A high-fat diet induces rapid changes in the mouse hypothalamic proteome

Abstract

Background: Prolonged over-consumption of a high-fat diet (HFD) commonly leads to obesity and insulin resistance. However, even 3 days of HFD consumption has been linked to inflammation within the key homeostatic brain region, the hypothalamus.

Methods: Mice were fed either a low-fat diet (LFD) or HFD containing 10% or 60% (Kcal) respectively from fat for 3 days. Mice were weighed, food intake measured and glucose tolerance calculated using intraperitoneal glucose tolerance tests (IPGTT). Proteomic analysis was carried out to determine if hypothalamic proteins were changed by a HFD. The direct effects of dietary fatty acids on mitochondrial morphology and on one of the proteins most changed by a HFD, dihydropyrimidinase-related protein 2 (DRP-2) a microtubule-associated protein which regulates microtubule dynamics, were also tested in mHypoE-N42 (N42) neuronal cells challenged with palmitic acid (PA) and oleic acid (OA).

Results: Mice on the HFD, as expected, showed increased adiposity and glucose intolerance. Hypothalamic proteomic analysis revealed changes in 104 spots after 3 days on HFD, which, when identified by LC/MS/MS, were found to represent 78 proteins mainly associated with cytoskeleton and synaptic plasticity, stress response, glucose metabolism and mitochondrial function. Over half of the changed proteins have also been reported to be changed in neurodegenerative conditions such as Alzheimer's disease. Also,in N42 neurons mitochondrial morphology and DRP-2 levels were altered by PA but not by OA.

Conclusion: These results demonstrate that within 3 days, there is a relatively large effect of HFD on the hypothalamic proteome indicative of cellular stress, altered synaptic plasticity and mitochondrial function, but not inflammation. Changes in N42 cells show an effect of PA but not OA on DRP-2 and on mitochondrial morphology indicating that long-chain saturated fatty acids damage neuronal function.

Keywords: High-fat diet; Hypothalamus; Mice; Neuronal plasticity; Proteomics.

Fig. 1

a Body weight of HFD mice was significantly higher than that of LFD mice after 2 and 3 days on diet b The intake of the HFD fed mice decreased after 1 day on diet but returned to LFD levels on days 2 and 3 (n = 6).c IPGTT in LFD and HFD fed mice after 3 days on diet. IPGTT was carried out as a non-recovery procedure as the effect of fasting and glucose administration can alter proteins for some time afterwards. Glucose levels at all time points tested was significantly higher in HFD fed mice (n = 8) (● HFD; ● LFD) as was AUC shown in (d). (* P < 0.05, **P < 0.01, ***P < 0.01)

Fig. 2

Heat map showing fold changes in proteins after 3 days of a HFD. Proteins are shown in multiples reflecting the number of spots which gave the same protein ID

Fig. 3

a Representative immunoblots showing changes in DRP-2 and beta-actin protein expression in response to fatty acid free BSA, PA and OA challenge in N42 cells b Quantification of DRP-2 bands normalised to beta-actin (n = 4 plates) BSA - bovine serum albumin, DRP-2 - dihydropyrimidinase-related protein 2, PA - palmitic acid, OA - oleic acid (* P < 0.05). c-e Representative fluorescence microscopy images of N42 hypothalamic neuronal mitochondria using MitoTracker® Red CMXRos. The red colour corresponds to mitochondria. Cells were challenged with, c fatty acid free BSA, d 200 μM PA and e 200 μM OA (Bar = 10 μm. Magnification = X100). f The percentage (%) area of the cell occupied by mitochondria after challenge. BSA - bovine serum albumin, PA - palmitic acid, OA - oleic acid (*P < 0.05, **P < 0.01, ***P < 0.001)