Cognitive impairment in metabolically-obese, normal-weight rats: identification of early biomarkers in peripheral blood mononuclear cells

Abstract

Background: Metabolically-obese, normal-weight (MONW) individuals are not obese in terms of weight and height but have a number of obesity-related features (e.g. greater visceral adiposity, insulin resistance, and increased risk of cardiovascular disease). The MONW phenotype is related to the intake of unbalanced diets, such as those rich in fat. Increasing evidence shows a relationship between high-fat diet consumption and mild cognitive impairment and dementia. Thus, MONW individuals could be at a greater risk of cognitive dysfunction. We aimed to evaluate whether MONW-like animals present gene expression alterations in the hippocampus associated with an increased risk of cognitive impairment, and to identify early biomarkers of cognitive dysfunction in peripheral blood mononuclear cells (PBMC).

Methods: Wistar rats were chronically fed with a 60% (HF60) or a 45% (HF45) high-fat diet administered isocalorically to control animals to mimic MONW features. Expression analysis of cognitive decline-related genes was performed using RT-qPCR, and working memory was assessed using a T-maze.

Results: High-fat diet consumption altered the pattern of gene expression in the hippocampus, clearly pointing to cognitive decline, which was accompanied by a worse performance in the T-maze in HF60 animals. Remarkably, Syn1 and Sorl1 mRNA showed the same expression pattern in both the hippocampus and the PBMC obtained at different time-points in the HF60 group, even before other pathological signs were observed.

Conclusions: Our results demonstrate that long-term intake of high-fat diets, even in the absence of obesity, leads to cognitive disruption that is reflected in PBMC transcriptome. Therefore, PBMC are revealed as a plausible, minimally-invasive source of early biomarkers of cognitive impairment associated with increased fat intake.

Keywords: Blood cells; Hippocampus; MCI; MONW.

Fig. 1

Effects of a control diet, a moderate high-fat diet (45% kcal from fats, HF45), and a very high-fat diet (60% kcal from fats, HF60) administered to male Wistar rats from the age of two months until the age of six months on working memory using spontaneous alternation paradigm in a T-maze. Food in HF groups was offered in isocaloric amounts to the NF group (control). Five trials per rat were performed and then % alternation was calculated per group. Data represent mean ± SD (n = 7). Statistics: * indicates that values from the HF groups are different from the control group (Student’s t test, p < 0.05). Additionally, a one-way ANOVA was performed followed by an LSD post hoc test; values not sharing a common letter (a, b) are significantly different (p < 0.05)

Fig. 2

Expression of cognitive impairment-related genes in the hippocampus in the groups described in Fig. 1. mRNA expression levels were measured by RT-qPCR. Data represent mean ± SD (n = 10) of ratios of specific mRNA levels normalized against Gdi1 and Rplp0 (used as reference genes). Data of the control (NF) group were set to 100%, serving as a reference to the rest of the values. Statistics: * indicates that values from the HF groups are different from the control group (Student’s t test, p < 0.05); additionally, p values indicate statistical trends towards significance. Furthermore, a one-way ANOVA was performed followed by an LSD post hoc test; values not sharing a common letter (a, b) are significantly different (p < 0.05); no letters = no statistical difference

Fig. 3

Expression of cognitive impairment-related genes in PBMC of male Wistar rats of three (a), five (b), and six (c) months of age submitted to a control (NF), HF45 or HF60 diet for four months. Food in the HF groups was offered in isocaloric amounts to the control group. mRNA expression levels were measured by RT-qPCR. Data represent mean ± SD (n = 10) of ratios of specific mRNA levels normalized against Itg1β (used as a reference gene). Data of the control (NF) group were set to 100%, serving as a reference to the rest of the values. Statistics: *indicates that values from the HF groups are different from the control group (Student’s t test, p < 0.05); additionally, p values indicate statistical trends towards significance. Furthermore, a one-way ANOVA was performed followed by an LSD post hoc test; values not sharing a common letter (a, b) are significantly different (p < 0.05); no letters = no statistical difference

Fig. 4

Protein levels of sortilin, synapsin I, and TMCC2 in the hippocampus in the groups described in Fig. 1 measured by Western Blot. Data represent mean ± SD (n = 5–7) of specific protein levels referred to beta actin (used as a reference gene). Data of the control group were set to 100%, serving as a reference to the rest of the values. Statistics: * indicates that values from the HF groups are different from the control group (Student’s t test, p < 0.05); additionally, p values indicate statistical trends towards significance. Furthermore, a one-way ANOVA was performed followed by an LSD post hoc test; values not sharing a common letter (a, b) are significantly different (p < 0.05); no letters = no statistical difference. Representative bands obtained in the Western Blot are shown: 15 μg of protein was loaded per lane. Beta actin was used as transfer and loading control

Fig. 5

Quantification of GFAP-stained cells in the hippocampus of male Wistar rats fed a control (NF), HF45 or HF60 diet from the age of two months until the age of six months, evaluated by immunohistochemistry. Food in HF groups was offered in isocaloric amounts to the NF group (control group). a Number of GFAP positive cells in the NF, HF45 and HF60 groups. GFAP-stained cells were counted from the whole glia present in the hippocampus samples, and referred to the glia area (positive cells/mm²). Data represent mean ± SD (n = 6 in control and HF60 groups; n = 5 in HF45 group). Statistics: P value indicates statistical trend towards significance (Student’s t-test). b An enlarged GFAP positive cell is shown as example of the specificity of the antibody. c Representative hippocampus sections immunostained for GFAP in the NF and HF60 groups. Insets: enlargement of the corresponding framed areas. *indicates GFAP-positive cells. Abbreviations: CA: cornu ammonis, G: glia. Scale bar: 200 μm and 50 μm for insets