2,017 research outputs found

    ER exit in physiology and disease

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    The biosynthetic secretory pathway is comprised of multiple steps, modifications and interactions that form a highly precise pathway of protein trafficking and secretion, that is essential for eukaryotic life. The general outline of this pathway is understood, however the specific mechanisms are still unclear. In the last 15 years there have been vast advancements in technology that enable us to advance our understanding of this complex and subtle pathway. Therefore, based on the strong foundation of work performed over the last 40 years, we can now build another level of understanding, using the new technologies available. The biosynthetic secretory pathway is a high precision process, that involves a number of tightly regulated steps: Protein folding and quality control, cargo selection for Endoplasmic Reticulum (ER) exit, Golgi trafficking, sorting and secretion. When deregulated it causes severe diseases that here we categorise into three main groups of aberrant secretion: decreased, excess and altered secretion. Each of these categories disrupts organ homeostasis differently, effecting extracellular matrix composition, changing signalling events, or damaging the secretory cells due to aberrant intracellular accumulation of secretory proteins. Diseases of aberrant secretion are very common, but despite this, there are few effective therapies. Here we describe ER exit sites (ERES) as key hubs for regulation of the secretory pathway, protein quality control and an integratory hub for signalling within the cell. This review also describes the challenges that will be faced in developing effective therapies, due to the specificity required of potential drug candidates and the crucial need to respect the fine equilibrium of the pathway. The development of novel tools is moving forward, and we can also use these tools to build our understanding of the acute regulation of ERES and protein trafficking. Here we review ERES regulation in context as a therapeutic strategy

    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

    Defining the immune phenotype of extremely early-onset type 1 diabetes

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    Type 1 diabetes is a lifelong autoimmune disease that is very rarely diagnosed in the first two years of life. We recently described a novel T1D phenotype, termed extremely early-onset T1D (here referred to as EXE-T1D) characterised by the development of T1D very early in life, which is associated with rapid and complete b-cell destruction. I therefore hypothesise that EXE-T1D may be the consequence of a more aggressive form of autoimmunity than seen in individuals who develop T1D at a later age. In this thesis this hypothesis was tested by comparing the immune phenotype of individuals with EXE-T1D and individuals with childhood or adulthood-onset T1D. Specifically, the frequency and phenotype of islet-specific T cells was assessed using sensitive FluoroSpot assays, and unbiased immune phenotyping was undertaken using five broad flow cytometry panels. Assessment of b-cell-specific T cell responses showed lower autoreactivity in the blood of EXE-T1D, and a decreased proportion of IL-10 responses close to diagnosis, suggestive of reduced immune regulation. Phenotypic characterisation of the circulating immune system identified increased frequencies of activated circulating follicular helper (aTfh) and peripheral helper (aTph) T cells co-expressing PD-1 and ICOS in EXE-T1D. Detailed examination of the phenotypic characteristics of these cells by single-cell transcriptional profiling revealed minor phenotypic differences between clinical cohorts, including a reduction in cell populations expressing heat shock proteins (HSP) and genes associated with an interferon (IFN) response. This study has revealed novel immune phenotypes associated with EXE-T1D which are currently being investigated as biomarkers of rapid b-cell destruction in larger cohorts of individuals with T1D. This work may therefore guide the selection of patients suitable for specific immune-based therapies to halt disease progression

    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

    Aldh1b1-mediated metabolism regulates pancreas progenitor differentiation and β-cell maturation

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    Pancreatic β-cells have a central function in the regulation of glucose homeostasis by releasing the blood sugar-lowering hormone insulin. Disruption of this process results in diabetes, which has a tremendous impact on the quality of life and requires lifelong treatment. Elucidating the mechanisms of pancreatic progenitor cell differentiation into fully functional β-cells will contribute to identifying the underlying reasons for β-cell dysfunction and to finding a cure for diabetes. Aldh1b1 was identified by our research group as a regulator of pancreas development and β-cell functionality. Aldh1b1 is a mitochondrial enzyme, expressed in all embryonic pancreas progenitors. Its expression is switched off during the process of differentiation and is undetectable in differentiated cells. Functional inactivation of Aldh1b1 in the mouse leads to premature differentiation of progenitor cells in the embryo and dysfunctional β-cells in the adult. However, the enzymatic function of Aldh1b1 in pancreas progenitors and how it ultimately affects β-cell functionality remained to be elucidated. In this study, I analyzed the role of Aldh1b1 in the metabolism of embryonic pancreas progenitor cells and its impact on chromatin structure and gene expression in both, progenitors and postnatal β-cells. Flow cytometry analysis of freshly isolated Aldh1b1 null embryonic pancreas progenitors showed a significant increase in ROS levels as well as a significant decrease in mitochondrial mass, whereas the mitochondrial membrane potential was not affected. To elucidate the impact of Aldh1b1 on cellular metabolism, I conducted metabolic flux experiments and untargeted metabolomics studies using FACS-isolated embryonic pancreas progenitors expanded in a 3D spheroid culture. Analyses following metabolic labeling with either 13C6-Glucose or 13C2-Glutamine showed that the absence of Aldh1b1 lead to an increase of the reductive glutamine metabolism towards citrate, a reaction that channels carbon units into the acetyl-CoA biosynthesis. However, the ACLy-dependent flux towards acetyl-coA synthesis was reduced and this was consistent with reduced expression of ACLy as well as the citrate transporter SLC25a1. A decrease in cellular acetyl-CoA would reduce histone acetylation. Untargeted metabolomics showed an increase in the concentration of S-adenosyl-methionine, suggesting increased DNA and histone methylation. Consistent with these findings, ATAC-Seq analyses on freshly isolated pancreatic progenitors showed reduced chromatin accessibility at genes implicated in chromatin organization, protein acetylation and histone modification. Transcription motif analysis showed that the affected genomic sites were mainly associated with the binding of Klf/Sp and Nrf1 transcription factors. Transcriptome analyses displayed that the expression of genes implicated in progenitor differentiation, ECM organization and transcriptional regulation was affected. Furthermore, transcriptome analyses of early postnatal β-cells uncovered early signs of oxidative stress and increased proliferation, thus providing the basis to explain the β-cell phenotype in Aldh1b1 null mice. I then used organotypic cultures of embryonic pancreata to investigate the connection between high ROS levels and aberrant differentiation in the Aldh1b1 null pancreata. Reducing ROS levels using NAC enabled the reversal of the aberrant transcription factor expression and increased viability of Aldh1b1 null explants, thus identifying high ROS levels as a driving force in this process. To investigate how persisting Aldh1b1 expression would affect progenitor differentiation, I generated ROSA26LSLAldh1b1, an inducible constitutive Aldh1b1 expression line. Progenitors with continuous Aldh1b1 expression avoided the endocrine cell fate, underscoring the importance of timely Aldh1b1 downregulation in the course of β-cell differentiation. Altogether, my work provides strong evidence for the role of Aldh1b1 as a metabolic regulator in the process of progenitor cell differentiation and identifies a link between metabolism and gene regulation through chromatin accessibility during development. Aldh1b1 inactivity causes defects in embryonic progenitor cells as well as postnatal β-cells and could therefore contribute, as genetic risk factor, to the development of hyperglycemia and diabetes later in life. Comprehending the mechanisms underlying the process of pancreas progenitor differentiation as well as the origins of β cell dysfunction should assist in the design of novel therapeutic interventions for diabetes

    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

    Decoding the genetic relationship between Alzheimer’s disease and type 2 diabetes: potential risk variants and future direction for North Africa

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    IntroductionAlzheimer’s disease (AD) and Type 2 diabetes (T2D) are both age-associated diseases. Identification of shared genes could help develop early diagnosis and preventive strategies. Although genetic background plays a crucial role in these diseases, we noticed an underrepresentation tendency of North African populations in omics studies.Materials and methodsFirst, we conducted a comprehensive review of genes and pathways shared between T2D and AD through PubMed. Then, the function of the identified genes and variants was investigated using annotation tools including PolyPhen2, RegulomeDB, and miRdSNP. Pathways enrichment analyses were performed with g:Profiler and EnrichmentMap. Next, we analyzed variant distributions in 16 worldwide populations using PLINK2, R, and STRUCTURE software. Finally, we performed an inter-ethnic comparison based on the minor allele frequency of T2D-AD common variants.ResultsA total of 59 eligible papers were included in our study. We found 231 variants and 363 genes shared between T2D and AD. Variant annotation revealed six single nucleotide polymorphisms (SNP) with a high pathogenic score, three SNPs with regulatory effects on the brain, and six SNPs with potential effects on miRNA-binding sites. The miRNAs affected were implicated in T2D, insulin signaling pathways, and AD. Moreover, replicated genes were significantly enriched in pathways related to plasma protein binding, positive regulation of amyloid fibril deposition, microglia activation, and cholesterol metabolism. Multidimensional screening performed based on the 363 shared genes showed that main North African populations are clustered together and are divergent from other worldwide populations. Interestingly, our results showed that 49 SNP associated with T2D and AD were present in North African populations. Among them, 11 variants located in DNM3, CFH, PPARG, ROHA, AGER, CLU, BDNF1, CST9, and PLCG1 genes display significant differences in risk allele frequencies between North African and other populations.ConclusionOur study highlighted the complexity and the unique molecular architecture of North African populations regarding T2D-AD shared genes. In conclusion, we emphasize the importance of T2D-AD shared genes and ethnicity-specific investigation studies for a better understanding of the link behind these diseases and to develop accurate diagnoses using personalized genetic biomarkers

    Insulin Secretory Granule biogenesis and VPS41

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    The pancreatic beta cells are not only the fundamental source of insulin production but also the predominant regulator of its storage and release upon appropriate stimuli to maintain glucose homeostasis. Insulin is stored in membrane-bounded structures called secretory granules (SGs), which are specialized secretory units of the regulated secretory pathway. Synthesized proinsulin as an inactive precursor form transports into the Golgi apparatus, where proinsulin is sorted and packaged into immature SGs. Soluble cargo proteins, membrane proteins, as well as ions are critical components for insulin production as well as insulin storage in pancreatic beta cells. Type 2 diabetes (T2D) is a prevalent and complex disease that is defined by chronic hyperglycemia and insulin resistance, which are strongly correlated with dysregulated insulin production machinery in beta cells. Recently, vacuolar protein sorting-associated protein 41 (VPS41) was identified as a regulator in the trafficking pathways of synthesized secretory proteins. In this thesis, I show that VPS41 plays a critical role in insulin storage capacity and insulin granule biogenesis in beta cells using both an in vitro rat insulinoma beta cell VPS41 knockout line and an in vivo mouse model with a conditional deletion of VPS41 in beta cells. I show for the first time that deletion of VPS41 in a mouse model leads to severe diabetes associated with extensive depletion of insulin in pancreatic beta cells. Together, my in vivo and in vitro data illustrate that VPS41 is a potent regulator of insulin secretory biology and is required for the maintenance of normal glucose homeostasis

    Dissecting the role of Aryl Hydrocarbon Receptor in adipose and vascular homeostasis

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    Aryl Hydrocarbon Receptor (AHR) is a ligand activated transcription factor, which was initially characterised as an environmental sensor, that mediates toxic effects in response to harmful chemicals. Early research therefore focussed on exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which elicit a sustained activation of the AHR pathway, rather than a transient one associated with natural ligands. More recently, AHR has been implicated in obesity. The incidence of obesity has increased dramatically over recent decades, and carries a high burden to health care systems, along with increased prevalence of cardiovascular disease and cancer. Continued work surrounding obesity has shown that thermogenesis contributes to energy balance, and is now an attractive therapeutic target to attempt to reduce obesity rates. Findings show that AHR is not required for activation, nor regulation of thermogenesis through the characterisation of the global AHR knock out (KO) and adipocyte-specific AHR KO mouse models when exposed to cold. Both AHR KO and adipocyte-specific KO animals are able to maintain body temperature when exposed to acute cold challenge. No differences were observed in oxygen consumption nor energy expenditure. These mice were also able to maintain body temperature upon fasting prior to cold exposure. Endothelial cells (ECs) were also selected as an important cell-type to investigate AHR function due to the severe vascular defects of global AHR KO animals. Vascular disease is again associated with a high burden on healthcare, especially in the ageing population and in individuals who are obese. A human model system of human umbilical vein endothelial cells (HUVECs) was used to investigate novel functions of AHR in ECs in vitro. Bulk RNA sequencing revealed that AHR activation caused a downregulation of many cell cycle-related pathways. Functional data using EdU staining also verified that activation of AHR with various concentrations of FICZ for 6Hrs caused a transient accumulation of cells in G0/1, whilst S and G2/M remained unchanged when compared to DMSO control. Further protein expression analysis at a single cell resolution using Operetta indicated that these effects were mediated through E2F. In addition, AHR was knocked down in HUVECs, which saw a decrease in cells at G1/0 compared to control siKD cells, and an increase in S phase when stained with EdU. Cells reached confluency more rapidly in siAHR group, compared to siKD control group using Incucyte. Increases in E2F were also observed using the Operetta system.Open Acces
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