Kylie Beale

Investigation of Structure-Activity Relationships of Oxyntomodulin (Oxm) Using Oxm Analogs

Oxyntomodulin (Oxm) is an intestinal peptide that inhibits food intake and body weight in rodents and humans. These studies used peptide analogs to study aspects of structure and function of Oxm, and the sensitivity of parts of the Oxm sequence to degradation. Analogs of Oxm were synthesized and studied using receptor binding and degradation studies in vitro. Their effects on food intake and conditioned taste avoidance were measured in vivo in rodents. Oxm breakdown by the enzyme dipeptidyl peptidase IV (DPPIV) was demonstrated in vitro and in vivo. In vitro degradation was reduced and in vivo bioactivity increased by inhibitors of DPPIV. Modifications to the N terminus of Oxm modulated binding to the glucagon-like peptide (GLP)-1 receptor and degradation by DPPIV. Modifications to the midsection of Oxm modulated binding to the GLP-1 receptor and degradation by neutral endopeptidase. These modifications also altered bioactivity in vivo. The C-terminal octapeptide of Oxm was shown to contribute to the properties of Oxm in vitro and in vivo but was not alone sufficient for the effects of the peptide. Elongation and acylation of the C terminus of Oxm altered GLP-1 receptor binding and duration of action in vivo, which may be due to changes in peptide clearance. An Oxm analog was developed with enhanced pharmaceutical characteristics, with greater potency and longevity with respect to effects on food intake. These studies suggest that Oxm is a potential target for antiobesity drug design.

Alarin stimulates food intake and gonadotrophin release in male rats.

BACKGROUND AND PURPOSE Alarin is a recently discovered member of the galanin peptide family encoded by a splice variant of galanin‐like peptide (GALP) mRNA. Galanin and GALP regulate energy homeostasis and reproduction. We therefore investigated the effects of alarin on food intake and gonadotrophin release.

Peripheral administration of prokineticin 2 potently reduces food intake and body weight in mice via the brainstem

Prokineticin 2 (PK2) has recently been shown to acutely reduce food intake in rodents. We aimed to determine the CNS sites and receptors that mediate the anorectic effects of peripherally administered PK2 and its chronic effects on glucose and energy homeostasis.

Augurin stimulates the hypothalamo-pituitary-adrenal axis via the release of corticotrophin-releasing factor in rats

Background and purpose:  The functional characterization of secreted peptides can provide the basis for the development of novel therapeutic agents. Augurin is a recently identified secreted peptide of unknown function expressed in multiple endocrine tissues, and in regions of the brain including the hypothalamus. We therefore investigated the effect of hypothalamic injection of augurin on the hypothalamo‐pituitary‐adrenal (HPA) axis in male Wistar rats.

Intracerebroventricular administration of vasoactive intestinal peptide inhibits food intake.

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide expressed throughout the peripheral and central nervous systems. VIP and the VIP receptor VPAC(2)R are expressed in hypothalamic nuclei involved in the regulation of energy homeostasis. VIP has been shown to be involved in the regulation of energy balance in a number of non-mammalian vertebrates. We therefore examined the effects of intracerebroventricular (ICV) administration of VIP on food intake, energy expenditure and activity in adult male Wistar rats. VIP administration caused a potent short lived decrease in food intake and an increase in activity and energy expenditure. The pathways potentially involved in the anorexigenic effects of VIP were investigated by measuring the release of neuropeptides involved in the regulation of food intake from hypothalamic explants treated with VIP. VIP significantly stimulated the release of the anorexigenic peptide alpha-melanocyte stimulating hormone (αMSH). These studies suggest that VIP may have an endogenous role in the hypothalamic control of energy homeostasis.

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

Objective In order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection. Design Monocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy. Results MOB, production of superoxide and bacterial killing in response to Escherichia coli were markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy. Conclusions Monocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.

Accurate Measurement of Body Weight and Food Intake in Environmentally Enriched Male Wistar Rats

Laboratory animals are crucial in the study of energy homeostasis. In particular, rats are used to study alterations in food intake and body weight. To accurately record food intake or energy expenditure it is necessary to house rats individually, which can be stressful for social animals. Environmental enrichment may reduce stress and improve welfare in laboratory rodents. However, the effect of environmental enrichment on food intake and thus experimental outcome is unknown. We aimed to determine the effect of environmental enrichment on food intake, body weight, behavior and fecal and plasma stress hormones in male Wistar rats. Singly housed 5–7‐week‐old male rats were given either no environmental enrichment, chew sticks, a plastic tube of 67 mm internal diameter, or both chew sticks and a tube. No differences in body weight or food intake were seen over a 7‐day period. Importantly, the refeeding response following a 24‐h fast was unaffected by environmental enrichment. Rearing, a behavior often associated with stress, was significantly reduced in all enriched groups compared to controls. There was a significant increase in fecal immunoglobulin A (IgA) in animals housed with both forms of enrichment compared to controls at the termination of the study, suggesting enrichment reduces hypothalamo‐pituitary‐adrenal (HPA) axis activity in singly housed rats. In summary, environmental enrichment does not influence body weight and food intake in singly housed male Wistar rats and may therefore be used to refine the living conditions of animals used in the study of energy homeostasis without compromising experimental outcome.

Quantification of rat kisspeptin using a novel radioimmunoassay.

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.

Cerebellin1 is a novel orexigenic peptide

Aim: Cerebellin1 (Cbln1) is highly expressed in the hypothalamus, a region of the brain involved in appetite regulation. However, the effects of Cbn1 on food intake are not known. The present study aimed to investigate the effect of Cbln1 on appetite regulation in rats.

OC-021 Monocyte oxidative burst defect is associated with susceptibility to infection in severe alcoholic hepatitis

Introduction Infection is a common cause of mortality in severe alcoholic hepatitis (SAH). Monocytes are innate immune cells that play a key role in fighting infection. We sought to characterise monocyte phenotype and function in SAH and relate defects to the development of infection. Method We analysed pre-treatment blood samples from 73 patients admitted to hospital suffering from SAH (DF >32), 34 healthy controls (HC) and 17 abstinent compensated cirrhotic patients (CLD). Flow cytometry was used to measure monocyte phenotype, cytokine production, phagocytosis and oxidative burst ex vivo . Production of superoxide (O2 -) was measured by luminometry and bacterial killing was assessed by counting viable E. coli colonies growing on agar plates after incubation with monocytes. Additionally, we evaluated the impact of in vivosteroid therapy on monocyte function via the Steroids or Pentoxyfilline for Alcoholic Hepatitis (STOPAH) clinical trial. Results The proportion of CD14+ +CD16+ + monocytes was increased in SAH and CLD vs HC (P < 0.003). These cells expressed elevated levels of activation marker HLA-DR (P < 0.01) and chemokine receptor CCR-5 (.01). In addition, they produced greater TNF-α in response to lipopolysaccharide vs CD14+ +CD16- cells (.03). Conversely, patrolling CD14+CD16+ + monocytes were reduced in SAH vs CLD and HC (.01). Phagocytosis was preserved in SAH for all subsets but monocyte oxidative burst (mOB) (P < 0.001), O2 -production (.02) and bacterial killing (.01) were markedly impaired vs CLD and HC. Importantly, pre-treatment mOB defect predicted the development of infection within the subsequent two weeks of sampling with area under receiver-operator curve of 0.86 (P < 0.02) (Figure 1). Accordingly, defective mOB was associated with death at 28 and 90 days (one-tailed .04 for both). Ex vivomOB was unchanged in patients who had received 7 days steroid therapy.Abstract OC-021 Figure 1 Conclusion Defective mOB is prevalent in patients with SAH and is strongly associated with the development of infection within the subsequent two weeks. In addition, CD14+ +CD16+ +monocytes are expanded in SAH and bear features suggestive of an inflammatory role in disease pathogenesis. Further work should seek the molecular mechanisms of these defects and components that might be amenable to therapeutic intervention. Disclosure of interest None Declared.