Elevated plasma levels of the branched-chain amino acid (BCAA) leucine are associated with obesity and insulin resistance (IR), and thus the propensity for type 2 diabetes mellitus development. ...However, other clinical studies suggest the contradictory view that leucine may in fact offer a degree of protection against metabolic syndrome. Aiming to resolve this apparent paradox, we assessed the effect of leucine supplementation on the metabolism of human hepatic HepG2 cells.
We demonstrate that pathophysiological leucine appears to be antagonistic to insulin, promotes glucose uptake (and not glycogen synthesis), but results in hepatic cell triglyceride (TG) accumulation. Further, we provide evidence that myostatin (MSTN) regulation of AMP-activated protein kinase (AMPK) is a key pathway in the metabolic effects elicited by excess leucine. Finally, we report associated changes in miRNA expression (some species previously linked to metabolic disease etiology), suggesting that epigenetic processes may contribute to these effects.
Collectively, our observations suggest leucine may be both 'friend' and 'foe' in the context of metabolic syndrome, promoting glucose sequestration and driving lipid accumulation in liver cells. These observations provide insight into the clinical consequences of excess plasma leucine, particularly for hyperglycemia, IR and nonalcoholic fatty liver disease (NAFLD).
E‐cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion‐dependent regulation of signaling has not been elucidated. We report that ...E‐cadherin can negatively regulate, in an adhesion‐dependent manner, the ligand‐dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand‐dependent activation in association with decreases in receptor mobility and in ligand‐binding affinity. E‐cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand‐dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein‐coupled receptors. EGFR regulation by E‐cadherin was associated with complex formation between EGFR and E‐cadherin that depended on the extracellular domain of E‐cadherin but was independent of β‐catenin binding or p120‐catenin binding. Transfection of E‐cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E‐cadherin. Abrogation of E‐cadherin regulation may contribute to the frequent ligand‐dependent activation of RTK in tumors.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Health issues associated with excessive caloric intake and sedentary lifestyle are driving a modern "epidemic" of liver disease. Initially presenting in the clinic as an excessive accumulation of fat ...within hepatocyte cells (steatosis), the progression to more severe non-alcoholic steatohepatitis (NASH) in which liver damage and inflammation are overt features, is becoming increasingly common. Often developing as a sequela of obesity, non-alcoholic fatty liver disease (NAFLD) arises in almost one-third of people initially carrying excess hepatic fat and is likely the result of the liver's limited capacity to cope with the modern-day levels of dietary fatty acids circulating in the blood. While routine imaging can readily assess the presence and level of "extra-hepatic fat", a proper diagnosis of disease progression to NASH is currently only possible by liver biopsy. A general reluctance to undergo such screening means that the prevalence of NASH is likely to be under reported and, thus, risk assessment for future metabolic syndrome (MetS) markedly compromised. The seemingly inevitable progression to overt insulin resistance that characterizes MetS may in part be the consequence of the body's attempt to cope with NAFLD by driving systemic insulin sensitivity and, thus, fatty acid breakdown. The potential significance of miRNAs in both physiological homeostasis and pathogenesis is increasingly appreciated and in the liver may contribute specifically to the regulation of lipid pathways and NAFLD progression. As such, they may have utility as molecular indicators for the accurate profiling of both initial risk and disease progression from simple steatosis to NASH, and further to fibrosis/cirrhosis.
Gestational diabetes mellitus (GDM), the most common metabolic complication of pregnancy, is influenced by the placenta, and its prevalence directly increases with obesity. Therefore, to define the ...aetiology of GDM requires that the confounding influence of obesity and the heterogeneous nature of the placenta impairing accurate quantitative studies be accounted for. Using laser capture microdissection (LCM), we optimized RNA extraction from human placental trophoblast, the metabolic cellular interface between mother and foetus. This allowed specific transcriptomic profiling of trophoblast isolated from GDM, and obese and normal human placentae. Genome-wide gene expression analysis was performed on the RNA extracted from the trophoblast of GDM and obese and normal placentae. Forty-five differentially expressed genes (DEGs) specifically discriminated GDM from matched obese subjects. Two genes previously linked with GDM, pregnancy specific beta-1 glycoprotein 6 (PSG6) and placental system A sodium-dependent transporter system (SLC38A1), were significantly increased in GDM. A number of these DEGs (8 ubiquitin-conjugating enzymes (UBE) splice variants (UBE2D3 variants 1, 3, 4, 5, 6, 7, and 9) and UBE2V1 variant 4)) were involved in RNA processing and splicing, and a significant number of the DEGs, including the UBE variants, were associated with increased maternal fasting plasma glucose. It is concluded that DEGs discriminating GDM from obese subjects were pinpointed. Our data indicate a biological link between genes involved in RNA processing and splicing, ubiquitination, and fasting plasma glucose in GDM taking into account obesity as the confounder.
Early life environments induce long-term changes in neurocognitive development and behaviour. In animal models, early environmental cues affect neuropsychological phenotypes via epigenetic processes ...but, as yet, there is little direct evidence for such mechanisms in humans.
We examined the relation between DNA methylation at birth and child neuropsychological outcomes in two culturally diverse populations using a genome-wide methylation analysis and validation by pyrosequencing.
Within the UK Southampton Women's Survey (SWS) we first identified 41 differentially methylated regions of interest (DMROI) at birth associated with child's full-scale IQ at age 4 years. Associations between HES1 DMROI methylation and later cognitive function were confirmed by pyrosequencing in 175 SWS children. Consistent with these findings, higher HES1 methylation was associated with higher executive memory function in a second independent group of 200 SWS 7-year-olds. Finally, we examined a pathway for this relationship within a Singaporean cohort (n = 108). Here, HES1 DMROI methylation predicted differences in early infant behaviour, known to be associated with academic success. In vitro, methylation of HES1 inhibited ETS transcription factor binding, suggesting a functional role of this site.
Thus, our findings suggest that perinatal epigenetic processes mark later neurocognitive function and behaviour, providing support for a role of epigenetic processes in mediating the long-term consequences of early life environment on cognitive development.
A highly conserved defence mechanism has evolved to protect cells from oxidative stress and xenobiotic exposure. A network of coupled xenobiotic metabolizing enzymatic reactions (XMEs) converts free ...oxidative radicals to less damaging metabolites, while efflux pumps remove toxins and XME derivatives from the cell. These mechanisms have been well studied in the contexts of hypoxia and Multidrug Resistance (MDR). Exposure of ruminants to fungal toxins leads to hepatotoxicosis and subsequent skin eczema (FE) depending upon toxic burden. Using toxin challenge in sheep we have investigated the potential for epigenetic regulation in cellular responses to xenobiotic exposure with a focus on the efflux protein ABCG2 which functions in Phase III of the defence mechanism. We show that ‘resistance’ to FE disease is positively associated with ABCG2 expression, and inversely correlated with DNA methylation state at CpG sites in the regulatory region of the ABCG2 gene. The analytical sensitivity provided by the Sequenom EpiTyper MS platform allows resolution of individual CpG sites varying significantly with disease progression, informing fine mapping of relevant transcription factor bindings which underpin this epigenetic response. Our findings indicate that epigenetic mechanisms are important to xenobiotic responses, suggest useful diagnostic markers and raise potential opportunities for disease remediation. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.
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►ABCG2 expression is positively associated with protection from xenobiotic exposure. ►ABCG2 is an epigenetic marker of xenobiotic exposure induced disease phenotype. ►Epigenomic variance in ABCG2 is at the level of single identified CpG sites. ►TF mapping suggests co-ordinated DNA and histone regulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Although IGF-I and insulin are important modulators of preimplantation embryonic physiology, the signalling pathways activated during development remain to be elucidated. As a model of ...preimplantation embryos, pluripotent mouse embryonic stem cells were used to investigate which receptor mediated actions of physiological concentrations of IGF-I and insulin on growth measured by protein synthesis. Exposure of mouse embryonic stem (ES) cells to 1.7 pM IGF-I or 1.7 nM insulin for 4 h caused approximately 25% increase in protein synthesis when compared with cells cultured in basal medium containing BSA. Dose-response studies showed 100-fold higher potency of IGF-I that pointed to the type 1 IGF receptor as the mediating receptor for both ligands. This was confirmed using an anti-type 1 IGF receptor-blocking antibody (αIR3). Both 1.7 pM IGF-I and 1.7 nM insulin increased phosphorylation of the type 1 IGF receptor and this increase was blocked by αIR3, but the insulin receptor was not phosphorylated. Finally, binding of either agonist led to downstream phosphorylation of ERK1/2 mitogen-activated protein kinase (MAPK) also via IGF-1R as this was blocked by αIR3. Together, these results suggest that IGF-I and insulin modulate ES cell physiology through binding to the type 1 IGF receptor and subsequent activation of MAPK pathway.
If the full potential of chromatin transfer (CT) technology is to be realized for both animal production and biomedical applications it is imperative that the efficiency of the reprogramming process ...be improved, and the potential for deleterious development be eliminated. Generation of the first cloned animals from adult somatic cells demonstrated that development is substantially an epigenetic process (Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH, 1997. Viable offspring derived from fetal and adult mammalian cells. Nature. 385(6619): 810–813.). In this study, we provide preliminary evidence that the epigenetic state of the donor cell, may be valuable in assessing potential cloning success. We have measured key indicators of cellular epigenetic state in both serially derived cell populations of the same genetic origin, but differing in epigenomic status, and in a distinct cohort of donor cell populations with diverse genetic origins and epigenomic status. Specifically, the relative abundance of particular histone modifications in donor populations prior to manipulation has been correlated with the measurable variance in reprogramming efficiencies observed following CT, as defined by the number of resulting live births and healthy progeny, and the concomitant incidence of deleterious growth measures (notably the appearance of large offspring syndrome (LOS)). Thus, we suggest that the likely outcome and relative success of cloning may be predictable based on the expression of discriminating histone marks present in the donor cell population before CT. This approach may provide the basis of a prognostic signature for the future evaluation and risk assessment of putative donor cells prior to CT, and thus increase future cloning success and alleviate the incidence of abnormal development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Babies born at lower gestational ages or smaller birthweights have a greater risk of poorer health in later life. Both the causes of these sub-optimal birth outcomes and the mechanism by which the ...effects are transmitted over decades are the subject of extensive study. We investigated whether a transcriptomic signature of either birthweight or gestational age could be detected in umbilical cord RNA.
The gene expression patterns of 32 umbilical cords from Singaporean babies of Chinese ethnicity across a range of birthweights (1698-4151 g) and gestational ages (35-41 weeks) were determined. We confirmed the differential expression pattern by gestational age for 12 genes in a series of 127 umbilical cords of Chinese, Malay and Indian ethnicity.
We found that the transcriptome is substantially influenced by gestational age; but less so by birthweight. We show that some of the expression changes dependent on gestational age are enriched in signal transduction pathways, such as Hedgehog and in genes with roles in cytokine signalling and angiogenesis. We show that some of the gene expression changes we report are reflected in the epigenome.
We studied the umbilical cord which is peripheral to disease susceptible tissues. The results suggest that soma-wide transcriptome changes, preserved at the epigenetic level, may be a mechanism whereby birth outcomes are linked to the risk of adult metabolic and arthritic disease and suggest that greater attention be given to the association between premature birth and later disease risk.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a ...vertebrate homolog of Zfh‐1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c‐Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c‐Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c‐Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c‐Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the α4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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