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Nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease among children and adolescents in the developed world. Betaine, as a methyl donor, recently has been demonstrated to exert its hepatoprotective effects through rectifying the genomic DNA hypomethylation state. However, whether betaine supplementation affects N6-methyladenosine (m(6)A) mRNA methylation in NAFLD is still unknown. We conducted the current study to investigate the effects of betaine supplementation during adolescence on high-fat diet-induced pathological changes in liver of mice, and we further identified the effects of betaine supplementation on expression of the fat mass and obesity-associated gene (FTO) and hepatic m(6)A mRNA methylation. Our results showed that betaine supplementation across adolescence significantly alleviated high-fat-induced impairment of liver function and morphology as well as ectopic fat accumulation. Surprisingly, no significant effects on serum TG and NEFA level, as well as fat mass, were observed in mice supplemented with betaine. We also found that high-fat diet upregulated ACC1 and FAS gene expression and downregulated HSL and ATGL gene expression. However, these alterations were rectified by betaine supplementation. Moreover, an m(6)A hypomethylation state and increased FTO expression were detected in mice fed with high-fat diet, while betaine supplementation prevented these changes. Our results suggested that betaine supplementation during adolescence could protect mice from high-fat-induced NAFLD by decreasing de novo lipogenesis and increasing lipolysis. Furthermore, a novel FTO-dependent function of m(6)A may involve in the hepatoprotective effects of betaine.

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Backgroundn-3 long chain polyunsaturated fatty acid (n-3 LC PUFA) increases β-oxidation and limits lipid accumulation in adipocytes. The current study was conducted to determine whether their precursor alpha-linolenic acid (ALA) could also exert the above effects and how AMP-activated protein kinase (AMPK) was involved.MethodsAMPKα1−/−, AMPKα2−/− mice and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD with ALA. Body weight was recorded weekly and serum was collected. Adipocytes size and expression of key players involved in mitochondrial biogenesis and lipid oxidation were also measured.ResultsOur results showed an elevated serum adiponectin level and a decreased leptin and insulin level in WT mice fed HFD with ALA when compared with WT mice fed HFD. In addition, dietary ALA decreased epididymal adiposity and adipocytes size in WT mice. At protein level, mitochondrial genes (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [PGC1α] and nuclear respiratory factor-1 [nrf1]) and β-oxidation related genes (carnitine palmitoyltransferase 1A [CPT1a] and peroxisome proliferator-activated receptor alpha [PPARα]) were upregulated by dietary ALA in epididymal fat of WT mice. Consistently, dietary ALA also increased mitochondrial genomic DNA copy numbers. Moreover, lipogenesis was repressed by dietary ALA, indicated by that expression of fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and stearoyl-CoA desaturase 1 (SCD1) were decreased. However, these aforementioned effects were abolished in the AMPKα1 and AMPKα2 knockout mice.ConclusionsOur results suggest that ALA could improve adipose tissue function and its anti-adipogenic effects are dependent on AMPK.

Selenium-enriched exopolysaccharides (EPS) produced by Enterobacter cloacae Z0206 have been proven to possess effect on reducing blood glucose level in diabetic mice. To investigate the specific mechanism, we studied the effects of oral supply with EPS on skeletal muscle glucose transportation and consumption in high-fat-diet-induced diabetic KKAy mice. We found that EPS supplementation increased expressions of glucose transporter 4 (Glut4), hexokinase 2 (hk2), phosphorylation of AMP-activated kinase subunit α2 (pAMPKα2), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and increased expression of characteristic protein of oxidative fibers such as troponin I and cytochrome c (Cytc). Furthermore, we found that EPS increased glucose uptake and expressions of pAMPKα2 and PGC-1α in palmitic acid (PA)-induced C2C12 cells. However, while EPS inhibited AMPKα2 with interference RNA (iRNA), effects of EPS on the improvement of glucose uptake diminished. These results indicated that EPS may improve skeletal muscle glucose uptake of diabetic KKAy mice through AMPKα2-PGC-1α pathway.

Abstract. Polysaccharides belong to a structurally diverse class of macromolecules, with the necessary flexibility for the precise regulatory mechanisms and high capacity for carrying biological information. On the basis of a previous study regarding the administration of selenium-enriched exopolysaccharides (Se-ECZ-EPS) produced by Enterobacter cloacae (E. cloacae) Z0206 which resulted in a reduction of blood glucose levels and showed significant anti-inflammatory and anti-diabetic effects, the present study was conducted to evaluate the effects and mechanism of EPS on the alleviation of fat inflammation in high-fat-diet (HFD) induced-diabetic KKAy mice. The HFD induced-diabetic KKAy mice were gavaged once daily with EPS (0.2 mg/g body weight) or distilled water, while the C57BL/6J mice were gavaged with distilled water. Six weeks later visceral adipose tissue (VAT) was collected for quantified polymerase chain reaction (qPCR) and western blot (WB) analysis. The results showed that following supplementation with EPS, interleukin (IL) 6, IL1β and tumor necrosis factor (TNF) α mRNA expression in VAT were significantly reduced, while Glut4, pAMPK and SirT1 protein expression were markedly increased when compared with KKAy mice gavaged with water. Furthermore, ATGL and HSL mRNA were also significantly decreased. Subsequently, 3T3-L1 adipocytes were treated with insulin to induce insulin resistance to determine the mechanism by which EPS affects inflammation. Following the treatment of adipocytes with 100 nM insulin for 8 h, IL6 and TNFα mRNA expression were significantly increased, while the content of glucose uptake and Glut4 protein expression were significantly decreased. When treated with 100 nM insulin and 0.1 mg/ml EPS, no significant change in IL6 and TNFα mRNA expression or glucose uptake were observed. However, when SirT1-siRNA or AMPKα1-siRNA was tranfected into the 3T3-L1 adipocytes prior to treatment with insulin and EPS, there was a significant increase in IL6 and TNFα mRNA abundance. In conclusion, VAT inflammation and lipolysis in HFD-induced KKAy mice were significantly decreased following EPS usage. Moreover, EPS may alleviate VAT inflammation primarily through the AMPK/SirT1 pathway.

n-3 Long-chain PUFA up-regulate intestinal lipid metabolism. However, whether these metabolic effects of PUFA on intestine are mediated by AMP-activated protein kinase (AMPK) remains to be elucidated. To determine the effects of α-linolenic acid (ALA) on intestinal fatty acid (FA) metabolism and whether these effects were affected by AMPK deletion, mice deficient in the catalytic subunit of AMPKα1 or AMPKα2 and wild-type (WT) mice were fed either a high-fat diet (HF) or HF supplemented with ALA (HF-A). The results showed that ALA supplementation decreased serum TAG content in WT mice. ALA also increased mRNA expression of genes (carnitine palmitoyltransferase 1a, acyl-CoA oxidase 1, medium-chain acyl-CoA dehydrogenase, cytochrome P450 4A10 and pyruvate dehydrogenase kinase isoenzyme 4a) involved in intestinal lipid oxidation and mRNA expression of TAG synthesis-related genes (monoacylglycerol O-acyltransferase 2, diacylglycerol O-acyltransferases 1 and 2) in WT mice. Consistent with these, expression levels of phosphorylated AMPKα1 and AMPKα2 were also increased in WT mice after ALA addition. However, in the absence of either AMPKα1 or AMPKα2, ALA supplementation failed to increase intestinal lipid oxidation. In addition, no significant effects of either diet (HF and HF-A) or genotype (WT, AMPKα1-/-and AMPKα2) on FA uptake in the intestine and faecal TAG output were observed. Our results suggest that AMPK is indispensable for the effects of ALA on intestinal lipid oxidation.