Auswahl der wissenschaftlichen Literatur zum Thema „Neuroendocrine signalling“

Somatostatin (SST) is a small peptide that exerts inhibitory effects on a wide range of neuroendocrine cells. Due to the fact that somatostatin regulates cell growth and hormone secretion, somatostatin receptors (SSTRs) have become valuable targets for the treatment of different types of neuroendocrine tumours (NETs). NETs are a heterogeneous group of tumours that can develop in various parts of the body, including the digestive system, lungs, and pituitary. NETs are usually slow growing, but they are often diagnosed in advanced stages and can display aggressive behaviour. The mortality rate of NETs is not outstandingly increased compared to other malignant tumours, even in the metastatic setting. One of the intrinsic properties of NETs is the expression of SSTRs that serve as drug targets for SST analogues (SSAs), which can delay tumour progression and downregulate hormone overproduction. Additionally, in many NETs, it has been demonstrated that the SSTR expression level provides a prognostic value in predicting a therapeutic response. Furthermore, higher a SSTR expression correlates with a better survival rate in NET patients. In recent studies, other epigenetic regulators affecting SST signalling or SSA–mTOR inhibitor combination therapy in NETs have been considered as novel strategies for tumour control. In conclusion, SST signalling is a relevant regulator of NET functionality. Alongside classical SSA treatment regimens, future advanced therapies and treatment modalities are expected to improve the disease outcomes and overall health of NET patients.

A family of insulin receptor substrate (IRS) proteins mediates the pleiotropic effects of insulin and insulin-like growth factor 1 (IGF-1) on cellular function by recruiting several intracellular signalling networks. Conventional murine knockout strategies have started to reveal distinct physiological roles for the IRS proteins. Deletion of Irsl produces a mild metabolic phenotype with compensated insulin resistance but also causes marked growth retardation. In contrast, mice lacking IRS-2 display nearly normal growth but develop diabetes owing to a combination of peripheral insulin resistance and β-cell failure. As well as the classical metabolic events regulated by insulin signalling pathways, studies in lower organisms have implicated insulin/IGF-1 signalling pathways in the control of food intake and reproductive function. Our analysis of IRS-2 knockout mice shows that female mice are infertile owing to defects in the hypothalamus, pituitary and gonad. IRS-2−1 mice have small, anovulatory ovaries with reduced numbers of follicles. Levels of the pituitary hormones luteinizing hormone and prolactin and gonadal steroids are low in these animals. Pituitaries of IRS-2−1 animals are decreased in size and contain reduced numbers of gonadotrophs. Additionally, IRS-2−1 females display increased food intake and develop obesity, despite elevated leptin levels, suggesting abnormalities in hypothalamic function. Coupled with recent observations that brain-specific deletion of the insulin receptor causes a similar phenotype, these findings implicate IRS signalling pathways in the neuroendocrine regulation of reproduction and energy homeostasis.

AimsBiliary neuroendocrine tumours (NETs) are rare and mostly exist as a component of mixed adenoneuroendocrine carcinomas (MANECs). Although the NET component in biliary MANECs is generally more malignant and clinically more important to the prognosis than the ordinary adenocarcinomatous component, the histogenesis of biliary NET has not been clarified. In this study, the role of the Notch1-Hes1 signalling axis in the histogenesis of biliary NETs was examined.MethodsImmunohistochemistry for Notch1, its ligand Jagged1 and Hes1 was performed using surgical specimens from 11 patients with biliary MANEC. Moreover, after the knock-down of Notch1 mRNA expression in a cholangiocarcinoma cell line, the expression of chromogranin A (a neuroendocrine marker) and Ascl1 (a neuroendocrine-inducing molecule inhibited by activated Hes1) was examined by quantitative PCR.ResultsHistological examination revealed that the adenocarcinomatous components were predominately located at the luminal surface of the MANEC and the majority of stromal invasion involved NET components. Ordinary adenocarcinomas and non-neoplastic biliary epithelium constantly expressed Notch1, Jagged1 and Hes1, but the expression of Notch1 and Hes1 was decreased or absent in NET components, suggesting interference with the Notch1-Hes1 signalling axis in biliary NET. Moreover, in the cholangiocarcinoma cell line in which the expression of Notch1 mRNA was knocked down, the mRNA expression of Ascl1 and chromogranin A was increased.ConclusionsThe Notch1-Hes1 signalling axis suppresses neuroendocrine differentiation and maintains tubular/acinar features in adenocarcinoma and non-neoplastic epithelium in the biliary tree. Moreover, a disruption of this signalling axis may be associated with the tumourigenesis of NETs in biliary MANEC.

Neuroendocrine tumours occur in some hereditary tumour syndromes, and the molecular pathophysiological mechanisms involved in these are also important in their sporadic counterparts which representing the majority of neuroendocrine tumours. These syndromes include multiple endocrine neoplasia type 1, von Hippel–Lindau syndrome, neurofibromatosis type 1 and tuberous sclerosis. All these follow an autosomal dominant inheritance. The primarily affected molecular pathways are Ras-MAPK signalling, hypoxia induced factor 1α, and mTOR signalling that are also involved in sporadic tumours and may even represent potential molecular targets of therapy. In this review, the major characteristics of hereditary tumour syndromes, their molecular genetics and the pathophysiological mechanisms involved in sporadic tumours are discussed. Orv. Hetil., 2013, 154, 1541–1548.

Whilst a number of neuroendocrine afferent signals are implicated in body-weight homeostasis, the major efferent pathway is the sympathetic nervous system (SNS), which affects both energy expenditure and substrate utilization. Thyroid hormones and their interactions with the SNS may also have a role to play. Some of the variability in resting energy expenditure can be explained by differences in SNS activity, and β-blockade can reduce energy expenditure and diet-induced thermogenesis in Caucasians. Excess energy intake leads to SNS activation and increased diet-induced thermogenesis. A relationship has also been demonstrated between spontaneous physical activity and SNS activity. In many animal models the SNS activates brown adipose tissue thermogenesis, hence increasing diet-induced thermogenesis and dissipating excess energy as heat. This effect is mediated via β3-adrenoceptors and activation of an uncoupling protein unique to brown adipose tissue. Homologous proteins have been identified in human tissues and may play a role in human energy expenditure. How the SNS is implicated in this process is unclear at present. β3-Adrenoceptor polymorphism has been associated both with lower resting energy expenditure in some populations and with reduced autonomic nervous system activity. SNS effects on substrate cycling may also play a role. In the development of obesity the effects of the SNS in promoting lipolysis and fat oxidation are likely to be at least as important as its effects on thermogenesis. β-Blockade has relatively small effects on energy expenditure, but more pronounced effects on reducing lipid oxidation, so tending to favour fat storage and weight gain. Low lipid oxidation is a risk factor for weight gain, and there is some evidence that low basal sympathetic nerve activity in muscle is associated with this process. Overall, the relationship between SNS activity and obesity is complex, with evidence of low SNS activity occurring in some, but not all, studies.

Little is known about the adaptor protein FAM159B. Recently, FAM159B was shown to be particularly expressed in neuroendocrine cells and tissues, such as pancreatic islets and neuroendocrine cells of the bronchopulmonary and gastrointestinal tracts, as well as in different types of neuroendocrine tumours. To gain insights into possible interactions of FAM159B with other proteins and/or receptors, we analysed the co-expression of FAM159B and various neuroendocrine-specific markers in the cancer cell lines BON-1, PC-3, NCI-h82, OH-1, and A431 and also in human pancreatic tissues and pancreatic neuroendocrine tumours. The markers included prominent markers of neuroendocrine differentiation, such as chromogranin A (CgA), neuron-specific enolase (NSE), synaptophysin (SYP), insulinoma-associated protein 1 (INSM1), neural cell adhesion molecule 1 (NCAM1), serotonin (5-HT), somatostatin-14/28 (SST), and several receptors that are typically expressed by neuroendocrine cells, such as dopamine receptor 2 (D2R), somatostatin receptor (SSTR) 1, 2, 3, 4 and 5, and regulator of G-protein signalling 9 (RGS9). FAM159B was expressed evenly throughout the cytosol in all five cancer cell lines. Immunocytochemical and immunohistochemical analyses revealed co-expression of FAM159B with SYP, INSM1, RGS9, D2R, SSTR2, SSTR3, SSTR4, and SSTR5 and strong overlapping co-localisation with NSE. Double-labelling and co-immunoprecipitation Western blot analyses confirmed a direct association between FAM159B and NSE. These results suggest the involvement of FAM159B in several intracellular signalling pathways and a direct or indirect influence on diverse membrane proteins and receptors.

Glucocorticoids contribute to obesity and metabolic syndrome; however, the mechanisms are unclear, and prognostic measures are unavailable. A systems level understanding of the hypothalamic–pituitary–adrenal (HPA)–leptin axis may reveal novel insights. Eighteen obese premenopausal women provided blood samples every 10 min over 24 h, which were assayed for cortisol, adrenocorticotropin releasing hormone (ACTH) and leptin. A published personalized HPA systems model was extended to incorporate leptin, yielding three parameters: (i) cortisol inhibitory feedback signalling, (ii) ACTH–adrenal signalling, and (iii) leptin–cortisol antagonism. We investigated associations between these parameters and metabolic risk profiles: fat and lean body mass (LBM; using dual-energy X-ray absorptiometry), and insulin resistance. Decreased cortisol inhibitory feedback signalling was significantly associated with greater fat (kg; p = 0.01) and insulin resistance ( p = 0.03) but not LBM. Leptin significantly antagonized cortisol dynamics in eight women, who exhibited significantly lower 24 h mean leptin levels, LBM and higher ACTH–adrenal signalling nocturnally (all p < 0.05), compared with women without antagonism. Traditional neuroendocrine measures did not predict metabolic health, whereas a dynamic systems approach revealed that lower central inhibitory cortisol feedback signalling was significantly associated with greater metabolic risk. While exploratory, leptin–cortisol antagonism may reflect a ‘neuroendocrine starvation’ response.

Although there is evidence of a significant rise of neuroendocrine neoplasms (NENs) incidence, current treatments are largely insufficient due to somewhat poor knowledge of these tumours. Despite showing differentiated features, NENs exhibit therapeutic resistance to most common treatments, similar to other cancers in many instances. Molecular mechanisms responsible for this resistance phenomenon are badly understood. We aimed at identifying signalling partners responsible of acquired resistance to treatments in order to develop novel therapeutic strategies. We engineered QGP-1 cells resistant to current leading treatments, the chemotherapeutic agent oxaliplatin and the mTor inhibitor everolimus. Cells were chronically exposed to the drugs and assessed for acquired resistance by viability assay. We used microarray-based kinomics to obtain highthroughput kinase activity profiles from drug sensitive vs resistant cells and identified ‘hit’ kinases hyperactivated in drug-resistant cells, including kinases from FGFR family, cyclin-dependant kinases and PKCs in oxaliplatin-resistant (R-Ox) QGP-1 cells. We then validated these ‘hit’ kinases and observed that ERK signalling is specifically enhanced in QGP-1 R-Ox cells. Finally, we assessed drug-resistant cells sensitivity to pharmacological inhibition of ‘hit’ kinases or their signalling partners. We found that FGFR inhibition markedly decreased ERK signalling and cell viability in QGP-1 R-Ox cells. These results suggest that the FGFR/ERK axis is hyperactivated in response to oxaliplatin-based chemotherapeutic strategy. Thus, this sensitive approach, based on the study of kinome activity, allows identifying potential candidates involved in drug resistance in NENs and may be used to broadly investigate markers of NENs therapeutic response.

I investigated the relationship between the HPA axis and endocrine factors to adipose tissue storage and appetite regulation during late pregnancy in rats. The responsiveness of the hypothalamic neurones regulating ACTH and hence corticosterone responses to insulin induced hypoglycaemia (IIH) and the orexigenic peptides NPY, orexin and ghrelin were investigated. IIH increased ACTH secretion similarly I both virgin and pregnant rats. Unlike most stressors IIH did not stimulate parvocellular CRH mRNA expression in pPVN neurones but it did increase AVP mRNA expression. The responsiveness of the HPA axis to the orexigenic peptides ghrelin, orexin and NPY given by i.c.v. injection was markedly reduced during late pregnancy. This was at least partly a result of reduced activation of the pPVN neurones, as revealed by reduced stimulation of FOS expression in the pPVN compared with virgin rats given these peptides. ACTH secretory responses were also strongly attenuated in late pregnant rats. In contrast all three orexigenic peptides increased food intake to a similar level in both virgin and pregnant rats. Thus neuroendocrine stress responses to central administration of orexin, NPY and ghrelin are absent during late pregnancy whilst ingestive behavioural responses remain intact. Changes in brain circuitry regulating appetite during late pregnancy were shown by increased FOS activation in the lateral hypothalamic area (LHA), ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Supraoptic and magnocellular PVN oxytocin responses to centrally administered NPY were reduced during late pregnancy. Endogenous opioids are involved in the attenuation of HPA axis responses to orexin and NPY during late pregnancy since pre-treatment with the opioid receptor antagonist naloxone reinstated the ACTH response and restored CRH and AVP mRNA responses. Naloxone administration revealed that endogenous opioids facilitate NPY-induced feeding in both virgin and late pregnant rats, but more importantly in late pregnant rats. Naloxone restored a FOS response in the PVN and SON in response to NPY in late pregnant rats indicating that oxytocin neurone responses to NPY are suppressed by endogenous opioids. Basal blood glucose levels were lower in late pregnant rats than in virgins. Ghrelin increased blood glucose levels similarly in both virgin and pregnant rats, whilst NPY and orexin increased blood glucose in only the virgin rats. In conclusion, neuroendocrine stress responses to orexin, ghrelin and NPY are reduced in pregnant rats and this was shown for orexin and NPY to be due to endogenous opioid restraint. Endogenous opioid mechanisms have opposite effects on neuroendocrine stress responses and feeding, which will enhance energy availability for the fetuses at this time. Intact HPA axis responses to IIH will ensure continued glucose supply.

Pancreatic neuroendocrine tumours (PNETs) are the most common neuroendocrine tumours diagnosed in humans and dogs. Due to the highly heterogeneous nature of these tumours, definitive data are still lacking over the molecular mechanisms involved in their cancerous behaviour. This study focused on insulinoma (INS), as it is the most commonly diagnosed PNET in human and veterinary oncology. INS is an insulin-producing tumour that causes a hypoglycaemic syndrome related to the excessive insulin production. In humans, it is often a small benign neoplasm readily curable by surgical resection whereas, in dogs, INS is often malignant. Despite current treatment modalities, malignant canine and human INS have a poor prognosis as patients tend to develop metastases in liver and lymph nodes that do not respond to current therapies. From a comparative oncology perspective, the close resemblance of canine and human malignant INS makes canine INS an interesting study model for human INS. Cancer stem cells (CSCs) are critical for the engraftment and chemoresistance of many tumours. Although CSCs have been isolated from a range of solid tumours, a comprehensive characterisation of INS CSCs has not yet been reported. In this study, it was confirmed that INS CSCs can be enriched and are potential targets for novel INS therapies. Highly invasive and tumourigenic human and canine INS CSCs were successfully isolated and exhibited greater resistance to chemotherapy, which may play a significant role in the poor prognosis of this disease. To date, the mechanisms by which tumours spread and the clinical causes of chemoresistance remain only partially understood. Here, RNA-sequencing analysis was performed over a small set of canine INS tumour samples in order to identify mechanisms involved in INS carcinogenesis through different stages of the disease. Preliminary data showed that distinct gene profiles characterised early and late stage of canine INS. Interestingly, differential gene expression and gene pathways analysis, highlighted that sets of genes involved in pancreatic embryogenesis and insulin secretion were overexpressed in canine primary INS lesions compared with normal pancreas. The Notch pathway is fundamental in pancreatic embryogenesis and it has been previously associated with carcinogenesis of neuroendocrine tumours and with the CSC phenotype. Protein analysis showed that the Notch pathway is activated in both human and canine INS CSCs, particularly when treated with chemotherapy, indicating that the Notch pathway may be involved in chemoresistance. Additionally, it was demonstrated that inhibition of the Notch pathway decreased INS CSCs' survival and chemoresistance, both in vitro and in vivo. These findings provide preclinical evidence that anti-Notch therapy may improve outcomes for patients with malignant INS.

This thesis shows that socially subordinate Arctic charr (Salvelinus alpinus) display elevated brain serotonergic (5-HT) and norepinephric activity along with a chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis, including elevated plasma concentrations of á-MSH. Furthermore, subordinate fish showed an inhibition of aggressive behaviour and darker body coloration, skin darkness being positively correlated with plasma á-MSH. Fish kept on dark background, and thus being darker in body colour, were less aggressive than conspecifics interacting on white background, supporting the hypothesis that skin darkening could signal social submission. The 5-HT_1A -receptor agonist 8-OH-DPAT stimulated HPI axis activity in non-stressed fish, but if administrated to stressed fish it inhibited HPI axis activity, suggesting that 5-HT_1A receptors may act as both post- and pre-synaptic receptors. 8-OH-DPAT also induced skin darkening in both non-stressed and stressed fish. Stimulation of brain dopaminergic activity by L-dopa treatment counteracted the stress-induced inhibition of aggressive behaviour, and stress related effects on brain 5-HT activity and plasma levels of cortisol. In conclusion, social subordination in Arctic charr results in skin darkening and an inhibition of aggressive behaviour. Stress-induced effects, that could be mediated by elevated brain 5-HT activity, and serve as a way of signalling social position and coping with stress.

Les Perturbateurs Endocriniens (PE) sont une classe de contaminants environnementaux d'origine anthropique qui affectent les organismes en perturbant leur système endocrinien. Notamment, le manque de compréhension détaillée des systèmes endocriniens et leur voie de signalisation empêche la compréhension de l’effet de PEs chez les invertébrés. Conformément, cette thèse vise à aborder la question de la perturbation endocrinienne chez les invertébrés marins en exploitant le développement larvaire de la moule méditerranéenne Mytilus galloprovincialis. En appliquant la logique de l’Adverse Outcome Pathway-AOP, le travail de cette thèse vise à caractériser le mécanisme d'action de PEs modèles BPA, TBBPA et TBT chez les larves de moule et à identifier de plausibles voies neuro-endocriniennes par lesquelles ils induisent leur effet biologique délétère. Les éléments neuroendocriniens régulant la biogenèse de la coquille larvaire ont été caractérisés et le processus morphogénétique établi comme sensible à la perturbation neuroendocrinienne. Les plausibles AOPs neuro-endocriniens qui pourraient être liées aux effets biologiques de PEs modèles ont été identifiées. En plus, l'AOP possible du TBT a établi une relation plausible entre la signalisation des Récepteurs Nucléaires et le système neuroendocrinien des larves de moule. Les résultats de cette thèse représentent des preuves essentielles qui aideront et feront avancer la compréhension actuelle des mécanismes de perturbation endocrinienne chez les organismes invertébrés
Endocrine Disruptors (EDCs) are a class of anthropogenic environmental contaminants that affect organisms by disrupting their endocrine system. Notably, the lack of detailed understanding of endocrine systems and their signaling pathway prevents understanding of the effect of PEs in invertebrates. Accordingly, this thesis aims to address the issue of endocrine disruption in marine invertebrates by exploiting the larval development of the Mediterranean mussel Mytilus galloprovincialis. By applying the logic of the Adverse Outcome Pathway-AOP, the work in this thesis attempted to characterize the mechanism of action of the model EDCs BPA, TBBPA and TBT in mussel larvae and to identify plausible neuroendocrine pathways by which they induce their deleterious biological effect. The neuroendocrine elements regulating the biogenesis of the larval shell have been characterized and the morphogenetic process established as sensitive to neuroendocrine disruption. Plausible neuroendocrine AOPs that could be linked to the biological effects of the model EDCs have been identified. In addition, the possible AOP of TBT has established a plausible relationship between nuclear receptor signaling and the neuroendocrine system of mussel larvae. The results of this thesis represent essential evidence that will help and advance the current understanding of endocrine disruption mechanisms in invertebrate organisms

Tese de Mestrado, Biologia Molecular e Genética, 2021, Universidade de Lisboa, Faculdade de Ciências
A glucose é um dos monossacarídeos mais importantes para a vida, uma vez que o nosso organismo a utiliza como uma fonte de energia indispensável ao funcionamento normal dos vários órgãos e tecidos que dela precisam. Para que esse uso aconteça, é necessário que a glucose passe da corrente sanguínea para o interior das células, sendo que esse transporte é possível graças a uma substância química concebida pelo organismo: a insulina. A insulina é uma hormona produzida pelas células β pancreáticas das ilhotas de Langerhans que é libertada das mesmas quando os níveis de “açúcar” na corrente sanguínea se encontram elevados. Contudo, na diabetes mellitus (DM), esta situação está alterada. A DM é uma doença crónica caracterizada pelo aumento desregulado dos níveis de glucose no sangue, fenómeno designado por hiperglicemia, e pela incapacidade do organismo em transportar toda a glucose proveniente dos alimentos, levando a um desequilíbrio nos seus níveis sanguíneos. Nos últimos dez anos, o impacto do envelhecimento da estrutura etária da população portuguesa (20-79 anos) levou a um aumento de cerca de dois pontos percentuais na taxa de prevalência da DM entre 2009 e 2018, o que equivale a um crescimento de cerca de 16,3%. Mundialmente, em 2019, 463 milhões de pessoas tinham DM (9,3% da população até à data), sendo que 134 milhões se encontravam entre os 65 e os 99 anos (19,4% da população contida nessa faixa etária). Em Portugal, a prevalência nessa faixa etária fixava-se nos 24%. Estima-se que essas prevalências cresçam a um ritmo alarmante, o que leva a que a DM seja considerada uma grande ameaça mundial para a saúde pública. O tipo mais comum de DM, a DM tipo 2 (T2DM), que ocorre em cerca de 90% da população mundial com DM, e cuja prevalência é mais elevada em indivíduos mais envelhecidos, resulta, numa fase inicial (pré-diabetes), de uma anomalia nos níveis de glucose em jejum e de uma intolerância a este carbohidrato. Após vários anos, as células do nosso organismo ficam incapazes de responder de forma correta à insulina, induzindo-se uma resistência a esta hormona que levará a um aumento nos seus níveis de produção, e eventualmente, à incapacidade das células β em compensar este desequilíbrio. Tudo isto provocará a ocorrência de complicações macrovasculares, tais como doença coronária e hipertensão arterial, e microvasculares, nomeadamente retinopatia e neuropatia, que podem causar complicações severas que diminuirão drasticamente a qualidade e esperança média de vida dos indivíduos com T2DM. Os tipos de cirurgia bariátrica são divididos em três grupos: 1) os principalmente restritivos, que incluem a banda gástrica ajustável e a gastrectomia vertical, e que diminuem a quantidade de alimentos ingeridos por diminuição do volume gástrico, 2) os principalmente disabsortivos, como a derivação biliopancreática e o duodenal switch, em que a perda de peso é altamente eficaz, uma vez que reduzem o trajeto no qual os nutrientes vão ser absorvidos, e 3) os que usam uma combinação de ambas as técnicas, também designados por procedimentos mistos, como é o caso do bypass gástrico em Y de Roux (RYGB). No RYGB, é criada uma pequena bolsa gástrica com cerca de trinta mililitros de capacidade que é ligada ao intestino delgado ao nível do jejuno, pelo que as regiões do intestino delgado proximal (i.e., duodeno e início do jejuno) nunca sofreriam a ação das secreções pancreática e biliar. Contudo, e completamente por acaso, a regressão da T2DM que se observou dias após este bypass gástrico colocam em evidência o intestino delgado proximal como um dos principais intervenientes da etiologia desta doença. Modificações dos níveis hormonais, principalmente ao nível do péptido semelhante ao glucagão 1 (GLP-1), e do microbioma intestinal têm sido estudadas para explicar esta regressão, mas os mecanismos moleculares subjacentes permanecem inconclusivos. Em condições normais, os processos de sinalização ao nível da parede intestinal que ocorrem em resposta a certos nutrientes ingeridos envolvem enterócitos, células enteroendócrinas (EEC’s), e uma rede de glias entéricas, nervos intrínsecos e extrínsecos que comunicam com o sistema nervoso central (CNS) e com o periférico. Para que haja propagação do sinal, é necessário que um neurónio pré-sináptico seja capaz de gerar e propagar um potencial de ação através do seu axónio, transmitindo-o por uma sinapse mediante a libertação de neurotransmissores. Esses neurotransmissores irão potenciar uma reação num neurónio pós-sináptico ou numa célula efetora, tais como as células musculares e a maioria das células exócrinas e endócrinas. Ao nível do CNS dos mamíferos em desenvolvimento, o principal neurotransmissor inibitório corresponde ao ácido γ-aminobutírico [nome IUPAC: ácido 4-aminobutanoico (GABA)], neurotransmissor esse que se encontra presente nos nervos entéricos e em EEC’s intestinais. Como tal, a neurotransmissão mediada pelo GABA considera-se envolvida 1) na regulação das funções fisiológicas ao nível do trato gastrointestinal, e 2) como sendo um mediador enteroendócrino que influencia a função desse trato. O GABA exerce a sua função através da ligação aos respetivos recetores, podendo os mesmos ser classificados em ionotrópicos (GABAA) ou metabotrópicos (GABAB). Dos dois, a informação relacionada com o recetor GABAB (GABABR) é a mais escassa, visto que 1) estes recetores foram dos últimos a ser caracterizados a nível molecular, e 2) apenas dois derivados agonísticos do GABA, baclofeno e ácido γ-hidroxibutirato, são capazes de se ligar ao lado ortostático da estrutura deste recetor. Contudo, os seus efeitos secundários, tais como dormência e tonturas, levam a que a administração destes derivados seja desfavorável em muitos casos de patologia. A nível molecular, os GABABRs consistem em duas subunidades, GABABR1 e GABABR2, sendo que o GABABR2 é também importante para o acoplamento das proteínas G, cuja sinalização é regulada por homo-oligómeros de subunidades auxiliares, aos quais se dá o nome de domínios de tetramerização dos canais de potássio (KCTD’s). KCTD8, KCTD12 e KCTD16 pertencem ao clado F de uma grande família de vinte e cinco proteínas humanas KCTD que atuam especificamente com os GABABR, regulando vias intracelulares a jusante via o acoplamento a proteínas G, nomeadamente os canais de iões cálcio dependentes de voltagem, os canais de iões potássio, e o adenilato ciclase. Mais concretamente, as proteínas KCTD interagem com um resíduo de tirosina-902 presente no terminal-C do domínio intracelular da subunidade GABABR2. No entanto, a distribuição do GABABR2 com estas proteínas específicas ao nível do duodeno e do início do jejuno, assim como o possível papel dessa interação no envelhecimento ou na progressão da T2DM, ainda são desconhecidas. Neste estudo, procedeu-se à extração do intestino delgado proximal proveniente de murganhos masculinos C57BL/6J que foram usados como modelos de envelhecimento e de pré-diabetes induzida por uma dieta rica em gordura (HFD). Esta região intestinal foi estudada sob a forma de secções do duodeno ou de rolinhos intestinais, secções essas que foram analisadas mediante dois ensaios imuno-histoquímicos: ligação por proximidade (PLA) e imunofluorescência (IF), que permitiram investigar a co-localização das proteínas KCTD com a subunidade GABABR2, e apenas IF, para analisar os níveis de expressão dos KCTD ao nível das células não gliais (presumivelmente neurais) e gliais marcadas com a proteína ácida fibrilar glial (GFAP), um dos melhores marcadores para simbolizar a ativação da glia entérica após situações de danos estruturais ou de stress ao nível do sistema nervoso entérico. Estas análises imuno-histoquímicas foram efetuadas na porção interior das vilosidades intestinais, à qual se dá o nome de lâmina própria. Os resultados obtidos por PLA e por IF demonstraram que, nos murganhos HFD, a expressão das proteínas KCTD8 e KCTD16 co-localizadas com o GABABR2 decresceu na ordem das 0.4 e 0.6 vezes respetivamente, quando comparada com os murganhos alimentados com uma dieta normal (NCD). No entanto, nos murganhos envelhecidos (20 meses de idade), todos os KCTD co-localizados com esta subunidade sofreram um aumento de cerca de 1.0 a 2.5 vezes na sua expressão, quando comparada com os murganhos jovens (4 meses). Estes resultados indicam que a sinalização potenciada pelo GABABR e dependente do KCTD8 e do KCTD16 sofre uma disrupção independente da idade em casos de pré-diabetes. Os estudos de IF revelaram que as células gliais que não expressam as proteínas KCTD sofreram um aumento geral da sua expressão em todos os murganhos, o que é indicativo de uma maior inflamação intestinal com a idade e com a pré-diabetes. Nas células não gliais (presumivelmente células neurais), observou-se um aumento de 50 vezes na expressão do KCTD12 ao nível dos murganhos HFD, e um decréscimo de 0.7 vezes para o KCTD16 nos murganhos envelhecidos. Finalmente, a expressão de todos os KCTD estudados ao nível das células gliais aumentou em todos os murganhos envelhecidos, mas decresceu 0.6 vezes para o KCTD16 nos murganhos HFD. A partir destes resultados, podemos inferir que a sinalização mediada pelo GABABR ao nível do intestino delgado proximal sofreu uma disrupção independente da idade ao nível dos modelos pré-diabéticos, sendo que a glia entérica, quando co-localizada com o KCTD16 (decréscimo nos seus níveis), pode estar envolvida nessa disrupção. Para além disso, o KCTD12 em células não gliais, pelo facto de apresentar uma expressão aumentada significativamente em condições pré-diabéticas, pode ser considerado, juntamente com o KCTD16, como um potencial alvo farmacológico para estudar a prevenção desta doença crónica.
Diabetes mellitus (DM) is a chronic disease characterized by hyperglycaemia and glucose intolerance. By 2019, 463 million people worldwide (9.3%) suffered from this disease. Type 2 diabetes mellitus (T2DM) accounts for about 90% of all DM cases, with higher prevalence in aged populations. This disease is characterized, at early stages (prediabetes), by impaired fasting glucose and glucose intolerance, and after several years, by insulin resistance, hyperglycaemia, and eventually impaired insulin secretion, leading to serious health complications. Current treatments only serve to regulate hyperglycaemia and attenuate progression of T2DM. However, by chance, remission of T2DM was observed within days after bariatric surgery involving duodenal/jejunal gut bypass, pointing to those bypassed regions as major culprits in its aetiology. Changes in gut hormones and gut microbiome could explain this observed remission, but the molecular mechanisms underlying it remain inconclusive. Gut signalling in response to ingested nutrients involves enterocytes, diverse enteroendocrine cells (EECs), and networks of enteric glia, intrinsic and extrinsic nerves that communicate with the peripheral and central nervous systems. γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter, is present in enteric nerves and EECs, implicating GABA-mediated neurotransmission in the regulation of physiological functions within the gastrointestinal tract. GABA mediates its effects via GABA receptors, including the heterodimeric and metabotropic GABAB receptors (GABABR), whose G protein signalling is regulated by homo-oligomers of auxiliary subunits termed potassium channel tetramerization domains (KCTDs). KCTD8, KCTD12 and KCTD16 function specifically with GABABR2, regulating downstream intracellular pathways via G protein coupling, including voltage-gated calcium ion channels, potassium ion channels, and adenylyl cyclase. However, the distribution of GABABR2 with specific KCTDs in the gut and their role in ageing or T2DM progression is still unknown. We extracted the proximal small intestine of young, old, and high-fat diet-induced prediabetic (HFD) C57BL/6J male mice, and used proximity ligation assay (PLA) and immunofluorescence (IF), to investigate KCTD proteins colocalized with GABABR2, and IF-only, to investigate KCTD expression in non-glial (presumed neural cells) and glial cells, in the interior of intestinal villi. PLA and IF results showed that expression of KCTD8 and KCTD16 colocalized with GABABR2 in HFD mice declined 0.4 and 0.6-fold respectively, when compared to mice on a normal chow diet (NCD). However, in old mice, all KCTDs colocalized with GABABR2 had a 1.0 to 2.5-fold increase, compared to young mice. These results reveal an age-independent disruption of KCTD8 and KCTD16 GABABR signalling in prediabetes. IF studies revealed that glial cells not expressing KCTDs were increased overall in both old and HFD mice, indicating enhanced gut inflammation. In non-glial cells (presumed neural cells), we observed a 50-fold increased expression of KCTD12 in prediabetic mice, but a 0.7-fold decrease of KCTD16 in old mice. Glial cell expression of all KCTDs studied increased in old mice, but decreased 0.6-fold for KCTD16 in prediabetic mice. From these results, we infer that proximal small intestine GABABR signalling is disrupted in prediabetes independent of age, with a possible involvement of decreased KCTD16-expressing enteric glia, and highlight KCTD12 and KCTD16 as potential pharmacological targets to study prediabetes prevention.

In the work described in this thesis, analytical methods for the detection and quantification of peptide hormones featuring on-line analyte concentration, post-separation tagging and HPLC-fluorescence detection were presented. These methods were used to detect and quantify calcitonin (CT) and its prohormones glycyllysyllysine-extended CT (CTGKK), glycyllysine-extended CT (CTGK) and glycine-extended CT (CT-G) for the first time, in DMS53 small cell lung carcinoma (SCLC) cell culture medium and lysate. Additionally, novel glycosylated versions of each species were also identified, suggesting the presence of a parallel biosynthetic pathway in DMS53. Extracellular but not intracellular levels of CT were reduced as a result of treatment with biosynthesis inhibitors, and it was suggested CT precursor flux through the glycosylated pathway acts as a bypass mechanism to maintain intracellular CT levels. Moreover, the up-regulation of extracellular levels of CT-related species in response to increased medium volume provided evidence of a homeostatic feedback loop maintaining extracellular CT concentrations. To interrogate the mechanism of this feedback, DMS53 cultures were treated with a specific human calcitonin receptor (hCTR) agonist, SUNB8155, to determine if the hCTR is involved in the regulation of CT. It was observed that the relative levels of extracellular CT increased with SUNB8155 treatment, but that the relative levels of the intracellular CT-related species were unchanged. This suggested that hCTR is expressed in DMS53, and that activation of the receptor influences the expression and biosynthetic processing of CT-related species. To investigate this hypothesis, hCTR was identified in DMS53 cells using reverse transcription PCR and Western blot analyses. Specifically, transcriptional and translational evidence of the isoform hCTR2 was identified. Thus, for the first time,hCTR activation was implicated in the up-regulation of CT. This suggested that a positive autocrine feedback loop was operating in DMS53, and based on the hCTR2 isoform, may be mediated by signal transduction through the cAMP- and Ca2+- dependent signalling pathways. To assess which signalling enzymes are activated by hCTR, signal transduction pathways were investigated using small molecule enzymes inhibitors, and their effects on the levels of CT-related species observed. It was observed that treatment of DMS53 cultures with the protein kinase C inhibitor, GF109203X had an effect on the levels of CT-related species in the medium. Again, the relative levels of the intracellular CT-related species were not changed by treatment with this inhibitor. This implicated PKC as a component of the hCTR signal transduction pathway. It was concluded that DMS53 cultures have mechanisms to maintain the intracellular and extracellular concentrations of CT-related species. The concentration of extracellular CT is regulated by a positive feedback mechanism, mediated by hCTR activation, and subsequent signalling involving PKC and AC. Treatment with biosynthetic and signalling inhibitors had no significant effect on the intracellular levels of CT-related species, demonstrating that DMS53 cultures prioritise tight control of intracellular concentrations over extracellular concentrations. With the methodology to detect and quantify peptide hormones in cell culture medium and lysate in hand, the generality of CT glycosylation was explored. Preliminary experiments successfully characterised the presence of glycosylated CT and CT-G in the medullary thyroid carcinoma cell line, TT. To broaden the range of detected hormones, HPLC-fluorescence methodology was developed to detect and quantify oxytocin (OT) and its precursors, and this methodology was used to investigate the presence of OT in the DMS79 SCLC cell line.

Multiple bidirectional interactions between stress neuroendocrine and innate immunity have been identified in previous research, but the extent of these interactions remain unresolved. This thesis thus aimed to explore the bidirectional interface between stress and innate immunity, by focusing on glucocorticoid and TLR4- MyD88 signalling. The research was undertaken in a series of 3 studies. Study 1 investigated the impact of baseline TLR4-MyD88 signalling on the neuroendocrine and behavioural responses to acute stress. Through examining stress responses in mice lacking Tlr4 or Myd88, this study found an intrinsic influence of TLR4-MyD88 signalling on both neuroendocrine and behavioural responses. Aadaptations to the feedforward and feedback pathways of glucocorticoid signalling were also identified. Studies 2 and 3 explored the effects of glucocorticoid signalling on innate immune function in immunocompetent cells. In these 2 studies, BV2 microglia-like cells, RAW264.7 macrophage-like cells and adult primary microglia were utilised. Study 2 demonstrated a biphasic innate immune response to glucocorticoids, where low concentrations of corticosterone pre-exposure primed, while a high stress-like concentration of corticosterone suppressed TLR4-NF-κB-IL-1β responses. Using pharmacological antagonists, it was further revealed that the priming effect on IL-1β was mediated by mineralocorticoid receptor (MR) signalling, while immunosuppressive actions were mediated by glucocorticoid receptor (GR) signalling. Study 3 further assessed glucocorticoid actions on non-cytokine innate immune responses via measurements of cell motility, cell death and danger-associated molecular pattern (DAMP)-related protein release. Here, a low concentration of corticosterone increased ATP-induced BV2 cell motility. Signalling via GR, a stress-like concentration of Corticosterone and Dexamethasone caused an increase in cytotoxicity and HMGB1 DAMP protein release. Collectively, the findings in this thesis provide support for multiple intrinsic connections between the stress neuroendocrine and TLR4 signalling pathway, both in vivo and in vitro. Further mechanistic insights such as GR and MR signalling were revealed, and the implications of this work to health and disease were also discussed.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017.

Ca2+ is an important mediator of stimulus-secretion coupling in beta cells of the pancreatic islets, which secrete insulin in response to elevation in plasma glucose concentration. I studied the actions of two lipids, arachidonic acid (AA) and cholesterol, on enzymatically-dissociated single beta cells of rat and mouse, using cytosolic Ca2+ ([Ca2+]i) measurement in conjunction with whole-cell patch-clamp techniques. AA, which is produced in the beta cell upon stimulation with either glucose or acetylcholine, was found to induce a large increase in [Ca2+]i that was dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. Part of the AA-mediated extracellular Ca2+ entry was due to Ca2+ influx through the arachidonate-regulated Ca2+ (ARC) channels, which have not previously been reported in beta cells. The AA-mediated intracellular Ca2+ release was a result of Ca2+ mobilization from multiple inositol trisphosphate (IP3)-sensitive intracellular stores, including the endoplasmic reticulum (ER) and an acidic Ca2+ store that is probably the secretory granules. Therefore, in beta cells, the AA-mediated Ca2+ signal may amplify the [Ca2+]i rise induced by insulin secretagogues. Cholesterol is an integral component of cellular membranes and an important regulator of cellular functions. However, elevation of cholesterol level in the pancreatic islets reduces glucose-stimulated insulin secretion. I found that cholesterol overload impairs the glucose-stimulated [Ca2+]i increase in beta cells by two major mechanisms: the first is a decrease in glucose-stimulated ATP production, which is partly mediated by a decrease in glucose uptake, and the second is the reduction of voltage-gated Ca2+ current density. These effects of cholesterol may partly account for the decreased insulin secretion that develops in patients with type II diabetes, who typically exhibit hypercholesterolemia. In summary, different lipids may mediate beneficial or detrimental effects on Ca2+ regulation in rodent pancreatic beta cells.

Once considered merely as a neurotransmitter, serotonin (5-HT) now enjoys a renewed reputation as an interlocutor in the dense and continuous dialogue between neuroendocrine and immune systems. In the last decades, a role has been depicted for serotonin and its derivatives as modulators of several immunological events, due to the expression of specific receptors or enzymes controlling 5-HT metabolism in diverse immune cell types. A growing body of evidence suggests that the effects of molecules belonging to the 5-HT pathways on the neuroimmune communication may be relevant in the clinical outcome of autoimmune/inflammatory pathologies of the central nervous system (CNS), such as multiple sclerosis, but also in Alzheimer’s disease, or in mood disorders and major depression. Moreover, since the predominance of 5-HT is produced by enterochromaffin cells of the gastrointestinal tract, where 5-HT and its derivatives are important mucosal signalling molecules giving rise to the so-called “brain-gut axis”, alterations in brain-gut communication are also involved in the pathogenesis and pathophysiology of several psychiatric and neurologic disorders. Here we illustrate how functional interactions between immune and neuronal cells are crucial to orchestrate tissue homeostasis and integrity, and the role of serotonin pathway components as pillars of the neuroimmune system.