Impaired de Novo Choline Synthesis Explains Why Phosphatidylethanolamine N-Methyltransferase-deficient Mice Are Protected from Diet-induced Obesity

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt^+/+ mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt^−/− mice did not. Compared with Pemt^+/+ mice, Pemt^−/− mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt^−/− mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt^−/− mice. Furthermore, Pemt^+/+ mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.     

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.     

The role of AMPKα2 in the HFD-induced nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic steatosis, inflammation and liver fibrosis and has become one of the leading causes of hepatocellular carcinoma and liver failure. However, the underlying molecular mechanism of hepatic steatosis and the progression to nonalcoholic steatohepatitis (NASH) are not fully understood. Herein, we discovered that AMPKα2 catalytic subunit showed reduced expression in the liver following high fat diet (HFD) feeding to mice. Importantly, knockout of AMPKα2 in mice aggravated NAFLD, hepatic steatosis, inflammation and fibrosis. On the other hand, hepatocyte-targeted overexpression of AMPKα2 prevented or reversed NAFLD indications. In vivo mechanistic studies revealed that increased phosphorylation of IKKα/β and NF-κB in HFD-fed AMPKα2^−/− mice compared to WT mice, and treatment of these mouse cohorts with an inhibitor of NF-κB signaling for 4 weeks, effectively attenuated the progression of steatohepatitis and metabolic disorder features. In summary, AMPKα2 provides a protective role in the process of hepatic steatosis to NASH progression through suppression of liver NF-κB signaling.     

Alpha-syntrophin null mice are protected from non-alcoholic steatohepatitis in the methionine-choline-deficient diet model but not the atherogenic diet model

Adipose tissue dysfunction contributes to the pathogenesis of non-alcoholic steatohepatitis (NASH). The adapter protein alpha-syntrophin (SNTA) is expressed in adipocytes. Knock-down of SNTA increases preadipocyte proliferation and formation of small lipid droplets, which are both characteristics of healthy adipose tissue. To elucidate a potential protective role of SNTA in NASH, SNTA null mice were fed a methionine-choline-deficient (MCD) diet or an atherogenic diet which are widely used as preclinical NASH models. MCD diet mediated loss of fat mass was largely improved in SNTA?/? mice compared to the respective wild type animals. Hepatic lipids were mostly unchanged while the oxidative stress marker malondialdehyde was only induced in the wild type mice. The expression of inflammatory markers and macrophage immigration into the liver were reduced in SNTA?/? animals. This protective function of SNTA loss was absent in atherogenic diet induced NASH. Here, hepatic expression of inflammatory and fibrotic genes was similar in both genotypes though mutant mice gained less body fat during feeding. Hepatic cholesterol and ceramide were strongly induced in both strains upon feeding the atherogenic diet, while hepatic sphingomyelin, phosphatidylserine and phosphatidylethanolamine levels were suppressed.SNTA deficient mice are protected from fat loss and NASH in the experimental MCD model. NASH induced by an atherogenic diet is not influenced by loss of SNTA. The present study suggests the use of different experimental NASH models to study the pathophysiological role of proteins like SNTA in NASH.     

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt^?/? mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt^?/? mice. Treatment with vitamin E (0.5?g/kg) for 3?weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt^?/? mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Betaine administration corrects ethanol-induced defective VLDL secretion

Our previous studies, demonstrating ethanol-induced alterations in phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine methyltransferase (PEMT) pathway, implicated a defect in very low-density lipoprotein (VLDL) secretion in the pathogenesis of hepatic steatosis. The objective of this study was to determine whether VLDL secretion was reduced by chronic ethanol consumption and whether betaine supplementation, that restores PEMT activity and prevents the development of alcoholic steatosis, could normalize VLDL secretion. The VLDL secretion in rats fed with control, ethanol and the betaine supplemented diets was determined using Triton WR-1339 to inhibit plasma VLDL metabolism. We observed reduced VLDL production rates in chronic alcohol-fed rats compared to control animals. Supplementation of betaine in the ethanol diet increased VLDL production rate to values significantly higher than those observed in the control diet-fed rats. To conclude, chronic ethanol consumption impairs PC generation via the PEMT pathway resulting in diminished VLDL secretion which contributes to the development of hepatic steatosis. By increasing PEMT-mediated PC generation, betaine results in increased fat export from the liver and attenuates the development of alcoholic fatty liver.     

Dissociation of diabetes and obesity in mice lacking orphan nuclear receptor small heterodimer partner

Mixed background SHP(-/-) mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP(-/-) mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP(-/-) mice. Compared with their C57BL/6 wild-type counterparts, SHP(-/-) mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP(-/-) mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.     

Hepatic steatosis in the absence of tumor necrosis factor in mice

Tumor necrosis factor (TNF) has pleiotropic effects including on hepatic metabolism. Here we investigated the effect of high cholesterol diet (1.25%) in TNF deficient mice. TNFα/β deficient mice developed hepatomegaly and extensive steatosis in the absence of steatohepatitis as compared to wild type mice. Saturated and unsaturated, prominently mono- but also poly-unsaturated fatty acids (MUFA, PUFA) prevailed in steatotic livers. Down-regulation of the cholesterol scavenger receptor B1 and reduced insulin induced phosphorylation of protein kinase B in cholesterol fed TNFα/β deficient mice likely contributed to the development of hepatic steatosis, which was accompanied by increased body weight and bone length. Steatosis was only present in TNFα/β double deficient mice, however not in single TNF deficient mice suggesting a redundant role of TNFα and TNFβ. In conclusion, high cholesterol diet causes an abnormal metabolic phenotype in the simultaneous absence of both TNFα and β signals. The presence of either TNFα or β alone is sufficient to reconstitute the control of lipid homeostasis.     

Involvement of G protein-coupled receptor kinase 2 (GRK2) in the development of non-alcoholic steatosis and steatohepatitis in mice and humans

Insulin resistance (IR) and obesity are important risk factors for non-alcoholic fatty liver disease (NAFLD). G protein-coupled receptor kinase 2 (GRK2) is involved in the development of IR and obesity in vivo. However, its possible contribution to NAFLD and/or non-alcoholic steatohepatitis (NASH) independently of its role on IR or fat mass accretion has not been explored. Here, we used wild-type (WT) or GRK2 hemizygous (GRK2±) mice fed a high-fat diet (HFD) or a methionine and choline-deficient diet (MCD) as a model of NASH independent of adiposity and IR. GRK2± mice were protected from HFD-induced NAFLD. Moreover, MCD feeding caused an increased in triglyceride content and liver-to-body weight ratio in WT mice, features that were attenuated in GRK2± mice. According to their NAFLD activity score, MCD-fed GRK2± mice were diagnosed with simple steatosis and not overt NASH. They also showed reduced expression of lipogenic and lipid-uptake markers and less signs of inflammation in the liver. GRK2± mice preserved hepatic protective mechanisms as enhanced autophagy and mitochondrial fusion and biogenesis, together with reduced endoplasmic reticulum stress. GRK2 protein was increased in MCD-fed WT but not in GRK2± mice, and enhanced GRK2 expression potentiated palmitic acid-triggered lipid accumulation in human hepatocytes directly relating GRK2 levels to steatosis. GRK2 protein and mRNA levels were increased in human liver biopsies from simple steatosis or NASH patients in two different human cohorts. Our results describe a functional relationship between GRK2 levels and hepatic lipid accumulation and implicate GRK2 in the establishment and/or development of NASH.     

Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

Nonalcoholic fatty liver disease (NAFLD) is a common cause of hepatic dysfunction. The disease spectrum ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The aim of this study was to identify metabolic differences in murine models of simple hepatic steatosis and NASH for the distinction of these NAFLD stages. For 12 weeks, male BALB/c mice were fed either a control or two different high-fat diets leading to hepatic steatosis and NASH, respectively. Metabolic differences were determined by independent component analysis (ICA) of nuclear magnetic resonance (NMR) spectra of lipophilic and hydrophilic liver extracts, and urine specimens. The results from ICA clearly discriminated the three investigated groups. Discriminatory biomarkers in the lipophilic liver extracts were free cholesterol, cholesterol ester and lipid methylene. Discrimination of the hydrophilic liver extracts was mainly mediated by betaine, glucose, and lactate, whereas in urine taurine, trimethylamine-N-oxide, and trimethylamine were the most discriminatory biomarkers. In conclusion, NMR metabolite fingerprinting of spot urine specimens may allow the noninvasive distinction of steatosis and NASH.     

Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent

The aims of this study were to determine whether combining features of a western lifestyle in mice with trans fats in a high-fat diet, high-fructose corn syrup in the water, and interventions designed to promote sedentary behavior would cause the hepatic histopathological and metabolic abnormalities that characterize nonalcoholic steatohepatitis (NASH). Male C57BL/6 mice fed ad libitum high-fat chow containing trans fats (partially hydrogenated vegetable oil) and relevant amounts of a high-fructose corn syrup (HFCS) equivalent for 1-16 wk were compared with mice fed standard chow or mice with trans fats or HFCS omitted. Cage racks were removed from western diet mice to promote sedentary behavior. By 16 wk, trans fat-fed mice became obese and developed severe hepatic steatosis with associated necroinflammatory changes. Plasma alanine aminotransferase levels increased, as did liver TNF-alpha and procollagen mRNA, indicating an inflammatory and profibrogenic response to injury. Glucose intolerance and impaired fasting glucose developed within 2 and 4 wk, respectively. Plasma insulin, resistin, and leptin levels increased in a profile similar to that seen in patients with NASH. The individual components of this diet contributed to the phenotype independently; isocaloric replacement of trans fats with lard established that trans fats played a major role in promoting hepatic steatosis and injury, whereas inclusion of HFCS promoted food consumption, obesity, and impaired insulin sensitivity. Combining risk factors for the metabolic syndrome by feeding mice trans fats and HFCS induced histological features of NASH in the context of a metabolic profile similar to patients with this disease. Because dietary trans fats promoted liver steatosis and injury, their role in the epidemic of NASH needs further evaluation.     

iPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.     

Acarbose attenuates experimental non-alcoholic steatohepatitis

The alpha-glucosidase inhibitor acarbose is beneficial in the prevention of type 2 diabetes. To determine whether it attenuates the commonly associated non-alcoholic steatohepatitis (NASH), we used an experimental NASH model. Rats were fed ad libitum a nutritionally adequate high fat diet (71% of calories as fat) with or without acarbose (200 mg/1000 calories) for 3 weeks. All rats given the high fat diet only developed typical NASH whereas acarbose attenuated several of the characteristic hepatic alterations of NASH: there was less steatosis and inflammation, with a significant reduction in the mRNA of the hepatic inflammatory cytokine TNF-alpha and of its protein. There was also a decrease in the CYP2E1 mRNA and in collagen, with similar trends for CYP2E1 protein and procollagen mRNA. Because acarbose attenuates many of the hepatic alterations associated with experimental NASH, it is now indicated to determine whether it exerts similar beneficial effects in patients afflicted by this disease.     

Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice

Partial leptin deficiency is not uncommon in the general population. We hypothesized that leptin insufficiency could favor obesity, nonalcoholic steatohepatitis (NASH), and other metabolic abnormalities, particularly under high calorie intake. Thus, mice partially deficient in leptin (ob/+) and their wild-type (+/+) littermates were fed for 4 mo with a standard-calorie (SC) or a high-calorie (HC) diet. Some ob/+ mice fed the HC diet were also treated weekly with leptin. Our results showed that, when fed the SC diet, ob/+ mice did not present significant metabolic abnormalities except for elevated levels of plasma adiponectin. Under high-fat feeding, increased body fat mass, hepatic steatosis, higher plasma total cholesterol, and glucose intolerance were observed in +/+ mice, and these abnormalities were further enhanced in ob/+ mice. Furthermore, some metabolic disturbances, such as blunted plasma levels of leptin and adiponectin, reduced UCP1 expression in brown adipose tissue, increased plasma liver enzymes, beta-hydroxybutyrate and triglycerides, and slight insulin resistance, were observed only in ob/+ mice fed the HC diet. Whereas de novo fatty acid synthesis in liver was decreased in +/+ mice fed the HC diet, it was disinhibited in ob/+ mice along with the restoration of the expression of several lipogenic genes. Enhanced expression of several genes involved in fatty acid oxidation was also observed only in ob/+ animals. Leptin supplementation alleviated most of the metabolic abnormalities observed in ob/+ fed the HC diet. Hence, leptin insufficiency could increase the risk of obesity, NASH, glucose intolerance, and hyperlipidemia in a context of calorie overconsumption.     

Diet-induced obesity and hepatic steatosis in L-Fabp / mice is abrogated with SF, but not PUFA, feeding and attenuated after cholesterol supplementation

Liver fatty acid (FA)-binding protein (L-Fabp), a cytoplasmic protein expressed in liver and small intestine, regulates FA trafficking in vitro and plays an important role in diet-induced obesity. We observed that L-Fabp(-/-) mice are protected against Western diet-induced obesity and hepatic steatosis. These findings are in conflict, however, with another report of exaggerated obesity and increased hepatic steatosis in female L-Fabp(-/-) mice fed a cholesterol-supplemented diet. To resolve this apparent paradox, we fed female L-Fabp(-/-) mice two different cholesterol-supplemented low-fat diets and discovered (on both diets) lower body weight in L-Fabp(-/-) mice than in congenic wild-type C57BL/6J controls and similar or reduced hepatic triglyceride content. We extended these comparisons to mice fed low-cholesterol, high-fat diets. Female L-Fabp(-/-) mice fed a high-saturated fat (SF) diet were dramatically protected against obesity and hepatic steatosis, whereas weight gain and hepatic lipid content were indistinguishable between mice fed a high-polyunsaturated FA (PUFA) diet and control mice. These findings demonstrate that L-Fabp functions as a metabolic sensor with a distinct hierarchy of FA sensitivity. We further conclude that cholesterol supplementation does not induce an obesity phenotype in L-Fabp(-/-) mice, nor does it play a significant role in the protection against Western diet-induced obesity in this background.     

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.