Carly E. Herbison

The Western Dietary Pattern Is Prospectively Associated With Nonalcoholic Fatty Liver Disease in Adolescence

OBJECTIVES:Poor dietary habits have been implicated in the development of nonalcoholic fatty liver disease (NAFLD); however, little is known about the role of specific dietary patterns in the development of NAFLD. We examined prospective associations between dietary patterns and NAFLD in a population-based cohort of adolescents.METHODS:Participants in the Western Australian Pregnancy Cohort (Raine) Study completed a food frequency questionnaire at 14 years and had liver ultrasound at 17 years (n=995). Healthy and Western dietary patterns were identified using factor analysis and all participants received a z-score for these patterns. Prospective associations between the dietary pattern scores and risk of NAFLD were analyzed using multiple logistic regression.RESULTS:NAFLD was present in 15.2% of adolescents. A higher Western dietary pattern score at 14 years was associated with a greater risk of NAFLD at 17 years (odds ratio (OR) 1.59; 95% confidence interval (CI) 1.17–2.14; P<0.005), although these associations were no longer significant after adjusting for body mass index at 14 years. However, a healthy dietary pattern at 14 years appeared protective against NAFLD at 17 years in centrally obese adolescents (OR 0.63; 95% CI 0.41–0.96; P=0.033), whereas a Western dietary pattern was associated with an increased risk of NAFLD.CONCLUSIONS:A Western dietary pattern at 14 years in a general population sample was associated with an increased risk of NAFLD at 17 years, particularly in obese adolescents. In centrally obese adolescents with NAFLD, a healthy dietary pattern may be protective, whereas a Western dietary pattern may increase the risk.

Association between the endothelin-1 gene Lys198Asn polymorphism blood pressure and plasma endothelin-1 levels in normal and pre-eclamptic pregnancy

Objective This study examined the frequency of the Lys198Asn polymorphism in the endothelin-1 (ET-1) gene in women with pre-eclampsia and normal pregnancy; and its contribution to levels of plasma ET-1 and blood pressure. Design and methods This was a retrospective study examining the frequency of the ET-1 Lys198Asn polymorphism in 72 proteinuric pre-eclamptics and 81 normal pregnant women. Height, weight, blood pressure and plasma ET-1 were measured antenatally and at 6 weeks post-partum. Using specific mutagenic primers, the frequency of the G/G (normal), G/T heterozygote and T/T (mutant) genotypes of the Lys198Asn polymorphism were examined. Results The polymorphism was not associated with pre-eclampsia. However, in the combined pregnant groups after correction for BMI and group, a significant effect of the T-allele (T/T,G/T) on systolic blood pressure was found (121 ± 1.5 mmHg compared with 116 ± 1.3 mmHg in the G/G homozygotes). A significant interaction was found between the T-allele and pregnancy in determining systolic blood pressure, so that the effect was no longer seen post-partum. Pregnant women with the T/T genotype had significantly elevated plasma ET-1 levels 5.8 pg/ml [confidence interval (CI) 3.7–9.1] compared with 3.1 pg/ml (CI 2.6–3.8) in the G/T heterozygotes and 3.6 pg/ml (CI 3.0–4.1) in the normal G/G homozygotes. Conclusion The Lys198Asn polymorphism does not directly contribute to the incidence of pre-eclampsia. However, the association of the T-allele with raised blood pressure and the T/T genotype with increased plasma ET-1 levels suggest that this polymorphism may interact with other genes or environmental factors to sensitize pregnant women to develop pre-eclampsia.

The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells

Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using (59)Fe-(125)I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe(2)Tf, Co(2)Tf, and Mn(2)Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co(2)Tf and Mn(2)Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1alpha protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 microM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15-20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.

Hepatic Iron Loading in Mice Increases Cholesterol Biosynthesis

Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up‐regulated with increasing hepatic iron (R2 between 0.602 and 0.164), including those of the rate‐limiting enzyme, 3‐hydroxy‐3‐methylglutarate‐coenzyme A reductase (Hmgcr; R2 = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R2 = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R2 = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol‐regulatory element binding factor 2 (Srebf2; R2 = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron. Conclusion: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity. (HEPATOLOGY 2010;)

Hepatic Lipase Gene −514 C/T Polymorphism and Premature Coronary Heart Disease

Background A common polymorphism in the hepatic lipase (HL) gene promoter, −514C/T, affecting enzyme activity, has been associated with alterations in plasma lipoprotein levels. However a relationship with coronary heart disease (CHD) is less well documented. Design and methods We studied HL −514 C/T in 562 Caucasian CHD patients aged under 50 years and in 642 Caucasian community recruited subjects without historical evidence of CHD. Results Male CHD subjects (n=490) had a 41% carrier rate for the C to T substitution, compared with 33% in corresponding controls (n=330), [OR=1.42 (95% CI:1.06–1.90), P < 0.02], T allele frequencies being 0.231 and 0.177 respectively [OR=1.39 (1.08–1.78), P < 0.01]. In male CHD subjects, the T allele was associated with higher HDL-cholesterol (HDL-C) (CC: 0.95 ± 0.24 (SD); CT: 1.04 ± 0.41; TT: 1.01 ± 0.20 mmol/l, P=0.02, ANOVA) but the trend was not significant in females. In male CHD patients the T allele was more frequently encountered in those with high (> 4.5 mmol/l) than in those with low triglycerides [68% vs. 39%, OR=3.13 (1.54–6.67), P=0.001]. In community control subjects, the T allele was associated with a trend to higher HDL-C levels, the significance varying between subgroups while, in males, serum total and LDL-cholesterol were significantly lower in T homozygotes than in the other two genotypes (LDL-C: 2.73 ± 0.63 vs. 3.56 ± 0.95 mmol/l; P=0.01). During the course of this study, a previously unreported promoter region polymorphism was found exclusively on −514C chromosomes (−592A/G, A allele frequency 0.108, 95% CI 0.09–0.126). It can lead to mistyping of C as T alleles in C/T heterozygotes, resulting in overestimation of −514 T homozygotes. Conclusions The T allele of the hepatic lipase −514 C/T polymorphism is associated with changes in plasma lipids. The superficially paradoxical predisposition to CHD in males is attributable to impairment of TG rich lipoprotein metabolism and reverse cholesterol transport.

Low intake of B-vitamins is associated with poor adolescent mental health and behaviour

OBJECTIVE The current prevalence of mental health problems in Western populations is approximately 20% and half of all adult mental health disorders are estimated to originate in adolescence. Diet plays an important role in modulating psychological wellbeing and B-vitamins are vital for the synthesis of neurotransmitters such as serotonin. We aimed to examine the relationship between B-group vitamins and adolescent mental health and behaviour. METHODS This is a cross-sectional analysis of the West Australian Pregnancy Cohort (Raine) Study. The 17-year follow-up included collection of a food frequency questionnaire allowing B-vitamin intake calculation. Mental health was assessed using the Youth Self Report (YSR) which measures total, internalising (withdrawn/depressed) and externalising (aggressive/delinquent) behaviour scores. Multiple linear regression was used to analyse associations between B-vitamins and mental health with adjustment for relevant confounders (n=709). RESULTS Lower intake of vitamins B1, B2, B3, B5, B6, and folate was associated with higher externalising behaviour scores (p ≤ 0.05). Reduced intake of vitamin B6 and folate was associated with higher internalising behaviour scores (p ≤ 0.05). CONCLUSIONS Poor nutrition may contribute to the pathogenesis of mental health problems in adolescence. The role of B-vitamins requires further investigation in randomised controlled trials.

The Role of Hfe in Transferrin-Bound Iron Uptake by Hepatocytes

HFE‐related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin‐bound iron via transferrin receptor (Tfr) 1 and Tfr1‐independent pathways (possibly Tfr2‐mediated). In this study, the role of Hfe in the regulation of hepatic transferrin‐bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non–iron‐loaded and iron‐loaded wild‐type mice were measured after incubation with 50 nM 125I‐Tf‐59Fe (Tfr1 pathway) and 5 μM 125I‐Tf‐59Fe (Tfr1‐independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real‐time polymerase chain reaction and western blotting, respectively. Tfr1‐mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron‐loaded wild‐type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1‐independent pathway was approximately 100‐fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1‐independent pathway. Conclusion: Tfr1‐mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1‐independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. Diferric transferrin up‐regulated hepatocyte Tfr2 protein expression but not iron uptake, suggesting that Tfr2 may have a limited role in the Tfr1‐independent pathway. (HEPATOLOGY 2008.)

Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis

BACKGROUND & AIMS Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. METHODS Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. RESULTS Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. CONCLUSIONS A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice.

Characterization and novel analyses of acute stress response patterns in a population-based cohort of young adults: Influence of gender, smoking, and BMI

Abstract Dysregulation of the biological stress response system has been implicated in the development of psychological, metabolic, and cardiovascular disease. Whilst changes in stress response are often quantified as an increase or decrease in cortisol levels, three different patterns of stress response have been reported in the literature for the Trier Social Stress Test (TSST) (reactive-responders (RR), anticipatory-responders (AR) and non-responders (NR)). However, these have never been systematically analyzed in a large population-based cohort. The aims of this study were to examine factors that contribute to TSST variation (gender, oral contraceptive use, menstrual cycle phase, smoking, and BMI) using traditional methods and novel analyses of stress response patterns. We analyzed the acute stress response of 798, 18-year-old participants from a community-based cohort using the TSST. Plasma adrenocorticotrophic hormone, plasma cortisol, and salivary cortisol levels were quantified. RR, AR, and NR patterns comprised 56.6%, 26.2%, and 17.2% of the cohort, respectively. Smokers were more likely to be NR than (RR or AR; adjusted, p < 0.05). Overweight and obese subjects were less likely to be NR than the other patterns (adjusted, p < 0.05). Males were more likely to be RR than NR (adjusted, p = 0.05). In addition, we present a novel AUC measure (AUCR), for use when the TSST baseline concentration is higher than later time points. These results show that in a young adult cohort, stress-response patterns, in addition to other parameters vary with gender, smoking, and BMI. The distribution of these patterns has the potential to vary with adult health and disease and may represent a biomarker for future investigation.

The impact of life stress on adult depression and anxiety is dependent on gender and timing of exposure

There is debate about the relative importance of timing of stressful events prenatally and over the life course and risk for subsequent depressive/anxious illness. The aim of this study was to examine the relative roles of prenatal stress and postnatal stress trajectories in predicting depression and anxiety in early adulthood in males and females. Exposure to life stress events was examined in the Western Australian Pregnancy Cohort (Raine) Study during pregnancy and ages 1, 2, 3, 5, 8, 10, 14, and 17 years. At age 20, offspring completed the Depression Anxiety Stress Scale. Prenatal stress and trajectories of stress events from age 1 to 17 were analyzed in linear regression analyses. Five postnatal stress trajectories were identified. In females, medium to high chronic stress exposure or exposure during puberty/adolescence predicted depression and anxiety symptoms while low or reduced stress exposure over the life course did not, after adjustment for relevant confounders. High stress early in pregnancy contributed to male depression/anxiety symptoms independent of postnatal stress trajectory. In females, postnatal stress trajectory was more important than prenatal stress in predicting depression/anxiety symptoms. Interventions focused on reducing and managing stress events around conception/pregnancy and exposure to chronic stress are likely to have beneficial outcomes on rates of depression and anxiety in adults.