A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

Exercise alone is not enough : weight loss also needs a healthy (Mediterranean) diet?

Objective: In the majority of exercise intervention studies, the aggregate reported weight loss is often small. The efficacy of exercise as a weight loss tool remains in question. The aim of the present study was to investigate the variability in appetite and body weight when participants engaged in a supervised and monitored exercise programme. ---------- Design: Fifty-eight obese men and women (BMI = 31·8 ± 4·5 kg/m2) were prescribed exercise to expend approximately 2092 kJ (500 kcal) per session, five times a week at an intensity of 70 % maximum heart rate for 12 weeks under supervised conditions in the research unit. Body weight and composition, total daily energy intake and various health markers were measured at weeks 0, 4, 8 and 12. ---------- Results: Mean reduction in body weight (3·2 ± 1·98 kg) was significant (P < 0·001); however, there was large individual variability (−14·7 to +2·7 kg). This large variability could be largely attributed to the differences in energy intake over the 12-week intervention. Those participants who failed to lose meaningful weight increased their food intake and reduced intake of fruits and vegetables. ---------- Conclusion: These data have demonstrated that even when exercise energy expenditure is high, a healthy diet is still required for weight loss to occur in many people.

Acute compensatory eating following exercise is associated with implicit hedonic wanting for food

The efficacy of exercise to promote weight loss could potentially be undermined by its influence on explicit or implicit processes of liking and wanting for food which in turn alter food preference. The present study was designed to examine hedonic and homeostatic mechanisms involved in the acute effects of exercise on food intake. 24 healthy female subjects were recruited to take part in two counterbalanced activity sessions; 50 min of high intensity (70% max heart rate) exercise (Ex) or no exercise (NEx). Subjective appetite sensations, explicit and implicit hedonic processes, food preference and energy intake (EI) were measured immediately before and after each activity session and an ad libitum test meal. Two groups of subjects were identified in which exercise exerted different effects on compensatory EI and food preference. After exercise, compensators (C) increased their EI, rated the food to be more palatable, and demonstrated increased implicit wanting. Compensators also showed a preference for high-fat sweet food compared with non-compensators (NC), independent of the exercise intervention. Exercise-induced changes in the hedonic response to food could be an important consideration in the efficacy of using exercise as a means to lose weight. An enhanced implicit wanting for food after exercise may help to explain why some people overcompensate during acute eating episodes. Some individuals could be resistant to the beneficial effects of exercise due to a predisposition to compensate for exercise-induced energy expenditure as a result of implicit changes in food preferences.

The malnutrition screening tool is a useful tool for identifying malnutrition risk in residential aged care

Background: The Malnutrition Screening Tool (MST) is a valid nutrition screening tool in the acute hospital setting but has not been assessed in residential aged care facilities. The aim of this secondary analysis was to determine whether the MST could be a useful nutrition screening tool when compared with a full nutrition assessment by Subjective Global Assessment (SGA) in the residential aged care setting. ----- Methods: Two hundred and eighty-five residents (29% male; mean age: 84 ± 9 years) from eight residential aged care facilities in Australia participated. A secondary analysis of data collected during a nutrition intervention study was conducted. The MST consists of two questions related to recent weight loss and appetite. While the MST was not specifically applied, weight loss and appetite information was available and an estimated MST score (0-5) calculated. Nutritional status was assessed by a research assistant trained in using SGA. ----- Results: Malnutrition prevalence was 42.8% (122 malnourished out of 285 residents). Compared to the SGA, the MST was an effective predictor of nutritional risk (sensitivity = 83.6%, specificity = 65.6%, positive predictive value = 0.65, negative predictive value =0.84). ----- Conclusions: The components of the MST have acceptable sensitivity and specificity suggesting it can play a valuable role in quickly identifying malnutrition risk in the residential aged care setting. Further prospective research using the MST tool against a broader array of objective and subjective nutritional parameters is required to confirm its validity as a screening tool in aged care settings.

Housing supply in Australia : the impact of the availability of development finance

Before the Global Financial Crisis many providers of finance had growth mandates and actively pursued development finance deals as a way of gaining higher returns on funds with regular capital turnover and re-investment possible. This was able to be achieved through high gearing and low presales in a strong market. As asset prices fell, loan covenants breached and memories of the 1990’s returned, banks rapidly adjusted their risk appetite via retraction of gearing and expansion of presale requirements. Early signs of loosening in bank credit policy are emerging, however parties seeking development finance are faced with a severely reduced number of institutions from which to source funding. The few institutions that are lending are filtering out only the best credit risks by way of constrictive credit conditions including: low loan to value ratios, the corresponding requirement to contribute high levels of equity, lack of support in non-prime locations and the requirement for only borrowers with well established track records. In this risk averse and capital constrained environment, the ability of developers to proceed with real estate developments is still being constrained by their inability to obtain project finance. This paper will examine the pre and post GFC development finance environment. It will identify the key lending criteria relevant to real estate development finance and will detail the related changes to credit policies over this period. The associated impact to real estate development projects will be presented, highlighting the significant constraint to supply that the inability to obtain finance poses.

Is it possible to dissociate ‘liking’ and ‘wanting’ for foods in humans? A novel experimental procedure

Berridge's model (e.g. [Berridge KC. Food reward: Brain substrates of wanting and liking. Neurosci Biobehav Rev 1996;20:1–25.; Berridge KC, Robinson T E. Parsing reward. Trends Neurosci 2003;26:507–513.; Berridge KC. Motivation concepts in behavioral neuroscience. Physiol Behav 2004;81:179–209]) outlines the brain substrates thought to mediate food reward with distinct ‘liking’ (hedonic/affective) and ‘wanting’ (incentive salience/motivation) components. Understanding the dual aspects of food reward could throw light on food choice, appetite control and overconsumption. The present study reports the development of a procedure to measure these processes in humans. A computer-based paradigm was used to assess ‘liking’ (through pleasantness ratings) and ‘wanting’ (through forced-choice photographic procedure) for foods that varied in fat (high or low) and taste (savoury or sweet). 60 participants completed the program when hungry and after an ad libitum meal. Findings indicate a state (hungry–satiated)-dependent, partial dissociation between ‘liking’ and ‘wanting’ for generic food categories. In the hungry state, participants ‘wanted’ high-fat savoury > low-fat savoury with no corresponding difference in ‘liking’, and ‘liked’ high-fat sweet > low-fat sweet but did not differ in ‘wanting’ for these foods. In the satiated state, participants ‘liked’, but did not ‘want’, high-fat savoury > low-fat savoury, and ‘wanted’ but did not ‘like’ low-fat sweet > high-fat sweet. More differences in ‘liking’ and ‘wanting’ were observed when hungry than when satiated. This procedure provides the first step in proof of concept that ‘liking’ and ‘wanting’ can be dissociated in humans and can be further developed for foods varying along different dimensions. Other experimental procedures may also be devised to separate ‘liking’ and ‘wanting’.

Cholecystokinin and leptin: their influence upon the eating behaviour and nutrient intake of dialysis patients

Background We have used serial visual analogue scores to demonstrate disturbances of the appetite profile in dialysis patients. This is potentially important as dialysis patients are prone to malnutrition yet have a lower nutrient intake than controls. Appetite disturbance may be influenced by accumulation of appetite inhibitors such as leptin and cholecystokinin (CCK) in dialysis patients. Methods Fasting blood samples were drawn from 43 controls, 50 haemodialysis (HD) and 39 peritoneal dialysis (PD) patients to measure leptin and CCK. Hunger and fullness scores were derived from profiles compiled using hourly visual analogue scores. Nutrient intake was derived from 3 day dietary records. Results Fasting CCK was elevated for PD (6.73 ± 4.42 ng/l vs control 4.99 ± 2.23 ng/l, P < 0.05; vs HD 4.43 ± 2.15 ng/l, P < 0.01). Fasting CCK correlated with the variability of the hunger (r = 0.426, P = 0.01) and fullness (r = 0.52, P = 0.002) scores for PD. There was a notable relationship with the increase in fullness after lunch for PD (r = 0.455, P = 0.006). When well nourished PD patients were compared with their malnourished counterparts, CCK was higher in the malnourished group (P = 0.004). Leptin levels were higher for the dialysis patients than controls (HD and PD, P < 0.001) with pronounced hyperleptinaemia evident in some PD patients. Control leptin levels demonstrated correlation with fullness scores (e.g. peak fullness, r = 0.45, P = 0.007) but the dialysis patients did not. PD nutrient intake (energy and protein intake, r = -0.56, P < 0.0001) demonstrated significant negative correlation with leptin. Conclusion Increased CCK levels appear to influence fullness and hunger perception in PD patients and thus may contribute to malnutrition. Leptin does not appear to affect perceived appetite in dialysis patients but it may influence nutrient intake in PD patients via central feeding centres.

The effect of graded levels of exercise on energy intake and balance in free-living men, consuming their normal diet

Objective: To assess the effect of graded increases in exercised-induced energy expenditure (EE) on appetite, energy intake (EI), total daily EE and body weight in men living in their normal environment and consuming their usual diets. Design: Within-subject, repeated measures design. Six men (mean (s.d.) age 31.0 (5.0) y; weight 75.1 (15.96) kg; height 1.79 (0.10) m; body mass index (BMI) 23.3(2.4) kg/m2), were each studied three times during a 9 day protocol, corresponding to prescriptions of no exercise, (control) (Nex; 0 MJ/day), medium exercise level (Mex; ~1.6 MJ/day) and high exercise level (Hex; ~3.2 MJ/day). On days 1-2 subjects were given a medium fat (MF) maintenance diet (1.6 ´ resting metabolic rate (RMR)). Measurements: On days 3-9 subjects self-recorded dietary intake using a food diary and self-weighed intake. EE was assessed by continual heart rate monitoring, using the modified FLEX method. Subjects' HR (heart rate) was individually calibrated against submaximal VO2 during incremental exercise tests at the beginning and end of each 9 day study period. Respiratory exchange was measured by indirect calorimetry. Subjects completed hourly hunger ratings during waking hours to record subjective sensations of hunger and appetite. Body weight was measured daily. Results: EE amounted to 11.7, 12.9 and 16.8 MJ/day (F(2,10)=48.26; P<0.001 (s.e.d=0.55)) on the Nex, Mex and Hex treatments, respectively. The corresponding values for EI were 11.6, 11.8 and 11.8 MJ/day (F(2,10)=0.10; P=0.910 (s.e.d.=0.10)), respectively. There were no treatment effects on hunger, appetite or body weight, but there was evidence of weight loss on the Hex treatment. Conclusion: Increasing EE did not lead to compensation of EI over 7 days. However, total daily EE tended to decrease over time on the two exercise treatments. Lean men appear able to tolerate a considerable negative energy balance, induced by exercise, over 7 days without invoking compensatory increases in EI.

No energy compensation at the meal following exercise indietary restrained and unrestrained women

The objective of this investigation was to compare the acute effects of exercise and diet manipulations on energy intake, between dietary restrained and unrestrained females. Comparisons of two studies using an identical 2 x 2 repeated-measures design (level of activity (rest or exercise) and lunch type (high-fat or low-fat)) including thirteen dietary unrestrained and twelve restrained females were performed. Energy expenditure during the rest session was estimated and the energy cost of exercise was measured by indirect calorimetry. Relative energy intake was calculated by subtracting the energy expenditure of the exercise session from the energy intake of the test meal. Post-meal hedonic ratings were completed after lunch. Energy intake and relative energy intake increased during high-fat conditions compared with the low-fat, independently of exercise (P < 0.001). There was a positive relationship between dietary restraint scores and energy intake or relative energy intake in the rest conditions only (r 0.54, P < 0.01). The decrease of relative energy intake between the rest and exercise conditions was higher in restrained than in unrestrained eaters (P < 0.01). These results confirm that a high-fat diet reversed the energy deficit due to exercise. There was no energy compensation in response to an acute bout of exercise during the following meal. In restrained eaters, exercise was more effective in creating an energy deficit than in unrestrained eaters. Exercise may help restrained eaters to maintain control over appetite.

Selective effects of acute exercise and breakfast interventions on mood and motivation to eat

The effects of exercise and breakfast manipulations on mood and motivation to eat were assessed in 11 healthy females who were regular exercisers and habitual breakfast eaters. The study involved a two by two repeated-measures design, with exercise (or no exercise) and a high-energy breakfast (or low-energy breakfast) as the repeated measures. The exercise or no-exercise session (0800 h) was followed by consumption of the low- or high-energy breakfast (0900 h). An ad libitum lunch test meal was provided 4 hours after the beginning of the exercise session (1200 h). Mood and motivation to eat were continuously tracked from 0800 until 1700 h by an electronic appetite ratings system (EARS). In general, morning subjective mood states (e.g., contentment) were significantly lower in the low-energy breakfast condition, but exercise reversed this effect. Exercise also significantly decreased feelings of lethargy, independent of the breakfast condition. Desire-to-eat and fullness ratings were significantly increased in the low-energy breakfast and high-energy breakfast conditions, respectively. Impairments of mood disappeared in the afternoon after consumption of an ad libitum lunch. In these healthy young adults, the condition inducing the largest energy deficit (exercise and low-energy breakfast) was not associated with the lowest mental states.

Ghrelin and obestatin concentrations during puberty: Relationships with adiposity, nutrition and physical activity

Ghrelin and obestatin are two peptides associated with appetite control and the regulation of energy balance in adults. It is intuitive that they have an important role in growth and development during puberty. Therefore, it is acknowledged that these peptides, in addition to others, form part of the substrate underlying energy homeostasis which in turn will contribute to body weight regulation and could explain changes in energy balance during puberty. Both peptides originate from the stomach; hence, it is intuitive that they are involved in generating signals from tissue stores which influence food intake. This could be manifested via alterations in the drive to eat (i.e. hunger), eating behaviors and appetite regulation. Furthermore, there is some evidence that these peptides might also be associated with physical activity behaviors and metabolism. Anecdotally, children and adolescents experience behavioral and metabolic changes during growth and development which will be associated with physiological changes.

Characterising the homeostatic and hedonic markers of the susceptible phenotype

Over the years, approaches to obesity prevention and treatment have gone from focusing on genetic and other biological factors to exploring a diversity of diets and individual behavior modification interventions anchored primarily in the power of the mind, to the recent shift focusing on societal interventions to design ";temptation-proof"; physical, social, and economic environments. In spite of repeated calls to action, including those of the World Health Organization (WHO), the pandemic continues to progress. WHO recently projected that if the current lifestyle trend in young and adult populations around the world persist, by 2012 in countries like the USA, health care costs may amount to as much as 17.7% of the GDP. Most importantly, in large part due to the problems of obesity, those children may be the first generation ever to have a shorter life expectancy than that of their parents. This work presents the most current research and proposals for addressing the pandemic. Past studies have focused primarly on either genetic or behavioral causes for obesity, however today's research indicates that a strongly integrated program is the best prospect for success in overcoming obesity. Furthermore, focus on the role of society in establishing an affordable, accessible and sustainable program for implementing these lifestyle changes is vital, particularly for those in economically challenged situations, who are ultimately at the highest risk for obesity. Using studies from both neuroscience and behavioral science to present a comprehensive overview of the challenges and possible solutions, The brain-to-society approach to obesity prevention focuses on what is needed in order to sustain a healthy, pleasurable and affordable lifestyle.

Physical activity and regulation of food intake: current evidence

Objective: The evidence was reviewed on how physical activity could influence the regulation of food intake by either adjusting the sensitivity of appetite control mechanisms or by generating an energy deficit that could adjust the drive to eat. Design: Interventionist and correlational studies that had a significant influence on the relationship between physical activity and food intake were reviewed. Interventionist studies involve a deliberate imposition of physical activity with subsequent monitoring of the eating response. Correlational studies make use of naturally occurring differences in the levels of physical activity (between and within subjects) with simultaneous assessment of energy expenditure and intake. Subjects: Studies using lean, overweight, and obese men and women were included. Results: Only 19% of interventionist studies report an increase in energy intake after exercise; 65% show no change and 16% show a decrease in appetite. Of the correlational studies, approximately half show no relationship between energy expenditure and intake. These data indicate a rather loose coupling between energy expenditure and intake. A common sense view is that exercise is futile as a form of weight control because the energy deficit drives a compensatory increase in food intake. However, evidence shows that this is not generally true. One positive aspect of this is that raising energy expenditure through physical activity (or maintaining an active life style) can cause weight loss or prevent weight gain. A negative feature is that when people become sedentary after a period of high activity, food intake is not “down-regulated” to balance a reduced energy expenditure. Conclusion: Evidence suggests that a high level of physical activity can aid weight control either by improving the matching of food intake to energy expenditure (regulation) or by raising expenditure so that it is difficult for people to eat themselves into a positive energy balance.

Effects of Sweetness and Energy in Drinks on Food Intake Following Exercise

Exercise is known to cause physiological changes that could affect the impact of nutrients on appetite control. This study was designed to assess the effect of drinks containing either sucrose or high-intensity sweeteners on food intake following exercise. Using a repeated-measures design, three drink conditions were employed: plain water (W), a low-energy drink sweetened with artificial sweeteners aspartame and acesulfame-K (L), and a high-energy, sucrose-sweetened drink (H). Following a period of challenging exercise (70% VO2 max for 50 min), subjects consumed freely from a particular drink before being offered a test meal at which energy and nutrient intakes were measured. The degree of pleasantness (palatability) of the drinks was also measured before and after exercise. At the test meal, energy intake following the artificially sweetened (L) drink was significantly greater than after water and the sucrose (H) drinks (p < 0.05). Compared with the artificially sweetened (L) drink, the high-energy (H) drink suppressed intake by approximately the energy contained in the drink itself. However, there was no difference between the water (W) and the sucrose (H) drink on test meal energy intake. When the net effects were compared (i.e., drink + test meal energy intake), total energy intake was significantly lower after the water (W) drink compared with the two sweet (L and H) drinks. The exercise period brought about changes in the perceived pleasantness of the water, but had no effect on either of the sweet drinks. The remarkably precise energy compensation demonstrated after the higher energy sucrose drink suggests that exercise may prime the system to respond sensitively to nutritional manipulations. The results may also have implications for the effect on short-term appetite control of different types of drinks used to quench thirst during and after exercise.