Zebrafish mutants provide insights into Apolipoprotein B functions during embryonic development and pathological conditions.

Apolipoprotein B (ApoB) is the primary protein of chylomicrons, VLDLs, and LDLs and is essential for their production. Defects in ApoB synthesis and secretion result in several human diseases, including abetalipoproteinemia and familial hypobetalipoproteinemia (FHBL1). In addition, ApoB-related dyslipidemia is linked to nonalcoholic fatty liver disease (NAFLD), a silent pandemic affecting billions globally. Due to the crucial role of APOB in supplying nutrients to the developing embryo, ApoB deletion in mammals is embryonic lethal. Thus, a clear understanding of the roles of this protein during development is lacking. Here, we established zebrafish mutants for 2 apoB genes: apoBa and apoBb.1. Double-mutant embryos displayed hepatic steatosis, a common hallmark of FHBL1 and NAFLD, as well as abnormal liver laterality, decreased numbers of goblet cells in the gut, and impaired angiogenesis. We further used these mutants to identify the domains within ApoB responsible for its functions. By assessing the ability of different truncated forms of human APOB to rescue the mutant phenotypes, we demonstrate the benefits of this model for prospective therapeutic screens. Overall, these zebrafish models uncover what are likely previously undescribed functions of ApoB in organ development and morphogenesis and shed light on the mechanisms underlying hypolipidemia-related diseases.

In utero exposure to phenanthrene induces hepatic steatosis in F1 adult female mice.

Environmental pollutants are thought to be a risk factor for the prevalence of hepatic steatosis. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, and human exposure is inevitable. In the present study, phenanthrene (Phe) was used as a representative PAH to investigate the effects of in utero exposure to PAH on hepatic lipid metabolism and the toxicological mechanism involved. Pregnant mice (C57BL/6J) were orally administered Phe (0, 60, 600 and 6000 µg kg-1 body weight) once every 3 days with 6 doses in total. F1 female mice aged 125 days showed significantly elevated hepatic lipid levels in the liver. The protein expression of hepatic peroxisome proliferator-activated receptors (PPARß and PPARγ) and retinoid X receptors (RXRs) was upregulated; the transcription of genes related to lipogenesis, such as srebp1 (encoding sterol regulatory element binding proteins), acca (acetyl-CoA carboxylase), fasn (fatty acid synthase) and pcsk9 (proprotein convertase subtilisin/kexin type 9), showed an upregulation, while the mRNA levels of the lipolysis gene lcat (encoding lecithin cholesterol acyl transferase) were downregulated. These results could be responsible for lipid accumulation. The promoter methylation levels of pparγ were reduced and were the lowest in the 600 µg kg-1 group, and the promoter methylation levels of lcat were significantly increased in all the Phe treatments. These changes were matched with the alterations in their mRNA levels, suggesting that prenatal Phe exposure could induce abnormal lipid metabolism in later life via epigenetic modification.

Maternal chocolate and sucrose soft drink intake induces hepatic steatosis in rat offspring associated with altered lipid gene expression profile.

AIM: According to the World Diabetes Foundation, there is an urgent need to investigate the impact of maternal health and nutrition during pregnancy to understand the background for the accelerating incidence of obesity and type 2 diabetes. In this study, we specifically concentrated on the role of overfeeding during different developmental periods. METHODS: Sprague-Dawley rats were offered chow or high-fat/high-sucrose diet (chow plus chocolate and soft drink) during gestation and lactation. At birth, offspring were randomly cross-fostered within each dietary group into small and normal litter sizes until weaning, giving four dietary groups. RESULTS: At postnatal day 1, offspring from high-fat/high-sucrose-fed dams were heavier and had increased hepatic triglycerides (TG), hepatic glycogen, blood glucose and plasma insulin compared with offspring from chow-fed dams. Hepatic genes involved in lipid oxidation, VLDL transport and insulin receptor were down-regulated, whereas FGF21 expression was up-regulated. Independent of postnatal litter size, offspring from high-fat/high-sucrose-fed dams aged 21 days had still increased hepatic TG and up-regulated FGF21 expression, while plasma insulin started to decrease. Litter size reduction in offspring from high-fat/high-sucrose-fed dams further increased body weight and adiposity, and up-regulated genes involved in hepatic mitochondrial lipid oxidation and VLDL transport compared with all other groups. Litter size reduction did not have any impact on body weight gain and adiposity in offspring born to chow-fed dams. CONCLUSION: Our results suggest that supplementation of chocolate and soft drink during gestation and lactation contributes to early onset of hepatic steatosis associated with changes in hepatic gene expression and lipid handling.

Developmental programming of pediatric nonalcoholic fatty liver disease: redefining the"first hit".

The incidence of pediatric nonalcoholic fatty liver disease has increased dramatically, and growing evidence indicates that the pathophysiology may be unique from the adult form, suggesting a role for early-life events. Recent radiologic techniques have now demonstrated that maternal obesity contributes to hepatic fat storage in newborn infants. In this review, we will explore how maternal obesity and a hyperlipidemic environment can initiate liver histopathogenesis in utero, including steatosis, mitochondrial dysfunction, oxidative stress, and inflammatory priming. Thus, early exposure to excess lipids may represent the "first hit" for the fetal liver, placing it on a trajectory toward future metabolic disease.

Early onset of fatty liver in growth-restricted rat fetuses and newborns.

Intrauterine growth-restricted (IUGR) newborns have increased risk of adult metabolic syndrome, including fatty liver. However, it is unclear whether the fatty liver development is "programmed" or secondary to the accompanying obesity. In this study, we examined hepatic lipid accumulation and lipid-regulatory factors (sterol regulatory element-binding protein-1c and fatty acid synthase) in IUGR and Control fetal (embryonic day 20; e20) and newborn (postnatal day 1; p1) rat pups. Notably, despite of in utero undernutrition state, IUGR fetuses demonstrated "fatty liver" with upregulation of these lipogenic indices at as early as e20. Both IUGR and Control newborns exhibited the same extent of massive increase in hepatic lipid content, whereas IUGR newborns continued to exhibit upregulated lipogenic indices. The persistent upregulation of the lipogenic indices in fetal and newborn IUGR suggests that fatty liver is gestationally programmed. Our study suggested that IUGR offspring were born with an altered metabolic life strategy of increased fuel/lipid storage which could be a distinct metabolic pathway of the thrifty phenotype.

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.

Long-term maternal high-fat feeding from weaning through pregnancy and lactation predisposes offspring to hypertension, raised plasma lipids and fatty liver in mice.

In rodents, adverse prenatal nutrition, such as a maternal diet rich in fat during pregnancy, enhances susceptibility of the offspring to hypertension, type 2 diabetes and other features of the human metabolic syndrome in adulthood. However, previous experimental studies were confined to short-term modifications of the maternal diet during pregnancy and/or lactation periods, a situation uncommon in humans. Moreover in humans, the offspring may also consume a high-fat diet, which may take them beyond the range to which their development has adapted them to respond healthily. We examined in C57 mice the effects on offspring of feeding their mothers a high-fat (HF) or standard chow (C) diet from weaning through pregnancy and lactation, and whether there are additive phenotypic effects of feeding the offspring an HF diet from weaning to adulthood (dam-offspring dietary group HF-HF). This group was compared with offspring from HF-fed dams fed a C diet from weaning to adulthood (HF-C) and offspring from C-fed mothers fed the C or HF diet (C-C and HF-C, respectively). HF-HF, HF-C and C-HF adult female offspring were heavier, fatter, and had raised serum cholesterol and blood pressure compared with C-C female offspring. We observed a similar trend in male offspring except for the HF-C group which was not heavier or fatter than male C-C offspring. Histology showed lipid vacuoles within hepatocytes in the HF-HF, HF-C and C-HF but not the CC offspring. Serum C-reactive protein was elevated in female (C-HF and HF-HF) but not in male offspring. Elevated blood pressure in the HF-C and C-HF groups was attenuated in the HF-HF group in males but not in females. These findings indicate that long-term consumption of an HF diet by the mother predisposes her offspring to developing a metabolic syndrome-like phenotype in adult life, although cardiovascular effects of an HF diet are related to sex specificity in the HF-HF group.

Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fish.

The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene.

Pediatric nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease related to excessive accumulation of hepatic fat, and represents a spectrum of liver disease ranging from fat accumulation alone (steatosis) to the more significant histologic finding of steatohepatitis. Nonalcoholic steatohepatitis is a progressive liver disease associated with increased risk of liver cirrhosis and cancer. NAFLD is becoming increasingly prevalent in the pediatric population in direct correlation with the emergence of childhood obesity as a significant pediatric health problem. The exact pathophysiology of NAFLD remains unclear, although the interplay of insulin resistance, oxidative stress, and release of proinflammatory cytokines are implicated in the process. The diagnostic workup and treatment for NAFLD and nonalcoholic steatohepatitis remains controversial. This review discusses current concepts regarding the natural history, pathophysiology, and management of pediatric patients with NAFLD.

Hepatic structural alteration in adult programmed offspring (severe maternal protein restriction) is aggravated by post-weaning high-fat diet.

The present study aimed to evaluate the effects of a post-weaning high-fat (HF) diet upon hepatic morphology in rats subjected to perinatal protein restriction. Pregnant Wistar rats were assigned to a normal-protein diet (NP; with 19 % of protein) or a low-protein (LP) diet (with 5 % of protein). At weaning, the following groups were formed: NP and NP-HF, males and females, which were fed standard chow and an HF diet, respectively. Likewise, LP rat dams originated LP and LP-HF offspring, both sexes. Euthanasia was performed at 6 months of age. Three-way ANOVA disclosed a three-factor interaction among sex, perinatal diet and HF diet in relation to body mass, retroperitoneal fat pad, liver mass:tibia length ratio, binucleation rate and hepatocyte area at 6 months old (P < 0.05). The high-fat diet intensified the effects of perinatal protein restriction concerning systolic blood pressure, genital fat pad and hepatocyte number (P < 0.05; two-way ANOVA). Furthermore, higher steatosis rates and insulin and leptin concentrations were found in males fed on the HF diet, indicating a sex-post-weaning diet interaction (P < 0.05; two-way ANOVA). Fetal programming and HF diet as a single stimulus caused mild hypertension at 3 months, an important reduction in hepatocyte number as well as stage 1 steatosis at 6 months. However, hypertension and hepatocyte number deficit were worsened and grade 2 steatosis occurred after exposure to the HF diet. All of these serve to highlight the paramount importance of intra-uterine conditions and postnatal diet quality when it comes to the pathogenesis of chronic diseases.