Timothy Wells

Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention

Circulating ghrelin elevates abdominal adiposity by a mechanism independent of its central orexigenic activity. In this study we tested the hypothesis that peripheral ghrelin induces a depot-specific increase in white adipose tissue (WAT) mass in vivo by GH secretagogue receptor (GHS-R(1a))-mediated lipolysis. Chronic iv infusion of acylated ghrelin increased retroperitoneal and inguinal WAT volume in rats without elevating superficial sc fat, food intake, or circulating lipids and glucose. Increased retroperitoneal WAT mass resulted from adipocyte enlargement probably due to reduced lipid export (ATP-binding cassette transporter G1 mRNA expression and circulating free fatty acids were halved by ghrelin infusion). In contrast, ghrelin treatment did not up-regulate biomarkers of adipogenesis (peroxisome proliferator-activated receptor-gamma2 or CCAAT/enhancer binding protein-alpha) or substrate uptake (glucose transporter 4, lipoprotein lipase, or CD36) and although ghrelin elevated sterol-regulatory element-binding protein 1c expression, WAT-specific mediators of lipogenesis (liver X receptor-alpha and fatty acid synthase) were unchanged. Adiposity was unaffected by infusion of unacylated ghrelin, and the effects of acylated ghrelin were abolished by transcriptional blockade of GHS-R(1a), but GHS-R(1a) mRNA expression was similar in responsive and unresponsive WAT. Microarray analysis suggested that depot-specific sensitivity to ghrelin may arise from differential fine tuning of signal transduction and/or lipid-handling mechanisms. Acylated ghrelin also induced hepatic steatosis, increasing lipid droplet number and triacylglycerol content by a GHS-R(1a)-dependent mechanism. Our data imply that, during periods of energy insufficiency, exposure to acylated ghrelin may limit energy utilization in specific WAT depots by GHS-R(1a)-dependent lipid retention.

Tissue-Specific Transgenic Knockdown of Fos-Related Antigen 2 (Fra-2) Expression Mediated by Dominant Negative Fra-2

ABSTRACT Fos-related antigen 2 (Fra-2) is a member of the Fos family of immediate-early genes, most of which are rapidly induced by second messengers. All members of this family act by binding to AP-1 sites as heterodimeric complexes with other proteins. However, each appears to have a distinct role. The role and biology of Fra-2 are less well understood than those of its relatives c-Fos, Fra-1, and FosB; moreover, Fra-2 target genes remain largely unknown, as does the basis of its selective effects on transcriptional activity. To pursue these issues, we created a transgenic rat line (NATDNF2) in which a dominant negative fra-2 (DNF2) gene is strongly expressed in the pineal gland; tissue selectivity was achieved by putting the DNF2 gene under the control of the rat arylalkylamineN-acetyltransferase (AANAT) regulatory region, which targets gene expression to a very restricted set of tissues (pineal gland ≫ retina). Expression of AANAT is normally turned on after the onset of darkness in the rat; as a result, pineal DNF2 expression occurs only at night. This was associated with marked suppression of the nocturnal increase in fra-2 mRNA and protein levels, indicating that DNF2 expression inhibits downstream effects of Fra-2, including the maintenance of high levels offra-2 gene expression. Analysis of 1,190 genes in the NATDNF2 pineal gland, including the AANAT gene, identified two whose expression is strongly linked to fra-2 expression: the genes encoding type II iodothyronine deiodinase and nectadrin (CD24).

Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy?

As mammalian genome projects move towards completion, the attention of molecular neuroscientists is currently moving away from gene identification towards both cell-specific gene expression patterns (neuronal transcriptions) and protein expression/interactions (neuronal proteomics). In the long term, attention will increasingly be directed towards experimental interventions which are able to question neuronal function in a sophisticated manner that is cognisant of both transcriptomic and proteomic organization. Central to this effort will be the application of a new generation of transgenic approaches which are now evolving towards an appropriate level of molecular, temporal and spatial resolution. In this review, we summarize recent developments in transgenesis, and show how they have been applied in the principal model species for neuroscience, namely rats and mice. Current concepts of transgene design are also considered together with an overview of new genetically-encoded tools including both cellular indicators such as fluorescent activity reporters, and cellular regulators such as dominant negative signalling factors. Application of these tools in a whole animal context can be used to question both basic concepts of brain function, and also current concepts of underlying dysfuction in neurological diseases.

Genetic targeting: the serotonin N-acetyltransferase promoter imparts circadian expression selectively in the pineal gland and retina of transgenic rats.

Abstract : The arylalkylamine N‐acetyltransferase (AA‐NAT) gene is strongly expressed in the rat primarily in the pineal gland ; low levels of expression are also found in the retina. AA‐NAT catalyzes the key regulatory step controlling rhythmic melatonin output : the acetylation of serotonin. In the rat, the AA‐NAT gene is expressed at night. This is controlled partly by cyclic AMP (cAMP) acting through a composite cAMP‐responsive element‐CCAAT site located upstream of the transcription start point. In the present study, we have extended our previous in vitro findings and found that additional elements in the 5′ flanking region and first intron play an important role in the regulation of the AA‐NAT gene. This led us to test the influence of an AA‐NAT 5′ flanking segment on the expression of the bacterial chloramphenicol acetyltransferase gene in a rat transgenic model. The results of this study clearly demonstrate that the segment of the AA‐NAT gene that encompasses the minimal promoter and the first intron is able to confer the highly specific pineal/retinal and time‐of‐day patterns of AA‐NAT gene expression. This advance also provides a tool that selectively targets genetic expression to pinealocytes and retinal photoreceptors, providing new experimental opportunities to probe gene expression in these tissues.

Vesicular osmometers, vasopression secretion and aquaporin-4: a new mechanism for osmoreception?

This review cites new evidence suggesting a link between the recently discovered membrane bound water-selective channel, aquaporin-4 (AQP4), and the mechanism of central osmoreception. AQP4 is found in a number of brain regions associated with the osmoregulation of vasopressin secretion and thirst, including the supraoptic nucleus (SON) and subfornical organ (SFO). AQP4 expression is restricted to ependymal cell membranes in the SFO and astrocyte membranes in the SON, especially perivascular end foot processes, suggesting that glial cells may correspond to Verneys hypothalamic vesicular osmometers. Information on osmotic status may thus be conveyed to the neuronal elements of the osmoreceptor complex by a neurone-glial interaction.

Ghrelin - defender of fat.

With one quarter of the population of the Western world now considered obese, it is essential that we understand the factors giving rise to elevated fat deposition. This review summarizes the cellular and molecular mechanisms governing the volume of white adipose tissue (WAT), and outlines the physiological signals that regulate these processes. Particular attention is given to the role of the gastric hormone, ghrelin, describing its actions in general and presenting detailed evidence of its role in regulating adipocyte biology. Combining this evidence with an analysis of the factors governing ghrelin secretion, leads to the hypothesis that during periods of food deprivation ghrelin acts as an energy deficit signal, defending the fat stored in responsive WAT against the forces of utilization. This scenario has clear implications for programmes of sustainable weight loss.

Short-term calorie restriction enhances adult hippocampal neurogenesis and remote fear memory in a Ghsr-dependent manner

Graphical abstract

Skeletal growth acceleration with growth hormone secretagogues in transgenic growth retarded rats: pattern-dependent effects and mechanisms of desensitization.

The transgenic growth retarded (Tgr) rat is the first genetic model of growth hormone (GH) deficiency whose growth can be accelerated with exogenous GH secretagogues (GHSs). In this study, we have demonstrated that GHS‐receptor (GHS‐R) mRNA expression in the arcuate nucleus of Tgr rats was not significantly different to that in wild‐type littermates. We have confirmed that GHS‐induced elevation in body weight gain was accompanied by acceleration of skeletal growth, and that the effects of the GHS, GHRP‐6, were both dose‐ and pattern‐dependent. The growth response with continuous infusion of GHRP‐6 was transient, accompanied by suppression of GH and corticosterone responses to bolus injection of GHRP‐6. This desensitization occurred without downregulation of arcuate GHS‐R mRNA expression, but was accompanied by elevated periventricular somatostatin mRNA expression. In contrast, pulsatile (3‐hourly) infusion of GHRP‐6 produced sustained growth and GH responses, which were accompanied by suppression of corticosterone responses and elevated arcuate GH‐releasing factor (GRF) mRNA expression. Skeletal growth was further accelerated by coinfusion of GRF, but significant depletion of pituitary GH stores suggested that this growth rate may not be sustainable. These experiments confirm the importance of the Tgr rat for investigating the growth promoting potential of the GHSs in the context of GH‐deficient dwarfism, and suggest that elevated somatostatin expression may mediate the suppression of the GRF‐GH and hypothalamo‐pituitary‐adrenal axes following continuous GHRP‐6 treatment.

A novel long-range enhancer regulates postnatal expression of Zeb2: implications for Mowat–Wilson syndrome phenotypes

The zinc-finger, E-box-binding homeobox-2 (Zeb2) gene encodes a SMAD-interacting transcription factor that has diverse roles in development and disease. Mutations at the hZeb2 locus cause Mowat-Wilson syndrome (MWS), a genetic disorder that is associated with mental retardation and other, case- and sex-dependent clinical features. Recent studies have detailed microRNA-mediated control of Zeb2, but little is known about the genomic context of this gene or of enhancer sequences that may direct its diverse functions. Here, we describe a novel transgenic rodent model in which Zeb2 regulatory sequence has been disrupted, resulting in a postnatal developmental phenotype that is autosomal dominant. The phenotype exhibits a genotype-by-sex interaction and manifests primarily as an acute attenuation of postnatal kidney development in males. Other aspects of embryonic and neonatal development, including neuronal, are unaffected. The transgene insertion site is associated with a 12 kb deletion, 1.2 Mb upstream of Zeb2, within a 4.1 Mb gene desert. A conserved sequence, derived from the deleted region, enhanced Zeb2 promoter activity in transcription assays. Tissue and temporal restriction of this enhancer activity may involve postnatal changes in proteins that bind this sequence. A control human/mouse VISTA enhancer (62 kb upstream of Zeb2) also up-regulated the Zeb2 promoter, providing evidence of a string of conserved distal enhancers. The phenotype arising from deletion of one copy of the extreme long-range enhancer indicates a critical role for this enhancer at one developmental stage. Haploinsufficiency of Zeb2 in this developmental context reflects inheritance of MWS and may underlie some sex-dependent, non-neural characteristics of this human inherited disorder.

Morphological Determinants of Femoral Strength in Growth Hormone‐Deficient Transgenic Growth‐Retarded (Tgr) Rats

The extent to which childhood GHD affects adult fracture risk is unclear. We measured femoral strength in adult transgenic growth‐retarded rats as a model of GHD. Long‐term, moderate GHD was accompanied by endocrine and morphometric changes consistent with a significant reduction in femoral strength.