Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus

Abstract

Chronic consumption of high-fat-and-fructose diets (HFFD) is associated with the development of insulin resistance (InsRes) and obesity. Systemic insulin resistance resulting from long-term HFFD feeding has detrimental consequences on cognitive performance, neurogenesis, and long-term potentiation establishment, accompanied by neuronal alterations in the hippocampus. However, diet-induced hippocampal InsRes has not been reported. Therefore, we investigated whether short-term HFFD feeding produced hippocampal insulin signaling alterations associated with neuronal changes in the hippocampus. Rats were fed with a control diet or an HFFD consisting of 10% lard supplemented chow and 20% high-fructose syrup in the drinking water. Our results show that 7 days of HFFD feeding induce obesity and InsRes, associated with the following alterations in the hippocampus: (1) a decreased insulin signaling; (2) a decreased hippocampal weight; (3) a reduction in dendritic arborization in CA1 and microtubule-associated protein 2 (MAP-2) levels; (4) a decreased dendritic spine number in CA1 and synaptophysin content, along with an increase in tau phosphorylation; and finally, (5) an increase in reactive astrocyte associated with microglial changes. To our knowledge, this is the first report addressing hippocampal insulin signaling, as well as morphologic, structural, and functional modifications due to short-term HFFD feeding in the rat.

Figure 1

Obesity and insulin resistance parameters. (A) Determination of: food consumption, water consumption, caloric intake, and weight gain in ctrl and high-fat-and-fructose diet (HFFD) rats. (B) Serum glucose concentration in fasted animals in basal state and after 15 minutes of an intraperitoneal insulin bolus administration of 5 U/kg (+INS). (C) Serum insulin and leptin levels in fasted animals; Spearman rank test showing a positive correlation between the concentration of insulin and leptin serum levels. Mean and s.e.m. are shown. n=10 to 12 rats per group for (A), n=6 to 10 rats per group for (B and C). n.s.: not significant,*P<0.05, **P<0.01 vs. ctrl.

Figure 2

Hippocampal insulin signaling assessment. Western blot of insulin signaling proteins: (A) phospho-Y1146-insulin receptor (IR), (B) phospho-Y608-insulin receptor substrate-1 (IRS-1), (C) phospho-S473-Akt and (D) phospho-T389-S6K. Representative western blots and densitometric analysis. Mean and s.e.m. are shown from 6 to 10 rats per group *P<0.05, **P<0.01 vs. ctrl.

Figure 3

Immunohistochemical and morphologic analysis of the hippocampus. (A) Wet weight measurements of dissected hippocampus and linear regression analysis showing a negative linear correlation between hippocampus weight and caloric intake. (B) Nissl staining in whole hippocampus reconstructions from × 10 images. (C) Sholl analysis in Golgi-stained CA1 neurons. Representative traces of ctrl and high-fat-and-fructose diet (HFFD) neurons along graphic description of Sholl analysis parameters (concentric circles: 20 μm). (D) Microtubule-associated protein 2 (MAP-2) immunohistochemistry in CA1 (A–D) and dentate gyrus (DG) (E–H). (E) MAP-2 densitometry. (F) Representative western blot of phospho-S199/S202-tau. Pyr, stratum pyramidale; Rad, stratum radiatum; Gcl, granular layer; H, hilus. Mean and s.e.m. are shown. n.s.: not significant n=10 rats per group for (A, E), n=4 rats per group for (B, D), n=3 to 5 neurons per rat; 4 rats per group for (C). Scale bar=100 μm in (C, D): a, b, e, and f; 20 μm in (D): c, d, g, and h. *P<0.005, **P<0.001 vs. ctrl.

Figure 4

Evaluation of synaptic markers. (A) Dendritic spine count on basal and apical dendrites of CA1 neurons. Representative Golgi-stained dendrites of ctrl and high-fat-and-fructose diet (HFFD) neurons. (B) Representative western blot and densitometry of synaptophysin. Mean and s.e.m. are shown. n=15 to 20 10 μm segments taken from 6 to 12 neurons per rat; 4 rats per group for (A), n=8 to 10 rats per group for (B). Scale bar: 1 μm in (A). *P<0.05 and **P>0.001 vs. ctrl.

Figure 5

Changes in hippocampal astrocytes and microglia. (A) Glial fibrillary acidic protein (GFAP) immunohistochemistry in CA1 (A–D) and dentate gyrus (DG) (E–J). (I and J)The detail of GFAP+ processes onto the granular layer of the DG is shown. (B) Number of GFAP+ cells per mm², and total astrocytic area (GFAP+) in CA1 and DG. (C) Iba1 immunohistochemistry in CA1 (A–F) and DG (G–L). Pyr, stratum pyramidale; Rad, stratum radiatum; Gcl, granular layer; H, hilus. Mean and s.e.m. are shown. n=4 rats per group. Scale bar: 100 μm in (A): a, b, e, f, i, and j; in (C): a, b, g, and h; 20 μm in (A): c, d, g, and h; in (C) c, d, e, f, i, j, k, and l. *P<0.05, **P>0.001 vs. ctrl.

Figure 6

Proposed integrative model of systemic and hippocampal alterations associated with insulin resistance and obesity caused by short-term high-fat and fructose feeding. Parameters/mechanisms assessed in this work are written in bold font, whereas known mechanisms reported in the literature are shown in plain font. MAP-2, microtubule-associated protein 2; IRS-1, insulin receptor substrate-1.