Short-term high-fat feeding induces a reversible net decrease in synaptic AMPA receptors in the hypothalamus

 Dietary obesity compromises brain function, but the effects of high-fat food on synaptic transmission in hypothalamic networks as well as their potential reversibility are yet to be fully characterized.

• In two experiments:
• Three days, but not one day of high fat diet (HFD) decreased the levels of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) that contain the subunits GluA1 and GluA2
• The effects are reversed by normal-chow feeding
• High-fat feeding increased energy intake, body weight, and serum concentrations of insulin, leptin, free fatty acids, and corticosterone

Food rich in saturated fat impairs cognitive performance and compromises synaptic plasticity

• Changes in neuronal function induced by fluctuations in the quantity and quality of nutrient supply are of high clinical relevance
• Being obese is associated with an increased risk of cognitive decline and dementia
• Synaptic strength is homeostatically regulated via synaptic scaling in response to global activity changes in individual cells or across larger neuronal ensembles
• A major mechanism underlying synaptic scaling and, hence, plasticity is the trafficking of postsynaptic glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs)
• AMPARs are the main ionotropic glutamate receptors mediating fast excitatory synaptic transmission in the mammalian brain, and their regulated trafficking underlies activity-triggered changes in transmission
• The predominantly expressed AMPAR subunits in the forebrain, including hypothalamus and neocortex, are GlluA1 and GluA2, while the other subunits are less abundant
• We hypothesized that high-fat feeding increases synaptic strength in the hypothalamus, but not in cortical brain regions, and, that these changes are reversible by returning animals to regular diet
• Since wakefulness and wakefulness are associated with synaptic strength loss and behavioral changes, we also account for sleep loss and behavior

Animals

• Experiments were conducted in male Wistar rats (RjHan:WI) aged 10-11 weeks and weighing 300-400 g at the start of experiments (Janvier, Le Genest-Saint-Isle, France).
• Animals were housed, and experiments were performed at controlled temperature (20 ± 2°C) and humidity (55 ± 10%), and a controlled 12 h/12 h light/dark cycle with light onset at 6 AM.
• Water and food were available ad libitum. Failures to groom and/or loss of more than 20% body weight were set as criteria of potential sickness and lead to euthanasia.

Experimental design and procedure

• After their arrival at the central animal facility, all animals were fed 9% fat standard chow and handled for seven days
• Food intake was assessed daily by weighing initial food supply and respective remains to calculate daily energy intake
• Wakefulness and sleep were assessed using standard visual examination
• Sleep was scored whenever the rat showed a typical sleep posture and stayed immobile for at least 10 s
• Blood samples were collected via cardiac puncture when the animal was sacrificed, and centrifuged at 3,000 × g for 10 min to obtain serum, followed by storage at −80°C until further analysis

Synaptoneurosome preparation

• Cortical and hypothalamic tissue was homogenized in a glass Teflon homogenizer in synaptic protein extraction reagent (Syn-PER; Thermo Scientific, Rockford, IL, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific).
• The homogenate was centrifuged at 1,200 × g for 10 minutes at 4°C to remove cell debris, and the supernatant (cytosolic fraction) was removed and resuspended in Syn-PER. The protein concentration was determined by bicinchoninic acid assay.

Western blot analysis of protein levels

• Samples were heat-denatured and equal amounts (30 μg) of samples from each animal were separated with SDS-polyacrylamide gel electrophoresis (5% (w/v) stacking and 8% separating gels, 1.5 mm) before electrophoretic transfer onto a 0.45-µm-pore nitrocellulose membrane (Carl Roth, Karlsruhe, Germany) using a semidry transfer system at 0.8 mA/cm2
• Membranes were first blocked for one hour at room temperature in freshly prepared 5% powdered non-fat milk in phosphate-buffered saline, and subsequently incubated overnight with primary antibodies with agitation at 4°C
• Primary antibodies were diluted in blocking buffer containing 0.1 % Tween 20
• Fluorescence images were obtained with a FUSION-FX7 imaging system
• Integrated (background-subtracted) signal intensity for each antibody band was quantified with ImageJ software, and normalized with reference to the β-actin band, which was used as a loading control

Analyses of leptin, insulin, free fatty acids and corticosterone

• serum concentrations were measured using Enzyme-Linked Immunosorbent Assay (ELISA) kits
• Free fatty acids (FFA) were determined using FFA quantification kit (Abcam, Germany).

Statistical Analysis

• All data are expressed as means ± SEM.
• Analyses were performed with GraphPad Prism (GraphPad Software, San Diego, CA, USA).
• Energy intake, body weight gain, time spent asleep/awake, and blood parameters were analyzed via analyses of variance (ANOVA) and unpaired t tests. A P value of <.05 was regarded as statistically significant.

HFD increases energy intake and after three days increases wakefulness

• All three groups of rats in experiment 1 consumed comparable amounts of normal chow during the five baseline days (P >.14; Fig. 1B). After the switch to HFD, both high-fat groups increased their energy intake on day 6 compared to that measured in controls on day 5 (F(2,6) = 17.5, P <.001).
• In experiment 2, energy intake increased in both groups during the three days of HFD (each P < 0.001); in the recovery control group it returned to baseline levels during the subsequent four recovery days.
• Changes in body weight followed those in energy intake, with comparable baseline values (>.20), increases upon HFD (>.05), and a subsequent return to baseline (P <.79 for comparison between days 8-12)

Three-day high-fat feeding reduces AMPAR levels in the hypothalamus

• We hypothesized that even short-term exposure to HFD can trigger global changes in hypothalamic glutamatergic signaling.
• Therefore, we hypothesized that AMPARs can be significantly reduced in the HF3D compared to normal chow.

High-fat feeding does not decrease cortical AMPAR levels

• In both experiments, three days of high-fat in comparison to normal-chow feeding appeared to increase rather than decrease levels of cortical GluA1/GluA2 AMPAR subunits (Fig. 4E, F, G, K, L, and M).
• Significant or trendwise signs of HF3D-induced increases in both experiments were restricted to GluA2 (P <.03) and Ser831 (P =.06) phosphorylation in the supernatant fraction.
• These findings robustly indicate that three-day high-fattening does not increase markers of synaptic plasticity in cortex.

Discussion

• The effect develops across the first three days after the transition from regular diet to HFD and is reversible by returning animals to a regular diet.
• Changes in the availability of nutrients like FFA in the body periphery and related neuronal and endocrine signals including insulin and leptin are constantly tracked by the brain, in particular hypothalamic ventromedial and arcuate nuclei [23,36].
• Against this background, our results indicate a distinct, reversible pattern of net synaptic scaling in association with short-term high-fat feeding [25,26] that precedes the manifestation of obesity and associated synaptic changes [35,37] and may underlie global regulatory efforts to counteract diet-induced body weight gain.

Reference

10.1016/j.jnutbio.2020.108516