Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

Molecular epidemiology of respiratory viruses in a paediatric cohort from Indonesia

Acute lower respiratory tract infections (ALRTIs) are a common cause of morbidity and mortality among children under 5 years of age and are found worldwide, with pneumonia as the most severe manifestation. Although the incidence of severe disease varies both between individuals and countries, there is still no clear understanding of what causes this variation. Studies of community-acquired pneumonia (CAP) have traditionally not focused on viral causes of disease due to a paucity of diagnostic tools. However, with the emergence of molecular techniques, it is now known that viruses outnumber bacteria as the etiological agents of childhood CAP, especially in children under 2 years of age. The main objective of this study was to investigate viruses contributing to disease severity in cases of childhood ALRTI, using a two year cohort study following 2014 infants and children enrolled in Bandung, Indonesia. A total of 352 nasopharyngeal washes collected from 256 paediatric ALRTI patients were used for analysis. A subset of samples was screened using a novel microarray pathogen detection method that identified respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and human rhinovirus (HRV) in the samples. Real-time RT-PCR was used both for confirming and quantifying viruses found in the nasopharyngeal samples. Viral copy numbers were determined and normalised to the numbers of human cells collected with the use of 18S rRNA. Molecular epidemiology was performed for RSV A and hMPV using sequences to the glycoprotein gene and nucleoprotein gene respectively, to determine genotypes circulating in this Indonesian paediatric cohort. This study found that HRV (119/352; 33.8%) was the most common virus detected as the cause of respiratory tract infections in this cohort, followed by the viral pathogens RSV A (73/352; 20.7%), hMPV (30/352; 8.5%) and RSV B (12/352; 3.4%). Co-infections of more than two viruses were detected in 31 episodes (defined as an infection which occurred more than two weeks apart), accounting for 8.8% of the 352 samples tested or 15.4% of the 201 episodes with at least one virus detected. RSV A genotypes circulating in this population were predominantly GA2, GA5 and GA7, while hMPV genotypes circulating were mainly A2a (27/30; 90.0%), B2 (2/30; 6.7%) and A1 (1/30; 3.3%). This study found no evidence of disease severity associated either with a specific virus or viral strain, or with viral load. However, this study did find a significant association with co-infection of RSV A and HRV with severe disease (P = 0.006), suggesting that this may be a novel cause of severe disease.

Maternal feeding practices and feeding behaviors of Australian children aged 12 to 36 months

Objectives To explore parents' perceptions of the eating behaviors and related feeding practices of their young children. Methods Mothers (N=740) of children aged 12 to 36 months and born in South Australia were randomly selected by birth date in four 6-month age bands from a centralized statewide database and invited to complete a postal questionnaire. Results Valid completed questionnaires were returned for 374 children (51% response rate; 54% female). Although mothers generally reported being confident and happy in feeding their children, 23% often worried that they gave their child the right amount of food. Based on a checklist of 36 specified items, 15% of children consumed no vegetables in the previous 24 hours, 11% no fruit and for a further 8% juice was the only fruit. Of 12 specified high fat/sugar foods and drinks, 11% of children consumed none, 20% one, 26% two, and 43% three or more. Six of eight child-feeding practices that promote healthy eating behaviors were undertaken by 75% parents 'often' or 'all of the time'. However, 8 of 11 practices that do not promote healthy eating were undertaken by a third of mothers at least ‘sometimes’. Conclusions In this representative sample, dietary quality issues emerge early and inappropriate feeding practices are prevalent thus identifying the need for very early interventions that promote healthy food preferences and positive feeding practices. Such programs should focus not just on the 'what', but also the 'how' of early feeding, including the feeding relationship and processes appropriate to developmental stage. Key words: Maternal feeding practices, infants, obesity

Determinants of infant and young child feeding practices in Bangladesh: Secondary data analysis of Demographic and Health Survey 2004

BACKGROUND: In Bangladesh, poor infant and young child feeding practices are contributing to the burden of infectious diseases and malnutrition. Objective. To estimate the determinants of selected feeding practices and key indicators of breastfeeding and complementary feeding in Bangladesh. METHODS: The sample included 2482 children aged 0 to 23 months from the Bangladesh Demographic and Health Survey of 2004. The World Health Organization (WHO)-recommended infant and young child feeding indicators were estimated, and selected feeding indicators were examined against a set of individual-, household-, and community-level variables using univariate and multivariate analyses. RESULTS: Only 27.5% of mothers initiated breastfeeding within the first hour after birth, 99.9% had ever breastfed their infants, 97.3% were currently breastfeeding, and 22.4% were currently bottle-feeding. Among infants under 6 months of age, 42.5% were exclusively breastfed, and among those aged 6 to 9 months, 62.3% received complementary foods in addition to breastmilk. Among the risk factors for an infant not being exclusively breastfed were higher socioeconomic status, higher maternal education, and living in the Dhaka region. Higher birth order and female sex were associated with increased rates of exclusive breastfeeding of infants under 6 months of age. The risk factors for bottle-feeding were similar and included having a partner with a higher educational level (OR = 2.17), older maternal age (OR for age > or = 35 years = 2.32), and being in the upper wealth quintiles (OR for the richest = 3.43). Urban mothers were at higher risk for not initiating breastfeeding within the first hour after birth (OR = 1.61). Those who made three to six visits to the antenatal clinic were at lower risk for not initiating breastfeeding within the first hour (OR = 0.61). The rate of initiating breastfeeding within the first hour was higher in mothers from richer households (OR = 0.37). CONCLUSIONS: Most breastfeeding indicators in Bangladesh were below acceptable levels. Breastfeeding promotion programs in Bangladesh need nationwide application because of the low rates of appropriate infant feeding indicators, but they should also target women who have the main risk factors, i.e., working mothers living in urban areas (particularly in Dhaka).