Betaine supplementation prevents fatty liver induced by a high-fat diet: effects on one-carbon metabolism

The purpose of this study was to examine the effects of betaine supplementation on the regulation of one-carbon metabolism and liver lipid accumulation induced by a high-fat diet in rats. Rats were fed one of three different liquid diets: control diet, high-fat diet and high-fat diet supplemented with betaine. The control and high-fat liquid diets contained, respectively, 35 and 71 % of energy derived from fat. Betaine supplementation involved the addition of 1 % (g/L) to the diet. After three weeks on the high-fat diet the rats had increased total liver fat concentration, liver triglycerides, liver TBARS and plasma TNF-α. The high-fat diet decreased the hepatic S-adenosylmethionine concentration and the S-adenosylmethionine/S-adenosylhomocysteine ratio compared to the control as well as altering the expression of genes involved in one-carbon metabolism. Betaine supplementation substantially increased the hepatic S-adenosylmethionine concentration (~fourfold) and prevented fatty liver and hepatic injury induced by the high-fat diet. It was accompanied by the normalization of the gene expression of BHMT, GNMT and MGAT, which code for key enzymes of one-carbon metabolism related to liver fat accumulation. In conclusion, the regulation of the expression of MGAT by betaine supplementation provides an additional and novel mechanism by which betaine supplementation regulates lipid metabolism and prevents accumulation of fat in the liver.     

Chromium attenuates high-fat diet-induced nonalcoholic fatty liver disease in KK/HlJ mice

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease associated with insulin resistance, oxidative stress, and inflammation. Evidence indicates that chromium has a role in the regulation of glucose and lipid metabolism and may improve insulin sensitivity. In this study, we report that chromium supplementation has a beneficial effect against NAFLD. We found that KK/HlJ mice developed obesity and progressed to NAFLD after feeding with high-fat diet for 8 weeks. High-fat-fed KK/HlJ mice showed hepatocyte injury and hepatic triglyceride accumulation, which was accompanied by insulin resistance, oxidative stress, and inflammation. Chromium supplementation prevented progression of NAFLD and the beneficial effects were accompanied by reduction of hepatic triglyceride accumulation, elevation of hepatic lipid catabolic enzyme, improvement of glucose and lipid metabolism, suppression of inflammation as well as resolution of oxidative stress, probably through enhancement of insulin signaling. Our findings suggest that chromium could serve as a hepatoprotective agent against NAFLD.     

Experimental evidence for therapeutic potential of taurine in the treatment of nonalcoholic fatty liver disease

The incidence of obesity is now at epidemic proportions and has resulted in the emergence of nonalcoholic fatty liver disease (NAFLD) as a common metabolic disorder that can lead to liver injury and cirrhosis. Excess sucrose and long-chain saturated fatty acids in the diet may play a role in the development and progression of NAFLD. One factor linking sucrose and saturated fatty acids to liver damage is dysfunction of the endoplasmic reticulum (ER). Although there is currently no proven, effective therapy for NAFLD, the amino sulfonic acid taurine is protective against various metabolic disturbances, including alcohol-induced liver damage. The present study was undertaken to evaluate the therapeutic potential of taurine to serve as a preventative treatment for diet-induced NAFLD. We report that taurine significantly mitigated palmitate-mediated caspase-3 activity, cell death, ER stress, and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high-sucrose diet, dietary taurine supplementation significantly reduced hepatic lipid accumulation, liver injury, inflammation, plasma triglycerides, and insulin levels. The high-sucrose diet resulted in an induction of multiple components of the unfolded protein response in the liver consistent with ER stress, which was ameliorated by taurine supplementation. Treatment of mice with the ER stress-inducing agent tunicamycin resulted in liver injury, unfolded protein response induction, and hepatic lipid accumulation that was significantly ameliorated by dietary supplementation with taurine. Our results indicate that dietary supplementation with taurine offers significant potential as a preventative treatment for NAFLD.     

Ketogenic Essential Amino Acids Modulate Lipid Synthetic Pathways and Prevent Hepatic Steatosis in Mice

Background

Although dietary ketogenic essential amino acid (KAA) content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated.

Methodology/Principal Findings

We designed a diet with a high ratio (E/N) of essential amino acids (EAAs) to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr) without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogensis (DNL) fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides.

Conclusion

Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways.     

Replacement of soybean oil by fish oil increases cytosolic lipases activities in liver and adipose tissue from rats fed a high-carbohydrate diets

Several studies have demonstrated that fish oil consumption improves metabolic syndrome and comorbidities, as insulin resistance, nonalcoholic fatty liver disease, dyslipidaemia and hypertension induced by high-fat diet ingestion. Previously, we demonstrated that administration of a fructose-rich diet to rats induces liver lipid accumulation, accompanied by a decrease in liver cytosolic lipases activities. In this study, the effect of replacement of soybean oil by fish oil in a high-fructose diet (FRUC, 60% fructose) for 8 weeks on lipid metabolism in liver and epididymal adipose tissue from rats was investigated. The interaction between fish oil and FRUC diet increased glucose tolerance and decreased serum levels of triacylglycerol (TAG), VLDL-TAG secretion and lipid droplet volume of hepatocytes. In addition, the fish oil supplementation increased the liver cytosolic lipases activities, independently of the type of carbohydrate ingested. Our results firmly establish the physiological regulation of liver cytosolic lipases to maintain lipid homeostasis in hepatocytes. In epididymal adipose tissue, the replacement of soybean oil by fish oil in FRUC diet did not change the tissue weight and lipoprotein lipase activity; however, there was increased basal and insulin-stimulated de novo lipogenesis and glucose uptake. Increased cytosolic lipases activities were observed, despite the decreased basal and isoproterenol-stimulated glycerol release to the incubation medium. These findings suggest that fish oil increases the glycerokinase activity and glycerol phosphorylation from endogenous TAG hydrolysis. Our findings are the first to show that the fish oil ingestion increases cytosolic lipases activities in liver and adipose tissue from rats treated with high-carbohydrate diets.     

Calcium supplementation relieves high-fat diet-induced liver steatosis by reducing energy metabolism and promoting lipolysis

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease affecting the health of many people worldwide. Previous studies have shown that dietary calcium supplementation may alleviate NAFLD, but the underlying mechanism is not clear. In this study investigating the effect of calcium on hepatic lipid metabolism, 8-week-old male C57BL/6J mice were divided into four groups (n = 6): (1) mice given a normal chow containing 0.5% calcium (CN0.5), (2) mice given a normal chow containing 1.2% calcium (CN1.2), (3) mice given a high-fat diet (HFD) containing 0.5% calcium (HFD0.5), and (4) mice fed a HFD containing 1.2% calcium (HFD1.2). To understand the underlying mechanism, cells were treated with oleic acid and palmitic acid to mimic the HFD conditions in vitro. The results showed that calcium alleviated the increase in triglyceride accumulation induced by oleic acid and/or palmitic acid in HepG2, AML12, and primary hepatocyte cells. Our data demonstrated that calcium supplementation alleviated HFD-induced hepatic steatosis through increased liver lipase activity, proving calcium is involved in the regulation of hepatic lipid metabolism. Moreover, calcium also increased the level of glycogen in the liver, and at the same time had the effect of reducing glycolysis and promoting glucose absorption. Calcium addition increased calcium levels in the mitochondria and cytoplasm. Taken together, we concluded that calcium supplementation could relieve HFD-induced hepatic steatosis by changing energy metabolism and lipase activity.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD)

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries and is considered the hepatic manifestation of metabolic syndrome. The hallmark of NAFLD is hepatic neutral lipid accumulation, mainly triacylglycerol, in the absence of significant ethanol consumption, viral infection or other specific etiologies. Hepatic lipid accumulation results from an imbalance between lipid availability (from circulating lipid uptake or de novo lipogenesis) and lipid disposal (via free fatty acid oxidation or triglyceride-rich lipoprotein secretion) and eventually triggers lipoperoxidative stress and hepatic injury. Each of these steps is altered in NAFLD, although to a different extent. Regulation of these pathways is complex and involves nuclear receptors, membrane transport proteins and cellular enzymes. We will review available data on different steps of hepatic lipid metabolism in NAFLD and recent advances in understanding molecular mechanisms underlying hepatic fat accumulation in these subjects.     

Chitosan reduces inflammation and protects against oxidative stress in a hyperlipidemic rat model: relevance to nonalcoholic fatty liver disease

Background

An altered lipid profile may lead to the development of inflammation and NAFLD (Non-alcoholic fatty liver disease). Although statins have a positive effect on blood lipid levels their long-term use is known to cause adverse effects, in this backdrop there is an interest in natural compounds which may affect lipid metabolism and prevent NAFLD. We have examined the effect of Chitosan on rats subjected to a high-fat diet.

Methods and results

Male Wistar middle aged rats (12–16 months) were treated with high-fat diet orally for two months for creating a NAFLD model. Rats were also supplemented with Chitosan (2% chitosan daily) for 2 months. We assessed the activity of antioxidant enzymes, the histopathological profile of the liver. Inflammatory cytokines and adiponectin levels were also measured in serum. HFD induced significant changes in liver tissue and inflammatory markers (Il-6, TNF- alpha, NF-KB). Chitosan treatment protected rats from HFD induced alterations.

Conclusions

The findings suggest that Chitosan can effectively improve liver lipid metabolism by normalizing cholesterol, triglyceride, lowering NF-KB expression, and protecting the liver from oxidative stress by improving hepatic function. Chitosan also regulates genes related to lipidemic stress i,e leptin and adiponectin.

     

Fish oil and 3-thia fatty acid have additive effects on lipid metabolism but antagonistic effects on oxidative damage when fed to rats for 50 weeks

The 3-thia fatty acid tetradecylthioacetic acid (TTA) is a synthetic modified fatty acid, which, similar with dietary fish oil (FO), influences the regulation of lipid metabolism, the inflammatory response and redox status. This study was aimed to penetrate the difference in TTA's mode of action compared to FO in a long-term experiment (50 weeks of feeding). Male Wistar rats were fed a control, high-fat (25% w/v) diet or a high-fat diet supplemented with either TTA (0.375% w/v) or FO (10% w/v) or their combination. Plasma fatty acid composition, hepatic lipids and expression of relevant genes in the liver and biomarkers of oxidative damage to protein were assessed at the end point of the experiment. Both supplements given in combination demonstrated an additive effect on the decrease in plasma cholesterol levels. The FO diet alone led to removal of plasma cholesterol and a concurrent cholesterol accumulation in liver; however, with TTA cotreatment, the hepatic cholesterol level was significantly reduced. Dietary FO supplementation led to an increased oxidative damage, as seen by biomarkers of protein oxidation and lipoxidation. Tetradecylthioacetic acid administration reduced the levels of these biomarkers confirming its protective role against lipoxidation and protein oxidative damage. Our findings explore the lipid reducing effects of TTA and FO and demonstrate that these bioactive dietary compounds might act in a different manner. The experiment confirms the antioxidant capacity of TTA, showing an improvement in FO-induced oxidative stress.     

Targeting the gut microbiota with resveratrol: a demonstration of novel evidence for the management of hepatic steatosis

Resveratrol is a natural polyphenol that has been reported to reduce the risk of obesity and nonalcoholic fatty liver disease (NAFLD). Recent evidence has demonstrated that the gut microbiota plays an important role in the protection against NAFLD and other metabolic diseases. The present study aimed to investigate the relationship between the gut microbiota and the beneficial effects of resveratrol on the amelioration of NAFLD in mice. We observed marked decreases in body weight and liver steatosis and improved insulin resistance in high-fat diet (HFD)-fed mice treated with resveratrol. Furthermore, we found that resveratrol treatment alleviated NAFLD in HFD-fed mice by improving the intestinal microenvironment, including gut barrier function and gut microbiota composition. On the one hand, resveratrol improved gut intestinal barrier integrity through the repair of intestinal mucosal morphology and increased the expression of physical barrier- and physiochemical barrier-related factors in HFD-fed mice. On the other hand, in HFD-fed mice, resveratrol supplementation modulated the gut bacterial composition. The resveratrol-induced gut microbiota was characterized by a decreased abundance of harmful bacteria, including Desulfovibrio, Lachnospiraceae_NK4A316_group and Alistipes, as well as an increased abundance of short-chain fatty acid (SCFA)-producing bacteria, such as Allobaculum, Bacteroides and Blautia. Moreover, transplantation of the HFDR-microbiota into HFD-fed mice sufficiently decreased body weight, liver steatosis and low-grade inflammation and improved hepatic lipid metabolism. Collectively, resveratrol would provide a potentially dietary intervention strategy against NAFLD through modulating the intestinal microenvironment.     

Serine prevented high-fat diet-induced oxidative stress by activating AMPK and epigenetically modulating the expression of glutathione synthesis-related genes

Serine deficiency has been observed in patients with nonalcoholic fatty liver disease (NAFLD). Whether serine supplementation has any beneficial effects on the prevention of NAFLD remains unknown. The present study was conducted to investigate the effects of serine supplementation on hepatic oxidative stress and steatosis and its related mechanisms. Forty male C57BL/6J mice (9 week-old) were randomly assigned into four groups (n = 10) and fed: i) a low-fat diet; ii) a low-fat diet supplemented with 1% (wt:vol) serine; iii) a high-fat (HF) diet; and iv) a HF diet supplemented with 1% serine, respectively. Palmitic acid (PA)-treated primary hepatocytes separated from adult mice were also used to study the effects of serine on oxidative stress. The results showed that serine supplementation increased glucose tolerance and insulin sensitivity, and protected mice from hepatic lipid accumulation, but did not significantly decreased HF diet-induced weight gain. In addition, serine supplementation protected glutathione (GSH) antioxidant system and prevented hypermethylation in the promoters of glutathione synthesis-related genes, while decreasing reactive oxygen species (ROS) in mice fed a HF diet. Moreover, we found that serine supplementation increased phosphorylation and S-glutathionylation of AMP-activated protein kinase α subunit (AMPKα), and decreased ROS, malondialdehyde and triglyceride contents in PA-treated primary hepatocytes. However, while AMPK activity or GSH synthesis was inhibited, the abovementioned effects of serine on PA-treated primary hepatocytes were not observed. Our results suggest that serine supplementation could prevent HF diet-induced oxidative stress and steatosis by epigenetically modulating the expression of glutathione synthesis-related genes and through AMPK activation.     

Body Fat Accumulation in Zebrafish Is Induced by a Diet Rich in Fat and Reduced by Supplementation with Green Tea Extract

Fat-rich diets not only induce obesity in humans but also make animals obese. Therefore, animals that accumulate body fat in response to a high-fat diet (especially rodents) are commonly used in obesity research. The effect of dietary fat on body fat accumulation is not fully understood in zebrafish, an excellent model of vertebrate lipid metabolism. Here, we explored the effects of dietary fat and green tea extract, which has anti-obesity properties, on body fat accumulation in zebrafish. Adult zebrafish were allocated to four diet groups and over 6 weeks were fed a high-fat diet containing basal diet plus two types of fat or a low-fat diet containing basal diet plus carbohydrate or protein. Another group of adult zebrafish was fed a high-fat diet with or without 5% green tea extract supplementation. Zebrafish fed the high-fat diets had nearly twice the body fat (visceral, subcutaneous, and total fat) volume and body fat volume ratio (body fat volume/body weight) of those fed low-fat diets. There were no differences in body fat accumulation between the two high-fat groups, nor were there any differences between the two low-fat groups. Adding green tea extract to the high-fat diet significantly suppressed body weight, body fat volume, and body fat volume ratio compared with the same diet lacking green tea extract. 3-Hydroxyacyl-coenzyme A dehydrogenase and citrate synthase activity in the liver and skeletal muscle were significantly higher in fish fed the diet supplemented with green tea extract than in those fed the unsupplemented diet. Our results suggest that a diet rich in fat, instead of protein or carbohydrate, induced body fat accumulation in zebrafish with mechanisms that might be similar to those in mammals. Consequently, zebrafish might serve as a good animal model for research into obesity induced by high-fat diets.     

A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening

Non-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously shown that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high-fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.     

Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.     

Long-term programming of skeletal muscle and liver lipid and energy metabolism by resveratrol supplementation to suckling mice

Metabolic programming by dietary chemicals consumed in early life stages is receiving increasing attention. We here studied long-term effects of mild resveratrol (RSV) supplementation during lactation on muscular and hepatic lipid metabolism in adulthood. Newborn male mice received RSV or vehicle from day 2–20 of age, were weaned onto a chow diet on day 21, and were assigned to either a high-fat diet (HFD) or a normal-fat diet on day 90 of age for 10 weeks. RSV-treated mice showed in adulthood protection against HFD-induced triacylglycerol accumulation in skeletal muscle, enhanced muscular capacities for fat oxidation and mitochondria activity, signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling in muscle, and increased fat oxidation capacities and a decreased capacity for lipogenesis in liver compared with controls. Thus, RSV supplementation in early postnatal life may help preventing later diet-related disorders linked to ectopic lipid accumulation in muscle and liver tissues.