Research Article
The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase α1 subunit

https://doi.org/10.1016/j.jnutbio.2015.08.014Get rights and content

Abstract

The current study was conducted to determine whether betaine could improve fatty acid oxidation, mitochondrial function and N6-methyladenosine (m6A) mRNA methylation in adipose tissue in high-fat-induced mice and how AMP-activated protein kinase α1 subunit (AMPKα1) was involved. AMPKα1 knockout mice and wild-type mice were fed either a low-fat diet, high-fat diet or high-fat diet supplemented with betaine in the drinking water for 8 weeks. Our results showed that mitochondrial genes (PGC1α) and β-oxidation-related genes (CPT1a) at protein level were increased in wild-type mice supplemented with betaine when compared with those in mice with high-fat diet. Betaine also decreased FTO expression and improved m6A methylation in adipose tissue of wild-type mice with high-fat diet. However, betaine failed to exert the abovementioned effects in AMPKα1 knockout mice. In adipocytes isolated from mice with high-fat diet, betaine treatment increased lipolysis and lipid oxidation. Moreover, betaine decreased FTO expression and increased m6A methylation. However, while AMPKα1 was knockdown, no remarkable changes in adipocytes were observed under betaine treatment. Our results indicated that betaine supplementation rectified mRNA hypomethylation and high FTO expression induced by high-fat diet, which may contribute to its beneficial effects on impaired adipose tissue function. Our results suggested that the AMPKα1 subunit is required for the beneficial effects of betaine on dysfunctional adipose tissue and m6A methylation. These results may provide the foundation for a mechanism that links m6A methylation status in RNA, AMPKα1 phosphorylation and dysfunctional adipose tissue induced by high-fat diet.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent disease which in most case is in association with insulin resistance, obesity and Type 2 diabetes [1]. Although evidence shows that NAFLD is caused by ectopic fat deposition within liver cells, the pathogenesis of NAFLD is still not clearly understood. Adipose tissue dysfunction is considered to be the central mechanism involved in the development of NAFLD [2], [3], as in normal situations adipose tissue is the major reservoir for lipids. Free fatty acids' (FFAs) influx from adipose tissue, due to uncontrolled lipolysis under the situation of insulin resistance, contributes to approximately 60% of liver triglycerides (TG) and plays a causal role in the development of obesity-related steatohepatitis in NAFLD [4], [5].

Betaine (trimethylglycine) is a natural component which serves as a methyl donor in the transmethylation of homocysteine and as an osmolyte maintaining fluid balance. For years, the hepatoprotective effects of betaine has been declared in a variety of experimental animal models of liver disease, mostly including alcoholic liver disease and NAFLD [6], [7], with different mechanisms involved. However, the detailed cellular and molecular mechanisms relating to liver diseases remain elusive. Recently, several studies demonstrated that betaine could normalize the aberrant DNA methylation in liver disease. Wang et al.[8] reported that betaine attenuated hepatic steatosis through elevating hepatic genomic methylation in mice fed with a high-fat (HF) diet. Moreover, studies also suggested that betaine exerted its hepatoprotective effect through rectification of impaired adipose tissue function and DNA methylation status [9], [10]. Recently, the role of N6-methyladenosine (m6A) mRNA methylation in adipogenesis has been declared [11], and we found that betaine could improve hepatic m6A methylation status [12]. However, whether betaine also affects m6A methylation in adipocytes and how m6A methylation is involved in the beneficial effects of betaine remains to be elucidated.

AMP-activated protein kinase (AMPK), as the cellular energy sensor, plays a key role in regulating adipocyte lipid metabolism [13]. Specially, the α1 catalytic subunit accounts for the most of the activity of this kinase in white adipose tissue [14], [15], [16]. And evidences suggest that AMPK is involved in the beneficial effects of betaine on the attenuation of hepatic steatosis [17], [18]. Moreover, since in adipocytes phosphorylation of AMPK increases in response to a decreased expression of FTO [19], which exerts a strong oxidative demethylation activity targeting m6A in nuclear RNA [20] and correlates with obesity, we hypothesize that betaine may increase m6A methylation and AMPK may involve in these process. Taken together, these results led us to investigate the effects of betaine supplementation on m6A methylation status of adipose tissue in HF diet-induced mice. And we focused our study on whether AMPKα1 was required for the effects of betaine on impaired adipose tissue function and m6A methylation in mRNA by using AMPKα1 knockout (KO) mice.

Section snippets

Animal model and experimental protocol

The C57BL/6 mice [wild-type (WT) mice] and AMPKα1flox/flox mice, Rosacre-ER mice were originally purchased from the Jackson laboratory, and AMPKα1 was KO after tamoxifen injection in AMPKα1flox/floxRosacre-ER mice. All animals were maintained with unrestricted access to water and food under controlled temperature (22 ± 1oC), humidity and air flow conditions, with a fixed 12-h light–dark cycle (light on from 0800 h to 2000 h).

AMPKα1 KO mice and WT mice (male mice at 8 weeks of age) were fed on either

Effects of betaine on body weight gain (BWG) and plasma TG content in WT and AMPKα1 KO mice

As shown in Table 1, compared with mice with LF diet, BWG was significantly increased in both WT and AMPKα1 KO mice with HF diet. However, although betaine supplementation significantly decreased BWG in WT mice, no such changes were observed in AMPKα1 KO mice. No significant difference in food intake (FI) was observed in mice of all treatments. Compared with mice with LF diet, plasma TG and FFA level in mice with HF were significantly increased. Interestingly, while betaine supplementation in

Discussion

In the current study, we provided evidence that betaine could improve adipose tissue function through increasing fatty acid oxidation and mitochondrial function, which contributed to a hepatoprotective effect in a mouse model of NAFLD induced by HF diet feeding. We also found that betaine could also rectify m6A hypomethylation status in RNA and high FTO expression in adipose tissue in mice induced by HF diet. Most importantly, our results suggested that the catalytic subunit α1 of AMPK was

Acknowledgements

This work was financially supported by National Basic Research Program of China (Grant No.2012CB124705) and the Special Fund for Cultivation and Breeding of New Transgenic Organism (Grant No. 2014ZX0800949B).

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      Changes in gene expression that have been revealed in rodents with insulin resistance are partially linked to the well-known properties of betaine, i.e., its action as a donor of methyl groups [9]. This assumption is supported by results indicating that betaine prevents mRNA hypomethylation in adipose tissue of mice fed an HFD [62]. Proper RNA and DNA methylation is vital for appropriate gene expression, while hypo- or hypermethylation disturbs this process [13].

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    Funding source: This work was financially supported by National Basic Research Program of China (Grant No.2012CB124705) and the Special Fund for Cultivation and Breeding of New Transgenic Organism (Grant No. 2014ZX0800949B).

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