Excessive Hepatic Lipid Accumulation Research Articles

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

A new mechanism of lipotoxicity: Calcium channel blockers as a treatment for nonalcoholic steatohepatitis?

A new mechanism of lipotoxicity: Calcium channel blockers as a treatment for nonalcoholic steatohepatitis?

Peroxisomes compensate hepatic lipid overflow in mice with fatty liver

Major causes of lipid accumulation in liver are increased import or synthesis or decreased catabolism of fatty acids. The latter is caused by dysfunction of cellular organelles controlling energy homeostasis, i.e., mitochondria. Peroxisomes also appear to be an important organelle in lipid metabolism of hepatocytes, but little is known about their role in the development of non-alcoholic fatty liver disease (NAFLD). To investigate the role of peroxisomes alongside mitochondria in excessive hepatic lipid accumulation, we used leptin-resistant db/db mice on C57BLKS background, a mouse model that develops hyperphagia-induced diabetes with obesity and NAFLD. Proteome and gene expression analyses along with lipid analyses in the liver revealed differential expression of genes related to lipid metabolism and β-oxidation, whereas genes for peroxisomal proteins were predominantly regulated. ConclusionOur investigations show that in fatty liver disease in combination with obesity and diabetes, the hepatocyte-protecting organelle peroxisome is altered. Hence, peroxisomes might indicate a stage of pre-NAFLD, play a role in the early development of NAFLD and appear to be a potential target for treatment and prevention of NAFLD.

Prevention and reversal of lipotoxicity-induced hepatic insulin resistance and steatohepatitis in mice by an antioxidant carotenoid, β-cryptoxanthin.

Excessive hepatic lipid accumulation promotes macrophages/Kupffer cells activation, resulting in exacerbation of insulin resistance and progression of nonalcoholic steatohepatitis (NASH). However, few promising treatment modalities target lipotoxicity-mediated hepatic activation/polarization of macrophages for NASH. Recent epidemiological surveys showed that serum β-cryptoxanthin, an antioxidant carotenoid, was inversely associated with the risks of insulin resistance and liver dysfunction. In the present study, we first showed that β-cryptoxanthin administration ameliorated hepatic steatosis in high-fat diet-induced obese mice. Next, we investigated the preventative and therapeutic effects of β-cryptoxanthin using a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat (CL) diet. After 12 weeks of CL diet feeding, β-cryptoxanthin administration attenuated insulin resistance and excessive hepatic lipid accumulation and peroxidation, with increases in M1-type macrophages/Kupffer cells and activated stellate cells, and fibrosis in CL diet-induced NASH. Comprehensive gene expression analysis showed that β-cryptoxanthin down-regulated macrophage activation signal-related genes significantly without affecting most lipid metabolism-related genes in the liver. Importantly, flow cytometry analysis revealed that, on a CL diet, β-cryptoxanthin caused a predominance of M2 over M1 macrophage populations, in addition to reducing total hepatic macrophage and T-cell contents. In parallel, β-cryptoxanthin decreased lipopolysaccharide-induced M1 marker mRNA expression in peritoneal macrophages, whereas it augmented IL-4-induced M2 marker mRNA expression, in a dose-dependent manner. Moreover, β-cryptoxanthin reversed steatosis, inflammation, and fibrosis progression in preexisting NASH in mice. In conclusion, β-cryptoxanthin prevents and reverses insulin resistance and steatohepatitis, at least in part, through an M2-dominant shift in macrophages/Kupffer cells in a lipotoxic model of NASH.

OP79 ADIPOKINES MEDIATE THE RELATIONSHIPS BETWEEN ABDOMINAL ADIPOSITY AND INSULIN RESISTANCE IN ASIAN ETHNIC GROUPS: SINGAPORE ADULT METABOLISM STUDY (SAMS)

Background: Nonalcoholic steatohepatitis (NASH) and insulin resistance frequently coexists in subjects with obesity and type 2 diabetes. NASH can be defined as a lipotoxic liver injury caused by excess body adiposity. Hepatic inflammation and/or fibrosis could be caused by lipid peroxidation preceded by excessive hepatic lipid accumulation. In our previous study, we developed a cholesteroland saturated fatty acid-induced model of lipotoxic NASH and revealed that hepatic oxidative stress and insulin resistance promotes hepatic inflammation and fibrosis [1,2]. Astaxanthin is a carotenoid compound that is known to be approximately 500 times more potent in inhibiting lipid peroxidation than vitamin E in vitro . In the present study, we examined the inhibitory effect of astaxanthin against the progression of NASH to clarify the significance of lipid peroxidation-mediated lipotoxicity and insulin resistance in the pathogenesis of NASH. Method: 7-week-old C57BL/6 mice were fed on a highcholesterol/high-fat diet (CL) or a CL diet containing 0.02% astaxanthin (CL + Ax), for a total of 12 weeks. The lipid peroxidation, insulin sensitivity, and inflammatory/stress signal were evaluated. Result: After 8 weeks of feeding, the levels of thiobarbituric acid reactive substances (TBARS) in liver were significantly increased and histological examination revealed hepatic steatosis and inflammation. At week 12, hepatic fibrosis was observed in mice fed CL diet. They showed hyperinsulinemia (CL1.4±0.3 vs normal chow (NC) 0.3±0.1ng/ml, p< 0.05) and glucose intolerance even though weight and adiposity were similar, indicating that mice fed on a CL diet developed NASH associated with insulin resistance. Astaxanthin administration markedly reduced lipid peroxidation and improved glucose intolerance and insulin sensitivity in CL group. CL group had 2.5-fold and 4.1-fold increases in hepatic triglyceride (TG) and total cholesterol (TC) levels compared to NC group; astaxanthin decreased hepatic TG and TC levels by 38% and 46%, respectively (all p< 0.05). The a-SMA positive cell, as an index of activated stellate cells, increased in CL group, but decreased significantly in CL+Ax group. Furthermore, CL treatment led to 1.5-fold and 6-fold increase in TGF-b and collagen I mRNA expression, and 4.3-fold increase in hydroxyproline content, whereas astaxanthin administration inhibited fibrosis, lowering the TGF-b and collagen I mRNA expression, and hydroxyproline content by 28%, 35%, and 24%, respectively (all p< 0.05). Astaxanthin administration in CL-fed mice led to enhanced insulin signal assessed by IRb and Akt phosphorylation in the liver. These findings were associated with reduction of ER stress (CHOP/GRP78), attenuation of MAPK (JNK/p38MAPK) and NF-kB activation in liver. Astaxanthin, however, showed little effect on insulin resistance and stress response in the adipose tissue in the CL-treated mice. Conclusion: Hepatic cholesterol and TG accumulation causes excessive lipid peroxidation and the development of insulin resistance, leading to hepatic inflammation and fibrosis. Astaxanthin, a potent antioxidant, inhibits the progression of NASH by attenuating lipid accumulation, lipid peroxidation, and insulin resistance. Reference(s) [1] Matsuzawa N et al, Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet. Hepatology 46: 1392–1403, 2007. [2] Ota T et al., Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis. Gastroenterology 132:282–293, 2007.

AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response

Lipid accumulation is a central event in the development of chronic metabolic diseases, including obesity and type 2 diabetes, but the mechanisms responsible for lipid accumulation are incompletely understood. This study was designed to investigate the mechanisms for excess nutrient-induced lipid accumulation and whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic lipid accumulation in excess nutrient-treated HepG2 cells and high fat diet (HFD)-fed mice. Exposure of HepG2 cells to high levels of glucose or palmitate induced the endoplasmic reticulum (ER) stress response, activated sterol regulatory element-binding protein-1 (SREBP-1), and enhanced lipid accumulation, all of which were sensitive to ER stress inhibitor and gene silencing of eukaryotic initiation factor 2α. The increases in ER stress response and lipid accumulation were associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling attenuated the ER stress response and lipid accumulation induced by high glucose or by deletion of tuberous sclerosis 2. In addition, AMPK activation prevented the mTORC1 activation, ER stress response, and lipid accumulation. This effect was mimicked or abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Finally, treatment of HFD-fed mice with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside inhibited the mTORC1 pathway, suppressed the ER stress response, and prevented insulin resistance and hepatic lipid accumulation. We conclude that activation of AMPK prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 and ER stress response.

Radix Hedysari polysaccharide suppresses lipid metabolism dysfunction in a rat model of non‑alcoholic fatty liver disease via adenosine monophosphate‑activated protein kinase pathway activation.

Oxidative stress and excess hepatic lipid accumulation contribute to non-alcoholic fatty liver disease. Radix Hedysari polysaccharides (RHP) have attracted interest due to their antioxidant properties and immunomodulatory effects. However, the effect of RHP on hepatic lipid metabolism remains to be elucidated. In the present study, the response of Sprague-Dawley rat livers to a high-fat diet and RHP treatment was investigated by evaluating body weight, liver histology, hepatic lipid content, adenosine monophosphate-activated protein kinase (AMPK) activity and lipid metabolism gene transcriptional profiles. The present study demonstrated that RHP ameliorated lipid metabolism disorders, regulated hepatic lipid content, improved liver inflammation and damage, activated AMPK via phosphorylation, upregulated peroxisome proliferator-activated receptor α and downregulated the mRNA expression of sterol regulatory element binding protein-1c in rat livers, which reduced lipogenesis and increased lipolysis. Taken together, these results suggested that RHP effectively ameliorates lipid metabolism disorders in rat livers; thus, RHP may be a potential therapeutic agent in the prevention of hepatic steatosis.

Fish oil prevents excessive hepatic lipid accumulation without inducing oxidative stress

We examined the effects of fish oil (FO) on high-cholesterol diet-induced hepatic lipid accumulation and oxidative stress. Female C57BL/6J mice were fed diets consisting of safflower oil (SO), 1 en% FO (1FO), 2 en% FO (2FO), or 20 en% FO (20FO) with or without 2 weight% (wt%) cholesterol (SO/CH, 1FO/CH, 2FO/CH, and 20FO/CH groups, respectively) for 8 weeks. The hepatic triacylglyceride levels were significantly lower in the 2FO/CH and 20FO/CH groups than in the SO/CH group. The hepatic mRNAs of fatty acid oxidation-related genes were upregulated and the fatty acid synthesis-related genes were downregulated by the FO feeding. Adverse effects were not observed in the plasma levels of indicators of oxidative stress in response to the consumption of FO up to 20 en%. These results suggest that FO consumption in the range of 2–20 en% prevents hepatic lipid accumulation, thus improving lipid metabolism without causing oxidative stress.

Effect of ω3 PUFA on diet induced non‐alcoholic fatty liver disease (NAFLD) development and progression in C57BL/6J mice

The incidence of NAFLD and non‐alcoholic steatohepatitis (NASH) has increased in parallel with obesity. While both are characterized by excessive hepatic lipid accumulation, NASH is defined as hepatic inflammation with hepatocellular damage. Our goal was to examine the effect of C20–22 ω3 PUFA on diet induced NASH in LDLR−/− C57BL/6J mice. Mice consumed the Western Diet (WD) supplemented with olive oil, eicosapentaenoic acid (20:5ω3; EPA), docosahexaenoic acid (22:6 ω3; DHA) or both EPA and DHA for 16 weeks. The WD induced a severe phenotype similar to human NASH; including robust induction of hepatosteatosis, inflammation (MCP1) and fibrosis (ProCol1α1). While dietary C20–22 ω3 PUFA failed to suppress hepatic steatosis, ω3 PUFA attenuated markers of inflammation, fibrosis, and liver damage (plasma ALT, AST). The ω3 PUFA effect on inflammation &amp; fibrosis correlated with the down regulation of multiple transcripts involved in toll‐like receptor signaling (TRL2, TRL4, CD14). Moreover, DHA was significantly more effective than EPA or the combination of EPA &amp; DHA at suppressing markers of inflammation and fibrosis associated with NASH. Thus, dietary ω3 PUFA attenuates inflammation &amp; fibrosis without suppressing hepatic lipid.Research Support: USDA Grant 2009‐65200‐05846

Melanocortin 4 Receptor–Deficient Mice as a Novel Mouse Model of Nonalcoholic Steatohepatitis

Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor-deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies.

Suboptimal maternal nutrition, during early fetal liver development, promotes lipid accumulation in the liver of obese offspring

Maternal nutrition during the period of early organ development can modulate the offspring's ability to metabolise excess fat as young adults when exposed to an obesogenic environment. This study examined the hypothesis that exposing offspring to nutrient restriction coincident with early hepatogenesis would result in endocrine and metabolic adaptations that subsequently lead to increased ectopic lipid accumulation within the liver. Pregnant sheep were fed either 50 or 100% of total metabolisable energy requirements from 30 to 80 days gestation and 100% thereafter. At weaning, offspring were made obese, and at ∼1 year of age livers were sampled. Lipid infiltration and molecular indices of gluconeogenesis, lipid metabolism and mitochondrial function were measured. Although hepatic triglyceride accumulation was not affected by obesity per se, it was nearly doubled in obese offspring born to nutrient-restricted mothers. This adaptation was accompanied by elevated gene expression for peroxisome proliferator-activated receptor γ (PPARG) and its co-activator PGC1α, which may be indicative of changes in the rate of hepatic fatty acid oxidation. In contrast, maternal diet had no influence on the stimulatory effect of obesity on gene expression for a range of proteins involved in glucose metabolism and energy balance including glucokinase, glucocorticoid receptors and uncoupling protein 2. Similarly, although gene expressions for the insulin and IGF1 receptors were suppressed by obesity they were not influenced by the prenatal nutritional environment. In conclusion, excess hepatic lipid accumulation with juvenile obesity is promoted by suboptimal nutrition coincident with early development of the fetal liver.

Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice

Excess hepatic lipid accumulation and oxidative stress contribute to nonalcoholic fatty liver disease (NAFLD). Thus, we hypothesized that the hypolipidemic and antioxidant activities of green tea extract (GTE) would attenuate events leading to NAFLD. Obese mice ( ob/ob; 5 weeks old, n=38) and their lean littermates ( n=12) were fed 0%, 0.5% or 1% GTE for 6 weeks. Then, hepatic steatosis, oxidative stress and inflammatory markers were measured. Obese mice, compared to lean controls, had greater hepatic lipids and serum alanine aminotransferase (ALT). GTE at 1% lowered ( P<.05) hepatic lipids and ALT in obese mice. The GTE-mediated attenuation in hepatic steatosis was accompanied by decreased mRNA expression of adipose sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl CoA desaturase-1, and hormone-sensitive lipase and decreased serum nonesterified fatty acid concentrations. Immunohistochemical data indicated that steatotic livers from obese mice had extensive accumulation of tumor necrosis factor-α (TNF-α), whereas GTE at 1% decreased hepatic TNF-α protein and inhibited adipose TNF-α mRNA expression. Hepatic total glutathione, malondialdehyde and Mn- and Cu/Zn-superoxide dismutase activities in obese mice fed GTE were normalized to the levels of lean littermates. Also, GTE increased hepatic catalase and glutathione peroxidase activities, and these activities were inversely correlated with ALT and liver lipids. Collectively, GTE mitigated NAFLD and hepatic injury in ob/ob mice by decreasing the release of fatty acids from adipose and inhibiting hepatic lipid peroxidation as well as restoring antioxidant defenses and decreasing inflammatory responses. These findings suggest that GTE may be used as an effective dietary strategy to mitigate obesity-triggered NAFLD.

NAFLD, Obesity, and Bariatric Surgery

NAFLD, Obesity, and Bariatric Surgery

L‐Carnitine and level of feed intake alter hepatic nutrient metabolism and tissue carnitine distribution in lactating dairy cows

Dairy cows are prone to excessive hepatic lipid accumulation around parturition. Carnitine stimulates fatty acid oxidation by bovine liver slices and may prevent or reduce hepatic lipid accumulation. Eight lactating cows were used in a replicated 4 × 4 Latin square design. Treatments were factorial combinations of feed intake (FI) restriction and abomasal carnitine (20 g/d) infusion: water infusion + ad libitum FI (WA), water infusion + restricted FI (WR), carnitine infusion + ad libitum FI (CA), carnitine infusion + restricted FI (CR). Each period of 14 d consisted of infusions on d 5–14, restricted FI imposed on d 10–14, and liver and muscle biopsies obtained on d 14 of each period. Milk was sampled on d 9 and 14. L-carnitine infusion increased the rate and proportion of palmitate oxidation and decreased the proportion of palmitate esterified by liver slices (P < 0.05). Liver triacylglycerol concentration tended to be lower (P < 0.10) in WA, CA, and CR (0.3% wet basis) than in WR (0.9%). Restricted FI reduced propionate conversion to CO2 by liver slices. Alanine conversion to CO2 was higher in CA versus other treatments. Conversion of alanine and propionate to glucose was unaffected by treatment. Liver carnitine was not altered in WR compared with WA, but was elevated in CR versus CA. Muscle carnitine was increased by carnitine infusion and restricted FI without interaction. Carnitine infusion increased milk carnitine content on d 9, while carnitine infusion and restricted FI increased milk carnitine content on d 14. L-Carnitine prevents hepatic lipid accumulation in feed-restricted lactating dairy cows. Supported by Lonza, Inc. (Allendale, NJ).

Lipid: The Unidentified Factor for Alleviating Fatty Liver Syndrome ,

Laying hens were fed diets based on either corn-soybean meal (CS) or wheat-soybean meal (WS). The WS diets were formulated to be isocaloric to the CS diet by supplementing with either corn oil, corn starch, or wheat starch. Hens fed the WS diets with either of the starches had significantly (P≤.01) higher percentages of hepatic lipid than those fed WS diets with corn oil. Values for hepatic lipid of hens fed the CS diets were intermediate to those of the wheat-based diets. The supplementation of fish meal or a selenium salt to supply .4 mg Se per kilogram of diet did not prevent or alleviate the problem of fatty livers caused by feeding diets composed mostly of wheat-soy and starch. The data revealed that wheat does not have an unidentified factor preventing FLS. The indication is that lipid at proper amounts in the diet acts through feed-back mechanisms to prevent excessive hepatic lipid accumulation that starches enhance.