Early impairments in the retina of rats fed with high fructose/high fat diet are associated with glucose metabolism deregulation but not dyslipidaemia

Abstract

Way of life changes such as high consumption of processed foods rich in fat and sugar and sedentary lifestyle are associated with the increasing prevalence of metabolic syndrome (MetS) that affects about 35% in the American population. MetS is the main risk factor for diabetes mellitus, which is associated with vascular changes in the retina. However, the early consequences of MetS in the retina are not well described. We therefore aimed at characterizing the early effects of a high fructose and high fat diet (HFHF) on the function and structure of the rat retina, and evaluate the associations with metabolic changes. Brown Norway rats of 6 weeks of age were fed for 8 days, 5 weeks or 13 weeks with HFHF diet, or a standard chow. After only 4 weeks of this diet, rats exhibited a reduction in cone photoreceptor sensitivity to light. Moreover, we observed that MetS significantly exacerbated laser-induced choroidal neovascularization by 72% and 67% 2 weeks and 3 weeks post laser treatment, respectively. These retinal abnormalities were associated with deregulation of glucose metabolism but not lipid metabolism. These data showed retinal modifications in HFHF-induced MetS in the rat, at very early stage of the disease.

Figure 1

Effect of HFHF diet on body weight gain and tissues. (A) Body weight (grams) evolution and (B) energy intake (MJ/rat) during the 13 weeks of nutritional experiments. (C) Percentage of total body fat per rat. (D) Percentage of lean mass per rat. (E) Percentage of total water per rat. Figures A and B: Values are means ± SEM. ANOVA followed by Bonferroni test is performed. Figures C to E: Values are means ± SD (n = 8). Mann-Whitney test was performed. Data with different * are significantly different at P < 0.05; ** at P < 0.01; *** at P < 0.001.

Figure 2

Effect of HFHF diet on plasma analytes. Fasted glycaemia (A, for 8 days of diet (n = 16); D, for 5 weeks of diet (n = 16) and G, for 13 weeks of diet (n = 8)), fasted lipids (mmol/L) (B, for 8 days of diet; E, for 5 weeks of diet and H, for 13 weeks of diet) and fasted insulin, leptine, IL1-β (C, for 8 days of diet) and TNF-α (F, for 5 weeks of diet and I, for 13 weeks of diet). Values are means ± SD (n = 16 for 8 days group, and n = 8 for 5 weeks and 13 weeks). Mann-Whitney test was performed. Data with different * are significantly different at P < 0.05; ** at P < 0.01; *** at P < 0.001; **** at P < 0.0001.

Figure 3

Glucose metabolism is impaired after 8 days of HFHF diet. Blood glucose (A1, for 8 days of diet; B1, for 5 weeks of diet and C1, for 13 weeks of diet) and plasma insulin values (A3, for 8 days of diet; B3, for 5 weeks of diet and C3, for 13 weeks of diet) at different times after intraperitoneal administration of glucose solution (2 g/kg body weight). Area under the curve (AUC) values for glucose (A2, for 8 days of diet; B2, for 5 weeks of diet and C2, for 13 weeks of diet) and insulin (A4, for 8 days of diet; B4, for 5 weeks of diet and C4, for 13 weeks of diet) concentrations. Blood glucose (A5, for 8 days of diet; B5, for 5 weeks of diet and C5, for 13 weeks of diet) values at different times after intraperitoneal administration of an insulin solution (0.5 U/kg body weight). Area under the curve (AUC) values for glucose concentrations (A6, for 8 days of diet; B6, for 5 weeks of diet and C6, for 13 weeks of diet). Results are the mean ± SD (n = 8 animals per group). *P < 0.05; **P < 0.01; ***P < 0.001 after Mann-Whitney and ANOVA test followed by Bonferroni test.

Figure 4

HFHF diet induces liver steatosis. (A) Hepatosomatic index. (B) Amount of lipids in the liver (milligrams) per gram of liver. Amount of different classes of fatty acids, saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (micrograms) per milligram of liver of rats fed during 8 days (C), 5 weeks (D) and 13 weeks (E) with HFHF or standard diet. Bars represent the mean ± SD of values obtained from n = 16 (8 days) and n = 8 (5 weeks and 13 weeks). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 after Mann-Whitney test.

Figure 5

HFHF diet triggers a decrease in cone photoreceptor sensitivity. 8.02 Hz Flicker electroretinographic data of HFHF or standard fed rats during 8 days (A), 5 weeks (B) and 13 weeks (C). Data of the amplitude (µV) of the electroretinographic response as a function of the light stimulus intensity (log(I)), (n = 8 per group). The first peak corresponds to the maximal response of rods and the second peak to the maximal response of cones. Bars represent the mean ± SEM of values obtained from n = 8 per group, *P < 0.05 after ANOVA test.

Figure 6

HFHF diet emphasizes laser-induced choroidal neovacularization (CNV). (A) Representative images of indocyanine green angiographies taken after 1, 2 and 3 weeks post laser impacts in standard or HFHF Bruch’s membrane. CNV corresponds to the filling of the new vessels as indicated by arrows on the figure. Indocyanine green reveals choroidal vascularization. (B) Semi-quantification of CNV (ratio between area of indocyanine green and optic disc area) at 1, 2 and 3 weeks after laser-induced CNV in rats fed during 5 weeks with either the standard or HFHF diets. Bars represent the mean ± SD of values obtained from n = 6. *P < 0.05 after Mann-Whitney test; Standard versus HFHF.

Figure 7

HFHF diet increases retinal gliosis. (A) Reading table. (B) Representative images of the GFAP IHC signals. (C) Double-blind coring of IHC signals associated with GFAP expression in Müller glial cells from strong to no signal. Bar: 100 µm. Values were obtaines from n = 6 eyes per group, 15 sections per eye. Chi-Square test was performed to determine if there was any association between diet and GFAP expression stage; ^**means P < 0.01. df = 13.74, 3.

Figure 8

Experimental procedure designed in the goal of reduce number of animal used and refine the procedure. GTT: glucose tolerance test, ITT: insulin tolerance test, ERG: electroretinography.