NIHRDataInsights/HRCSData huggingface dataset:
Title: The role of the immune cell transcription factor T-bet in metabolic physiology and pathophysiology.
Funder: Medical Research Council

Abstract: Building on our own substantial preliminary data which suggests a role for the immune system in obesity-associated inflammation, this proposal aims to investigate the role for the immune cell transcription factor T-bet in adipose tissue inflammation, metabolic physiology and pathophysiology. To date, no immune specific transcription factor has been described to have a role in this process. We will determine impact of T-bet on energy homeostasis and adipose tissue inflammation by undertaking a series of in vivo and ex vivo studies involving manipulation of the T-bet axis. We will examine the effect of high fat diet on the susceptibility to obesity and its metabolic complications and correlate the findings with adipose immune infiltration/inflammation in various models of T-bet deficiency. The metabolic response to low and high fat diet will involve weekly measurements of food intake and body weight, together with periodic evaluation of hormonal profiles (by ELISA) and evaluation of glucose homeostasis by glucose and insulin tolerance testing. Such in vivo measures will be correlated with other measurements ex vivo. Body fat levels and distribution will be quantified by measuring the weight of the different adipose tissue depots. A variety of immune cell populations within the different adipose depots will be phenotyped using multiparameter flow cytometry. Further measurements of immune and adipose gene expression and production of anti-inflammatory and pro-inflammatory cytokines and other molecules involved in immune cell trafficking will also be evaluated using quantitative PCR, organ culture and ELISAs. Additional in vivo models, including adoptive T cell transfer studies, will enable us to map the beneficial effect of T bet deficiency found in our preliminary work to the adaptive or innate immune system and explore potential downstream molecular pathways.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Dyslipidaemia and Hepatic Steatosis: Distinct Roles of DGAT2 and DGAT1 in Hepatocytes and Enterocytes
Funder: Medical Research Council

Abstract: Two major risk factors for cardiometabolic risk are the triglyceride content of the liver (steatosis) and dyslipidaemia, particularly hypertriglyceridaemia which is caused primarily by hepatic and intestinal over-secretion of TAG-rich lipoproteins. Post-prandial lipaemia associates particularly strongly with the development and progression of athersclerosis. DGAT1 and DGAT2 belong to different protein families; although they catalyse the identical final and dedicated reaction of triglyceride synthesis from diglyceride, they appear to have different functions, as they do not compensate for each other in knock-out mice. The effects of the absence of DGAT1 from mouse hepatocytes in culture on these parametes will be studied using wild-type and Dgat 1-/- mice. Analysis of the size and triglyceride stereo-isomeric compositon of VLDL particles will be compared with those of cytosolic and endoplasmic reticular lumen lipid droplets. We will use siRNA-mediated gene silencing and selective inhibitors of human DGAT1 and DGAT2 on human-derived cell types (HepG2 and CaCo2 cells) to investigate the effects of the speciic inhibition of the two enzymes on TG synthesis and partitioning between intracellular accumulation and secretion and the interaction with lipogenic and lipase enzyme activities. We will use these models and primary hepatocytes prepared from DGAT1-/- mice to test the hypotheses (i) that DGAT2 acts upstream of DGAT1, (ii) that it is dependent on DAG substrate that incorporates de novo synthesised and desaturated fatty acids, (iii) that DGAT2 is specialised primarily for the net synthesis of triglycerides whereas DGAT1 is involved in the rescue and remodelling of triglyceride molecules, and provision of TAG for the full lipidation and maturation of the nascent VLDL and chylomicron particles, and (iv) that TAG-DAG substrate cycling occurs in both the cytosolic and ER lumenal compartments.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Investigation of the target of metformin
Funder: Medical Research Council

Abstract: The biguanide metformin is the drug of choice for treatment of type 2 diabetes (T2D) and is under investigation for use in a variety of other age-related disease states. Used in T2D, metformin is associated with reduced risk of cardiovascular and other mortality when compared with other treatment regimes. Around one third of individuals prescribed metformin discontinue use shortly after starting the drug due to gastrointestinal side effects and therefore it would be highly desirable to develop metformin-like compounds with similar properties. Unfortunately however, the mechanism of action of metformin remains incompletely understood and the identity of its proximal target, which would greatly aid development of such drugs, is unknown. In the current work we wish to follow up new evidence that we have obtained that an important direct target of metformin is copper. Previously, we investigated how metal ions can modify the activity of drugs and in our preliminary studies we have gathered strong evidence that copper, whose interaction with metformin has been known for over a century, is required for specific cellular actions of metformin that are linked to its beneficial therapeutic effects. In the current study we aim to elucidate the precise role of copper in metformin action using unique chemical tools and a variety of techniques including western blotting, gene expression analysis and microscopy. This research will establish which effects of metformin are copper-dependent, and of these which depend on metformin's unusual 'pseudoaromatic' metal-binding mode. Research to identify the target of metformin will ultimately facilitate development of new drugs for the treatment of diseases associated with age-related metabolic dysfunction, consistent with MRC's strategic research priority "Living a long and healthy life"


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: METABOLIC AND ENDOCRINE
Title: Preclinical in vivo assessment of a combination of protein and cell therapies to prevent human islet transplant rejection as a cure for Type 1 diabete
Funder: Medical Research Council

Abstract: At the end of this project, which tests a strategy for promoting islet survival in a novel pre-clinical model, we should be in a position to translate our findings straight to the clinic. The first major objective is to refine the humanised mouse model already established in our laboratory, so that the results obtained are more representative of those seen in vivo in humans. We will do this by using NSG mice expressing HLA-DR4 molecules but lacking H-2. Once reconstituted with human DR4-expressing CD34+ cells, these mice, which are already available to us, should mount immune responses that are highly similar to those in humans. The second objective is to transplant human islet cells under the kidney capsule (we already have more than one year of experience in this approach) and study the immune responses to the transplanted islets, in particular to define the impact that complement activation has on these responses. The third and final objective will be to expand regulatory T cells by applying protocols that are currently in use in the laboratory and inject these cells in the DR4-NSG mice, in combination with the pre-treatment of islets with complement inhibitor (APT070). Our prediction is that this combination will lead to indefinite islet survival.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Resolution of type II diabetes following RNY gastric bypass surgery: can gene expression profiling reveal the underlying dynamic mechanisms?
Funder: Medical Research Council

Abstract: The rising prevalence of type 2 diabetes (T2D) represents a major challenge for healthcare provision worldwide. Bariatric surgery is the most effective treatment for morbid obesity and certain operations, such as the Roux-en-Y (RNY) gastric bypass, also have dramatic effects on T2D, including complete long-term remission of the condition in some 40% of patients prior to significant weight loss. Investigation of such T2D remission has the potential to highlight possible biomarkers for stratification of patients likely to show greatest benefit from such approaches, to shed new light on the underlying mechanisms of diabetes risk and protection in morbid obesity, and to identify possible new drug targets for treatment of diabetes. We will conduct RNAseq on 3 peripheral blood samples (taken immediately before surgery, 2 days after surgery, 6 months after surgery) from each of 3 groups of 50 patients undergoing RNY: non-diaebetic; diabetics who show resolution immediately following surgery; and non-resolving diabetics. The resulting dataset will be examined for differences between those with and without T2D at baseline, and differences between those T2D subjects who experienced resolution of their diabetes and those who did not. This will yield a set of transcripts putatively associated with protection from/risk of diabetes in the context of morbid obesity and another set putatively discriminating between those whose T2D does or does not resolve. These findings will be replicated in a larger set of RNY patients (2,800 total) using droplet digital PCR. We will then carry-out whole-genome genotyping and conduct eQTL analysis of transcripts of interest and seek associations of EQTL loci with diabetes resolution and, in large-scale population cohorts, with quantitative traits relevant to T2D.


Research activity codes: 6.4 SURGERY;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: MICA. Therapy for the body and breath malodour disorder Trimethylaminuria (TMAU)
Funder: Medical Research Council

Abstract: Trimethylaminuria (TMAU) is an inherited disorder caused by mutations in the FMO3 gene. A lack of active FM03 means affected individuals cannot convert odorous trimethylamine (TMA), produced from breakdown of dietary choline by gut bacteria, to the non-odorous N-oxide. TMAU individuals excrete large amounts of TMA in all bodily excretions, including breath. Social isolation, ridicule and limited employment prospects are experienced by those with TMAU, which translates to a low quality of life; high levels of depression, suicide and divorce occur in this population. We will carry out preclinical animal studies to test a therapeutic for TMAU. We have established very promising early proof of concept data in a rodent model indicating that this therapeutic approach is both realistic and potentially of great use to humans suffering from this condition. The end point measurements of efficacy of the treatment are the amounts of TMA and TMA N-oxide excreted in urine, which are a measure of the severity of TMAU in humans. Affected individuals with TMAU excrete greater than 10 uM TMA /mmol creatinine (> 10% of total TMA excreted as unmetabolised TMA, calculated as a ratio of TMA/TMAO). Severity of TMAU correlates with levels of urinary TMA, this will be our main endpoint indicator in the proposed preclinical studies. Male mice, >6-weeks of age, are natural knockouts for FMO3 and excrete large amounts of urinary TMA. Female mice will be subjected to a choline challenge. Mice will be dosed i.v. or s.c. with the therapeutic. Initially, we will assess route of delivery; therapeutic window of dosage; half-life of therapeutic; pharmacokinetics. A longer-term study, using GLP therapeutic, prepared to the standard for human clinical studies, will determine bioavailability, biodistribution, toxicity and pathology.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: METABOLIC AND ENDOCRINE
Title: Enhancement of pancreatic beta cell generation in vitro and in vivo by post-translational regulation of Neurogenin3
Funder: Medical Research Council

Abstract: Diabetes, currently one of the UK's most pressing public health problems, has the potential to be treated successfully by pancreatic islet transplantation, but islet supply is a critical issue that must first be addressed. Stem cells (either ES or iPS) could act as a source for beta cell replacement. However, major obstacles remain in that stem cells must be induced to adopt functional beta cell fate and maintain that fate indefinitely when transplanted back into the patient. This may be achieved by recapitulating developmental events that occur during pancreas formation. Endocrine pancreas and beta cell formation is ultimately controlled by the activity of the key proneural bHLH transcription factor Ngn3. We have identified multi-site phosphorylation as critical post-translational modifications of Ngn3 and we hypothesise that rational manipulation of Ngn3 phospho-status will enhance Ngn3 activity, promoting stable, more mature beta cell formation. Specifically we will: 1) Characterise the contribution of distinct SP sites to phosphoregulation of Ngn3 in the rapidly analysed in vivo model, Xenopus. 2) Use RNA-mediated transfection to over-express Ngn3 and phospho-mutant Ngn3 in human ES cells induced to differentiate into pancreatic progenitor cells in vitro, comparing the efficiency of beta cell generation and maturation. To assay engraftment and functionality of human islet cells so generated. This work will be conducted in collaboration with Prof. Douglas Melton, Harvard Stem Cell Institute. 3) Differentiate ES cell lines derived from our Ngn3eYFP and 6S-ANgn3eYFP mice towards an endocrine pancreas fate and to compare both efficiency of endocrine cell differentiation and the endocrine subtypes so generated. 4) Generate inducible mES cell lines over-expressing wild-type and phosphomutant Ngn3 and use genome-wide ChIP seq and RNA seq to qualitatively and quantitatively compare transcriptional profiles to identify differentially regulated targets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Molecular analysis of the protein machinery underlying insulin secretion
Funder: Medical Research Council

Abstract: Regulated exocytosis is a highly orchestrated cascade of protein-protein interactions culminating in the fusion of cargo containing vesicles with the plasma membrane. In beta-cells the primary cargo is insulin, which is released as part of glucose homeostasis. In common with other highly specialised secretory cells the secretory apparatus is composed of the SNARE proteins present on the plasma membrane (tSNARE) and the cargo-containing vesicle (vSNARE). It is clear that the SNARE proteins act in a highly coordinated manner and that disruption of these proteins results in a marked decrease in insulin release. However, how these proteins are organised spatially, how they move on the plasma membrane and where they interact relative to secretory vesicles in beta cells remains unknown. Recently it was reported that the SNARE proteins in beta-cells can be directly affected by the high extracellular glucose concentrations encountered during Type 2 diabetes. This project will examine at the molecular level the organisation and interactions of the plasma membrane tSNAREs and how this is altered under chronic elevated glucose concentrations. Specifically we will utilise advanced super-resolution single molecule approaches to observe the spatial organisation, molecular dynamics and interactions of large cohorts of tSNAREs in intact beta-cells. We will examine the underlying molecular basis for the observed organisation and analyse how different tSNARE isoforms, known to be involved in different phases of insulin secretion are organised. Finally we will investigate how this organisation is altered under chronic elevated extracellular glucose concentrations. Our extensive preliminary data, demonstrating all of the required techniques are in place in the laboratory, shows the feasibility of the project. Ultimately this could lead to new methods of diagnosis and therapeutic treatments for Type 2 diabetes.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: A novel model of AMPK-mediated obesity involving the gamma2 subunit
Funder: Medical Research Council

Abstract: Obesity is a global public health problem. Current pharmacological options for obesity are limited by poor efficacy in terms of weight loss and a preponderance of adverse reactions when used chronically. An unprecedented need now exists to better understand the biological mechanisms of obesity and identify suitable molecular substrates for safe and efficacious long-term pharmacotherapeutics. An attractive target in this regard is the mammalian AMP-activated protein kinase (AMPK). AMPK is a conserved serine-threonine kinase that acts as an energy sensor (through binding of adenine nucleotides via its gamma subunit) and integrator of metabolic signals at cellular and whole-body level. Activation of AMPK switches on ATP-generating catabolic processes (e.g. fatty acid oxidation), whilst repressing anabolic ATP-consuming pathways (e.g. lipid synthesis). Accordingly, short-term in vivo administration of AMPK activators is associated with beneficial metabolic effects in experimental obesity models. However, the chronic sequelae of these drugs remain unexplored. We previously identified activating mutations in AMPK's regulatory gamma2 subunit as the cause of a rare human cardiomyopathy. In seeking to better understand the nature of these mutations, we generated the first gene-targeted murine model bearing an activating mutation in gamma2. Surprisingly, whilst inducing only subtle cardiac abnormalities, mutant mice develop striking adult-onset obesity associated with hyperphagia on a normal diet. In this proposal we seek to define the mechanisms underlying the systemic phenotype of this model and, in doing so, aim to elucidate the diverse consequences of chronic in vivo AMPK activation. Given intense academic and pharmaceutical interest into the potential of AMPK activation as a viable therapeutic strategy for the epidemic of metabolic disease, a better understanding of the long-term risk-benefit profile of chronic AMPK activation, as exemplified herein, is essential.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Investigation into the role of IL-33-responsive natural helper cells in adipose tissue inflammation during obesity
Funder: Medical Research Council

Abstract: Obesity is accompanied by chronic, low-grade inflammation that is thought to contribute to type 2 diabetes and cardiovascular disease. Under normal conditions in adipose tissue, immune effector cells reside alongside adipocytes, and help maintain metabolic homeostasis. However, during obesity, this delicate balance can be disrupted leading to excessive activation of resident immune cells and infiltration of new immune cells, such as T cells, NK cells and monocytes, which along with the adipocytes themselves secrete a variety of pro-inflammatory cytokines. Recent studies demonstrate that the infiltrating T cells display a T helper (Th)-1 pattern of activation, and that an imbalance may exist in obese adipose between resident Th2 cells and T regulatory (Treg) cells that fail to limit an expanding pool of infiltrating pro-inflammatory Th1 cells. Natural helper cells (NHC) are a new population of non-NK cell, RORyt-independent cells that can express a cytokine profile similar to Th2 cells in response to activation with IL-33 or IL-25, and have recently been identified in adipose tissue. Importantly, the identification of resident NHCs in adipose tissue raises questions about their potential involvement in affecting Th2 aspects of the regulation of metabolic homeostasis. To investigate how these cells impact on metabolic and immune responses within adipose tissue I propose to study, in depth, the kinetics of NHC recruitment, and chemotactic signals involved, in adipose tissue collected from obese mice and obese patients with type 2 diabetes undergoing bariatric surgery. The mechanistic action of NHC activation on inflammatory and metabolic function will be studied using in vitro culture systems Finally, using a combination of gain and loss of function approaches we plan to examine the importance of NHC regulation of metabolic processes in obesity and diabetes in vivo. These studies will identify the immunometabolic processes that are regulated by NHCs in vivo.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Ligand bias at the GLP-1 receptor as a means of favouring cytoprotection
Funder: Medical Research Council

Abstract: One of the key pathophysiological mechanisms in type 2 diabetes is apoptosis of beta cells and consequent failure to secrete insulin. Glucagon-like peptide 1 (GLP-1) prevents apoptosis and therefore holds promise as a cytoprotective therapy, but dosage is limited by nausea. It is now known that beta-arrestin recruitment to the GLP-1 receptor, which mediates the anti-apoptotic response, can be preferentially triggered by different GLP-1 mimetics. A GLP-1 analogue now needs to be identified which optimally stimulates the beta-arrestin pathway in order to enhance its cytoprotective effects. This will be achieved as follows: 1) To determine the optimum peptide sequence for cytoprotection, I will screen a library of synthetic GLP-1-like peptides for ability to rescue MIN6 cells from glucolipotoxic cell death. By correlating sequence with cytoprotection, advantageous sequences will be identified and combined. 2) To confirm the beta-arrestin pathway is responsible for better cytoprotection, I will compare the effect of a small number of peptides on intermediates in the beta-arrestin and other pathways using Western blotting and other in vitro techniques, including in human islets. 3) I will then demonstrate effectiveness in vivo by investigating the ability of promising peptides to protect against streptozotocin-induced beta cell damage in a mouse model.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: METABOLIC AND ENDOCRINE