Metabolite and gene expression profiles suggest a putative mechanism through which high dietary carbohydrates reduce the content of hepatic betaine in <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>

Background

High-carbohydrate diets (HCD) are favoured by the aquaculture industry for economic reasons, but they can produce negative impacts on growth and induce hepatic steatosis. We hypothesised that the mechanism behind this is the reduction of hepatic betaine content.

Objective

We further explored this mechanism by supplementing betaine (1%) to the diet of a farmed fish Megalobrama amblycephala.

Methods

Four diet groups were designed: control (CD, 27.11% carbohydrates), high-carbohydrate (HCD, 36.75% carbohydrates), long-term betaine (LBD, 35.64% carbohydrates) and short-term betaine diet (SBD; 12 weeks HCD?+?4 weeks LBD). We analysed growth performance, body composition, liver condition, and expression of genes and profiles of metabolites associated with betaine metabolism.

Results

HCD resulted in poorer growth and liver health (compared to CD), whereas LBD improved these parameters (compared to HCD). HCD induced the expression of genes associated with glucose, serine and cystathionine metabolisms, and (non-significantly, p?=?.20) a betaine-catabolizing enzyme betaine-homocysteine-methyltransferase; and decreased the content of betaine, methionine, S-adenosylhomocysteine and carnitine. Betaine supplementation (LBD) reversed these patterns, and elevated betaine-homocysteine-methyltransferase, S-adenosylmethionine and S-adenosylhomocysteine (all p?≤?.05).

Conclusion

We hypothesise that HCD reduced the content of hepatic betaine by enhancing the activity of metabolic pathways from glucose to homocysteine, reflected in increased glycolysis, serine metabolism, cystathionine metabolism and homocysteine remethylation. Long-term dietary betaine supplementation improved the negative impacts of HCD, inculding growth parameters, body composition, liver condition, and betaine metabolism. However, betaine supplementation may have caused a temporary disruption in the metabolic homeostasis.

     

<Emphasis Type="Italic">Agaricus bisporus</Emphasis> supplementation reduces high-fat diet-induced body weight gain and fatty liver development

Obesity is a global epidemic characterized not only by excessive fat deposition but also by important complications such as nonalcoholic liver steatosis. Beneficial antiobesogenic effects have been described for some mushrooms. The current study aimed to demonstrate the protective effect of Agaricus bisporus (AB) supplementation against the metabolic alterations induced by high-fat-diet (HFD) feeding. Eight-week-old C57BL/6J mice were fed for 10 weeks with one of the following diets: (1) control diet (n?=?7), (2) HFD (n?=?7), (3) HFD supplemented with 5% AB (n?=?9), and (4) HFD supplemented with 10% AB (n?=?9). A pair-fed group was also included for the 10% AB group (n?=?6). The impact of AB supplementation on food intake, body weight gain, and liver and fat pad weights was examined. Biochemical, histological, and molecular parameters were also analyzed. Dietary supplementation with 10% AB reduced the HFD-induced increase in body, epididymal, and mesenteric fat weights (p?<?0.01, p?<?0.05, and p?<?0.05, respectively). Supplementation with AB also reduced liver damage in a dose-dependent manner (p?<?0.01 and p?<?0.001). This effect was confirmed by histological analysis that showed that liver steatosis was markedly reduced in mice fed with AB. The beneficial properties of 10% AB supplementation appear to be mediated through a decrease in food intake and via stimulation of mesenteric and hepatic free-fatty acid beta-oxidation, along with a decrease in epidydimal and hepatic expression of CD36. In conclusion, supplementation with AB prevents excessive body weight gain and liver steatosis induced by HFD consumption.     

Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoter and elevating genomic methylation in mice fed a high-fat diet

Aberrant DNA methylation contributes to the abnormality of hepatic gene expression, one of the main factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Betaine is a methyl donor and has been considered to be a lipotropic agent. However, whether betaine supplementation improves NAFLD via its effect on the DNA methylation of specific genes and the genome has not been explored. Male C57BL/6 mice were fed either a control diet or high-fat diet (HFD) supplemented with 0%, 1% and 2% betaine in water (wt/vol) for 12 weeks. Betaine supplementation ameliorated HFD-induced hepatic steatosis in a dose-dependent manner. HFD up-regulated FAS and ACOX messenger RNA (mRNA) expression and down-regulated PPARα, ApoB and MTTP mRNA expression; however, these alterations were reversed by betaine supplementation, except ApoB. MTTP mRNA expression was negatively correlated with the DNA methylation of its CpG sites at −184, −156, −63 and −60. Methylation of these CpG sites was lower in both the 1% and 2% betaine-supplemented groups than in the HFD group (averages; 25.55% and 14.33% vs. 30.13%). In addition, both 1% and 2% betaine supplementation significantly restored the methylation capacity [S-adenosylmethionine (SAM) concentration and SAM/S-adenosylhomocysteine ratios] and genomic methylation level, which had been decreased by HFD (0.37% and 0.47% vs. 0.25%). These results suggest that the regulation of aberrant DNA methylation by betaine might be a possible mechanism of the improvements in NAFLD upon betaine supplementation.     

Antioxidative effect of CLA diet and endurance training in liver and skeletal muscles of rat

The purpose of this study is to investigate the effects of conjugated linoleic acid (CLA) supplementation and endurance exercise on the oxidative/anti-oxidative status in rat liver and skeletal muscles. Sprague-Dawley male rats were randomly divided into HS (high-fat diet sedentary group, n = 8), CS group (CLA supplemented sedentary group, n = 8), and CE group (CLA supplemented exercise group, n = 8). For CLA supplementation, 1.0% CLA was substituted for dietary fat. For endurance exercise, the rats swam for 60 min a day, 5 days a week for 8 weeks. The MDA content, Cu, Zn-SOD and Mn-SOD expression in the soleus muscle (SOM) of the CE group improved significantly compared to the HS (p < 0.01) and CS groups (p < 0.05). Moreover, Mn-SOD expression in both the SOM and extensor digitorum longus muscle (EDL) of the CS were enhanced significantly compared to the HS (p < 0.05). From these results, it was suggested that CLA supplementation under the endurance exercise condition may improve the oxidative status by decreasing the MDA content via potential scavenging of Cu,Zn-SOD, and Mn-SOD protein in red muscle, respectively. Therefore, our study demonstrated long-term endurance exercise with CLA supplementation plays a crucial role for maintenance of antioxidative properties in the skeletal muscle of rat.     

Maternal high-fat diet during pregnancy and lactation affects hepatic lipid metabolism in early life of offspring rat

We investigated whether maternal over-nutrition during pregnancy and lactation affects the offspring’s lipid metabolism at weaning by assessing liver lipid metabolic gene expressions and analysing its mechanisms on the development of metabolic abnormalities. Female Sprague–Dawley rats were fed with standard chow diet (CON) or high-fat diet (HFD) for 8 weeks, and then continued feeding during gestation and lactation. The offspring whose dams were fed with HFD had a lower birth weight but an increased body weight with impaired glucose tolerance, higher serum cholesterol, and hepatic steatosis at weaning. Microarray analyses showed that there were 120 genes differently expressed between the two groups. We further verified the results by qRT-PCR. Significant increase of the lipogenesis (Me1, Scd1) gene expression was found in HFD (P<0.05), and up-regulated expression of genes (PPAR-α, Cpt1α, Ehhadh) involved in β-oxidation was also observed (P<0.05), but the Acsl3 gene was down-regulated (P<0.05). Maternal over-nutrition could not only primarily induce lipogenesis, but also promote lipolysis through an oxidation pathway as compensation, eventually leading to an increased body weight, impaired glucose tolerance, elevated serum cholesterol and hepatic steatosis at weaning. This finding may provide some evidence for a healthy maternal diet in order to reduce the risk of metabolic diseases in the early life of the offspring.     

A betaine aldehyde dehydrogenase gene in quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>): structure,phylogeny, and expression pattern

Betaine aldehyde dehydrogenase (BADH) is widely considered as a key enzyme in glycine betaine metabolism in higher plants. Several paralogous genes encoding different isozymes of BADH have been identified and characterized in some plants; however, until now, only limited information is available about BADH genes in quinoa (Chenopodium quinoa). Here, we report the molecular cloning, structural organization, phylogenetic evolution, and expression profile of a BADH gene (CqBADH1) from quinoa. The translated putative CqBADH1 protein included five conserved features of the ALDH Family 10. Comparisons between the cDNA and genomic sequences revealed that the CqBADH1 gene contained 15 exons and 14 introns. Comparative screening of introns in homologous genes demonstrated that the number and position of the BADH introns were highly conserved among the BADH genes in Amaranthaceae plants and in other more distantly related plant species. A phylogenetic analysis showed that CqBADH1 had the closest relationship with a protein from Atriplex canescens and belonged to the ALDH10 family. Expression profile analyses indicated that CqBADH1 was expressed only in root, and showed time-dependent expression profiles under NaCl-stress condition. Moreover, in quinoa, NaCl stress led to increased levels of CqBADH1 mRNA accompanied by the accumulation of glycine betaine. This is the first study to describe a BADH gene in quinoa.     

Mapping liver fat female-dependent quantitative trait loci in collaborative cross mice

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the western world, with spectrum from simple steatosis to non-alcoholic steatohepatitis, which can progress to cirrhosis. NAFLD developments are known to be affected by host genetic background. Herein we emphasize the power of collaborative cross (CC) mouse for dissecting this complex trait and revealing quantitative trait loci (QTL) controlling hepatic fat accumulation in mice. 168 female and 338 male mice from 24 and 37 CC lines, respectively, of 18–20 weeks old, maintained on standard rodent diet, since weaning. Hepatic fat content was assessed, using dual DEXA scan in the liver. Using the available high-density genotype markers of the CC line, QTL mapping associated with percentage liver fat accumulation was performed. Our results revealed significant fatty liver accumulation QTL that were specifically, mapped in females. Two significant QTLs on chromosomes 17 and 18, with genomic intervals 3 and 2 Mb, respectively, were mapped. A third QTL, with a less significant P value, was mapped to chromosome 4, with genomic interval of 2 Mb. These QTLs were named Flal1–Flal3, referring to Fatty Liver Accumulation Locus 1–3, for the QTLs on chromosomes 17, 18, and 4, respectively. Unfortunately, no QTL was mapped with males. Searching the mouse genome database suggested several candidate genes involved in hepatic fat accumulation. Our results show that susceptibility to hepatic fat accumulations is a complex trait, controlled by multiple genetic factors in female mice, but not in male.     

Dissociation of Systemic Glucose Homeostasis from Triacylglyceride Accumulation by Reduced <Emphasis Type="Italic">Acsl6</Emphasis> Expression in Skeletal Muscle

Long chain acyl-CoA synthetase (ACSL) is an enzyme that activates fatty acids before they are further metabolized. ACSL6 is the one of main ACSL isoforms exclusively expressed in skeletal muscle, but the consequences of the suppression of this gene in systemic glucose homeostasis has yet to be reported. Hence, we investigated the roles of ACSL6 gene in glucose tolerance and TAG distribution in physiological conditions. Eight-week-old male C57BL/6J mice were administered with control or Acsl6 siRNAs and then fed with either AIN-93 control diet or high fat diet. At seven days after the first siRNA injection, oral glucose tolerance tests and TAG quantification were performed. In vivo administration of Acsl6 siRNA decreased Acsl6 expression only in skeletal muscle under AIN-93 or a high fat diet. However Acsl6 siRNA injection to animals increased TAG accumulation in the liver without the change of Acsl6 expression. Atelocollagen mediated Acsl6 suppression enhanced whole-body glucose tolerance coinciding with decreased TAG accumulation in skeletal muscle of mice fed an AIN-93 diet. However, the improved glucose tolerance by Acsl6 reduction was ablated by high fat diet. Moreover reduced Acsl6 did not alter the phosphorylation of insulin signaling proteins in skeletal muscle. These results suggest that Acsl6 reduction in skeletal muscle enhances glucose homeostasis and dissociates the insulin responses from TAG accumulation in skeletal muscle.     

Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 +/- 0.5 g (means +/- SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system.     

Lipid metabolism in adipose tissue and liver from diet-induced obese rats: a comparison between <Emphasis Type="Italic">Wistar</Emphasis> and <Emphasis Type="Italic">Sprague-Dawley</Emphasis> strains

Some researchers have proposed important variations in adipose tissue among different strains of rats and mice in response to a high-caloric (hc) diet, but data concerning the mechanisms underlying these differences are scarce. The aim of the present research was to characterize different aspects of triacylglycerol (TG) metabolism and clock genes between Sprague-Dawley and Wistar rats. For this purpose, 16 male Sprague-Dawley and 16 male Wistar rats were divided into four experimental groups (n?=?8) and fed either a normal-caloric (nc) diet or a hc diet for 6 weeks. After sacrifice, liver and epididymal, perirenal, mesenteric, and subcutaneous adipose tissue depots were dissected, weighed and immediately frozen. Liver TG content was quantified, RNA extracted for gene expression analysis and fatty acid synthase enzyme activity measured. Two-way ANOVA and Student’s t test were used to perform the statistical analyses. Under hc feeding conditions, Wistar rats were more prone to fat accumulation in adipose tissue, especially in the epididymal fat depot, due to their increased lipogenesis and fatty acid uptake. By contrast, both strains of rats showed similarly fatty livers after hc feeding. Peripheral clock machinery seems to be a potential explanatory mechanism for Wistar and Sprague-Dawley strain differences. In conclusion, Wistar strain seems to be the best choice as animal model in dietary-induced obesity studies.     

DJ-1 deficiency alleviates steatosis in cultured hepatocytes

Imbalance in lipid metabolism within hepatocytes can lead to hepatosteatosis, which is a cause of numerous hepatic dysfunctions. Previous studies have demonstrated the roles of DJ-1 in Parkinson’s disease, diet-induced oxidative stress, obesity, and diabetes. Although recent studies have shown that DJ-1 is involved in metabolic complications, the roles of DJ-1 in steatosis are largely unknown. Therefore, the aim of the current study was to elucidate the potential roles of DJ-1 in hepatosteatosis in vitro. Normal rat liver cells Clone 9 (C9) were treated with 1 mM oleic acid (OA) for 24 h for establishment of a steatosis model, after which various biochemical parameters, including triglyceride, total cholesterol, and free fatty acid contents, were determined after knockdown with Dj1- specific siRNA. Silencing of Dj1 prevented hepatic steatosis by suppressing expression of hepatic lipogenic markers, which was confirmed by immunoblotting and real-time PCR analysis. Up-regulation of mitochondrial and β- oxidation-associated proteins showed potential to reduce fat accumulation in the liver. Silencing of Dj1 resulted in alleviation of steatosis by reducing lipogenesis and improving mitochondrial biogenesis. Further in vivo studies will be required to understand the molecular mechanism behind the role of DJ-1 in steatosis.     

Mice lacking myotubularin-related protein 14 show accelerated high-fat diet-induced lipid accumulation and inflammation

The phosphoinositide phosphatase, myotubularin-related protein 14 (MTMR14), has been reported to play an important role in the regulation of muscle performance, autophagy, and aging in mice. We previously showed that MTMR14-knockout (KO) mice gain weight earlier than their wild-type (WT) littermates even on a normal chow diet (NCD), suggesting that this gene might also be involved in regulating metabolism. In the present study, we evaluated the effect of MTMR14 deficiency on high-fat diet (HFD)-induced obesity, lipid accumulation, metabolic disorders, and inflammation in WT and MTMR14-KO mice fed with NCD or HFD. To this end, MTMR14-KO mice fed with HFD showed significantly increased body weight, blood glucose levels, serum triglyceride (TG) levels, and total cholesterol (TC) levels as compared to their age-matched WT control. Additionally, lipid accumulation also increased in the KO mice. Simultaneously, the expression of metabolism-associated genes (Glut4, adiponectin, and leptin) was different in the liver, muscle, and fatty tissue of MTMR14-KO mice fed with HFD. More importantly, the expression of several inflammation-associated genes (TNF-α, IL-6, IL-1β, and MCP-1) dramatically increased in the liver, muscle, and fatty tissue of MTMR14-KO mice relative to control. Taken together, these results suggest that MTMR14 deficiency accelerates HFD-induced metabolic dysfunction and inflammation. Furthermore, the results showed that exacerbated metabolic dysfunction and inflammation may be regulated via the PI3K/Akt and ERK signaling pathways.     

Sex differences in hepatic one-carbon metabolism

Background

There are large differences between men and women of child-bearing age in the expression level of 5 key enzymes in one-carbon metabolism almost certainly caused by the sex hormones. These male-female differences in one-carbon metabolism are greatly accentuated during pregnancy. Thus, understanding the origin and consequences of sex differences in one-carbon metabolism is important for precision medicine.

Results

We have created a mathematical model of hepatic one-carbon metabolism based on the underlying physiology and biochemistry. We use the model to investigate the consequences of sex differences in gene expression. We give a mechanistic understanding of observed concentration differences in one-carbon metabolism and explain why women have lower S-andenosylmethionine, lower homocysteine, and higher choline and betaine. We give a new explanation of the well known phenomenon that folate supplementation lowers homocysteine and we show how to use the model to investigate the effects of vitamin deficiencies, gene polymorphisms, and nutrient input changes.

Conclusions

Our model of hepatic one-carbon metabolism is a useful platform for investigating the mechanistic reasons that underlie known associations between metabolites. In particular, we explain how gene expression differences lead to metabolic differences between males and females.

     

Molecular characterisation of tumour necrosis factor alpha and its potential connection with lipoprotein lipase and peroxisome proliferator-activated receptors in blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

Tumour necrosis factor alpha (TNF-α) is one kind of cytokines which is related to inflammation and lipid metabolism. TNF-α cDNA was cloned from the liver of blunt snout bream (Megalobrama amblycephala) through real-time polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of TNF-α covered 1467 bp, with an open reading frame (ORF) of 723 bp, which encodes 240 amino acids. It possessed the TNF family signature IIIPDDGIYFVYSQ. After the lipopolysaccharide (LPS) challenge test, a graded tissue-specific expression pattern of TNF-α was observed and there was high expression abundance in the kidney, brain and liver. After 8 weeks feeding trial, liver samples, two groups fed with 6% and 11% lipid levels, were collected. The results showed that, for fish fed with high-fat diet, the triglyceride of serum and lipid content of liver were elevated. Furthermore, TNF-α and peroxisome proliferator-activated receptors (PPARα, β) mRNA expression of fish fed 11% lipid diet were significantly up-regulated (p?<?0.05). Lipoprotein lipase (LPL) and PPARγ mRNA expression of fish fed 11% lipid lever diet were significantly decreased compared to those of fish fed 6% (p?<?0.05). The differences between the various expression of related genes in the high and low fat groups demonstrated that TNF-α played a key role in lipid metabolism, which may have an influence on fat metabolism through reducing fat synthesis and strengthening the β-oxidation of fatty acid. These discrepancies warrant further research.     

Betaine reduces β-amyloid-induced paralysis through activation of cystathionine-β-synthase in an Alzheimer model of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background

The neurodegenerative disorder Alzheimer’s disease is caused by the accumulation of toxic aggregates of β-amyloid in the human brain. On the one hand, hyperhomocysteinemia has been shown to be a risk factor for cognitive decline in Alzheimer’s disease. On the other hand, betaine has been demonstrated to attenuate Alzheimer-like pathological changes induced by homocysteine. It is reasonable to conclude that this is due to triggering the remethylation pathway mediated by betaine-homocysteine-methyltransferase. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, to test whether betaine is able to reduce β-amyloid-induced paralysis in C. elegans. This model expresses human β-amyloid 1–42 under control of a muscle-specific promoter that leads to progressive, age-dependent paralysis in the nematodes.

Results

Betaine at a concentration of 100 μM was able to reduce homocysteine levels in the presence and absence of 1 mM homocysteine. Simultaneously, betaine both reduced normal paralysis rates in the absence of homocysteine and increased paralysis rates triggered by addition of homocysteine. Knockdown of cystathionine-β-synthase using RNA interference both increased homocysteine levels and paralysis. Additionally, it prevented the reducing effects of betaine on homocysteine levels and paralysis.

Conclusion

Our studies show that betaine is able to reduce homocysteine levels and β-amyloid-induced toxicity in a C. elegans model for Alzheimer’s disease. This effect is independent of the remethylation pathway but requires the transsulfuration pathway mediated by cystathionine-β-synthase.

     

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon

Background

Obesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.

Methods

C57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log₂ fold change of ≥1 or ≤?1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.

Results

High-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.

Conclusions

Our data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.