Effect of dietary fat and the circadian clock on the expression of brain-derived neurotrophic factor (BDNF)

https://doi.org/10.1016/j.mce.2016.04.015Get rights and content

Highlights

  • Bdnf mRNA oscillates robustly in brain and liver.

  • Bdnf mRNA does not show a phase advance under restricted feeding (RF).

  • Clock knockdown in hippocampal neurons up-regulates Bdnf mRNA.

  • Clock knockdown in hepatocytes down-regulates Bdnf mRNA.

  • RF combined with HFD leads to high AMPK and mBDNF levels.

Abstract

Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophin in the brain and its decreased levels are associated with the development of obesity and neurodegeneration. Our aim was to test the effect of dietary fat, its timing and the circadian clock on the expression of BDNF and associated signaling pathways in mouse brain and liver. Bdnf mRNA oscillated robustly in brain and liver, but with a 12-h shift between the tissues. Brain and liver Bdnf mRNA showed a 12-h phase shift when fed ketogenic diet (KD) compared with high-fat diet (HFD) or low-fat diet (LFD). Brain or liver Bdnf mRNA did not show the typical phase advance usually seen under time-restricted feeding (RF). Clock knockdown in HT-4 hippocampal neurons led to 86% up-regulation of Bdnf mRNA, whereas it led to 60% down-regulation in AML-12 hepatocytes. Dietary fat in mice or cultured hepatocytes and hippocampal neurons led to increased Bdnf mRNA expression. At the protein level, HFD increased the ratio of the mature BDNF protein (mBDNF) to its precursor (proBDNF). In the liver, RF under LFD or HFD reduced the mBDNF/proBDNF ratio. In the brain, the two signaling pathways related to BDNF, mTOR and AMPK, showed reduced and increased levels, respectively, under timed HFD. In the liver, the reverse was achieved. In summary, Bdnf expression is mediated by the circadian clock and dietary fat. Although RF does not affect its expression phase, in the brain, when combined with high-fat diet, it leads to a unique metabolic state in which AMPK is activated, mTOR is down-regulated and the levels of mBDNF are high.

Introduction

The circadian clock, located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus, generates endogenous rhythms of ∼24 h. Light perceived by the retina synchronizes the SCN clock and leads to the generation of exactly 24-h rhythms (Reppert and Weaver, 2002). Similar clocks are found in peripheral tissues, such as the liver, intestine and adipose tissue (Ando et al., 2005, Froy and Chapnik, 2007, Zvonic et al., 2006). The clock mechanism in the brain and peripheral tissues comprises two loops. The positive loop consists of the CLOCK and BMAL1 heterodimer that mediates transcription of tissue-specific genes and those of the negative feedback loop. The negative feedback loop consists of the PERIOD (PERs) and CRYPTOCHROME (CRYs) proteins that inhibit CLOCK:BMAL1-mediated transcription (Reppert and Weaver, 2002). The circadian clock regulates metabolism and disrupted rhythms lead to hyperphagia, diabetes, obesity (Froy, 2010, Marcheva et al., 2010, Oishi et al., 2005, Turek et al., 2005) and neurodegeneration (Yesavage et al., 2011).

Restricted feeding (RF) limits the time of food availability without caloric restriction, i.e., the food is available for 3–5 h every day at the same time (Sherman et al., 2011). RF causes uncoupling between the SCN and peripheral tissues. Numerous pathways usually synchronized by the SCN alter their expression to the time of food availability (Damiola et al., 2000). We have shown that long-term RF increases the amplitudes of clock gene expression, and the expression and activity of catabolic factors and decreases the expression of pro-inflammatory factors, all of which improve health (Sherman et al., 2011, Sherman et al., 2012).

Brain-derived neurotrophic factor (BDNF), the most abundant neurotrophin in the brain, promotes neuronal differentiation/maturation in the developing central nervous system. In addition, it positively impacts the survival and maintenance of neuronal functions, and decreased BDNF levels could be associated with the development of neurodegenerative diseases (Allen et al., 2013, Connor et al., 1997, Laske et al., 2006, Montano et al., 2010, Skaper, 2012). Aside from its effect on neuron survival, BDNF and its cognate receptor, TrkB, are involved in the regulation of energy balance and glucose homeostasis in the central nervous system. Perturbed BDNF signaling in the brain triggers hyperphagia and obesity in mice, suggesting that BDNF acts as an anorexigenic signaling molecule (Rios et al., 2001). BDNF is initially synthesized as a precursor (proBDNF), which is subsequently cleaved to generate mature BDNF (mBDNF) (Nagappan et al., 2009). BDNF action during memory is mediated by activating the translation machinery through the engagement of mammalian target of rapamycin (mTOR) and its downstream target P70S6 kinase (P70S6K) (Slipczuk et al., 2009). mTOR activation leads to AMP-activated protein kinase (AMPK) inhibition (Jiang et al., 2008a, Jiang et al., 2008b, Nguyen et al., 2013). In skeletal muscle, overproduction of BDNF increases fat oxidation in an AMPK-dependent manner, which, in turn, leads to the phosphorylation and inactivation of acetyl CoA carboxylase (ACC) (Matthews et al., 2009), the rate limiting enzyme in fatty acid synthesis. In addition, activation of brain AMPK results in inhibition of mTOR and its target P70S6K, both of which are activated by BDNF (Ishizuka et al., 2013). The relationship between AMPK and BDNF signaling in other peripheral tissues, such as the liver, remains open, despite its role in whole body metabolism.

Ad libitum high-fat diet (AL-HFD), rich in fat (∼45%) and carbohydrates, leads to decreased BDNF levels (Liu et al., 2014, Park et al., 2010). In addition, AL-HFD leads to changes in behavioral rhythmicity and disrupted circadian gene expression within the hypothalamus and liver and to disrupted cycling of hormones involved in fuel utilization (Barnea et al., 2010). We have recently shown that combining RF with HFD was able to counteract the harmful effects of AL-HFD by improving the functionality of key metabolic proteins and synchronizing the circadian clock and their target genes (Sherman et al., 2012). We hypothesize that dietary fat will disrupt BDNF expression and RF may rectify this effect. In this study, we tested the effect of dietary fat, its timing and the circadian clock on the expression of BDNF and associated signaling pathways in mouse brain and liver.

Section snippets

Animals, treatments, and tissues

Four-week-old male C57BL/6 mice were housed in a temperature- and humidity-controlled facility (23–24 °C, 60% humidity). Mice were entrained to a light-dark cycle of 12 h light and 12 h darkness (LD) for two weeks with food available ad libitum (AL). After two weeks, mice were fed AL low-fat diet (AL-LFD), AL high-fat diet (AL-HFD), restricted feeding (RF) LFD (RF-LFD), RF-HFD, ketogenic diet (KD) or RF-KD for 8 weeks. The HF diet was based on soybean oil and palm stearin (fatty acid

Bdnf mRNA exhibits a circadian expression

As decreased BDNF levels are associated with obesity and neurodegeneration and these pathologies are characterized by disrupted circadian rhythms, we analyzed the circadian expression of Bdnf mRNA under various levels of fat in the diet. Mice were fed ad libitum high-fat diet (AL-HFD) and ketogenic diet (AL-KD) and were compared to low-fat diet (AL-LFD) for 2 months. Bdnf mRNA was analyzed at 6 time-points around the circadian cycle in brain and liver tissues. Under all treatments, Bdnf mRNA

Discussion

In this study, we show that Bdnf expression is mediated by the circadian clock. Whereas in hepatocytes, clock knock-down down-regulated Bdnf expression, in hippocampal cells, its expression was up-regulated. It has been shown that the Bdnf promoter has several E-box enhancer sequences, the binding sites for the CLOCK:BMAL1 heterodimer (Jiang et al., 2008a, Jiang et al., 2008b). Similarly to our results, it has been shown that in hippocampus these enhancer sequences inhibit Bdnf expression (

Conflict of interest

Authors declare no conflict of interest.

Author contributions

Conceived and designed the experiments: OF, YG. Performed the experiments: MD, CB, YG, NC. Analyzed the data: YG, MD, CB, NC OF. Contributed reagents/materials/analysis tools: YG, NC. Wrote the paper: YG, OF.

Acknowledgements

This study was supported by the Israel Science Foundation (1044/12) and Strauss Group Ltd.

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