Obesity is currently a public health problem worldwide. This problem, which nowadays can be called an epidemic, is caused by various factors such as the intake of foods with high caloric content coupled with physical inactivity and genetics, which leads to other diseases, particularly type 2 diabetes and cardio-vascular disease [1
]. Recently, some carbohydrates have been of particular interest in the field of obesity because they are involved in lipid and carbohydrate metabolism, as in the case of fructans including agave fructans coming from Agave
plants from Mexico [3
]. Agave fructans are non-reducing carbohydrates formed by a complex mixture. They present a highly branched structure that includes β(2→1) and β(2→6) linkages and are called agavins [4
]. Importantly, it has been found that differences in the effect of prebiotics may be due to the molecular conformation, degree of polymerization and the species of each fructan [5
]. Fructans are resistant to hydrolysis by all human digestive enzymes but are fermented by the intestinal microbiota forming short-chain fatty acids (SCFAs), which are known to have important implications in host health [8
]. Moreover, fructans may also act as scavengers of reactive oxygen species [9
], decreasing inflammation and improving redox status. However, the roles of fructans in the brain have been unexplored until now. In this study, we focused on the hippocampus, frontal cortex and cerebellum because they are cortical areas particularly susceptible to diet-induced impairment, especially the hippocampus as it is an area involved in learning and memory [10
Elevated intake of saturated fat and simple sugars increase incidence of cognition disruption and neurodegenerative diseases including Alzheimer’s disease [10
], as well as impairing hippocampal synaptic plasticity and cognitive abilities such as learning and memory through brain derived neurotrophic factor (BDNF) [12
]. BDNF is a neurotrophin that facilitates neurogenesis, neuroprotection, neuroregeneration, cell survival, and synaptic plasticity, as well as formation, retention, and recall of memory in the hippocampus and frontal cortex [15
]. In rodents, BDNF acts as an anorexigenic factor, its postnatal reduction leads to hyperphagia and obesity [17
]. Interestingly, the administration of fructo-oligosaccharides and galacto-oligosaccharides increased hippocampal BDNF and N
-aspartate receptor subunit (NR1) expression in rodents possibly through the involvement of gut hormones [18
Glial derived neurotrophic factor (GDNF) is a potent promoter of neuronal survival in the central nervous system and peripheral nervous system. It has been shown to have effect on the number of cell populations including sensory and autonomic ganglia, Purkinje cells of the cerebellum, hippocampal neurons, as well as noradrenergic, serotoninergic, and cholinergic neurons [15
]. Several studies have shown that chronic hypothalamic or nigrostriatal expression of GDNF in rodents and primates can induce weight loss in animals with age-related obesity and prevent weight gain in young animals [19
]. Recently, a novel role of GDNF on the regulation of high-fat diet-induced obesity through increased energy expenditure has been noted [22
On the other hand, oxidative stress is defined as an imbalance between production of oxygen-free radicals and antioxidant defense mechanisms, leading to a cascade of reactions in which lipids, proteins, and DNA may be damaged [23
]. A high-fat diet (HFD) consumption led to increased brain oxidative damage through induction of lipid peroxidation in the hippocampus [24
] and protein carbonyls [26
]. Many studies have shown the prebiotic effect of fructans in obesity both in animals and humans [5
], though the neurobiological effects of fructans intake have not been explored. To increase neurotrophic factors and reduce oxidative stress in the brain caused by high-fat diet consumption, a dietary supplementation with agavins is proposed. The aim of this work is to investigate the impact on body weight gain occurring with agave fructans supplementation. We focused on the influence of agavins on neurotrophic factors and oxidative stress levels in the brain of obese mice under a high-fat diet.
3.1. Animals and Diets
Forty male C57BL/6 mice were purchased from UPEAL (Mexico City, Mexico). Mice were housed individually in cages with access to plastic nest-boxes, tunnels and nest building material. Tunnels and nest-boxes were rearranged once a week and replaced with new ones once a week, to promote a stimulating environment. Once a week dirty sawdust was removed and replaced with new sawdust, and the entire cage was replaced for a cleaned cage. Mice were handled as little as possible.
The mice had a 12-h light/dark cycle and were given free access to diet and water. After a week adaptation period, animals were distributed into 4 experimental groups (n =
10). Thus, mice were fed a standard diet (SD) (Picolab rodent diet, 5053, LabDiet®
, Richmond, IN, USA) (group 1) or a high-fat diet (HFD) (DIO rodent purified diet with 60% energy from fat-blue 58Y1, Test Diet®
, Richmond, IN, USA) (group 2), and two groups supplemented daily with 5% (HFD/A5) (group 3) or 10% (HFD/A10) (group 4) with agavins (Ingredion, Guadalajara, Mexico) in water (5 or 10 g/100 g of diet, respectively) as previously described [6
]. Mice were treated during 10 weeks. Food intake, taking into account spillage, was recorded daily for 10 weeks. Water consumption was also recorded daily to analyze prebiotic intake. The mean daily energy intake (kJ/day) was calculated as follows: food intake (g) X energy value of diet (kJ/g). The energy value for the STD diet was 14.26 kJ/g and the HFD was 21.33 kJ/g.
The experimental protocol and all animal procedures were approved and conducted according to the Guidelines of the Institutional Care and Use of Laboratory Animals Committee from SIACUAL system of CINVESTAV-Mexico (Permit Number: 0092-14) in accordance with current Mexican legislations (NOM-062-ZOO-1999).
3.2. Measurement of Body Weight Gain and Glucose, Triglyceride and Total Cholesterol Serum Levels
The body weight of each mice was assessed with an analytical balance and recorded every seven days until the end of the experiment. Blood samples were obtained from the caudal vein every 15 days until the end of the experiment and immediately used for the quantitative determination of glucose, triglycerides, and cholesterol (Accutrend, Roche Diagnostics©, Mannheim, Germany) with the Accutrend Plus meter (Roche Diagnostics©) (according to the manufacturer´s instructions). At the end of the experiment mice were sacrificed by cervical dislocation and the hippocampus, frontal cortex and cerebellum were dissected and stored in 300–500 µL of Buffer A: HEPES 10 mM, pH 7.9, Nonidet P-40 (Sigma-Aldrich, Toluca, Mexico) at 0.6%, NaCl (Fermont, Mexico City) 150 mM, EDTA) 1 mM and complete Mini Protease Inhibitor (Roche Diagnostics©); at −20 °C prior to processing.
3.3. Quantification of Short-Chain Fatty Acids (SCFAs) in Feces
To individually quantify short-chain fatty acids (SCFAs) we collected mice feces once a week for the whole experiment. SCFAs were measured with an HP 5890 series II gas chromatograph (Hewlett-Packard, Waldbronn, Germany) equipped with an HP-20 M column and a flame ionization detector as described in [6
] with a minor modifications. Briefly, 0.2 g of feces were weighed and acidified using 70 µL of H2
and 480 µL of water and centrifuged at 14,000 rpm for 10 min. SCFAs were extracted by shaking with 0.2 mL of diethyl ether and finally centrifuged at 14,000 rpm for 5 min. One µL of the organic phase was injected directly into the capillary column. The initial temperature was 80 °C and the final temperature was 200 °C, using He and N2
as the carrier gases and O2
as combustion gas. Samples were analyzed in duplicates. Calibration curves of acetic, propionic and butyric acids were used to carry out SCFAs quantification in the samples.
3.4. Tissue Homogenization and Measurement of Protein Concentration
Brain samples were homogenized in a 300–500 µL of buffer A in a silent crusher device (Roche Diagnostics©) at 4°C. Protein concentration of samples was determined using the bicinchoninic acid method as described in Franco-Robles et al. [58
]. Briefly, a 1:20 dilution of samples was performed. Bicinchoninic acid (Sigma-Aldrich) (0.2 g) was dissolved into 5 mL of H2
O to one ELISA plate of 96 wells. Two hundred µL of CuSO4
(Fermont, Mexico City, Mexico) (4%) and 5.2 mL of micro reagent A were added. In the wells 0, 2, 4, 6 and 8 µL of BSA were loaded and adjusted to 100 mL with H2
O. Eight µL of the samples were then loaded in their respective wells, all measurements were done in triplicate. One hundred µL of the working solution were added to each well and incubated at room temperature for 2–4 h and the plate was read at 540 nm. The plate was covered to prevent evaporation.
3.5. Measurement of BDNF and GDNF Levels
In the total homogenate of mice hippocampus, frontal cortex and cerebellum, BDNF and GDNF levels were measured using the commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions for BDNF (Mouse BDNF ELISA Kit, MyBioSource, San Diego, CA, USA; catalog number MBS355435) and GDNF (GDNF Mouse ELISA Kit, abcam®, Cambridge, MA, USA; catalog number ab171178).
3.6. Determination of Thiobarbituric Acid Reactive Substances (TBARS) in the Brain
In the total homogenate of mice hippocampus, frontal cortex and cerebellum, the MDA (malondialdehyde)-TBA adduct formed from the reaction of MDA in samples with TBA were quantified with the OxiSelect™ TBARS Assay Kit (MDA Quantitation) (Cell Biolabs, INC., San Diego, CA, USA; catalog number STA-330) according to the manufacturer’s instructions. Five hundred µg of total homogenate were used. Concentrations were expressed as nmol TBARS/mg of protein in brain tissue.
3.7. Measurement of Oxidized Protein
In the total homogenate of mice hippocampus, frontal cortex and cerebellum, the quantification of carbonyls content was performed as described by Franco-Robles et al. [58
]. Five hundred µg of total homogenate were used. Carbonyl-DNPH Mix 2 was used as standard control (Supelco, Bellefonte, PA, USA). Concentrations were expressed as nmol carbonyls/mg of protein in brain tissue.
3.8. Statistical Analysis
Statistical analysis was performed using the software Statistica 8 (StatSoft Inc, Tulsa, OK, USA). Data obtained from mice were analyzed with one-way ANOVA and ANOVA repeated measures followed by Tukey’s tests for the difference between groups. Data are represented as the means ± standard error of the means (SEM). Values were considered statistically significant if p < 0.05.