changes in specific ganglioside content occurs within photoreceptor cells during postnatal maturation, to reach an end stage characterized by a predominance of GD3 in the outer retina and only trace amounts of less complex gangliosides. ¹ The increase in GD3 content corresponds to the period between 10 and 30 days after birth when the outer segments, photoreceptor cells, synaptic cells, and rhodopsin kinase in the rat retina become functionally active. ^{2 3} Although most gangliosides are localized in the inner retinal membranes, GD3 is primarily found in photoreceptors in the outer retina, ^{1 4} where it may play a major role in increasing membrane permeability and fluidity. ^{5 6} Because GD3 is the most prevalent ganglioside in fully mature mammalian retinas, ^{1 7} it can be used as a biological marker to evaluate the stage of retinal development. 

Dietary long-chain polyunsaturated fatty acids (LCPs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) influence the lipid composition of retinal membranes, particularly during developmental stages. ^{8 9 10 11 12 13} Dietary DHA alters the lipid composition of neuronal tissues in retina and brain, affecting the turnover time for rhodopsin in photoreceptor membranes. ^{8 11 13} Dietary DHA increases DHA levels and levels of other very long-chain fatty acids in rod outer segment membranes. ^{11 14 15} Inclusion of DHA and AA in infants’ formulas improves their visual development and acuity, ^{9 10 12 14 16} but the biological basis for this effect has not been completely discovered. 

Gangliosides found in the brain are known to play a role in neuronal function including neurite outgrowth, axon generation, and synapse formation. ^{17 18 19} Because the retina is a part of the CNS, ²⁰ the localization of gangliosides in the retina may also be crucial to the structure of photoreceptor membranes in the early-development stage. ^{2 7 21} Administration of the ganglioside GM1 to animals has shown protective effects against retinal ischemia, ²² probably by protecting against the loss of membrane permeability and fatty acids. ²³ This lipid also improved visual function in animals that underwent a graded crush of the optic nerve. ²⁴ The influence of dietary lipids on retinal ganglioside composition during development is poorly understood. Human milk contains higher ganglioside and LCP content than bovine milk or infant formulas. ^{25 26} Because neonates and infants consume gangliosides and LCPs from milk, it is necessary to understand the fate of dietary gangliosides and LCPs for retinal development. Thus, the present study was conducted to determine whether dietary LCPs and gangliosides alter the ganglioside profile in developing rat retina. 

Materials and Methods

Animals and Diets

This study was approved by the University of Alberta Animal Ethics Committee and adheres to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two experimental diets or a control diet were fed to male weanling (18-day-old, 40 ± 4.5 g) Sprague-Dawley rats for 2 weeks. Animals had free access to water and one of three semipurified diets—the first containing 20% (wt/wt) fat (control diet). The control diet was formulated as 80% basal diet plus 20% fat as a triglyceride blend (Table 1) , reflecting the overall fat composition of infant formula. ²⁷ The LCP-enriched diet (LCP diet) was formulated by including 1% AA (20:4n-6; Martek Biosciences, Irvine, CA) and 0.5% DHA (22:6n-3; Martek Biosciences) by weight as triglyceride in the control diet. The ganglioside-enriched diet (GG diet) was formulated by including the gangliosides (70%–80% GD3, 9% GD1b, 5% GM3, 6% other gangliosides; New Zealand Dairy Foods, Ltd., Auckland, New Zealand) at a level of 0.1% by weight of total fat. The GG diet also contained 0.25% (wt/wt of fat) phospholipid and <0.002% (wt/wt of fat) cholesterol. The ganglioside-enriched lipid also contained 60% to 70% lactose and 10% to 12% minerals, and the level of these amounts was adjusted in the basal diet. Body weight and food intake were measured every other day throughout the experimental period. 

Collection of Retina, Lipid Extraction, and Ganglioside Separation

After decapitation of the animals, whole retinas were removed. All samples were weighed and kept in a −70°C freezer until analysis. Total lipids were extracted using the Folch method. ²⁸ Gangliosides were extracted into the Folch upper-phase solution. ²⁸ The lower organic phase was washed twice with chloroform-methanol-water (3:48:47, by vol) and the upper phase extracts combined. Gangliosides were purified by passing the upper phase extract through C₁₈ cartridges (Sep-Pak; Waters Corp., Milford, MA) preconditioned with 10 mL methanol, 20 mL chloroform-methanol (2:1, vol/vol), and 10 mL methanol. ²⁹ Cartridges were then washed with 20 mL of distilled water to remove salts and water-soluble contaminants. Gangliosides were eluted with 5 mL methanol and 20 mL chloroform-methanol (2:1, vol/vol), dried under N₂ gas, and then redissolved with 500 μL of chloroform-methanol (2:1, vol/vol). Gangliosides were stored at −70°C until analysis. 

Analysis of Ganglioside Content

Measurement of total gangliosides as ganglioside-bound N-acetyl neuraminic acid (GG-NANA) was performed as described by Suzuki. ³⁰ An aliquot of the purified ganglioside sample was dried under N₂ gas and dissolved with 0.5 mL each of distilled H₂O and resorcinol-HCl. ³¹ The purple-blue color developed by heating was extracted into butyl acetate-butanol (85:15, vol/vol). Optical density was read at 580 nm. Commercial NANA (Sigma-Aldrich, St. Louis, MO) was used as a standard. 

Individual gangliosides were separated by silica gel high-performance thin layer chromatography (HPTLC; Whatman Inc., Clifton, NJ) in a solvent system of chloroform-methanol-0.2% (wt/vol) CaCl₂·2H₂O (55:45:10, by vol) and identified using the ganglioside standards GM3, GM2, and GD3 and a bovine brain ganglioside mixture (Alexis, San Diego, CA). Individual gangliosides were recovered and measured as described earlier. 

Analysis of Phospholipid Content

Phospholipids were separated from total retinal lipids by TLC on silica gel (gel G; Fisher Scientific, Pittsburgh, PA) by using chloroform-methanol-water, (65:35:6, by vol). Individual phospholipids were resolved on silica gel H TLC plates, by using chloroform-methanol-2-propanol-0.2% KOH-triethylamine (45:13.5:37.5:9:27, by vol) and identified by comparison to authentic phospholipid standards (Sigma-Aldrich, St. Louis, MO). Plates were visualized by 0.1% anilinonaphthalene sulfonic acid under UV exposure. Lipid fractions were recovered, and lipid phosphate was measured according to the method of Itoh et al. ³²  

Statistical Analysis

Six retinas from three animals were pooled to constitute one replicate to analyze retinal lipids because of the minute amount of lipids in the retina. Data are expressed as the mean ± SD of six replicates (n = 6) except for individual phospholipid analysis (n = 5). Significant differences between the control group and experimental groups were determined by one-way analysis of variance (ANOVA) on computer (SAS, ver. 8.2; SAS Institute Inc., Cary, NC) Significant effects of diet treatment were determined by the Duncan multiple-range test at a significance level of P < 0.05. 

Results

Animal Growth and Diet Consumption

The initial and final body weight of animals and food consumption after 2 weeks of feeding them the experimental diets was not significantly different among the control, LCP, and GG diet groups. Retinal weight was not affected by dietary treatment (20.6 ± 2.4, 19.7 ± 1.8, 19.8 ± 2.0 mg/retina for the control, LCP, and GG diets, respectively). 

Ganglioside Content and Composition

Animals fed the GG diet showed higher levels of total gangliosides in the retina (by 39%; P < 0.0008) when compared with animals fed the control diet (Fig. 1A) . The absolute amount of total ganglioside was 2.71 ± 0.35, 3.11 ± 0.25, and 3.76 ± 0.44 μg/retina with the control, the LCP, and the GG diets, respectively. Feeding animals either the LCP or the GG diet caused an increase in the relative percentage of GD3 in the retina in comparison with feeding animals the control diet (by 19% and 13%, respectively; Table 2 ). The composition of GM3, GM1, GD1a, GD1b, and GT1b was not changed by either experimental diet (Table 2) . 

Phospholipid Content and Composition

Feeding animals the LCP diet, but not the GG diet, reduced total phospholipid content in the retina compared with animals fed the control diet (Fig. 1B) . The absolute amount of total phospholipids was 15.3 ± 1.97, 11.09 ± 1.01, and 14.65 ± 1.36 μg/retina with the control, the LCP, and the GG diets, respectively. Animals fed either the LCP or the GG diet showed lower levels of phosphatidylinositol and lyso-phosphatidylethanolamine (PE) and higher levels of phosphatidylserine and phosphatidylcholine (PC) than did animals fed the control diet (Table 3) . PE and sphingomyelin (SM) were not changed by either diet treatment. 

Discussion

The present study demonstrates for the first time that dietary gangliosides and LCPs can modify the ganglioside composition in the developing retina. Although dietary LCPs have long been known to modify the lipid classes and the composition of phospholipids in the developing retina ^{13 35} and to improve visual function, ^{9 36} an explanation for these effects at a metabolic level based on interaction between LCP and gangliosides has not been revealed. In this study, we found that animals fed the LCP diet showed higher relative levels of GD3, but total ganglioside content was not changed, compared with animals fed the control diet. The effect of dietary LCPs on the proportional increase of GD3 may suggest that dietary LCPs influence activity of GD3 synthase, an enzyme in the outer retina necessary for the synthesis of GD3 from GM3. ³⁷ Although the difference is not significant, the lower level of GM3 may explain a potential activation of GD3 synthase by the LCP diet. Further study with a larger sample size is needed to demonstrate whether dietary LCP activates GD3 synthase in the retina. Trafficking of DHA-containing PL from the trans-Golgi network to the retina outer segment is accompanied with rhodopsin during biogenesis of frog photoreceptor membrane biogenesis. ³⁸ Sphingolipids, including gangliosides, are enriched in microdomains called lipid rafts or caveolae, which are important domains for lipid trafficking. ^{38 39 40} It is reasonable to assume that gangliosides including GD3 in response to the LCP diet are enriched in the Golgi apparatus ⁴¹ and participate in lipid trafficking from the Golgi apparatus to the retina outer membranes, ⁴² probably with rhodopsin. ³⁸ Thus, further study is needed to determine whether the increased level of GD3 in response to the LCP diet influences the intracellular trafficking of DHA and rhodopsin during retinal development. This potential interaction may suggest a mechanism that explains the improvement in visual acuity associated with dietary LCP. 

In the retina of the rat, the outer segments, photoreceptor cells, synaptic cells, and rhodopsin kinase become functionally active between 10 days and 1 month after birth, ^{2 3} during which time GD3 becomes the predominant ganglioside. ^{1 4 7} GD3 in the outer retina is involved in increasing membrane permeability and fluidity ^{5 6} and is enriched in differentiated retinas. ^{1 7} Because animals used in the present study were fed for 2 weeks starting from 17 days of age, this study suggests that dietary LCP and gangliosides may stimulate retinal maturation and development by increasing GD3 content. 

Concurrent with changes in the ganglioside profile was an alteration in retinal phospholipid composition attributed to both the GG and the LCP diets. Both diets were associated with decreases in the relative amounts of phosphatidylinositol and PE, and increase in phosphatidylserine and PC (Table 2) . There was no effect of the GG diet on total phospholipids, whereas the LCP diet was associated with a decrease in total retinal phospholipids (Fig. 1) . Phospholipid turnover alters electric surface potential by affecting calcium and cation concentration in retinal rod outer segments ⁴³ and is tightly regulated by light and phosphorylation-dephosphorylation reactions. ⁴⁴ Thus, compositional changes in retinal phospholipids in response to dietary gangliosides or LCPs may affect light adaptation and activation of protein kinases, which ultimately may lead to enhanced development of retinal function in neonates. 

In summary, this study demonstrates that dietary LCP and gangliosides modify metabolism of gangliosides and phospholipids in developing retinal membranes. The present experiment suggests that a small physiologic amount of LCP or gangliosides have profound effects on the lipid profile of membranes in the retina. Photoreceptor cells contain approximately 25% of the total gangliosides present in the whole retina. ¹ Photoreceptor cells consist predominantly of GD3 (45%–50% of total gangliosides) compared with other gangliosides. ² This suggests that GD3 is important for visual function through photoreceptor cells by membrane structure ⁶ and activity of signaling molecules. ⁴⁵ Gangliosides stabilize membrane, ²³ protect against injuries, ²² and enhance visual function ²⁴ after retinal damage. The biological activity of gangliosides in the diet seemed high, as GD3 content in structural components of the retina was rapidly altered. Alterations in the ganglioside profile in the retina by LCP and GG diet may provide a means to discover mechanisms in relation to visual function in the developing retina. Further investigation is needed to determine whether dietary gangliosides and/or LCPs enhance retina development and visual function in neonates and alter ganglioside content of other neuronal cell types. 

²  Current affiliation: Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.

 Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

 Submitted for publication December 7, 2004; revised February 17, 2005; accepted March 1, 2005.

 Disclosure: E.J. Park, None; M. Suh, None; M.T. Clandinin, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Composition of Experimental Diets

	Control	LCP	GG
Basal diet* (g/100g)	80.0	80.0	80.0
Triglyceride, †	20.0 (100), §	20.0 (100)	19.9 (98.6)
AA	—	0.2 (1.0)	Tr, ‡
DHA	—	0.1 (0.5)
Ganglioside	—	—	0.02 (0.1)
Phospholipid	—	—	0.05 (0.25)
Cholesterol	—	—	Tr (0.002)

*  The composition of the basal diet is described by Clandinin and Yamashiro. ²⁷

†  The fatty acid composition of the triglyceride fed was similar to that of an infant formula. ³³

‡  Trace amount.

§  Values in parenthesis represent the percentage of total fat.

Figure 1.

 

Total level of (A) gangliosides and (B) phospholipids in the retinas of control and treatment groups. Data are the mean ± SD results of six replicates. Letters represent a significant difference between groups at levels of P < 0.0008 (a) and P < 0.001 (b), respectively.

Table 2.

 

The Composition of Gangliosides in the Retina of Animals Fed Different Diets^*

Ganglioside (%)	Control	LCP	GG	Diet Effect (P)
GM3, †	7.6 ± 2.8	5.8 ± 3.2	7.7 ± 2.3
GM1	7.3 ± 1.8	6.8 ± 2.0	7.7 ± 2.2
GD3	25.0 ± 1.8b	29.8 ± 1.7a	28.2 ± 3.5a	0.01
GD1a	14.3 ± 3.3	15.4 ± 3.5	16.1 ± 1.8
GD1b	19.1 ± 2.2	19.0 ± 42.	16.7 ± 1.0
GT1b, ‡	26.9 ± 2.9	23.2 ± 3.1	23.6 ± 1.3

*  Data are percentage of ganglioside-bound NANA content (μg/retina) in the ganglioside fraction (means ± SD of six replicates). Values in a row with different superscripts are significantly different (P < 0.01).

†  Nomenclature of gangliosides is described by Svennerholm. ³⁴

‡  GT1b fraction included GQ1b fraction because of close proximity during TLC separation.

Table 3.

 

The Composition of Phospholipids in the Retina of Animals Fed Different Diets

Phospholipid (%)	Control	LCP	GG	Diet Effect (P)
PE†	35.2 ± 4.7	33.4 ± 1.5	34.4 ± 2.3
PI	7.4 ± 1.6a	5.1 ± 1.3b	4.6 ± 0.7b	0.01
PS	2.3 ± 0.4b	2.8 ± 0.4a	3.1 ± 0.1a	0.01
LPE	8.3 ± 0.6a	6.6 ± 0.4b	6.4 ± 1.0b	0.001
PC	40.4 ± 3.3b	45.5 ± 2.8a	45.6 ± 2.7a	0.05
SM	6.5 ± 1.3	6.6 ± 2.2	6.0 ± 0.7

 Data are percentage of phospholipid phosphate content (μg/retina) in the phospholipid fraction (means ± SD of six replicates). Values in a row with different superscripts are significantly different (P < 0.05, P < 0.01, or P < 0.001).