Dietary treatment of fatty liver: High dietary protein content has an antisteatotic and antiobesogenic effect in mice.

Few studies have assessed the effect of changing ratios of dietary macronutrients on fat accumulation in adipose tissue and organs such as the liver in a 3×n(n≥3) factorial design. We investigated the effects of 7 diets from a single manufacturer containing 11-58en% protein (casein), 0-81en% carbohydrates (CHO; sucrose, maltrodextrin-10 and corn starch), and 8-42en% fat (triheptanoin, olive oil or cocoa butter) in C57BL/6J mice, a good model for diet-induced obesity and fatty liver. The diets were fed for 3weeks to wild-type and hyperlipidemic male and female mice. Caloric intake was mainly determined by dietary fat. Body weight, liver lipid and cholesterol content, NFκB activation, and fat-pad size decreased only in mice fed a high-protein diet. A high dietary protein:CHO ratio reduced plasma FGF21 concentration, and increased liver PCK1 protein content and plasma triglyceride concentration. The dietary protein:CHO ratio determined hepatic expression of Pck1 and Ppargc1a in males, and Fgf21 in females, whereas the dietary CHO:fat ratio determined that of Fasn, Acaca1, and Scd1 in females. Hepatic glycogen content was determined by all three dietary components. Both hepatic PCK1 and plasma FGF21 correlated strongly and inversely with hepatic TG content, suggesting a key role for PCK1 and increased gluconeogenesis in resolving steatosis with a high-protein diet, with FGF21 expression reflecting declining cell stress. We propose that a diet containing ~35en% protein, 5-10en% fat, and 55-60en% carbohydrate will prevent fatty liver in mice without inducing side effects.

The odd-carbon medium-chain fatty triglyceride triheptanoin does not reduce hepatic steatosis.

BACKGROUND & AIMS: Non-alcoholic fatty-liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Previously, we showed that a high-protein diet minimized diet-induced development of fatty liver and even reversed pre-existing steatosis. A high-protein diet leads to amino-acid catabolism, which in turn causes anaplerosis of the tricarboxylic-acid (TCA) cycle. Therefore, we hypothesized that anaplerosis of the TCA cycle could be responsible for the high-protein diet-induced improvement of NAFLD by channeling amino acids into the TCA cycle. Next we considered that an efficient anaplerotic agent, the odd-carbon medium-chain triglyceride triheptanoin (TH), might have similar beneficial effects. METHODS: C57BL/6J mice were fed low-fat (8en%) or high-fat (42en%) oleate-containing diets with or without 15en% TH for 3 weeks. RESULTS: TH treatment enhanced the hepatic capacity for fatty-acid oxidation by a selective increase in hepatic Ppara, Acox, and Cd36 expression, and a decline in plasma acetyl-carnitines. It also induced pyruvate cycling through an increased hepatic PCK1 protein concentration and it increased thermogenesis reflected by an increased Ucp2 mRNA content. TH, however, did not reduce hepatic lipid content. CONCLUSION: The comparison of the present effects of dietary triheptanoin with a previous study by our group on protein supplementation shows that the beneficial effects of the high-protein diet are not mimicked by TH. This argues against anaplerosis as the sole explanatory mechanism for the anti-steatotic effect of a high-protein diet.

High-protein diets prevent steatosis and induce hepatic accumulation of monomethyl branched-chain fatty acids.

The hallmark of nonalcoholic fatty liver disease is steatosis of unknown etiology. To test how dietary protein decreases steatosis, we fed female C57BL/6 J mice low-fat (8 en%) or high-fat (42 en%) combined with low-protein (11 en%), high-protein (HP; 35 en%) or extra-high-protein (HPX; 58 en%) diets for 3 weeks. The 35 en% protein diets reduced hepatic triglyceride, free fatty acid, cholesterol and phospholipid contents to ~50% of that in 11 en% protein diets. Every additional 10 en% protein reduced hepatic fat content ~1.5 g%. HP diets had no effect on lipogenic or fatty acid-oxidizing genes except Ppargc1α (+30%), increased hepatic PCK1 content 3- to 5-fold, left plasma glucose and hepatic glycogen concentration unchanged, and decreased inflammation and cell stress (decreased Fgf21 and increased Gsta expression). The HP-mediated decrease in steatosis correlated inversely with plasma branched-chain amino-acid (BCAA) concentrations and hepatic content of BCAA-derived monomethyl branched-chain fatty acids (mmBCFAs) 14-methylpentadecanoic (14-MPDA; valine-derived) and, to a lesser extent, 14-methylhexadecanoic acid (isoleucine-derived). Liver lipid content was 1.6- to 1.8-fold higher in females than in males, but the anti-steatotic effect of HP diets was equally strong. The strong up-regulation of PCK1 and literature data showing an increase in phosphoenolpyruvate and a decline in tricarboxylic acid cycle intermediates in liver reveal that an increased efflux of these intermediates from mitochondria represents an important effect of an HP diet. The HP diet-induced increase in 14-MPDA and the dietary response in gene expression were more pronounced in females than males. Our findings are compatible with a facilitating role of valine-derived mmBCFAs in the antisteatotic effect of HP diets.

A high-protein diet is anti-steatotic and has no pro-inflammatory side effects in dyslipidaemic APOE2 knock-in mice.

High-protein (HP) diets are effective anti-steatotic treatment options for patients with non-alcoholic fatty liver disease, but whether these diets also decrease steatosis in hyperlipidaemic conditions is not known. The aim of the present study was to determine the effects of a HP diet on hepatic steatosis and inflammation in hyperlipidaemic mice. Hyperlipidaemic male and female APOE2 knock-in (APOE2ki) mice were fed a semi-synthetic low-protein (LP) or HP diet in combination with a low-fat diet or a high-fat diet for 3 weeks. The HP diets reduced hepatic fat and cholesterol concentrations to 40-55 % of those induced by the corresponding LP diets and attenuated hepatic inflammation mildly. The VLDL-associated plasma cholesterol concentrations decreased to 60-80 %, but those of TAG increased 3-4-fold. APOE2-mediated restriction of fat import into the liver did not modify the effects of a HP diet previously observed in wild-type mice. Female APOE2ki mice exhibited a higher expression of lipogenic, cholesterol-synthesising, inflammatory and cell-stress genes than wild-type female or male APOE2ki mice, but a similar response to HP diets. Low Apob expression and unchanged plasma APOB100 concentrations suggest that HP diets increase the plasma concentrations of TAG by slowing their clearance. The decrease in plasma leptin and hepatic fat and glycogen concentrations and the increase in fatty acid-oxidising gene and phosphoenolpyruvate carboxykinase 1 protein expression suggest a HP diet-mediated increase in mitochondrial metabolism. In conclusion, a HP diet reduces hepatic lipid content in dyslipidaemic mice and lowers the activation status of inflammatory cells in the liver.

Prevention and reversal of hepatic steatosis with a high-protein diet in mice.

UNLABELLED: The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)(2) diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C0]/[C16+C18] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma-glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C16:1n-7]/[C16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). CONCLUSION: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.

Atheroprotective effect of dietary walnut intake in ApoE-deficient mice: involvement of lipids and coagulation factors.

INTRODUCTION: Consumption of n-3 polyunsaturated fatty acids (PUFA) and antioxidant polyphenols is considered to decline the risk of cardiovascular disease. MATERIALS AND METHODS: To provide an explanation for this cardioprotective effect, we performed an intervention study with proatherogenic Apoe(-/-) mice which were fed during eight weeks with a high fat diet supplemented with either walnuts (rich in n-3 PUFA and antioxidant compounds), walnut oil (with n-3 PUFA only) or sunflower oil as a control (12 mice per group). RESULTS: Feeding walnuts, but not walnut oil, caused a 55% reduction in atherosclerotic plaque development in the aortic arch in comparison to the control diet. This was associated with reduced staining of plaques for CD36, a scavenger receptor expressed by macrophages. Feeding mice with walnuts also lowered plasma levels of triglycerides, cholesterol and prothrombin with 36%, 23% and 21 %, respectively, compared to control diet. In addition, accumulation of lipids in the liver was decreased, while plasma antioxidant capacity was increased. On the other hand, feeding mice with walnut oil did not provoke significant changes in these parameters in comparison to the control diet. Platelet activation and thrombus formation under flow remained unchanged with either diet. CONCLUSIONS: In Apoe(-/-) mice on high fat diet, intake of dietary walnut (but not walnut oil) beneficially influences lipid metabolism and atherosclerotic plaque development, with no more than limited effects on platelet and coagulation function.

Dietary cholesterol, female gender and n-3 fatty acid deficiency are more important factors in the development of non-alcoholic fatty liver disease than the saturation index of the fat.

BACKGROUND: The central feature of NAFLD is a disturbed fatty-acid metabolism with hepatic lipid accumulation. However, the factors that determine the severity of NAFLD, including the role of nutrition, gender, and plasma lipid levels, remain to be determined. METHODS: High-fat diets (42 en% fat), containing 0.2% cholesterol, were fed to male and female wild-type and hyperlipidemic APOE2ki C57BL/6J mice for three weeks. The fats were, in order of decreasing saturation, fractionated palm fat (fPF; ~95%), cocoa butter (CB; ~60%), olive oil (OO; ~15%), sunflower oil (SO; ~12%), and high-oleic-acid sunflower oil (hoSO; ~7%). Plasma and liver triglycerides (concentration and composition), liver inflammation (Ccl2, Cd68, Tnf-α mRNA), and infiltration of macrophages (Cd68, Cd11b immunohistochemistry) and neutrophils (Mpo) were quantified. RESULTS: Addition of cholesterol to a low-fat diet decreased plasma HDL and increased (V)LDL levels in APOE2ki mice. Plasma cholesterol levels in female, but not male APOE2ki mice correlated significantly with inflammation. Kupffer cells of inflamed livers were swollen. Wild-type mice refused the highly saturated fPF diet. The high-fat CB, OO, and SO diets induced hyperglycemia and a 2-fold increase in hepatic fat content in male, but not female wild-type mice (in females, hepatic fat content was similar to that in males fed a high-fat diet). All high-fat diets induced macrovesicular setatosis. APOE2ki mice were protected against high-fat diet-induced steatosis and hyperglycemia, except when fed a hoSO diet. This diet caused a 5-fold increase in liver triglyceride and mead-acid content, and an increased expression of lipogenic genes, suggesting a deficiency in poly-unsaturated fatty acids. Irrespective of the composition of the high-fat diet, oleic acid was the main triglyceride component of liver fat in wild-type and APOE2ki mouse livers. Liver inflammation was dependent on genotype (APOE2ki > wild type), gender (female > male), and cholesterol content (high > low) of the diet, but not on dietary fat composition. CONCLUSIONS: Dietary cholesterol plays a determining, independent role in inflammation, especially in female mice. The fatty-acid saturation of the diet hardly affected hepatic steatosis or inflammation.