Investigating How Calcium Diffusion Affects Metabolic Oscillations and Synchronization of Pancreatic Beta Cells

Diabetes is a disease characterized by improper concentrations of blood glucose due to irregular insulin production or sensitivity. Coupled in islets of Langerhans within the pancreas, β-cells are responsible for the production and regulation of insulin based on changes in glucose levels. Using the Dual Oscillator Model (DOM), we will examine how calcium handling between individual pancreatic β-cells affects the synchronization of metabolic oscillations within electrically coupled islets. Calcium permeability was implemented into the DOM, and numerical solutions of the system were obtained via MATLAB using a modified ordinary differential equation solver for stiff systems and the Automatic Differentiation for MATLAB software. We developed a synchronization index to quantitatively describe the synchronization of variables between nearest neighboring cells and throughout the islet as a whole. We considered how calcium permeability between heterogeneous cells affects the behavior of metabolic oscillations and their synchronization. In particular, we examined fructose-1, 6-bisphosphate. In our study metabolic oscillations were always maintained. We also showed that, for low to moderate levels of electrical coupling, calcium permeability increased the synchronization index, but increasing calcium permeability had little effect on synchronization when cells were already strongly synchronized with strong electrical coupling. Heterogeneity due to glucose influx or initial state of the cells had similar synchronization results

Genetic Regulators Of Toxicity In A Mouse Model Of Amyotrophic Lateral Sclerosis And A Worm Model Of Anoxic Injury

The equilibrium between energy consumption and energy production defines the metabolic rate of an organism. This homeostatic balance is tightly regulated by a variety of sophisticated processes that occur within and between cells and tissues. These processes also allow animals to tolerate some deviation from baseline by recruiting adaptive mechanisms to bring cells and organisms back to homeostasis. Temporary changes in the organism’s environment, such as alterations in ambient temperature, oxygen levels and infections are examples of conditions where animals must have a healthful adaptive metabolic response, allowing them to sustain the duration of the stress. However, in conditions of chronic disease or long-term stress, these adaptive mechanisms can no longer be protective and may even contribute to the damage incurred by the animal. Therefore, metabolism can either be healthful and adaptive to stressors, or stressors can induce pathogenic metabolic changes in an organism. In this body of work, I explore this bidirectional relationship between external stresses and organismal metabolism. In chapter 2, I investigate the contribution of hypermetabolism (energy production \u3e energy consumption) in a mouse model of the disease amyotrophic lateral sclerosis (ALS). While hypermetabolism is a feature of ALS, it is not known if it contributes to disease pathogenesis. In a mouse model of ALS, I genetically induce hypometabolism to determine if this change alters disease progression. In chapter 3, I study the role of neuropeptides in regulating hypometabolic tolerance to extreme oxygen deprivation. Here, I employ C. elegans, a genetically tractable soil-dwelling nematode, as a model. Worms can tolerate long durations of anoxia by lowering their metabolic rate, and loss of neuropeptide signaling can further increase its ability to tolerate this stress. I investigate various aspects of neuropeptide regulation of this phenotype. Together, these projects demonstrate the role of metabolism in health, disease and stress, and suggest that inter-cellular and inter-tissue communication is a critical aspect of metabolic homeostasis

Regulation of Murine Physiology and Metabolism by the Adipocyte Circadian Clock through Sphingosine Kinase 1

The adipocyte, widely known for its large lipid droplets, is a crucial signaling cell at the crux of metabolism. Programs within the adipocyte itself may have massive impacts upon the body. For example, the internal cell-autonomous circadian clock within the adipocyte regulates such lipid droplet-associated processes as lipolysis (triacylglycerol breakdown) and lipogenesis (lipid uptake, triacylglycerol synthesis), which influence circulating lipids, thus impacting other organs. Circadian rhythms (molecular, metabolic, and behavioral patterning over a day) and lipid droplets are both highly evolutionarily conserved biological phenomena that also happen to be intertwined in the system about which we care today, the global human obesity pandemic. Excessive adiposity is a hallmark of obesity, and humans live in an extraordinarily artificially-lit world, which serves to disrupt circadian clocks, thereby disrupting metabolism. Characterized by their unique sphingosine backbone, sphingolipids, which may derive, in part, from the excess of adiposity and lipid free fatty acids, are well known to affect metabolic disease phenotypes. As the Cowart lab has shown in the past, and as shown in this work, sphingosine kinase 1 (SPHK1) and its enzymatic product sphingosine-1-phosphate (S1P) affect cellular signaling pathways that directly influence metabolism. SPHK1 is a lipid kinase which phosphorylates sphingosine (an N-acylated amino alcohol) to S1P, a bioactive autocrine and paracrine signaling lipid, to influence various processes including cellular survival, angiogenesis, endothelial permeability, immune cell trafficking, and oncogenesis. Since SPHK1 is highly involved in metabolism, such as glucose metabolism, and since metabolism is a circadian-governed process, we hypothesized that SPHK1 is involved in the regulation of the adipocyte circadian clock, which may impact circulating metabolites, such as glucose, fatty acids, and adipokines, thereby affecting weight gain and glucose tolerance. Using a wide variety of techniques and methodologies including RNA sequencing, mass spectrometry coupled with lipidomics and proteomics, chromatin immunoprecipitation (ChIP), protein co-immunoprecipitation, microscopy, gene and protein expression analysis, we conclude that novel properties of SPHK1 and S1P elicit changes in nuclear chromatin remodeling events, leading to circadian clock disruption and subsequent impairment of key metabolic physiological parameters in the mouse. To our knowledge, his is the first report of a lipid metabolite directly affecting the circadian clock

Linking dietary intake, circadian biomarkers, and clock genes on obesity: A study protocol

BackgroundThe prevalence of obesity continues to rise, and although this is a complex disease, the screening is made simply with the value of the Body Mass Index. This index only considers weight and height, being limited in portraying the multiple existing obesity phenotypes. The characterization of the chronotype and circadian system as an innovative phenotype of a patient's form of obesity is gaining increasing importance for the development of novel and pinpointed nutritional interventions. ObjectiveThe present study is a prospective observational controlled study conducted in Portugal, aiming to characterize the chronotype and determine its relation to the phenotype and dietary patterns of patients with obesity and healthy participants. MethodsAdults with obesity (study group) and healthy adults (control group), aged between 18 and 75, will be enrolled in this study. Data will be collected to characterize the chronotype, dietary intake, and sleep quality through validated questionnaires. Body composition will also be assessed, and blood samples will be collected to quantify circadian and metabolic biomarkers. DiscussionThis study is expected to contribute to a better understanding of the impact of obesity and dietary intake on circadian biomarkers and, therefore, increase scientific evidence to help future therapeutic interventions based on chronobiology, with a particular focus on nutritional interventions

White adipose tissue and circadian rhythm dysfunctions in obesity : Pathogenesis and available therapies

A combined neuroendocrine, metabolic, and chronobiological view can help to better understand the multiple and complex mechanisms involved in obesity development and maintenance, as well as to provide new effective approaches for its control and treatment. Indeed, we have currently updated data on the whole adipogenic process involved in white adipose tissue (WAT) mass expansion, namely due to a mechanism whereby WAT cells become hypertrophic, thus inducing a serious local (WAT) inflammatory condition that in turn, will impair not only the cross-talk between the hypothalamus and the WAT, but also favoring the development of deep and widespread neuroendocrine-metabolic dysfunction. Moreover, we also have revisited the circadian clock genes involved in dysfunctional WAT mass expansion and the mechanisms that may lead to obesity development, including early metabolic dysfunctions, enhanced oxidative stress and distorted energy homeostasis. The epigenetic changes of clock genes driving metabolic disease and obesity development have also been included in this review. Finally, we have also underlined the relevance of metabolic homeostasis regulation by central and peripheral organ clocks, sleep disturbances, nutrients, and feeding time, as key factors in obesity development as well as both, classical and chronotherapeutic approaches for its prevention and treatment.Centro de Endocrinología Experimental y Aplicad

Blood Glucose Levels

The main source of energy for the body is glucose. Its low blood concentrations can cause seizures, loss of consciousness and death. Long lasting high glucose levels can cause blindness, renal failure, cardiac and peripheral vascular disease, and neuropathy. Blood glucose concentrations need to be maintained within narrow limits. The process of maintaining blood glucose at a steady state is called glucose homeostasis. This is achieved through a balance of the rate of consumption of dietary carbohydrates, utilization of glucose by peripheral tissues, and the loss of glucose through the kidney tubule. The liver and kidney also play a role in glucose homeostasis. This book aims to provide an overview of blood glucose levels in health and diseases

The role of GPR120 in diet induced obesity, mood dysregulation, and microglial function

L'obésité est un facteur de risque majeur pour le développement de maladies psychiatriques, telles que le trouble dépressif majeur et la schizophrénie. Une consommation excessive de graisses saturées est bien connue pour provoquer non seulement des troubles métaboliques, mais également des comportements anormaux chez les modèles animaux et les humains. Les acides gras saturés augmentent l'inflammation dans divers tissus. Plusieurs études ont démontré que l'activation de la microglie en tant qu'acteur central de la neuroinflammation joue un rôle crucial dans l'anxiété liée à l'inflammation et les comportements dépressifs dans les cas d'obésité. En outre, il existe de plus en plus de preuves démontrant l'effet bénéfique de la supplémentation en acides gras polyinsaturés n-3 (AGPI n-3) sur les comportements cognitifs, anxieux et dépressifs. Nous avons précédemment montré que la supplémentation en AGPI n-3 via l'administration d'huile de poisson (FO) a des effets anxiolytiques sur les souris rendues obèses par une diète riche en gras saturés (SHFD). De plus, l'activation du récepteur couplé aux protéines G 120 (GPR120), un récepteur des AGPI n-3, dans le cerveau, attenue l'anxiété et les comportements dépressifs induits par la SHFD. Cependant, les mécanismes par lesquels GPR120 régule les changements induits par la SHFD dans les comportements liés à l'humeur ne sont toujours pas compris. Dans la présente étude, nous avons étudié le rôle du GPR120 dans les troubles anxieux et dépressifs reliés à la neuroinflammation. Parmi plusieurs types de cellules neurales, la microglie exprime fortement GPR120. Un agoniste de GPR120 (Compound A; CpdA) réduit la production et la libération de cytokines inflammatoires (IL-1B, IL-6, MCP1 et TNF-α) induites par les lipopolysaccharides (LPS) dans la microglie en culture. D'autre part, l'administration centrale de CpdA par injection intracérébroventriculaire (ICV) améliore le comportement anxieux et de malaise suite à l’injection de LPS systémique in vivo. De plus, l'acide eicosapentaénoique (EPA) et l'acide docosahexaénoique (DHA) sont des AGPI n-3 et des agonistes naturels du GPR120. L'EPA et le DHA suppriment l'inflammation dans un modèle de culture de microglies primaires, telle qu'évaluée par l'expression et la sécrétion de cytokines. Ces résultats suggèrent que l'activation du GPR120 contribue à l'amélioration de l'anxiété et des comportements de type dépressif liés à l'inflammation grâce à la régulation de la microglie.Obesity is a major risk factor for the development of psychiatric diseases, such as major depressive disorder and anxiety. Excess intake of saturated fat is well known to cause not only metabolic diseases, but also abnormal behavior in humans and animal models. Saturated fatty acids enhance peripheral and central inflammation. Several studies have demonstrated that microglia activation as a central player in neuroinflammation plays a crucial role in inflammation-related anxiety and depressive-like behaviors in the case of obesity. Also, there is increasing evidence to demonstrate the beneficial effect of n-3 PUFAs supplementation on cognitive, anxiety and depressive-like behaviors. Previously, we reported that the n-3 PUFAs supplementation by the administration of fish oil (FO) has demonstrated anxiolytic effects on saturated high fat diet (SHFD)-induced obese mice and that, the activation of G-protein coupled receptor 120 (GPR120), a lipid sensor for n-3 poly-unsaturated fatty acids (n-3 PUFAs), in the brain rescued SHFD-induced anxiety and depressive-like behaviors. However, it is still unclear how GPR120 regulates SHFD-induced changes in mood-related behaviors. In the present study, we focused on the role of GPR120 on neuroinflammation in inflammation-related anxiety and depressive-like behaviors. Amongst the several types of brain cells, microglia demonstrated a high expression of GPR120. Compound A (CpdA), a selective agonist of GPR120, reduced lipopolysaccharide (LPS)-induced inflammatory cytokine (IL-1B, IL-6, MCP1, and TNF-a) expression and release in cultured microglia. Additionally, central administration of cpdA via intracerebroventricular (ICV) injection ameliorated neuroinflammation and systemic LPS injection-induced anxiety-like and sickness behavior in vivo. Furthermore, n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as natural agonists of GPR120 suppressed inflammation in primary cultured microglia as assessed by cytokine expression. These results suggest that GPR120 activation contributes to the amelioration of inflammation-related anxiety and depressive-like behaviors through the regulation of microglia

Carboxymethyl cellulose-based cryogels as scaffolds for pancreatic and skeletal muscle tissue engineering

[eng] Diabetes incidence highly increased in the last years. According to IDF (International Diabetes Federation), 463 million people suffered this disease in 2019. The estimations of diabetic people highly increase in the upcoming years, rising approximately to 700 million diabetic patients in 2045 [1]. Type 2 diabetes (T2D) is the most common type of diabetes, representing 90% of diabetic patients. It occurs when the body becomes resistant to insulin. Body insulin resistance confirms that T2D is not only a pancreatic disease, as there are many other tissues involved, like liver, adipose tissue, or skeletal muscle. This last has a significant implication in glucose-insulin homeostasis as it is one of the main glucose-consuming organs in the body. Nowadays, to study how two tissues crosstalk between them, animal testing is the gold standard. However, the unmatching physiological behaviors compared to humans, the variability between different animals, ethical dilemmas, and the need to go for more personalized medicine activates the search for other suitable alternatives. At this point, Organs-on-a-chip appeared as a valid alternative. Organs-on-a-chip (OOC) are 3D bioengineered microfluidic cell culture platforms to simulate microphysological environments of an organ or its specific functions. Nowadays, to engineer the tissues for OOC applications, encapsulating cells inside hydrogels is the most common technique. Its beneficial properties include high water content, mechanical adjustability, and moldability to generate the desired architectures [2]. However, its small porosity limits nutrient and oxygen diffusion through it [3]. This problem is a significant limitation when pancreatic islets are encapsulated inside hydrogels due to their size (~100 μm of diameter). Pancreatic islets are cell aggregations composed of many different cells as insulin-secreting cells (Beta-cells) or glucagon-secreting cells (alpha-cells). Similarly, skeletal muscle tissue is generally encapsulated in small bundles. Skeletal muscle is a highly aligned and multinucleated tissue formed from the fusion of single cells, called myoblasts, into multinucleated cells, called myotubes. Cryogels have been proposed as a valid alternative to overcome these limitations. Cryogels are fabricated by crosslinking a prepolymer solution at sub-zero temperatures, so while the material crosslinks, water freezes, generating the desired micropore architecture. After thawing, cryogels are sponge-like scaffolds with microporous structure, high interconnected porosity, high diffusivity, fine-tuned properties, and desired internal pore architecture. This thesis developed two cryogel scaffolds made of gelatin and carboxymethylcellulose with different pore architectures to engineer pancreatic and skeletal muscle tissues. Here, we proved that the achieved pore architecture fits with the prerequisites to engineer each tissue. Moreover, the mechanical and physical properties of each scaffold highly resemble the 3D microenvironment of each tissue. In pancreatic tissue, we generate a random pore cryogel to aggregate beta-cells to form pseudoislets. We proved that these engineered pseudoislets are viable, functional responding correctly to the glucose and improving insulin response compared to monolayer results. In the skeletal muscle approach, we could develop a highly aligned pore architecture to prompt cell alignment and cell fusion. Moreover, we incorporate carbon nanotubes to enhance the electrical conductivity of the scaffold, so by applying electrical pulse stimulation, we could improve the early steps of the myogenic maturation.[cat] La incidència de la diabetis ha augmentat considerablement en els últims anys. Segons l’IDF (International Diabetes Federation), al 2019 hi havia 463 milions de persones que patien diabetis i les estimacions estimen un augment considerable de casos, arribant als 700 milions de persones diabètiques cap al 2045 [1]. Entre els diferents tipus de diabetis, la diabetis tipus 2, és la que té major incidència en la població, corresponent al 90% dels casos de pacients amb diabetis. Aquest tipus de diabetis, succeeix quan el cos es torna resistent a la insulina. Aquesta resistència a la insulina per part dels teixits perifèrics ens prova que la diabetis no és només una malaltia del pàncreas, sinó que hi ha altres teixits relacionats, com el fetge, el teixit adipós o el múscul esquelètic. Aquest últim té un factor molt rellevant en la homeòstasi de la insulina i la glucosa, ja que és un dels principals teixits consumidors de glucosa. La interacció, però, entre aquest dos teixits encara presenta molts interrogants. Actualment, per estudiar com dos teixits interactuen entre ells, el testeig animal és el mètode més confiable. No obstant, presenta certes limitacions, com la poca similitud en quan a l’activitat dels illots, la variabilitat fisiològica entre diferents animals, dilemes ètics o la necessitat d’encarar la recerca cap a una medicina més personalitzada. Aquesta finalitat és el que ha portat als científics a buscar alternatives a l’experimentació animal. Entre moltes, una de les més prometedores són els anomenats Òrgans-en-un-xip, plataformes 3D de cultiu cel·lular combinades amb microfluídica i biomaterials que permeten simular les funcions específiques d’un òrgan. Per tal de generar el teixit dins d’aquesta plataforma, l’encapsulació de cèl·lules dins de hidrogels és la tècnica més utilitzada, degut al seu alt contingut d’aigua, la seva adaptabilitat mecànica o la possibilitat de generar una certa estructura geomètrica [2]. No obstant, la seva petita porositat, limita la difusió homogènia d’oxigen i de nutrients dins seu [3]. Aquest problema creix quan es volen encapsular illots pancreàtics en bastides d’hidrogel, degut a la seva mida (~100 μm de diàmetre). Els illots pancreàtics són agregacions de varis tipus diferent de cèl·lules, on destaquen les cèl·lules secretores de insulina (cèl·lules beta) i les secretores de glucagó (cèl·lules alfa). Per altre costat, el teixit muscular s’encapsula en petits constructes per tal d’imitar l’estructura d’aquest. El múscul esquelètic és un teixit altament alineat, amb cèl·lules multi nucleades, anomenades miotubs, que s’obtenen a partir de la fusió de cèl·lules soles, anomenades mioblasts. Per tal de solucionar aquests problemes, els criogels han aparegut com a alternativa. Els criogels, estan fabricats a temperatures sota zero, així mentres el polímer crosslinca es formen cristalls de gel. Un cop formada la matriu, la bastida es descongela i aquests cristalls es desfaran, deixant pas a espais buits, anomenats pors. Aquests, seran els que posteriorment li donaran la l’estructura porosa, altament interconnectada, amb alta permeabilitat i amb una arquitectura de pors determinada a la nostra bestida. En aquesta tesi s’han desenvolupat dos bastides de cel·lulosa carboxymetilada diferents seguint la tècnica de la criogelificació. Cada bastida ha estat dissenyada per tenir una distribució i una arquitectura de pors diferent d’acord amb la necessitat i propietat del teixit que es vulgui generar. A més, les propietats físiques i mecàniques de les dos bastides tenen alta semblança amb les propietats físiques i mecàniques de la matriu extracel·lular de cada teixit. Per el teixit pancreàtic, s’ha generat una bastida amb un diàmetre de pors similar als illots pancreàtics, per tal que, sembrant cèl·lules beta, aquestes formin pseudoillots similars als illots fisiològics. A més, s’ha demostrat que aquests illots tenen el diàmetre i la arquitectura desitjada, són viables i capaços de respondre a diferents nivells de glucosa. A més, s’ha demostrat que aquestes cèl·lules agregades en forma de pseudoillots responen millor a la glucosa que les cèl·lules configurades en distribució dispersa. En el cas del múscul esquelètic, s’ha desenvolupat una bastida amb una arquitectura de pors altament alineada per promoure l’alineament cel·lular i la fusió cel·lular. A més, s’han pogut incorporar nanotubs de carboni per millorar les propietats elèctriques de la vestida. D’aquesta manera, aplicant pulsos elèctrics per estimular el teixit, s’han pogut millorar les etapes primerenques de la maduració miogènica