7,255 research outputs found

    Principles of generalization for sensorimotor cerebellar learning

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    Adaptations in physiological and neuronal function during diet-induced obesity

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    Obesity significantly increases the risk of developing chronic conditions including type II diabetes, cardiovascular disease, and some cancers. The rate of obesity has tripled globally since 1975, which is in part due to the sudden prevalence and overconsumption of palatable high-fat diets (HFDs). Obesity profoundly perturbs the neural control of energy balance, affecting diverse cell types within the hypothalamus. However, an incomplete understanding of how HFD impacts the regulation of energy balance hinders our ability to more effectively treat obesity. In this thesis, I describe the physiological and neuronal response to HFD feeding in rodents. We identified that HFD exposure elevates the body weight set point, which is initially driven by a transient hyperphagia. This hyperphagia coincides with increased excitatory transmission to lateral hypothalamic orexin (ORX) neurons, which regulate acute food intake. This suggests that ORX neurons may be involved in the initial hyperphagia, implicating them in the development of obesity. As HFD prolongs, body weight gain slows and reaches a new steady state regardless of age at the start, duration of feeding, or palatability of the diet. This sustained weight coincides with increased synaptic contacts to melanin-concentrating hormone (MCH) neurons, which promote weight gain and food intake, likely contributing to the maintenance of obesity. The molecular mechanism underlying the establishment of a new set point remains elusive. During HFD feeding, the presence of a chronic low-grade hypothalamic inflammation exacerbates weight gain, therefore we reasoned that inflammatory factors could modulate appetite-promoting neurons to maintain a new set point. We found that the inflammatory mediator prostaglandin E2 (PGE2) activate MCH neurons via its EP2 receptor (EP2R). Suppressing PGE2-EP2R on MCH neurons partially protects against excess weight gain and fat accumulation in the liver during HFD feeding. This mechanism could contribute to the maintenance of an elevated body weight set point in during diet-induced obesity. Without long-term treatment options in face of the increasing rates of obesity, we are in desperate need of novel interventions. In the future, we hope that targeting EP2R on MCH neurons can lower body weight set point and aid in combatting obesity

    Principles of generalization for sensorimotor cerebellar learning

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    The KINGS mouse as a model of beta cell endoplasmic reticulum (ER) stress and sex differences in diabetes.

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    Background: The KINGS mouse is a novel model of beta cell endoplasmic reticulum (ER) stress which shows stark sex differences in diabetes, with males developing overt and progressive hyperglycaemia whilst females are protected. Beta cell ER stress has been implicated in many types of diabetes and underpins numerous factors known to drive beta cell failure. Sex differences also exist in diabetes in humans with premenopausal women having a lower diabetes incidence compared to men. Further characterisation of the KINGS mice may provide valuable insight into these phenomena. Aims: The objectives of this thesis were to 1) further characterise beta cell ER stress and associated cellular response in the KINGS mice, 2) investigate the influence of sex hormones and beta cell ER stress manipulation on glycaemic control in the KINGS mice and 3) investigate whether diabetes development can be prevented in the male KINGS mice. Methods: Western blotting and immunofluorescent staining were used to investigate the expression of ER stress and unfolded protein response (UPR) markers in KINGS islets, as well as beta cell turnover and mass. To determine the influence of oestradiol on the KINGS phenotype, endogenous oestradiol was removed from female mice via ovariectomy, and exogenous oestradiol was delivered to male KINGS mice through implantation of oestradiol- containing capsules. A western diet was used to exacerbate beta cell ER stress in female KINGS mice, whilst liraglutide administration, TUDCA administration and removal of endogenous testosterone (via orchidectomy) was used in an attempt to reduce ER stress and prevent diabetes in the male KINGS mice. For all in vivo studies, glycaemic control was assessed through blood glucose concentration monitoring, glucose tolerance testing and insulin tolerance testing. Results: Male KINGS mice developed diabetes by 5-6 weeks of age whereas female KINGS mice were protected, in line with previous studies. Protein markers of ER stress and the UPR were observed in KINGS islets from 4 weeks of age and a sex difference was observed in expression profiles with males largely showing an increased expression of markers. Despite this, we did not observe a loss of beta cell mass in either male or female KINGS mice. However, subtle changes in beta cell proliferation and apoptosis in the male KINGS mice are suggestive of mild changes to beta cell turnover which may contribute to diabetes development. A western diet exacerbated beta cell ER stress in female KINGS mice, however this only led to a mild impairment in glycaemic control which was not as severe as that seen in male KINGS mice. This may suggest that even under conditions of further ER stress, female mice are still able to respond adaptively. Removal of endogenous oestradiol also exacerbated beta cell ER stress, however again this was only associated with a subtle impairment in glycaemic control. On the contrary, exogenous oestradiol delivery in the male KINGS mice prevented the development of overt diabetes. Treatment with liraglutide was used in an attempt to alleviate ER stress in the male KINGS mice. Although liraglutide prevented the development of diabetes and reduced blood glucose concentrations once diabetes was established, this protection only lasted during the treatment window and cessation of treatment was associated with increases in blood glucose concentrations. In addition, liraglutide had no effect on beta cell ER stress levels. Treatment with TUDCA, a chemical chaperone previously found to reduce beta cell ER stress, had no impact on blood glucose concentrations in the KINGS mice. However, removal of endogenous testosterone through orchidectomy prevented the development of overt diabetes. Conclusion: In this study we have confirmed that the KINGS mutation drives beta cell ER stress and that sex differences exist in beta cell response to this. Interestingly, an adaptive response to beta cell ER stress was still maintained in female KINGS mice when ER stress was exacerbated through a western diet. We also found that whilst oestradiol likely contributes in-part to sex differences in diabetes, it cannot be the sole mediator and other factors must be involved. Indeed, we found that endogenous testosterone removal prevented the development of diabetes in male mice. Liraglutide treatment also prevented diabetes development in male mice, however this was likely to be mediated through mechanisms unrelated to beta cell ER stress. Further study is required to investigate how testosterone removal and liraglutide protect male mice.</div

    Adolescent intermittent ethanol (AIE) produces lasting, sex-specific changes in rat body fat independent of changes in white blood cell composition

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    Early initiation of alcohol use during adolescence, and adolescent binge drinking are risk factors for the development of alcohol use disorder later in life. Adolescence is a time of rapid sex-dependent neural, physiological, and behavioral changes as well as a period of heightened vulnerability to many effects of alcohol. The goal of the present studies was to determine age-related changes in blood (leukocyte populations) and body composition across adolescence and early adulthood, and to investigate whether adolescent intermittent ethanol (AIE) exposure would alter the trajectory of adolescent development on these broad physiological parameters. We observed significant ontogenetic changes in leukocyte populations that were mirrored by an age-related increase in cytokine expression among mixed populations of circulating leukocytes. Despite these developmental changes, AIE did not significantly alter overall leukocyte numbers or cytokine gene expression. However, AIE led to sex-specific changes in body fat mass and fat percentage, with AIE-exposed male rats showing significantly decreased fat levels and female rats showing significantly increased fat levels relative to controls. These changes suggest that while AIE may not alter overall leukocyte levels, more complex phenotypic changes in leukocyte populations could underlie previously reported differences in cytokine expression. Coupled with long-term shifts in adipocyte levels, this could have long-lasting effects on innate immunity and the capacity of individuals to respond to later immunological and physiological threats

    Identifying alterations in adipose tissue-derived islet GPCR peptide ligand mRNAs in obesity: implications for islet function

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    In addition to acting as an energy reservoir, white adipose tissue is a vital endocrine organ involved in the modulation of cellular function and the maintenance of metabolic homeostasis through the synthesis and secretion of peptides, known as adipokines. It is known that some of these secretory peptides play important regulatory roles in glycaemic control by acting directly on islet β-cells or on insulin-sensitive tissues. Excess adiposity causes alterations in the circulating levels of some adipokines which, depending on their mode of action, can have pro-inflammatory, pro-diabetic or anti-inflammatory, anti-diabetic properties. Some adipokines that are known to act at β-cells have actions that are transduced by binding to G protein- coupled receptors (GPCRs). This large family of receptors represents ~35% of all current drug targets for the treatment of a wide range of diseases, including type 2 diabetes (T2D). Islets express ~300 GPCRs, yet only one islet GPCR is currently directly targeted for T2D treatment. This deficit represents a therapeutic gap that could be filled by the identification of adipose tissue-derived islet GPCR peptide ligands that increase insulin secretion and overall β-cell function. Thus, by defining their mechanisms of action, there is potential for the development of new pharmacotherapies for T2D. Therefore, this thesis describes experiments which aimed to compare the expression profiles of adipose tissue-derived islet GPCR peptide ligand mRNAs under lean and obese conditions, and to characterise the functional effects of a selected candidate of interest on islet cells. Visceral fat depots were retrieved from high-fat diet-induced and genetically obese mouse models, and from human participants. Fat pads were either processed as whole tissue, or mature adipocyte cells were separated from the stromal vascular fraction (SVF) which contains several other cell populations, including preadipocytes and macrophages. The expression levels of 155 islet GPCR peptide ligand mRNAs in whole adipose tissue or in isolated mature adipocytes were quantified using optimised RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols. Comparisons between lean and obese states in mice models and humans revealed significant modifications in the expression levels of several adipokine mRNAs. As expected, mRNAs encoding the positive control genes, Lep and AdipoQ were quantifiable, with the expression of Lep mRNA increasing and that of AdipoQ mRNA decreasing in obesity. Expression of Ccl4 mRNA, encoding chemokine (C-C motif) ligand 4, was significantly upregulated in whole adipose tissue across all models of obesity compared to their lean counterparts. This coincided with elevated circulating Ccl4 peptide levels. This increase was not replicated in isolated mature adipocytes, indicating that the source of upregulated Ccl4 expression in obesity was the SVF of adipose tissue. Based on this significant increase in Ccl4 mRNA expression within visceral fat and its undetermined effects on β-cell function, Ccl4 was selected for further investigation in MIN6 β-cells and mouse islets. PRESTO-Tango β-arrestin reporter assays were performed to determine which GPCRs were activated by exogenous Ccl4. Experiments using HTLA cells expressing a protease-tagged β- arrestin and transfected with GPCR plasmids of interest indicated that 100ng/mL Ccl4 significantly activated Cxcr1 and Cxcr5, but it was not an agonist at the previously identified Ccl4-target GPCRs Ccr1, Ccr2, Ccr5, Ccr9 and Ackr2. RNA extraction and RT-qPCR experiments using MIN6 β-cells and primary islets from lean mice revealed the expression of Cxcr5 mRNA in mouse islets, but it was absent in MIN6 β-cells. The remaining putative Ccl4 receptors (Ccr1, Ccr2, Ccr5, Ccr9, Cxcr1 and Ackr2) were either absent or present at trace levels in mouse islets and MIN6 β-cells. Recombinant mouse Ccl4 protein was used for functional experiments at concentrations of 5, 10, 50 and 100ng/mL, based on previous reports of biological activities at these concentrations. Trypan blue exclusion testing was initially performed to assess the effect of exogenous Ccl4 on MIN6 β-cell viability and these experiments indicated that all concentrations (5-100ng/mL) were well-tolerated. Since β-cells have a low basal rate of apoptosis, cell death was induced by exposure to the saturated free fatty acid, palmitate, or by a cocktail of pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ). In MIN6 β-cells, Ccl4 demonstrated concentration-dependent protective effects against palmitate-induced and cytokine-induced apoptosis. Conversely, while palmitate and cytokines also increased apoptosis of mouse islets, Ccl4 did not protect islets from either inducer. Quantification of bromodeoxyuridine (BrdU) incorporation into β-cell DNA indicated that Ccl4 caused a concentration-dependent reduction in proliferation of MIN6 β-cells in response to 10% fetal bovine serum (FBS). In contrast, immunohistochemical quantification of Ki67-positive mouse islet β-cells showed no differences in β-cell proliferation between control- and Ccl4-treated islets. Whilst the number of β-cells and δ-cells were unaffected, α- cells were significantly depleted by Ccl4 treatment. Exogenous Ccl4 had no effect on nutrient- stimulated insulin secretion from both MIN6 β-cells and primary mouse islets. The 3T3-L1 preadipocyte cell line was used to assess potential Ccl4-mediated paracrine and/or autocrine signalling within adipose tissue. Ccl4 did not alter the mRNA expression of Pparγ, a master regulator of adipocyte differentiation, but did significantly downregulate the mRNA expression of the crucial adipogenic gene, adiponectin. Oil Red O staining and Western blotting were performed to assess lipid accumulation, and insulin and lipolytic signalling, respectively, and these experiments indicated that the observed Ccl4-induced decrease in adiponectin expression failed to correlate with any changes in adipocyte function. In summary, these data demonstrated anti-apoptotic and anti-proliferative actions of the adipokine, Ccl4, on MIN6 β-cells that were not replicated in mouse islets. The absence of any anti-apoptotic, insulin secretory and/or pro-proliferative effects of Ccl4 in islet β-cells suggests that it is unlikely to play a role in regulating β-cell function via crosstalk between adipose tissue and islets. The divergent functional effects highlight that whilst MIN6 cells are a useful primary β-cell surrogate for some studies, primary islets should always be used to confirm physiological relevance. On the other hand, significant α-cell depletion following Ccl4 treatment suggests a cell-specific function within the islets. Furthermore, Ccl4 impaired adiponectin mRNA expression in adipocytes, although, how adipocyte function is affected as a result requires further investigation. Collectively, these data have contributed increased understanding of the role of obesity in modifying the expression of adipose tissue-derived islet GPCR peptide ligands

    A novel rhein-huprine hybrid ameliorates disease-modifying properties in preclinical mice model of Alzheimer's disease exacerbated with high fat diet

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    Background: Alzheimer's disease (AD) is characterized by a polyetiological origin. Despite the global burden of AD and the advances made in AD drug research and development, the cure of the disease remains elusive, since any developed drug has demonstrated effectiveness to cure AD. Strikingly, an increasing number of studies indicate a linkage between AD and type 2 diabetes mellitus (T2DM), as both diseases share some common pathophysiological features. In fact, β-secretase (BACE1) and acetylcholinesterase (AChE), two enzymes involved in both conditions, have been considered promising targets for both pathologies. In this regard, due to the multifactorial origin of these diseases, current research efforts are focusing on the development of multi-target drugs as a very promising option to derive effective treatments for both conditions. In the present study, we evaluated the effect of rhein-huprine hybrid (RHE-HUP), a synthesized BACE1 and AChE inhibitor, both considered key factors not only in AD but also in metabolic pathologies. Thus, the aim of this study is to evaluate the effects of this compound in APP/PS1 female mice, a well-established familial AD mouse model, challenged by high-fat diet (HFD) consumption to concomitantly simulate a T2DM-like condition. Results: Intraperitoneal treatment with RHE-HUP in APP/PS1 mice for 4 weeks reduced the main hallmarks of AD, including Tau hyperphosphorylation, Aβ42 peptide levels and plaque formation. Moreover, we found a decreased inflammatory response together with an increase in different synaptic proteins, such as drebrin 1 (DBN1) or synaptophysin, and in neurotrophic factors, especially in BDNF levels, correlated with a recovery in the number of dendritic spines, which resulted in memory improvement. Notably, the improvement observed in this model can be attributed directly to a protein regulation at central level, since no peripheral modification of those alterations induced by HFD consumption was observed. Conclusions: Our results suggest that RHE-HUP could be a new candidate for the treatment of AD, even for individuals with high risk due to peripheral metabolic disturbances, given its multi-target profile which allows for the improvement of some of the most important hallmarks of the disease

    Immunological mechanisms in the pathogenesis of type 1 diabetes

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    Type 1 diabetes is a chronic autoimmune disease, in which insulin secreting beta cells in the pancreas are destroyed. The disease pathogenesis can be divided into an asymptomatic preclinical phase and symptomatic disease. The preclinical phase is characterised by the appearance of autoantibodies targeting pancreatic islet antigens and clinical onset of disease happens after the amount of functional beta cells becomes too low to sustain glucose homeostasis. While several genetic and environmental type 1 diabetes risk factors have been identified, the exact mechanism of the autoimmune process leading to the disease remains unknown. Additionally, strong heterogeneity in the disease pathogenesis has been observed and different possible pathways, or endotypes, to type 1 diabetes are suspected. The focus of this thesis was to investigate factors leading to heterogeneity in type 1 diabetes pathogenesis. Several type 1 diabetes predisposing genetic polymorphisms, including the loci NRP1, INS, UBASH3A and STAT4, were found to associate with specific phases of disease pathogenesis. Moreover, other disease risk polymorphisms, like PTPN22 and INS, associated significantly with suspected type 1 diabetes endotypes defined through the first appearing islet autoantibody. The autoimmune risk variant of PTPN22 was also associated with elevated total and naïve regulatory T cell frequencies. No gene expression differences could be detected in individual genes between children positive for multiple type 1 diabetes associated autoantibodies and their healthy controls in monocytes and monocyte-depleted peripheral blood mononuclear cells. However, gene sets relating to viral responses and a type I interferon response were upregulated in monocytes of multiple autoantibody positive children, compared to healthy controls. These data lend support to heterogeneity of type 1 diabetes with multiple possible pathways to disease onset.Immunologiset mekanismit tyypin 1 diabeteksen patogeneesissa Tyypin 1 diabetes on krooninen autoimmuunisairaus, jossa insuliinia tuottavat haiman betasolut tuhoutuvat. Taudinkulku voidaan jakaa oireettomaan prekliiniseen vaiheeseen ja oireiseen sairauteen. Prekliinisessä vaiheessa ilmaantuu autovasta-aineta haiman saarekesoluja vastaan ja sairauden kliininen puhkeaminen tapahtuu toiminnallisten betasolujen määrän laskiessa liian pieneksi sokeritasapainon ylläpitämiseen. Vaikka useita tyypin 1 diabetekselle altistavia perimä- ja ympäristötekijöitä tunnetaan, tautiin johtavan autoimmuniiprosessin tarkkaa mekanismia ei tunneta. Taudinkulussa on lisäksi havaittu suurta vaihtelevuutta ja useiden rinnakkaisten tyypin 1 diabetekseen johtavien tautimekanisminen tai taudin endotyyppien arvellaan olevan mahdollisia. Tässä väitöskirjassa keskityttiin tyypin 1 diabeteksen patogeneesin heterogeniaan johtavien tekijöiden tutkimiseen. Useiden tyypin 1 diabetekselle altistavien geneettisten polymorfismien, kuten NRP1, INS, UBASH3A ja STAT4, havaittiin assosioituvan tiettyihin taudin patogeneesin vaiheisiin. Lisäksi toiset taudin riskipolymorfismit, kuten PTPN22 ja INS, assosioituivat merkittävästi ensimmäisen ilmestyneen autovasta-aineen mukaan määritettyihin, oletettuihin taudin endotyyppeihin. PTPN22-geenin autoimmuunitaudeille altistava riskivariantti assosioitui myös kohonneisiin regulatoristen T-solujen määriin kokonaispopulaatiossa ja naiiveissa soluissa. Useita tyypin 1 diabetekseen liitettyjä autovasta-aineita kehittäneiden lasten monosyyteissä ja monosyytittömissä muissa perifeerisen veren mononukleaarisissa soluissa ei havaittu yksittäisten geenien välisiä ekspressioeroja terveisiin kontrollilapsiin verrattuna. Virusvasteisiin ja tyypin I interferonivasteeseen liittyvät geeniryhmien ekspressiotasot olivat kuitenkin voimistuneet useita autovasta-aineita kehittäneiden lasten monosyyteissä terveisiin kontrolleihin verrattuna. Nämä tulokset tukevat näkemystä tyypin 1 diabeteksen heterogeniasta ja useista mahdollisista poluista taudin puhkeamiseen
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