Modulation of carotid body activity as a therapeutic intervention in metabolic diseases

A diabetes tipo 2 é uma das doenças crónicas mais comuns no mundo, cuja prevalência continua a aumentar. Em 2045 estima-se que esta doença afete cerca de 629 milhões de pessoas no mundo. A diabetes tipo 2 é caracterizada pela resistência periférica à insulina, o anormal metabolismo hepático de glucose e a falha progressiva das células beta do pâncreas. Existem vários fármacos disponíveis para o tratamento da diabetes tipo 2 contudo, com o tempo, o controlo da glucose deteriora-se progressivamente e, mesmo com ajustes na medicação, uma proporção considerável de indivíduos continua pouco controlado. Deste modo, é essencial o desenvolvimento de novas estratégicas terapêuticas para o controlo da diabetes tipo 2. Nos últimos anos, o corpo carotídeo (CB), um quimoreceptor periférico que deteta alterações de O2, CO2 e pH no sangue, tem também sido descrito como um sensor metabólico envolvido no controlo da homeostasia energética. De facto, foi descrito que o CB está envolvido na génesis da resistência à insulina e hipertensão induzidos pelas dietas hipercalóricas. Este trabalho teve como objetivo avaliar o papel do CB no controlo da homeostasia da glucose e investigar uma possível metodologia para modular a atividade do CB com o intuito de encontrar um novo tratamento para a diabetes tipo 2. O capítulo I introduz conceitos gerais na diabetes tipo 2, tais como, a sinalização da insulina, a homeostasia da glucose e as opções terapêuticas para o tratamento da diabetes tipo 2. Para além disso, conceitos relacionados com a função do CB e o papel do ATP e da adenosina na neurotransmissão do CB, bem como, o seu papel como sensor metabólico serão também abordados. No capítulo II estão descritos os objetivos gerais e específicos do presente trabalho. No capítulo III demonstrou-se que a ressecção do nervo do seio carotídeo (CSN), o nervo sensitivo do CB, restaura a sensibilidade à insulina em dois modelos animais de prediabetes, um efeito que se mantém com a continuação da administração das dietas hipercalóricas. Para além disso, foi observado que a ressecção do CSN normaliza a atividade do sistema nervo simpático, a pressão arterial, a função endotelial, o perfil lipídico e os níveis plasmáticos de glucose e insulina. Foi também descrito que o mecanismo pelo qual é restaurada a homeostasia da glucose envolve uma melhoria na captação de glucose no fígado e no tecido adiposo perientérico bem como, o restauro das vias de sinalização da insulina no músculo esquelético e no tecido adiposo. No capítulo IV foi descrito que a modulação bioelectrónica do CSN através da aplicação de uma corrente alternada de alta frequência (KHFAC) restaura a sensibilidade à insulina e a tolerância à glucose num modelo animal de diabetes tipo 2. Para além disso, observou-se que estes efeitos são reversíveis após o término do estímulo elétrico de alta frequência. Assim, este trabalho suporta o potencial terapêutico da medicina bioelectrónica na diabetes tipo 2. Uma abordagem farmacológica poderá também ser utilizada para modular a atividade do CSN. Desta forma, no capítulo V foi estudado o papel do ATP e da adenosina na atividade do CSN no basal e em resposta à hipóxia. Observou-se que a frequência de descarga do CSN está aumentada num modelo animal de prediabetes, sendo este efeito modulado pelo ATP e pela adenosina. Tendo em conta que a adenosina contribui mais do que o ATP para gerar atividade do CSN na hipóxia moderada, enquanto que o ATP tem um papel preponderante durante a hipóxia intensa e, sabendo que hipóxias intensas são menos frequentes em situações fisiológicas, é sugerido que a modulação da sinalização do ATP no CB poderá ser um alvo terapêutico para o tratamento da diabetes tipo 2. Finalmente, no capítulo VI, é apresentada uma discussão geral e integrada da presente tese de Doutoramento. Em conclusão, os resultados apresentados neste trabalho contribuem para reforçar que a modulação do CB/CSN representa uma nova estratégia terapêutica na diabetes tipo 2.Type 2 diabetes (T2D) is one of the most common chronic diseases, whose prevalence continues to increase, being expected to affect 629 million people in the world in 2045. The principal defects in T2D are peripheral insulin resistance, abnormal hepatic glucose metabolism and progressive pancreatic beta cell failure. Despite the several different drugs available for T2D treatment, over time, glucose control deteriorates progressively and even with the rearrange of medication, a sizeable proportion of individuals remain poorly control. Therefore, is crucial the development of new therapeutic strategies to control this epidemic. In the last years, the carotid body (CB), a peripheral chemoreceptor that sense changes in blood O2, CO2 and pH, have also been described as a metabolic sensor implicated in the control of energy homeostasis. In fact, it was described that CB overactivity is involved in the genesis of insulin resistance and hypertension induced by the hypercaloric diets. The aims of the present work were to investigate the role of CB in the control of glucose homeostasis and to search a method/approach to modulate CB activity aiming to treat T2D. Chapter I introduces general concepts in T2D field, as the insulin signaling, glucose homeostasis and the therapeutic options for T2D treatment. Additionally, fundamental concepts of CB function and the role of ATP and adenosine in the CB neurotransmission, as well as, the role of CB as a metabolic sensor are also addressed. In chapter II are described the general and specific aims of the present work. In chapter III it was demonstrated that the carotid sinus nerve (CSN), the CB sensitive nerve, resection restores the insulin sensitivity in two prediabetes animal models, an effect that was maintained even when the animals were continuously fed hypercaloric diets. Moreover, it was also demonstrated that CSN resection normalized systemic sympathetic nervous system activity, blood pressure, endothelial function, lipid profile and plasma glucose and insulin levels. Additionally, the mechanism behind the repair of glucohomeostasis involves an improvement in glucose uptake in the liver and perienteric adipose tissue and a restored insulin signaling pathways in skeletal muscle and adipose tissue. In chapter IV it was demonstrated that the bioelectronic modulation of the CSN by using the kilohertz frequency alternating current (KHFAC) is capable to restore the insulin sensitivity and the glucose tolerance in a diet-induced early stage T2D animal model. Furthermore, it was also described that these effects were reversed after discontinuation of the electrical stimulus. This work support a potential role for bioelectronic medicines in the treatment of T2D. Another approach that could be used to modulate the CSN activity is a pharmacological approach. In chapter V, it was explored the role of ATP and adenosine on the basal and CSN chemosensory activity evoked by hypoxia. It was shown that the CSN frequency of discharge is overactivated in a prediabetes animal model, being this effect modulated by ATP and adenosine. Since adenosine contributes more than ATP to generate CSN activity in moderate hypoxia, while ATP shows a more preponderant role during intense hypoxia and knowing that intense hypoxias are less prone to occur, it is suggested that the modulation of ATP signaling in the CB could be a therapeutic target to treat T2D. Finally, in chapter VI, it is presented a general and integrated discussion of this PhD thesis. In conclusion, the data present in this work contribute to strengthen that the modulation of CB/CSN activity represents a novel therapeutic approach for T2D.Fundação para a Ciência e Tecnologia (FCT – Portugal)Fundo Social Europeu (FSE)Programa Operacional Capital Humano (POCH)Galvani Bioelectronics (former GlaxoSmithKline Bioelectronics R&D)Ministry of Economy and Competitiveness and the European Fund for Regional Development (MINECO/FEDER)Institute of Health Carlos III (Espanha

implications for metabolic diseases

Funding: Joana F. Sacramento and Fatima O. Martins are funded by contracts from the Portuguese Foundation for Science and Technology with reference CEEC IND/02428/2018 and CEECIND/04266/2017, respectivelyNeuro-immune communication has gained enormous interest in recent years due to increasing knowledge of the way in which the brain coordinates functional alterations in inflammatory and autoimmune responses, and the mechanisms of neuron-immune cell interactions in the context of metabolic diseases such as obesity and type 2 diabetes. In this review, we will explain how this relationship between the nervous and immune system impacts the pro- and anti-inflammatory pathways with specific reference to the hypothalamus-pituitary-adrenal gland axis and the vagal reflex and will explore the possible involvement of the carotid body (CB) in the neural control of inflammation. We will also highlight the mechanisms of vagal anti-inflammatory reflex control of immunity and metabolism, and the consequences of functional disarrangement of this reflex in settlement and development of metabolic diseases, with special attention to obesity and type 2 diabetes. Additionally, the role of CB in the interplay between metabolism and immune responses will be discussed, with specific reference to the different stimuli that promote CB activation and the balance between sympathetic and parasympathetic in this context. In doing so, we clarify the multivarious neuronal reflexes that coordinate tissue-specific responses (gut, pancreas, adipose tissue and liver) critical to metabolic control, and metabolic disease settlement and development. In the final section, we will summarize how electrical modulation of the carotid sinus nerve may be utilized to adjust these reflex responses and thus control inflammation and metabolic diseases, envisioning new therapeutics horizons.publishersversionpublishe

Synthesis of b-substituted alanines via Michael addition of nucleophiles to dehydroalanine derivatives

Several b-substituted alanines are synthesised in high yields by a Michael addition of nucleophiles to N-acyl,N-tert-butyloxycarbonyl-dehydroalanine methyl ester, using mild reaction conditions and simple work-up procedures. The same method can be applied to dipeptides containing dehydroalanine

Cafeína e resistência à insulina: qual o papel da adenosina?

O seguinte prémio: - Prémio Hargreaves 2012, Guarino MP, Ribeiro MJ, Sacramento JF, Antunes DD, Conde SV, Chronic caffeine intake reverses age-induced insulin resistance in the rat: effects on skeletal muscle glut4 expression and AMPK activity, Sociedade Portuguesa de Diabetologia (SPD) e Jaba Recordati.A diabetes do tipo 2 é um grave problema de saúde das sociedades ocidentais estando associado ao estilo de vida moderno. A cafeína é um antagonista não selectivo dos receptores de adenosina que quando administrada de forma aguda e crónica possui efeitos contrários na sensibilidade à insulina. Neste trabalho pretendeu-se investigar o(s) subtipo(s) de receptore(s) de adenosina que medeiam os efeitos da administração aguda e crónica de cafeína na sensibilidade à insulina. A administração aguda e crónica de diferentes antagonistas dos receptores de adenosina (cafeína; DPCPX, antagonista A1; SCH58261, antagonista A2A; MRS1754, antagonista A2B) foi testada em ratos Wistar controlo e submetidos a uma dieta rica em sacarose (HSu). Nestes animais foi avaliada a sensibilidade à insulina, a pressão arterial, glicémia basal, insulinémia, gordura visceral e a expressão de transportadores Glut4 e AMPK no músculo-esquelético. Observou-se que a administração aguda de cafeína diminuiu a sensibilidade à insulina de uma maneira dose-dependente, efeito este mediado pelos receptores A1 e A2B e, envolvendo um decréscimo dos Glut4 no músculo-esquelético. A administração aguda dos antagonistas dos receptores de adenosina não modificou a pressão arterial, a glicémia e insulinémia. Quanto à administração crónica dos diferentes antagonistas dos receptores de adenosina observou-se que: em animais controlo apenas o MRS1754 modificou a sensibilidade à insulina; nos animais submetidos a dieta HSu que o DPCPX e o MRS1754 aumentaram a sensibilidade à insulina, sendo o efeito deste último significativo; o MRS1754 reverteu a hipertensão nos animais HSu; o DPCPX reverteu o aumento de gordura visceral. A administração crónica de cafeína não alterou a expressão de Glut4 e fosforilação da AMPKα1Thr172 no músculo-esquelético. Em conclusão, o efeito da administração aguda de cafeína na sensibilidade à insulina é mediado pelos receptores A1 e A2B. Por sua vez, os efeitos da cafeína crónica na pressão arterial e na sensibilidade à insulina parecem ser mediados pelos receptores A2B, não se podendo excluir um efeito dos receptores A1 e/ou A2A na sensibilidade à insulina

High yielding synthesis of dehydroamino acid and dehydropeptide derivatives

By using a dimethylaminopyridine (DMAP) catalysed reaction of b-hydroxyamino acid derivatives with tert-butylpyrocarbonate [(Boc)2O], dehydroamino acid derivatives are obtained in high yields. The same methodology applied to dipeptides with a b-hydroxyamino acid residue gives the corresponding dipeptides with a dehydroamino acid residue

Exploring the mediators that promote carotid body dysfunction in type 2 diabetes and obesity related syndromes

Carotid bodies (CBs) are peripheral chemoreceptors that sense changes in blood O2, CO2, and pH levels. Apart from ventilatory control, these organs are deeply involved in the homeostatic regulation of carbohydrates and lipid metabolism and inflammation. It has been described that CB dysfunction is involved in the genesis of metabolic diseases and that CB overactivation is present in animal models of metabolic disease and in prediabetes patients. Additionally, resection of the CB-sensitive nerve, the carotid sinus nerve (CSN), or CB ablation in animals prevents and reverses diet-induced insulin resistance and glucose intolerance as well as sympathoadrenal overactivity, meaning that the beneficial effects of decreasing CB activity on glucose homeostasis are modulated by target-related efferent sympathetic nerves, through a reflex initiated in the CBs. In agreement with our pre-clinical data, hyperbaric oxygen therapy, which reduces CB activity, improves glucose homeostasis in type 2 diabetes patients. Insulin, leptin, and pro-inflammatory cytokines activate the CB. In this manuscript, we review in a concise manner the putative pathways linking CB chemoreceptor deregulation with the pathogenesis of metabolic diseases and discuss and present new data that highlight the roles of hyperinsulinemia, hyperleptinemia, and chronic inflammation as major factors contributing to CB dysfunction in metabolic disorders.publishersversionpublishe

Primary Effects on Skeletal Muscle

The present study was supported by the Portuguese Society of Diabetes. JS was supported by a PhD Grant from the Portuguese Foundation for Science and Technology, PD/BD/105890/2014.Epidemiological studies showed that chronic caffeine intake decreased the risk of type 2 diabetes. Previously, we described that chronic caffeine intake prevents and reverses insulin resistance induced by hypercaloric diets and aging, in rats. Caffeine has several cellular mechanisms of action, being the antagonism of adenosine receptors the only attained with human coffee consumption. Here, we investigated the subtypes of adenosine receptors involved on the effects of chronic caffeine intake on insulin sensitivity and the mechanisms and sex differences behind this effect. Experiments were performed in male and female Wistar rats fed either a chow or high-sucrose (HSu) diet (35% of sucrose in drinking water) during 28 days, to induce insulin resistance. In the last 15 days of diet the animals were submitted to DPCPX (A1 antagonist, 0.4 mg/kg), SCH58261 (A2A antagonist, 0.5 mg/kg), or MRS1754 (A2B antagonist, 9.5 μg/kg) administration. Insulin sensitivity, fasting glycaemia, blood pressure, catecholamines, and fat depots were assessed. Expression of A1, A2A, A2B adenosine receptors and protein involved in insulin signaling pathways were evaluated in the liver, skeletal muscle, and visceral adipose tissue. UCP1 expression was measured in adipose tissue. Paradoxically, SCH58261 and MRS1754 decreased insulin sensitivity in control animals, whereas they both improved insulin response in HSu diet animals. DPCPX did not alter significantly insulin sensitivity in control or HSu animals, but reversed the increase in total and visceral fat induced by the HSu diet. In skeletal muscle, A1, A2A, and A2B adenosine receptor expression were increased in HSu group, an effect that was restored by SCH58261 and MRS1754. In the liver, A1, A2A expression was increased in HSu group, while A2B expression was decreased, being this last effect reversed by administration of MRS1754. In adipose tissue, A1 and A2A block upregulated the expression of these receptors. A2 adenosine antagonists restored impaired insulin signaling in the skeletal muscle of HSu rats, but did not affect liver or adipose insulin signaling. Our results show that adenosine receptors exert opposite effects on insulin sensitivity, in control and insulin resistant states and strongly suggest that A2 adenosine receptors in the skeletal muscle are the majors responsible for whole-body insulin sensitivity.publishersversionpublishe

Clinical Implications for Carotid Body Neuromodulation

Funding Information: This study was partially supported by the GlaxoSmithKline Bioelectronics R&D ?Innovation Challenge. JS and BM were supported by a contract and a Ph.D. Grant from the Portuguese Foundation for Science and Technology Reference CEECIND/02428/2018 and PD/BD/128336/2017, respectively. This study received funding from the Fundação para a Ciência e Tecnologia (FCT) (UIDB/05704/2020).Chronic carotid sinus nerve (CSN) electrical modulation through kilohertz frequency alternating current improves metabolic control in rat models of type 2 diabetes, underpinning the potential of bioelectronic modulation of the CSN as a therapeutic modality for metabolic diseases in humans. The CSN carries sensory information from the carotid bodies, peripheral chemoreceptor organs that respond to changes in blood biochemical modifications such as hypoxia, hypercapnia, acidosis, and hyperinsulinemia. In addition, the CSN also delivers information from carotid sinus baroreceptors—mechanoreceptor sensory neurons directly involved in the control of blood pressure—to the central nervous system. The interaction between these powerful reflex systems—chemoreflex and baroreflex—whose sensory receptors are in anatomical proximity, may be regarded as a drawback to the development of selective bioelectronic tools to modulate the CSN. Herein we aimed to disclose CSN influence on cardiovascular regulation, particularly under hypoxic conditions, and we tested the hypothesis that neuromodulation of the CSN, either by electrical stimuli or surgical means, does not significantly impact blood pressure. Experiments were performed in Wistar rats aged 10–12 weeks. No significant effects of acute hypoxia were observed in systolic or diastolic blood pressure or heart rate although there was a significant activation of the cardiac sympathetic nervous system. We conclude that chemoreceptor activation by hypoxia leads to an expected increase in sympathetic activity accompanied by compensatory regional mechanisms that assure blood flow to regional beds and maintenance of hemodynamic homeostasis. Upon surgical denervation or electrical block of the CSN, the increase in cardiac sympathetic nervous system activity in response to hypoxia was lost, and there were no significant changes in blood pressure in comparison to control animals. We conclude that the responses to hypoxia and vasomotor control short-term regulation of blood pressure are dissociated in terms of hypoxic response but integrated to generate an effector response to a given change in arterial pressure.publishersversionpublishe

NEVOS RECORRENTES – UMA REVISÃO DE 5 ANOS

The development of a melanocytic lesion at the site where it had been previously excised can place clini- cally and histologically some problems because of the differential diagnosis with melanoma, named by some authors as 'pseudomelanoma. In this study, the authors intend to carry out a comparative analysis between the clinical and the pathologic features of recurrent nevi and primary melanocytic lesions.KEYWORDS – Nevus, Pigmented; Melanoma.A persistência de uma lesão melanocítica, em local onde tenha sido previamente excisado um nevo me- lanocítico pode colocar, clínica e histologicamente, problemas no diagnóstico diferencial com melanoma, designado por alguns autores como ‘pseudomelanoma’. Neste estudo, os autores pretendem realizar uma análise comparativa entre os achados clínicos e histopatológicos das lesões melanocíticas primárias e dos nevos recorrentes. Procura-se também avaliar eventuais factores predisponentes para este fenómeno.PALAVRAS-CHAVE – Nevo recorrente; Pseudomelanoma; Melanoma; Regressão

High fat diet blunts the effects of leptin on ventilation and on carotid body activity

Funding : This study was supported by the Portuguese Foundation for Science and Technology grant PTDC/SAU-ORG/111417/2009 to S.C. and by Grants BFU2015-70616R from MINECO-FEDER, Spain. J.F.S. and B.F.M. are supported by PhD Grants from FCT, PD/BD/105890/2014 and PD/BD/128336/2017, respectively.Leptin plays a role in the control of breathing, acting mainly on central nervous structures. Leptin receptor is expressed in the carotid body (CB) and this finding has been associated with a putative physiological role of leptin in the regulation of CB function. Since, the CBs are implicated in energy metabolism herein we tested the effects of different concentrations of leptin administration on ventilatory parameters and on carotid sinus nerve (CSN) activity in control and high-fat (HF) diet fed rats, in order to clarify the role of leptin in ventilation control in metabolic disease states. We also investigated the expression of leptin receptors and the neurotransmitters involved in leptin signalling in the CBs. We found that in non-disease conditions, leptin increases minute ventilation both in basal and hypoxic conditions. However, in the HF model, the effect of leptin in ventilatory control is blunted. We also observed that HF rats display an increased frequency of CSN discharge in basal conditions that is not altered by leptin, in contrast to what is observed in control animals. Leptin did not modify intracellular Ca2+ in CB chemoreceptor cells, but it produced an increase in the release of adenosine from the whole CB. We conclude that CBs represent an important target for leptin signalling, not only to coordinate peripheral ventilatory chemoreflexive drive, but probably also to modulate metabolic variables. We also concluded that leptin signalling is mediated by adenosine release and that HF diets blunt leptin responses in the CB, compromising ventilatory adaptation. This article is protected by copyright. All rights reserved.publishersversionpublishe