A Mixture of Pure, Isolated Polyphenols Worsens the Insulin Resistance and Induces Kidney and Liver Fibrosis Markers in Diet-Induced Obese Mice

Abstract

Obesity is a worldwide epidemic with severe metabolic consequences. Polyphenols are secondary metabolites in plants and the most abundant dietary antioxidants, which possess a wide range of health effects. The most relevant food sources are fruit and vegetables, red wine, black and green tea, coffee, virgin olive oil, and chocolate, as well as nuts, seeds, herbs, and spices. The aim of this work was to evaluate the ability of a pure, isolated polyphenol supplementation to counteract the pernicious metabolic effects of a high-fat diet (HFD). Our results indicated that the administration of pure, isolated polyphenols under HFD conditions for 26 weeks worsened the glucose metabolism in diet-induced obese mice. The data showed that the main target organ for these undesirable effects were the kidneys, where we observed fibrotic, oxidative, and kidney-disease markers. This work led us to conclude that the administration of pure polyphenols as a food supplement would not be advisable. Instead, the ingestion of complete "whole" foods would be the best way to get the health effects of bioactive compounds such as polyphenols.

Keywords: antioxidants; food matrix; insulin resistance; kidney disease; obesity; polyphenols.

Figure 1

Polyphenol dietary supplementation increased the Kcal intake of HFD-only mice. (a) The graph represents the weight-gain mean between the beginning and the end of the dietary intervention (26 weeks). (b) The graph represents the food-intake average (Kcal) during the nutritional intervention. Data are presented as the mean ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001. The p-values were determined by using a one-way ANOVA test and a Tukey’s multiple tests correction. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.

Figure 2

The polyphenol supplementation produced extra weight gain in HFD + Pol murine kidneys, as compared to HFD-only murine kidneys. The graph represents the tissue weight (g) of different tissues. Data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001. The p-values were determined by using a one-way ANOVA test and a Tukey’s multiple tests correction. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.

Figure 3

Polyphenol dietary supplementation worsened the glucose metabolism caused by an HFD. (a) AUC of the plasma glucose levels after i.p. administration of glucose (1.5 g/kg body weight (b.w.)) in standard-chow-diet, HFD, and HFD + Pol mice groups from the GTTs performed at weeks 7, 14, and 20; (b) AUC of the plasma glucose levels after i.p. administration of insulin (0.75 UI/kg b.w) in standard chow-diet, HFD, and HFD + Pol mice groups from the ITTs performed at weeks 8, 15, and 21; (c) Fasting blood glucose levels after 6 h of fasting. Data are presented as the mean ± SEM. ** p < 0.01; *** p < 0.001; **** p < 0.0001. $$$$ p < 0.0001. The p-values for each week were determined by using a one-way ANOVA test and a Tukey’s multiple tests correction. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.

Figure 4

Polyphenol-supplemented HFD increased the oxidative stress markers in the kidneys. (a) Malondialdehyde (MDA) levels in the kidneys of standard-chow-fed, HFD-only, and HFD + Pol mice measured by the TBARS assay. (b) Relative mRNA levels of Sod, Cat, and Gsr. Data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01. The p-values were determined by using a one-way ANOVA test and a Tukey’s multiple tests correction. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.

Figure 5

HFD + Pol upregulated the expression of fibrosis and kidney-damage markers. (a) Relative mRNA levels of several fibrosis, oxidative stress and kidney damage markers, kidney injury molecule-1(kim1), fibronectin-1, carbohydrate-responsive element-binding protein b (Chrebpb), glucose transpoorter1 (Glut1), thioredoxin-interacting protein (Txnip), Osteopontin (Opn), Transforming growth factor beta-1 (Tgfb), Nuclear factor erythroid 2-related factor 2 (Nrf2), Lipocalin 2 (Lcn2) and Adiponectin. (b) Protein levels of LCN2 measured by ELISA in the kidney. Data are presented as the mean ± SEM. * p < 0.05. The p-values for the ELISA assay and Glut1, Txnip, Opn, Tgfb and Nrf2 genes were determined by using a one-way ANOVA test and Tukey’s multiple tests correction. For genes showing different variances like Kim-1, Fibronectin-1, Cherbpa, Cherbpb, Lcn2 and Adiponectin a one-way ANOVA with Brown-Forsythe and Welch’s correction and a Dunnett’s comparisons tests were performed. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.

Figure 6

HFD + Pol upregulated the expression of fibronectin in the liver. (a) Hepatic TG content. The concentration of TG (ng/uL) was measured in the livers of chow, HFD and HFD + Pol mice. (b) Relative mRNA levels of different genes to evaluate the general state of the livers, fatty acid synthase (Fasn), sterol regulatory element binding protein, (Srebp1c), Cell death activator CIDE-3, (Fsp27), Binding immunoglobulin protein (Bip) and C/EBP Homologous Protein (Chop). (b) Protein levels of LCN2 measured by ELISA in the kidney. Data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; **** p < 0.0001. The p-values for the TG assay and Fasn gene were determined by using a one-way ANOVA test and a Tukey’s multiple tests correction. For genes showing different variances like Fsp27B, Fibronectin-1, Srebp1c, Chop, Lcn2 and Bip a one-way ANOVA with Brown-Forsythe and Welch’s correction and a Dunnett’s comparisons tests were performed. Chow Diet n = 8; HFD = 11; HFD + Pol = 10.