AP1 transcription factors are required to maintain 1 the peripheral taste system

The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance

Regulator of G Protein Signaling-21 (RGS21) in Peripheral Taste Physiology.

The gustatory system subjects ingested food to ‘quality control’ that prevents consumption of harmful compounds while also regulating nutrient intake. A better understanding of the physiological regulation of taste will enhance our ability to facilitate the appropriate consumption of nutrients and improve overall health. Bitter, sweet, and umami tastes are detected by a family of G protein-coupled receptors (GPCRs) that associate with heterotrimeric G proteins and initiate intracellular signaling cascades after activation by tastant binding. ‘Regulators of G protein Signaling’ (RGS proteins) act as Ga-directed GTPase-accelerating proteins (GAPs) and thereby accelerate inactivation of GPCR-mediated signaling. Rgs21 is selectively expressed in tastantresponsive tissue, suggesting it likely facilitates the inactivation of the taste transduction pathway. We have assessed taste responses in Rgs21 knockout mice: bitterant, sweetener, and umami responses (metabotropic, Type II cell responses) are blunted in the absence of RGS21, whereas aversion to sour (ionotropic, Type III cell response) is unchanged. Notably, appetitive responses to NaCl are blunted in Rgs21-deficient mice as well, suggesting transduction of NaCl taste involves a GPCR and/or G protein signaling in Type II taste receptor cells. We suspect that RGS21 loss leads to hyperactivity of GPCRs in taste receptor cells, eventually causing prolonged desensitization and/or downregulation. Further work is needed to test this hypothesis and thus elucidate the mechanism(s) by which RGS21 affects peripheral taste signaling, including appetitive salt taste (a taste modality traditionally considered the exclusive domain of ionotropic signal transduction)

A comparison of the palatability of racemic praziquantel and its two enantioseparated isomers in yellowtail kingfish Seriola lalandi (Valenciennes, 1833)

The bitterness of racemic praziquantel (Rac-PZQ) constrains its use as an in-feed treatment against monogenean flukes in finfish aquaculture. Evidence exists in mammals that the R-(-) enantiomer of PZQ is less bitter than the S-(+) enantiomer. If fish exhibit this same response, then the recently described techniques for the large-scale resolution of R-(-)-PZQ from Rac-PZQ could facilitate the wide-spread application of this effective anthelmintic compound via feed. The hypothesis that yellowtail kingfish Seriola lalandi would find R-(-)-PZQ more palatable than Rac-PZQ and S-(+)-PZQ was tested in four trials. During the first three trials, the palatability of diets top-coated with 10 g kg-1 of Rac-PZQ or its two enantioseparated isomers were compared in small (85-160 g) and large (1.2 kg) yellowtail kingfish. A fourth trial compared the palatability of R-(-)-PZQ and Rac-PZQ at dietary inclusion levels of 2.5, 5.0 and 10.0 g kg-1 in small yellowtail kingfish (170 g). Ingestion data showed that R-(-)-PZQ to be no more palatable than either Rac-PZQ or S-(+)-PZQ to yellowtail kingfish, regardless of size. Indeed, evidence suggested that the S-(+)-PZQ to be slightly more palatable than both R-(-)-PZQ and Rac-PZQ. From these data, we hypothesize that the strong smell of R-(-)-PZQ (which was not present in S-(+)-PZQ) is an equally important determinant to palatability as taste in yellowtail kingfish. Results demonstrate that dietary inclusion level is a more important determinant to palatability than PZQ chirality; however, administration of R-(-)-PZQ may still be advantageous if it is demonstrated to be the only enantiomer efficacious against monogeneans

Neural processing of basic tastes in healthy young and older adults - an fMRI study

AbstractAgeing affects taste perception as shown in psychophysical studies, however, underlying structural and functional mechanisms of these changes are still largely unknown. To investigate the neurobiology of age-related differences associated with processing of basic tastes, we measured brain activation (i.e. fMRI-BOLD activity) during tasting of four increasing concentrations of sweet, sour, salty, and bitter tastes in young (average 23years of age) and older (average 65years of age) adults. The current study highlighted age-related differences in taste perception at the different higher order brain areas of the taste pathway. We found that the taste information delivered to the brain in young and older adults was not different, as illustrated by the absence of age effects in NTS and VPM activity. Our results indicate that multisensory integration changes with age; older adults showed less brain activation to integrate both taste and somatosensory information. Furthermore, older adults directed less attention to the taste stimulus; therefore attention had to be reallocated by the older individuals in order to perceive the tastes. In addition, we considered that the observed age-related differences in brain activation between taste concentrations in the amygdala reflect its involvement in processing both concentration and pleasantness of taste. Finally, we state the importance of homeostatic mechanisms in understanding the taste quality specificity in age related differences in taste perception

Sweet Taste Is Complex: Signaling Cascades and Circuits Involved in Sweet Sensation

Sweetness is the preferred taste of humans and many animals, likely because sugars are a primary source of energy. In many mammals, sweet compounds are sensed in the tongue by the gustatory organ, the taste buds. Here, a group of taste bud cells expresses a canonical sweet taste receptor, whose activation induces Ca2+ rise, cell depolarization and ATP release to communicate with afferent gustatory nerves. The discovery of the sweet taste receptor, 20 years ago, was a milestone in the understanding of sweet signal transduction and is described here from a historical perspective. Our review briefly summarizes the major findings of the canonical sweet taste pathway, and then focuses on molecular details, about the related downstream signaling, that are still elusive or have been neglected. In this context, we discuss evidence supporting the existence of an alternative pathway, independent of the sweet taste receptor, to sense sugars and its proposed role in glucose homeostasis. Further, given that sweet taste receptor expression has been reported in many other organs, the physiological role of these extraoral receptors is addressed. Finally, and along these lines, we expand on the multiple direct and indirect effects of sugars on the brain. In summary, the review tries to stimulate a comprehensive understanding of how sweet compounds signal to the brain upon taste bud cells activation, and how this gustatory process is integrated with gastro-intestinal sugar sensing to create a hedonic and metabolic representation of sugars, which finally drives our behavior. Understanding of this is indeed a crucial step in developing new strategies to prevent obesity and associated diseases

The effects of the Endoluminal Duodeno-Jejunal bypass liner on eating behaviour in humans

Background: The Endoluminal Duodeno-Jejunal Bypass Liner (DJBL) is a thin, flexible, sleeve-like device, made of a single use 60cm fluoropolymer. The DJBL is inserted endoscopically through the mouth and anchored to the proximal small intestine to acts as a physical barrier between the walls of the duodenal and the food ingested. The DJBL is currently being used for the treatment of diabetes in patients with obesity. Therefore, this device offers the unique opportunity to apply a reductionist approach and interrogates the contribution of bypassing the proximal bowel in the regulation of eating behaviour. This is the first study to assess eating behaviour in DJBL patients using direct and indirect measures of behaviour. Aims: To assess whether the DJBL affects eating behaviour 6-months post intervention compared to Best Medical Practice for the treatment of obesity and Type 2 Diabetes. Objective: To investigate the effect of DJBL on: 1. Food choices and calories intake 2.  Eating behaviour 3. The sensory domain of taste. 4. The appetitive behaviour subdomain of the hedonic ingestive motivation domain. 5. The consummatory behaviour subdomain of the hedonic ingestive motivation domain. Methods: This was a randomised controlled study of 42 subjects (23 DJBL, 19 SMT) with Type 2 Diabetes Mellitus who receive the DJBL device or standard medical therapy alone. All patients (40% female) were studied at baseline and followed up for 6-months post intervention. Food choices and calories intake were assessed using Food Diaries, Food Frequency Questionnaire, and 24hr Diet Recall. Psychology and personality traits linked to eating behaviour were assessed with questionnaires, whereas appetite and hunger scores were assessed with Visual Analogue Scales. The intensity of sweet taste stimuli was measured using (a direct behavioural technique) to determine the taste detection threshold using the method of constant stimuli. The appetitive reward of sweet taste stimuli was assessed using a progressive ratio task (a direct behavioural technique). Finally, the consummatory reward value of taste was assessed using visual analogue scales (indirect behaviour technique). Results: 1. Total food intake reduced from at 6-months reduced albeit not significantly and DJBL patients had a modest healthier shift in food preferences. 2. A shift towards healthier eating behaviour and psychological factors was found, which was specific to the treatment type. However, no change in the reported appetite ratings was found. 3. No changes in sucrose detection threshold after DJBL. 4. No change in the appetitive reward value of sweet and fatty tastant after DJBL. 5. No change in the consummatory reward value of sweet taste after DJBL. Conclusion: I conclude, that despite not adding extra benefits on total weight loss, the DJBL could potentially make weight loss an easier task due to the modest changes in food preferences and eating behaviour and psychological traits. In addition, the DJBL did not affect any of the taste dimensions. Therefore, the bypass of the proximal small bowel is not behind the changes in eating behaviour observed post RYGB or that RYGB alters eating behaviour via a combined/synergistic effect of the multiple components and the profound changes in the GI tract. This study contributes to the clinical benefits of the use of DJBL for weight loss and also to the research field on the physiological mechanisms behind RYGB operation.Open Acces

Immortalisation, characterisation and differentiation of temperature sensitive cell lines from the Olfactory Neuroepithelium

Bibliography: p. 194-210.Embryonic olfactory neuroepithelium provides a useful experimental system for the study of olfactory neurogenesis. As a substrate for experimental neural cell biology, olfactory neuroepithelium is of unique interest since, unlike other neural cells, olfactory neurons are continually replaced - a feature that is dictated by their direct exposure to the damaging external environment. Basal cells in the olfactory placode are the source of this replacement. Each olfactory neuron expresses only one or a few of the many olfactory receptors that are encoded by the large array of olfactory genes. Despite this limited cellular display of receptors, vertebrates are able to distinguish many thousands of different odorants, implying a complicated need for perceptive neurological processing of signals coming from individual olfactory neurons. To study the events that take place during the differentiation of neuronal precursors - a process that sustains a diverse receptor repertoire - I felt that lines of conditionally immortalised cells that could be induced to differentiate would provide useful reagents. In this thesis I describe my successful attempts to immortalise olfactory cell lines from the neuroepithelium of E 10.5 mouse embryos. I used a conditionally immortalising retrovirus that included the coding sequence for the temperature-sensitive SY 40 large T antigen. Integration of this retrovirus into the genome of cells allowed continuous proliferation at the permissive temperature of 33°C. A shift to the nonpermissive temperature of 39°C inactivated the SV40 large T antigen, the cells ceased proliferation and differentiation commenced. Sixty cell lines were derived of which four were chosen for further characterisation. These four cell lines (OP6, OP27, OP47 and OP55) were clonally derived and were immortalised rather than transformed. They continued to express the SV40 large T antigen at 33°C but lost expression at 39°C concomitant with cessation of proliferation. When the OP cells were shifted to 39°C in the absence or presence of the morphogen, retinoic acid, morphological changes ensued that were consistent with the development of neuronal characteristics. The OP6, OP27 and the OP47 cells became phase-bright with neuritic extensions. The OP55 cells were the exception in that they did not develop extensions but instead differentiated to form compact epithelial islands when grown in DM-10 medium but not in RA medium. Differentiation of the OP cells at 39°C was further documented by the induced expression of a number of markers demonstrated by RT-PCR and/or immunocytochemistry. The OP cells differentiated at 39°C in DM-10 and in retinoic acid-containing medium to express olfactory receptor transcripts. Cloning and sequencing showed that each cell line expressed a single receptor type but that different receptors were expressed by different cell lines. Sequencing revealed that the receptors cloned from the OP27 cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55 had greatest homology to rat-Olf3 olfactory receptor. The transcripts induced in OP6 and the OP47 cells showed greatest homology with Gus58 - a taste receptor homologous to olfactory receptors. Sequences obtained from OP6, OP47 and OP55 cells were not 100% identical to published receptors and could thus represent members of different subfamilies. Interestingly, induced OP55 cells also expressed mRNA for clusterin - a molecule that has no homology with olfactory receptor transcripts but is involved in differentiation during embryogenesis

Appetite and Satiety Control-Gut Mechanisms

The prevalence of obesity and its comorbidities, particularly type 2 diabetes, cardiovascular and hepatic disease and certain cancers, continues to rise worldwide. Paradoxically, despite an increasingly obesogenic environment, particularly in Western societies, undernutrition is also extremely common. The application of novel, sophisticated techniques, particularly related to imaging and molecular biology, has substantially advanced our understanding of the mechanisms controlling appetite and energy intake. This has led to a redefinition of many concepts, including the relative importance of central versus peripheral mechanisms, recognising that the gastrointestinal (GI) tract, particularly gut hormones, plays a critical role. Given the major advance in knowledge in the field, this Special Issue provides a comprehensive overview of the GI mechanisms underlying the regulation of appetite and energy intake, as a series of definitive reviews by international authorities. The reviews address gut-related mechanisms, including nutrient sensing, gut hormones and GI motility, gut-brain communication, including the roles of the vagus and the modulation of reward perception, the roles of diet and the microbiota, as well as the abnormalities associated with eating disorders, specifically obesity and anorexia of ageing, and the beneficial effects of bariatric surgery. The reviews cover both preclinical research and studies in humans, and are complemented by a number of important original papers

Goat’s vs cow’s milk consumption: Analysis of feeding behaviour, brain activation and gene expression in laboratory animals

Milk is a complex and highly nutritive food. In Western societies, cow’s milk (CM) is most commonly consumed, but recent years have generated interest in milk from other species, especially in goat’s milk (GM). Importantly, select physical and chemical properties of milk are species-dependent and – thus – so are the physiological consequences of consumption of milk sourced from specific species. For example, variation between GM and CM protein impacts digestibility and gastrointestinal processes. Consumption of GM vs CM differentially affects levels of blood hormones regulating energy balance. Furthermore, some conflicting results on acceptability of GM- and CM-based foods have been reported, and it is unclear to what extent habituation to a specific milk type underpins these parameters. To add to the confusion, CM and GM are typically consumed and, therefore, studied as modified milk products, with one of the typical compositional alterations being done to the protein fraction in which the natural 20:80 whey:casein ratio is changed to resemble the 60:40 ratio of human milk. One of the most fundamental gaps in our knowledge regarding CM vs GM relates to the acceptability, palatability and satiating properties of these milks and to appetite-controlling brain processes triggered by CM and GM consumption. Thus, in this doctoral project, I sought to examine whether GM and CM diets elicit unique feeding responses in laboratory rodents and whether the presumed appetite differences are associated with changes in neuronal activation and/or gene expression in key central regions regulating food intake. In Specific Aim 1 of the project, I conducted a comprehensive investigation of short-term intake and palatability profiles of GM- and CM-based liquid and solid diets in mice and rats. Consumption was studied in no-choice and choice scenarios, including meal microstructure. Feeding experiments were followed by qPCR analysis of expression of relevant genes in the energy balance-related hypothalamus and brain stem, and in the nucleus accumbens, which regulates eating for palatability. I found that GM and CM are palatable to juvenile, adult, and aged rodents. Given a choice, animals prefer GM- to CM-based diets. Analysis of meal microstructure using licking patterns points to enhanced palatability of and, possibly, greater motivation toward GM over CM. Most profound changes in gene expression after GM vs. CM were associated with the brain systems driving consumption for reward. The results allow me to conclude that, while both GM and CM are palatable, GM is preferred over CM by animals, and this preference is driven by central mechanisms controlling eating for pleasure. In Specific Aim 2 of the thesis, I investigated the impact of whey enhancement in GM protein fraction on appetite and feeding-related brain processes. The shift from the natural whey:casein ratio of ~20:80 in animal milks is done to match the 60:40 ratio of human milk. Studies show that 20:80 versus 60:40 whey:casein milks differently affect glucose metabolism and hormone release. It is unknown whether the 20:80-to-60:40 ratio adjustment affects appetite and brain processes related to food intake. In this set of studies I focused on the impact of the 20:80 vs 60:40 whey:casein content in GM on food intake and feeding-related brain mechanisms in laboratory mice. I found that the 20:80 whey:casein GM formulation was consumed less avidly and was less preferred than the 60:40 GM in short-term choice and no-choice paradigms. The qPCR analyses in the hypothalamus and brain stem revealed that the 20:80 whey:casein GM intake upregulated genes involved in early termination of feeding and in an interplay between reward and satiety, such as MC3R, OXT, POMC and GLP1R. The 20:80 versus 60:40 whey:casein GM intake differently affected brain neuronal activation (assessed through c-Fos, an immediate-early gene product) in the nucleus of the solitary tract, area postrema, ventromedial hypothalamic nucleus and supraoptic nucleus. Overall, the findings show that whey enhancement in GM promotes overconsumption of GM in no-choice and choice scenarios and that this increased appetite for the 60:40 GM is reflected by changes in neuronal activation and gene expression relevant to feeding regulatory mechanisms. Specific Aim 2 results showing preference for whey-enhanced GM and corresponding changes in c-Fos and gene expression, do not predetermine whether the preference for the 60:40 milk would be retained if - instead of a highly palatable GM - a somewhat less preferred CM was used. Thus, in Specific Aim 3, I replicated the aforementioned feeding, gene expression and c-Fos analyses using CM with the 20:80 vs 60:40 whey:casein. I found that mice exhibited preference for the 60:40 over 20:80 whey:casein CM. This preference for the 60:40 CM was retained even when animals had simultaneous access to the 20:80 GM. Consumption of similar quantities of 20:80 CM vs 60:40 CM differently affected c-Fos in the paraventricular, dorsomedial, arcuate and lateral hypothalamic nuclei and in the nucleus of the solitary tract in the brain stem and relative gene expression (melanocortin and opioid transcripts). It can be concluded that the 60:40 whey:casein milks are more preferred regardless of the species from which the milk was derived, indicating that whey:casein ratio influences preference. Mechanistic commonalities in the whey:casein ratio changes in CM vs GM include the hindbrain neuronal activity changes. Differences in hypothalamic c-Fos and gene expression as well as differences in no-choice feeding paradigms indicate that milk type (GM vs CM) influences some aspects of feeding processes driven by the shift in the whey:casein ratio. Overall, the data presented in this thesis indicate that GM is generally more preferred and it has higher acceptance than CM in laboratory animal models. This phenomenon is reflected by unique changes in feeding-related brain processes induced by GM vs CM. Whey enhancement increases preference toward milk and this effect on consumption is more profound than the effect of the species from which the milk was derived. In a broader context, one has to consider, however, that whey enhancement’s impact on feeding, brain activation and molecular responses might – if sustained over a longer time period - have metabolic consequences