Fructose Consumption Affects Glucocorticoid Signaling in the Liver of Young Female Rats

Abstract

The effects of early-life fructose consumption on hepatic signaling pathways and their relation to the development of metabolic disorders in later life are not fully understood. To investigate whether fructose overconsumption at a young age induces alterations in glucocorticoid signaling that might contribute to development of metabolic disturbances, we analysed glucocorticoid receptor hormone-binding parameters and expression of its target genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) and lipid metabolism (lipin-1), as well as redox and inflammatory status in the liver of female rats subjected to a fructose-rich diet immediately after weaning. The fructose diet increased hepatic corticosterone concentration, 11β-hydroxysteroid dehydrogenase type 1 level, glucocorticoid receptor protein level and hormone-binding activity, as well as lipin-1 level. The expression of glucose-6-phosphatase was reduced in fructose-fed rats, while phosphoenolpyruvate carboxykinase remained unaltered. The fructose-rich diet increased the level of fructose transporter GLUT2, while the expression of fructolytic enzymes fructokinase and aldolase B remained unaltered. The diet also affected pro-inflammatory pathways, but had no effect on the antioxidant defence system. In conclusion, a fructose-rich diet applied immediately after weaning promoted lipogenesis and enhanced hepatic glucocorticoid signaling, possibly to protect against inflammatory damage, but without an effect on gluconeogenesis and antioxidant enzymes. Yet, prolonged treatment might ultimately lead to more pronounced metabolic disturbances.

Keywords: antioxidant enzymes; fructose-fed rat; glucocorticoid receptor; inflammation; lipin-1; lipogenesis.

Figure 1

The effects of a fructose-rich diet on plasma and liver corticosterone concentrations, and the level of hepatic 11βHSD1. Groups: control (C), fructose-fed (F). Corticosterone concentrations in plasma (a) and liver (b) were measured by EIA kit. 11βHSD1 protein level in hepatic microsomal fraction (c) was measured by Western blot. Relative integrated optical density of the immunoreactive bands was assessed by ImageQuant software, normalized to β-actin, and expressed as fold of the control. The values represent the means ± SEM (n = 9). Statistical significance of differences between experimental groups: * p < 0.05.

Figure 2

In vitro dexamethasone binding to glucocorticoid receptor (GR) from the liver of female rats subjected to a fructose-rich diet. Groups: control (C), fructose-fed (F). To measure dexamethasone binding to GR, aliquots of cytosols were incubated (18 h, 0 C) with increasing concentrations of [³H]dexamethasone (1–80 nM) in the presence and absence of 100-fold molar excess of unlabeled dexamethasone. Unbound [³H]dexamethasone was removed by dextran-charcoal. The saturation curves (a) were fitted to determine (b) the number of receptor binding sites (B_max) and (c) equilibrium dissociation constant (K_d). Relative binding potential (d) was calculated as the B_max/K_d ratio. The values represent the mean ± SEM (n = 9). All measurements were done in triplicate. Statistical significance of differences between experimental groups: * p < 0.05; ** p < 0.01.

Figure 3

In vitro dexamethasone binding to hepatic GR in the presence and absence of thioprotector. Groups: control (C), fructose-fed (F). Cytosols were incubated (1 h, on ice) in the absence or in the presence of 10 mM DTT and subsequently incubated (18 h, on ice) with 80 nM [³H]dexamethasone in the absence and in the presence of 100-fold excess of the radioinert dexamethasone. Unbound [³H]dexamethasone was removed by dextran-charcoal and specific binding was calculated by subtracting non-specific from total binding. The values represent the mean ± SEM (n = 9). All measurements were done in triplicate.

Figure 4

The effects of a fructose-rich diet on the level of GR, lipin-1 PEPCK, and G6Pase in the liver. Groups: control (C), fructose-fed (F). (a) GR protein level in hepatic cytosols and nucleosols, and (b) lipin-1 protein level in hepatic whole cell extracts, were measured by Western blot. Relative integrated optical density of the immunoreactive bands was assessed by ImageQuant software, normalized to equal load controls and expressed as fold of the control. The levels of PEPCK (c) and G6Pase (d) mRNA were determined by qPCR and expressed as fold of the control. The values represent the means ± SEM (n = 9). Statistical significance of differences between experimental groups: * p < 0.05.

Figure 5

The effects of a fructose-rich diet on the level of GLUT2, fructokinase, and aldolase B in the liver. Groups: control (C), fructose-fed (F). (a) GLUT2 protein level in the hepatic whole cell extracts was measured by immunoblotting. Relative integrated optical density of the immunoreactive bands corresponding to GLUT2 was assessed by ImageQuant software, normalized to β-actin, and expressed as fold of the control. Representative Western blot is shown. The level of fructokinase (b) and aldolase B (c) mRNA relative to β-actin mRNA was determined by SYBR Green real-time PCR and expressed as fold of the control. The values represent the means ± SEM (n = 9). Statistical significance (Student’s t-test) of differences between experimental groups: *** p < 0.001.

Figure 6

The effects of a fructose-rich diet on the level of NFkB, TNFα, and IL6 in the liver. Groups: control (C), fructose-fed (F). (a) NFkB protein level in hepatic cytosols and nucleosols was measured by Western blot. Relative integrated optical density of the immunoreactive bands corresponding to NFkB was assessed by ImageQuant software, normalized to equal load controls, and expressed as fold of the control. (b) The level of TNFα and IL6 mRNA relative to HPRT mRNA was determined by TaqMan real-time PCR and expressed as fold of the control. The values represent the means ± SEM (n = 9). Statistical significance of differences between experimental groups: ** p < 0.01.

Figure 7

The effects of a fructose-rich diet on the level of antioxidant enzymes in the liver. Hepatic whole cell extracts (50 µg protein) were subjected to SDS-PAGE and Western blotting. Relative integrated optical density was assessed by ImageQuant software. β-actin was used as loading control. Representative Western blots and relative quantification of antioxidant enzyme protein levels of control (C) and fructose-fed rats (F) are shown. Values are means ± SEM (n = 9) and are presented as fold of the control. SOD1, cytoplasmic copper-zinc superoxide dismutase; SOD2, mitochondrial manganese superoxide dismutase: CAT, catalase; GSH-Px, glutathione peroxidase; GSH-Red, glutathione reductase.