Yi-Chun Loraine Tung

The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate–Dependent Nucleic Acid Demethylase

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate–dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization.

mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development.

Hypothalamic-Specific Manipulation of Fto, the Ortholog of the Human Obesity Gene FTO, Affects Food Intake in Rats

Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Ftos actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.

Role for the obesity-related FTO gene in the cellular sensing of amino acids

SNPs in the first intron of FTO (fat mass and obesity associated) are strongly associated with human obesity. While it is not yet formally established that this effect is mediated through the actions of the FTO protein itself, loss of function mutations in FTO or its murine homologue Fto result in severe growth retardation, and mice globally overexpressing FTO are obese. The mechanisms through which FTO influences growth and body composition are unknown. We describe a role for FTO in the coupling of amino acid levels to mammalian target of rapamycin complex 1 signaling. These findings suggest that FTO may influence body composition through playing a role in cellular nutrient sensing.

Novel Leptin-Regulated Genes Revealed by Transcriptional Profiling of the Hypothalamic Paraventricular Nucleus

Leptin plays a major role in coordinating the integrated response of the CNS to changes in nutritional state. Neurons within the paraventricular nucleus (PVN) of the hypothalamus express leptin receptors and receive dense innervation from leptin receptor-expressing neurons in the arcuate nucleus. To obtain new insights into the effects of circulating leptin on PVN function, we compared global transcriptional profiles of laser-captured PVN from ad libitum fed mice versus 48 h fasted mice receiving either sham or leptin treatment intraperitoneally. Five hundred twenty-seven PVN-expressed genes were altered by fasting in a manner that was at least partially reversible by leptin. Consistent with previous reports, thyrotrophin releasing hormone mRNA levels were decreased by fasting but restored to fed levels with leptin treatment. mRNA levels of oxytocin, vasopressin, and somatostatin were also reduced by fasting and restored by leptin. Given the known effects of leptin on synaptic remodeling, it is notable that, among the top 15 genes that were positively regulated by leptin, five have been implicated in synaptic function and/or plasticity (basigin, apolipoprotein E, Gap43, GABAA receptor-associated protein, and synuclein-γ). Pathway analysis identified oxidative phosphorylation, in particular, genes encoding complex 1 proteins that play a role in ubiquinone biosynthesis, to be the predominant gene set that was significantly regulated in a leptin-dependent manner. Thus, in addition to its effects on the expression of a broad range of neuropeptides, leptin may also exert more general influences on synaptic function in, and the bioenergetic state of, the PVN.

From GWAS to biology: lessons from FTO

Genome‐wide association studies have been very powerful, uncovering potentially new biology that would not have been possible using a candidate gene approach. A prime example of this is the gene FTO (fat mass and obesity associated), which first came to light in 2007, when single nucleotide polymorphisms in its first intron were robustly associated with body mass index and obesity. Subsequently, as it became clear that this association with body weight, and increasingly food intake, was replicable across multiple populations and different age groups, attention was turned to studying the biology of FTO, about which absolutely nothing was known. This review focuses on the genetic and biochemical approaches as well as animal models that have been used by us and others since 2007 to try and uncover the complex biology of FTO.

A truncation mutation in TBC1D4 in a family with acanthosis nigricans and postprandial hyperinsulinemia

Tre-2, BUB2, CDC16, 1 domain family member 4 (TBC1D4) (AS160) is a Rab-GTPase activating protein implicated in insulin-stimulated glucose transporter 4 (GLUT4) translocation in adipocytes and myotubes. To determine whether loss-of-function mutations in TBC1D4 might impair GLUT4 translocation and cause insulin resistance in humans, we screened the coding regions of this gene in 156 severely insulin-resistant patients. A female presenting at age 11 years with acanthosis nigricans and extreme postprandial hyperinsulinemia was heterozygous for a premature stop mutation (R363X) in TBC1D4. After demonstrating reduced expression of wild-type TBC1D4 protein and expression of the truncated protein in lymphocytes from the proband, we further characterized the biological effects of the truncated protein in 3T3L1 adipocytes. Prematurely truncated TBC1D4 protein tended to increase basal cell membrane GLUT4 levels (P = 0.053) and significantly reduced insulin-stimulated GLUT4 cell membrane translocation (P < 0.05). When coexpressed with wild-type TBC1D4, the truncated protein dimerized with full-length TBC1D4, suggesting that the heterozygous truncated variant might interfere with its wild-type counterpart in a dominant negative fashion. Two overweight family members with the mutation also manifested normal fasting glucose and insulin levels but disproportionately elevated insulin levels following an oral glucose challenge. This family provides unique genetic evidence of TBC1D4 involvement in human insulin action.

Differential effects of α-, β- and γ2-melanocyte-stimulating hormones on hypothalamic neuronal activation and feeding in the fasted rat

Abstract Hypothalamic pro-opiomelanocortin neurones have an established role in the control of feeding. While pro-opiomelanocortin is the precursor for at least three melanocortin peptides, α-, β- and γ-melanocyte-stimulating hormone (MSH), it has been widely assumed that α-MSH is the predominant ligand involved. We compared the effects of centrally administered α-, β- and γ2-MSH on hypothalamic neuronal activation and on food intake in rats fasted for 48 h. Significant reductions in food intake were seen with α-MSH (first hour) and γ2-MSH (second hour) but not with β-MSH. The pattern of neuronal activation, assessed by the detection of early growth response factor-1 protein, showed considerable overlap; all three melanocortins activated cells in the arcuate, ventromedial, paraventricular, periventricular and supraoptic nuclei, as well as the preoptic area. α-MSH and β-MSH produced activation in the dorsomedial nuclei while γ2-MSH was only weakly active here. Retrograde labelling by systemic Fluorogold injection revealed that many cells activated by MSH compounds in the arcuate, paraventricular, periventricular and supraoptic nuclei (but not dorsomedial or ventromedial) project outside the blood–brain barrier and are therefore likely to include neuroendocrine cells. Desacetyl-α-MSH, which has previously been reported to lack effects on feeding, produced no discernible neuronal activation in the hypothalamus. Our finding that both the pattern of neuronal activation and the distribution of neuroendocrine cells activated in response to these closely related peptides show only partial overlap suggests that, in addition to common pathways, there may exist distinct hypothalamic circuits activated by different pro-opiomelanocortin products. The slower time course of γ2-MSH- versus α-MSH-induced suppression of feeding provides further support for the notion that the biological responses to individual melanocortin peptides may involve distinct neuronal mechanisms.

Diet-induced gene expression of isolated pancreatic islets from a polygenic mouse model of the metabolic syndrome

Aims/hypothesisNumerous new genes have recently been identified in genome-wide association studies for type 2 diabetes. Most are highly expressed in beta cells and presumably play important roles in their function. However, these genes account for only a small proportion of total risk and there are likely to be additional candidate genes not detected by current methodology. We therefore investigated islets from the polygenic New Zealand mouse (NZL) model of diet-induced beta cell dysfunction to identify novel genes and pathways that may play a role in the pathogenesis of diabetes.MethodsNZL mice were fed a diabetogenic high-fat diet (HF) or a diabetes-protective carbohydrate-free HF diet (CHF). Pancreatic islets were isolated by laser capture microdissection (LCM) and subjected to genome-wide transcriptome analyses.ResultsIn the prediabetic state, 2,109 islet transcripts were differentially regulated (>1.5-fold) between HF and CHF diets. Of the genes identified, 39 (e.g. Cacna1d, Chd2, Clip2, Igf2bp2, Dach1, Tspan8) correlated with data from the Diabetes Genetics Initiative and Wellcome Trust Case Control Consortium genome-wide scans for type 2 diabetes, thus validating our approach. HF diet induced early changes in gene expression associated with increased cell-cycle progression, proliferation and differentiation of islet cells, and oxidative stress (e.g. Cdkn1b, Tmem27, Pax6, Cat, Prdx4 and Txnip). In addition, pathway analysis identified oxidative phosphorylation as the predominant gene-set that was significantly upregulated in response to the diabetogenic HF diet.Conclusions/interpretationWe demonstrated that LCM of pancreatic islet cells in combination with transcriptional profiling can be successfully used to identify novel candidate genes for diabetes. Our data strongly implicate glucose-induced oxidative stress in disease progression.

Actions of leptin on growth hormone secretagogue-responsive neurones in the rat hypothalamic arcuate nucleus recorded in vitro.

In the arcuate nucleus, the growth hormone (GH) secretagogue (GHS)‐responsive cells include a subpopulation of the neuropeptide Y (NPY) neurones. It is not known whether these include the orexigenic NPY population that are inhibited by the satiety hormone, leptin. Thus we investigated whether (i) the arcuate nucleus cells electrically excited by GHS are inhibited by leptin and (ii) chronic central leptin infusion alters GHS‐induced Fos expression. Of 36 cells recorded from a trimmed hypothalamic slice containing arcuate nucleus, 13 cells were excited by the nonpeptide GHS, CP‐459,599. The predominant response of these cells to leptin was inhibitory: six inhibited, three excited and four unresponsive. Similar responses were observed in a population of arcuate cells recorded from a preparation in which synaptic transmission was blocked, suggesting that leptin acts directly on a subpopulation of GHS‐responsive neurones. Intracerebroventricular infusion of leptin for 1 week did not alter the number of cells expressing Fos following GHS administration. Thus, while leptin does not appear to influence the central actions of GHS to induce immediate early gene expression, it does act directly on a subpopulation of cells excited by GHS, eliciting mostly inhibitory but also some excitatory responses. It will be interesting to discover the consequences of leptins inhibitory effects on the hypothalamic circuits excited by GHS, particularly since leptin paradoxically has a stimulatory effect on GH secretion, presumed to reflect a suppression of central NPY pathways.