Item Published version Open AccessSex and gender differences in developmental programming of metabolism.(Elsevier BV, 2018-09) Dearden, Laura; Bouret, Sebastien G; Ozanne, Susan E; Dearden, Laura [0000-0002-0804-074X]; Ozanne, Susan [0000-0001-8753-5144]BACKGROUND: The early life environment experienced by an individual in utero and during the neonatal period is a major factor in shaping later life disease risk-including susceptibility to develop obesity, diabetes, and cardiovascular disease. The incidence of metabolic disease is different between males and females. How the early life environment may underlie these sex differences is an area of active investigation. SCOPE OF REVIEW: The purpose of this review is to summarize our current understanding of how the early life environment influences metabolic disease risk in a sex specific manner. We also discuss the possible mechanisms responsible for mediating these sexually dimorphic effects and highlight the results of recent intervention studies in animal models. MAJOR CONCLUSIONS: Exposure to states of both under- and over-nutrition during early life predisposes both sexes to develop metabolic disease. Females seem particularly susceptible to develop increased adiposity and disrupted glucose homeostasis as a result of exposure to in utero undernutrition or high sugar environments, respectively. The male placenta is particularly vulnerable to damage by adverse nutritional states and this may underlie some of the metabolic phenotypes observed in adulthood. More studies investigating both sexes are needed to understand how changes to the early life environment impact differently on the long-term health of male and female individuals. Item Published version Open AccessThe WDR11 complex facilitates the tethering of AP-1-derived vesicles.(Springer Science and Business Media LLC, 2018-02-09) Navarro Negredo, Paloma; Edgar, James R; Manna, Paul T; Antrobus, Robin; Robinson, Margaret S; Edgar, James R [0000-0001-7903-8199]Vesicluar transport of proteins from endosomes to the trans-Golgi network (TGN) is an essential cellular pathway, but much of its machinery is still unknown. A screen for genes involved in endosome-to-TGN trafficking produced two hits, the adaptor protein-1 (AP-1 complex), which facilitates vesicle budding, and WDR11. Here we demonstrate that WDR11 forms a stable complex with two other proteins, which localises to the TGN region and does not appear to be associated with AP-1, suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rather than the TGN, where it may in turn recruit the golgin binding partner TBC1D23. We propose that the complex acts together with TBC1D23 to facilitate the golgin-mediated capture of vesicles that were generated using AP-1. Item Accepted version Open AccessGWAS for BMI: a treasure trove of fundamental insights into the genetic basis of obesity.(Springer Science and Business Media LLC, 2018-08) Speakman, JR; Loos, RJF; O'Rahilly, S; Hirschhorn, JN; Allison, DB; Speakman, JR [0000-0002-2457-1823]; Loos, RJF [0000-0002-8532-5087]; O'Rahilly, S [0000-0003-2199-4449]Muller et al.  have provided a strong critique of the Genome-Wide Association Studies (GWAS) of body-mass index (BMI), arguing that the GWAS approach for the study of BMI is flawed, and has provided us with few biological insights. They suggest that what is needed instead is a new start, involving GWAS for more complex energy balance related traits. In this invited counter-point, we highlight the substantial advances that have occurred in the obesity field, directly stimulated by the GWAS of BMI. We agree that GWAS for BMI is not perfect, but consider that the best route forward for additional discoveries will likely be to expand the search for common and rare variants linked to BMI and other easily obtained measures of obesity, rather than attempting to perform new, much smaller GWAS for energy balance traits that are complex and expensive to measure. For GWAS in general, we emphasise that the power from increasing the sample size of a crude but easily measured phenotype outweighs the benefits of better phenotyping. Item Accepted version Open AccessDay-and-Night Closed-Loop Insulin Delivery in a Broad Population of Pregnant Women With Type 1 Diabetes: A Randomized Controlled Crossover Trial.(American Diabetes Association, 2018-07) Stewart, Zoe A; Wilinska, Malgorzata E; Hartnell, Sara; O'Neil, Leanne K; Rayman, Gerry; Scott, Eleanor M; Barnard, Katharine; Farrington, Conor; Hovorka, Roman; Murphy, Helen R; Rayman, Gerry [0000-0003-3331-7015]; Murphy, Helen R [0000-0001-6876-8727]OBJECTIVE: Despite advances in technology, optimal glucose control remains elusive and neonatal complications remain ubiquitous in type 1 diabetes (T1D) pregnancy. Our aim was to examine the safety, efficacy, and longer-term feasibility of day-and-night closed-loop insulin delivery. RESEARCH DESIGN AND METHODS: We recruited 16 pregnant women (mean [SD]: age 32.8 [5.0] years, T1D duration 19.4 [10.2] years, HbA1c 8.0% [1.1], and BMI 26.6 [4.4] kg/m2) to an open-label, randomized, crossover trial. Participants completed 28 days of closed-loop and sensor-augmented pump (SAP) insulin delivery separated by a washout period. Afterward, participants could continue to use the closed-loop system up to 6 weeks postpartum. The primary end point was the proportion of time with glucose levels within the target range (63-140 mg/dL). RESULTS: The proportion of time with glucose levels within target was comparable during closed-loop and SAP insulin delivery (62.3 vs. 60.1% [95% CI -4.1 to 8.3]; P = 0.47). Mean glucose and time spent hyperglycemic >140 mg/dL also did not differ (131.4 vs. 131.4 mg/dL [P = 0.85] and 36.6 vs. 36.1% [P = 0.86], respectively). During closed-loop, fewer hypoglycemic episodes occurred (median 8 [range 1-17] vs. 12.5 [1-53] over 28 days; P = 0.04) and less time at <63 mg/dL (1.6 vs. 2.7%; P = 0.02). Hypoglycemia <50 mg/dL (0.24 vs. 0.47%; P = 0.03) and low blood glucose index (1.0 vs. 1.4; P = 0.01) were lower. Less nocturnal hypoglycemia (2300-0700 h) during closed-loop therapy (1.1 vs. 2.7%; P = 0.008) and a trend toward higher overnight time in target (67.7 vs. 60.6%; P = 0.06) were found. CONCLUSIONS: Closed-loop insulin delivery was associated with comparable glucose control and significantly less hypoglycemia than SAP therapy. Larger, longer-duration multicenter trials are now indicated to determine clinical efficacy of closed-loop insulin delivery in T1D pregnancy and the impact on neonatal outcomes. Item Published version Open AccessPCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.(Elsevier BV, 2018-05-21) Haider, Afreen; Wei, Yu-Chen; Lim, Koini; Barbosa, Antonio D; Liu, Che-Hsiung; Weber, Ursula; Mlodzik, Marek; Oras, Kadri; Collier, Simon; Hussain, M Mahmood; Dong, Liang; Patel, Satish; Alvarez-Guaita, Anna; Saudek, Vladimir; Jenkins, Benjamin J; Koulman, Albert; Dymond, Marcus K; Hardie, Roger C; Siniossoglou, Symeon; Savage, David B; Koulman, Albert [0000-0001-9998-051X]; Hardie, Roger [0000-0001-5531-3264]; Savage, David [0000-0002-7857-7032]Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis. Item Accepted version Open AccessA Protocol for Producing the Maternal Low-Protein Rat Model: A Tool for Preclinical Proteomic Studies.(Springer International Publishing, 2017) Ma, Dan; Ozanne, Susan E; Guest, Paul C; Ozanne, Susan [0000-0001-8753-5144]Epidemiological studies have shown that periods of poor nutrition during pregnancy can lead to an increased risk of metabolic and psychiatric disorders in the offspring. In addition, some individuals with disorders such as schizophrenia show signs of impaired insulin signalling. These findings indicate that there may be a link between metabolism and neuronal function and they also provide supporting evidence for neurodevelopmental origins of mental illness. This chapter gives a detailed protocol for generation of the maternal low-protein (LP) rat model, which leads to metabolic disturbances in the offspring. This model has been used in proteomic studies of several disorders including schizophrenia. Item Accepted version Open AccessPulsed SILAC as a Approach for miRNA Targets Identification in Cell Culture.(Springer New York, 2017) Duque-Guimarães, Daniella E; de Almeida-Faria, Juliana; Ong, Thomas Prates; Ozanne, Susan E; Ozanne, Susan [0000-0001-8753-5144]Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) comprises a variation of the classical SILAC proteomic methodology that enables the identification of short-term proteomic responses such as those elicited by micro RNAs (miRNAs). Here, we describe a detailed pSILAC protocol for global identification and quantification of protein translation alterations induced by a miRNA using 3T3-L1 pre-adipocytes as a model system. Item Accepted version Open AccessMultiplex Biomarker Approaches in Type 2 Diabetes Mellitus Research.(Springer New York, 2017) Ozanne, Susan E; Rahmoune, Hassan; Guest, Paul C; Ozanne, Susan [0000-0001-8753-5144]Type 2 diabetes mellitus is a multifactorial condition resulting in high fasting blood glucose levels. Although its diagnosis is straightforward, there is not one set of biomarkers or drug targets that can be used for classification or personalized treatment of individuals who suffer from this condition. Instead, the application of multiplex methods incorporating a systems biology approach is essential in order to increase our understanding of this disease. This chapter reviews the state of the art in biomarker studies of human type 2 diabetes from a proteomic and metabolomic perspective. Our main focus was on biomarkers for disease prediction as these could lead to early intervention strategies for the best possible patient outcomes. Item Open AccessA gut-brain axis regulating glucose metabolism mediated by bile acids and competitive fibroblast growth factor actions at the hypothalamus(Elsevier, 2018-02) Blouet, C; Chua, Streamson; Marcelin, Genevieve; Schwartz, Gary; Liu, Shun-Mei; Jeong, Jae Hoon; Jo, Young Hwan; Blouet, Clemence [0000-0002-1752-1270]Objective: Bile acids have been implicated as important regulators of glucose metabolism via activation of FXR and GPBAR1. We have previously shown that FGF19 can modulate glucose handling by suppressing the activity of hypothalamic AGRP/NPY neurons. As bile acids stimulate the release of FGF19/FGF15 into the circulation, we pursued the potential of bile acids to improve glucose tolerance via a gut-brain axis involving FXR and FGF15/FGF19 within enterocytes and FGF receptors on hypothalamic AGRP/NPY neurons. Methods: A 5-day gavage of taurocholic acid, mirroring our previous protocol of a 5-day FGF19 treatment, was performed. Oral glucose tolerance tests in mice with genetic manipulations of FGF signaling and melanocortin signaling were used to define a gut-brain axis responsive to bile acids. Results: The taurocholic acid gavage led to increased serum concentrations of taurocholic acid as well as increases of FGF15 mRNA in the ileum and improved oral glucose tolerance in obese (ob/ob) mice. In contrast, lithocholic acid, an FXR antagonist but a potent agonist for GPBAR1, did not improve glucose tolerance. The positive response to taurocholic acid is dependent upon an intact melanocortinergic system as obese MC4R-null mice or ob/ob mice without AGRP did not show improvements in glucose tolerance after taurocholate gavage. We also tested the FGF receptor isoform necessary for the bile acid response, using AGRP:Fgfr1-/- and AGRP:Fgfr2-/- mice. While the absence of FGFR1 in AGRP/NPY neurons did not alter glucose tolerance after taurocholate gavage, manipulations of Fgfr2caused bidirectional changes depending upon the experimental model. We hypothesized the existence of an endogenous hypothalamic FGF, most likely FGF17, that acted as a chronic activator of AGRP/NPY neurons. We developed two short peptides based on FGF8 and FGF17 that should antagonize FGF17 action. Both of these peptides improved glucose homeostasis after a 4-day course of central and peripheral injections. Significantly, daily average blood glucose from continuous glucose monitoring was reduced in all tested animals but glucose concentrations remained in the euglycemia range. Conclusions: We have defined a gut-brain axis that regulates glucose metabolism mediated by antagonistic fibroblast growth factors. From the intestine, bile acids stimulate FGF15 secretion, leading to activation of the FGF receptors in hypothalamic AGRP/NPY neurons. FGF receptor intracellular signaling subsequently silences AGRP/NPY neurons, leading to improvements of glucose tolerance that are likely mediated by the autonomic nervous system. Finally, short peptides that antagonize homodimeric FGF receptor signaling within the hypothalamus have beneficial effects on glucose homeostasis without inducing hypoglycemia. These peptides could provide a new mode of regulating glucose metabolism. Item Accepted version Open AccessPOMC: The Physiological Power of Hormone Processing.(American Physiological Society, 2018-10-01) Harno, Erika; Gali Ramamoorthy, Thanuja; Coll, Anthony P; White, Anne; Coll, Anthony [0000-0003-2594-7463]Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon. Item Accepted version Open AccessA Pharmacogenetic Approach to the Treatment of Patients With PPARG Mutations.(American Diabetes Association, 2018-06) Agostini, Maura; Schoenmakers, Erik; Beig, Junaid; Fairall, Louise; Szatmari, Istvan; Rajanayagam, Odelia; Muskett, Frederick W; Adams, Claire; Marais, A David; O'Rahilly, Stephen; Semple, Robert K; Nagy, Laszlo; Majithia, Amit R; Schwabe, John WR; Blom, Dirk J; Murphy, Rinki; Chatterjee, Krishna; Savage, David B; Semple, Robert K [0000-0001-6539-3069]; Murphy, Rinki [0000-0002-0043-2423]; Savage, David B [0000-0002-7857-7032]Loss-of-function mutations in PPARG cause familial partial lipodystrophy type 3 (FPLD3) and severe metabolic disease in many patients. Missense mutations in PPARG are present in ∼1 in 500 people. Although mutations are often binarily classified as benign or deleterious, prospective functional classification of all missense PPARG variants suggests that their impact is graded. Furthermore, in testing novel mutations with both prototypic endogenous (e.g., prostaglandin J2 [PGJ2]) and synthetic ligands (thiazolidinediones, tyrosine agonists), we observed that synthetic agonists selectively rescue function of some peroxisome proliferator-activated receptor-γ (PPARγ) mutants. We report on patients with FPLD3 who harbor two such PPARγ mutations (R308P and A261E). Both PPARγ mutants exhibit negligible constitutive or PGJ2-induced transcriptional activity but respond readily to synthetic agonists in vitro, with structural modeling providing a basis for such differential ligand-dependent responsiveness. Concordant with this finding, dramatic clinical improvement was seen after pioglitazone treatment of a patient with R308P mutant PPARγ. A patient with A261E mutant PPARγ also responded beneficially to rosiglitazone, although cardiomyopathy precluded prolonged thiazolidinedione use. These observations indicate that detailed structural and functional classification can be used to inform therapeutic decisions in patients with PPARG mutations. Item Published version Open AccessIL-36γ Is a Strong Inducer of IL-23 in Psoriatic Cells and Activates Angiogenesis.(Frontiers Media SA, 2018) Bridgewood, Charlie; Fearnley, Gareth W; Berekmeri, Anna; Laws, Philip; Macleod, Tom; Ponnambalam, Sreenivasan; Stacey, Martin; Graham, Anne; Wittmann, Miriam; Bridgewood, Charlie [0000-0001-6797-4633]; Fearnley, Gareth [0000-0002-9602-5071]; Berekmeri, Anna [0000-0001-5894-8812]; Macleod, Tom [0000-0002-7903-2990]; Ponnambalam, Sreenivasan [0000-0002-4452-7619]; Wittmann, Miriam [0000-0003-2328-4926]The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence. Item Published version Open AccessLeptin mediates the increase in blood pressure associated with obesity.(Elsevier BV, 2014-12-04) Simonds, Stephanie E; Pryor, Jack T; Ravussin, Eric; Greenway, Frank L; Dileone, Ralph; Allen, Andrew M; Bassi, Jaspreet; Elmquist, Joel K; Keogh, Julia M; Henning, Elana; Myers, Martin G; Licinio, Julio; Brown, Russell D; Enriori, Pablo J; O'Rahilly, Stephen; Sternson, Scott M; Grove, Kevin L; Spanswick, David C; Farooqi, I Sadaf; Cowley, Michael A; Keogh, Julia [0000-0002-0399-4114]; Henning, Elana [0000-0002-0399-4114]; O'Rahilly, Stephen [0000-0003-2199-4449]; Farooqi, Ismaa [0000-0001-7609-3504]Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species. Item Accepted version Open AccessCerebral arterial bolus arrival time is prolonged in multiple sclerosis and associated with disability.(SAGE Publications, 2014-01) Paling, David; Thade Petersen, Esben; Tozer, Daniel J; Altmann, Daniel R; Wheeler-Kingshott, Claudia AM; Kapoor, Raju; Miller, David H; Golay, Xavier; Tozer, Daniel [0000-0002-0404-3214]Alterations in the overall cerebral hemodynamics have been reported in multiple sclerosis (MS); however, their cause and significance is unknown. While potential venous causes have been examined, arterial causes have not. In this study, a multiple delay time arterial spin labeling magnetic resonance imaging sequence at 3T was used to quantify the arterial hemodynamic parameter bolus arrival time (BAT) and cerebral blood flow (CBF) in normal-appearing white matter (NAWM) and deep gray matter in 33 controls and 35 patients with relapsing-remitting MS. Bolus arrival time was prolonged in MS in NAWM (1.0±0.2 versus 0.9±0.2 seconds, P=0.031) and deep gray matter (0.90±0.18 versus 0.80±0.14 seconds, P=0.001) and CBF was increased in NAWM (14±4 versus 10±2 mL/100 g/min, P=0.001). Prolonged BAT in NAWM (P=0.042) and deep gray matter (P=0.01) were associated with higher expanded disability status score. This study demonstrates alteration in cerebral arterial hemodynamics in MS. One possible cause may be widespread inflammation. Bolus arrival time was longer in patients with greater disability independent of atrophy and T2 lesion load, suggesting alterations in cerebral arterial hemodynamics may be a marker of clinically relevant pathology. Item Accepted version Open AccessLong-term effectiveness and safety of metreleptin in the treatment of patients with generalized lipodystrophy.(Springer Science and Business Media LLC, 2018-06) Brown, Rebecca J; Oral, Elif A; Cochran, Elaine; Araújo-Vilar, David; Savage, David B; Long, Alison; Fine, Gregory; Salinardi, Taylor; Gorden, Phillip; Savage, David [0000-0002-7857-7032]PURPOSE: The purpose of this study is to summarize the effectiveness and safety of metreleptin in patients with congenital or acquired generalized lipodystrophy. METHODS: Patients (n = 66) aged ≥6 months had lipodystrophy, low circulating leptin, and ≥1 metabolic abnormality (diabetes mellitus, insulin resistance, or hypertriglyceridemia). Metreleptin dose (once or twice daily) was titrated to a mean dose of 0.10 mg/kg/day with a maximum of 0.24 mg/kg/day. Means and changes from baseline to month 12 were assessed for glycated hemoglobin (HbA1c), fasting triglycerides (TGs), and fasting plasma glucose (FPG). Additional assessments included the proportions of patients achieving target decreases in HbA1c or fasting TGs at months 4, 12, and 36, medication changes, and estimates of liver size. Treatment-emergent adverse events (TEAEs) were recorded. RESULTS: Significant mean reductions from baseline were seen at month 12 for HbA1c (-2.2%, n = 59) and FPG (-3.0 mmol/L, n = 59) and mean percent change in fasting TGs (-32.1%, n = 57) (all p ≤ 0.001). Reductions from baseline over time in these parameters were also significant at month 36 (all p < 0.001, n = 14). At month 4, 34.8% of patients had a ≥1% reduction in HbA1c and 62.5% had a ≥30% reduction in fasting TGs; at month 12, 80% of patients had a ≥1% decrease in HbA1c or ≥30% decrease in TGs, and 66% had a decrease of ≥2% in HbA1c or ≥40% decrease in TGs. Of those on medications, 41%, 22%, and 24% discontinued insulin, oral antidiabetic medications, or lipid-lowering medications, respectively. Mean decrease in liver volume at month 12 was 33.8% (p < 0.001, n = 12). Most TEAEs were of mild/moderate severity. CONCLUSIONS: In patients with generalized lipodystrophy, long-term treatment with metreleptin was well tolerated and resulted in sustained improvements in hypertriglyceridemia, glycemic control, and liver volume. Item Published version Open AccessARTHRITIS IN DIABETES: PATHOGENESIS OF CHARCOT ARTHROPATHY(The British Editorial Society of Bone & Joint Surgery) Coll, Anthony Patrick; Johnson-Lynn, Sarah; McCaskie, Andrew; Robinson, Andrew HN; McCaskie, Andrew [0000-0001-6476-0832]Charcot neuroarthropathy is a rare but serious complication of peripheral neuropathy, causing progressive destruction of the bones and joints of the foot leading to deformity, altered biomechanics and increased risk of ulceration. Management is complicated by a lack of consensus on diagnostic criteria and an incomplete understanding of the pathogenesis. In this review, we consider recent insights into the development of Charcot neuroarthropathy. It is likely to be dependent on several interconnected factors. These may include a genetic pre-disposition in combination with diabetic neuropathy. This leads to decreased neuropeptides (NO and CGRP), which may affect the normal coupling of bone formation / resorption and increased levels of RANKL, potentiating osteoclastogenesis. Repetitive unrecognised trauma due to neuropathy increases levels of pro-inflammatory cytokines (IL1β, IL6, TNFα) which could also contribute to increased bone resorption, on a background of a system biased towards inflammation with increased autoimmune reactivity and a profile of monocytes primed to transform into osteoclasts (CD14). Increased blood glucose and loss of circulating RAGE, leading to increased non-enzymatic glycation of collagen and accumulation of AGEs in the tissues of the foot, may also contribute to the pathological process. An understanding of the relative contributions of each of these mechanisms and a final common pathway for the development of Charcot arthropathy are still lacking. Item Published version Open AccessNeuroarthropathy in diabetes: pathogenesis of Charcot arthropathy.(British Editorial Society of Bone & Joint Surgery, 2018-05) Johnson-Lynn, SE; McCaskie, AW; Coll, AP; Robinson, AHN; McCaskie, Andrew [0000-0001-6476-0832]; Coll, Anthony [0000-0003-2594-7463]Charcot neuroarthropathy is a rare but serious complication of diabetes, causing progressive destruction of the bones and joints of the foot leading to deformity, altered biomechanics and an increased risk of ulceration. Management is complicated by a lack of consensus on diagnostic criteria and an incomplete understanding of the pathogenesis. In this review, we consider recent insights into the development of Charcot neuroarthropathy. It is likely to be dependent on several interrelated factors which may include a genetic pre-disposition in combination with diabetic neuropathy. This leads to decreased neuropeptides (nitric oxide and calcitonin gene-related peptide), which may affect the normal coupling of bone formation and resorption, and increased levels of Receptor activator of nuclear factor kappa-B ligand, potentiating osteoclastogenesis. Repetitive unrecognized trauma due to neuropathy increases levels of pro-inflammatory cytokines (interleukin-1β, interleukin-6, tumour necrosis factor α) which could also contribute to increased bone resorption, in combination with a pre-inflammatory state, with increased autoimmune reactivity and a profile of monocytes primed to transform into osteoclasts - cluster of differentiation 14 (CD14). Increased blood glucose and loss of circulating Receptor for Advanced Glycation End-Products (AGLEPs), leading to increased non-enzymatic glycation of collagen and accumulation of AGLEPs in the tissues of the foot, may also contribute to the pathological process. An understanding of the relative contributions of each of these mechanisms and a final common pathway for the development of Charcot neuroarthropathy are still lacking. Cite this article: S. E. Johnson-Lynn, A. W. McCaskie, A. P. Coll, A. H. N. Robinson. Neuroarthropathy in diabetes: pathogenesis of Charcot arthropathy. Bone Joint Res 2018;7:373-378. DOI: 10.1302/2046-3758.75.BJR-2017-0334.R1. Item Published version Open AccessPharmacokinetics of insulin aspart in pregnant women with type 1 diabetes: every day is different.(American Diabetes Association, 2014-06) Goudie, Robert JB; Lunn, David; Hovorka, Roman; Murphy, Helen R; Goudie, Robert [0000-0001-9554-1499]; Hovorka, Roman [0000-0003-2901-461X]Rapid-acting insulin analogs are increasingly used during type 1 diabetic pregnancy. They may assist women to safely optimize glucose control (1,2), but little is known about their pharmacokinetics and reproducibility in pregnancy. Using a unique data set of 1,300 plasma insulin samples collected under strictly observed experimental conditions, we explored the relationship between aspart pharmacokinetics and clinical and demographic factors. We also assessed reproducibility both within and between pregnant women using continuous subcutaneous insulin infusion (CSII). Item Published version Open AccessRapid model exploration for complex hierarchical data: application to pharmacokinetics of insulin aspart.(Wiley, 2015-10-15) Goudie, Robert JB; Hovorka, Roman; Murphy, Helen R; Lunn, David; Goudie, Robert [0000-0001-9554-1499]; Hovorka, Roman [0000-0003-2901-461X]We consider situations, which are common in medical statistics, where we have a number of sets of response data, from different individuals, say, potentially under different conditions. A parametric model is defined for each set of data, giving rise to a set of random effects. Our goal here is to efficiently explore a range of possible 'population' models for the random effects, to select the most appropriate model. The range of possible models is potentially vast, because the random effects may depend on observed covariates, and there may be multiple credible ways of partitioning their variability. Here, we consider pharmacokinetic (PK) data on insulin aspart, a fast acting insulin analogue used in the treatment of diabetes. PK models are typically nonlinear (in their parameters), often complex and sometimes only available as a set of differential equations, with no closed-form solution. Fitting such a model for just a single individual can be a challenging task. Fitting a joint model for all individuals can be even harder, even without the complication of an overarching model selection objective. We describe a two-stage approach that decouples the population model for the random effects from the PK model applied to the response data but nevertheless fits the full, joint, hierarchical model, accounting fully for uncertainty. This allows us to repeatedly reuse results from a single analysis of the response data to explore various population models for the random effects. This greatly expedites not only model exploration but also cross-validation for the purposes of model criticism. © 2015 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. Item Published version Open AccessA J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.(Elsevier BV, 2017-12-14) Amin-Wetzel, Niko; Saunders, Reuben A; Kamphuis, Maarten J; Rato, Claudia; Preissler, Steffen; Harding, Heather P; Ron, David; Preissler, Steffen [0000-0001-7936-9836]; Harding, Heather [0000-0002-7359-7974]; Ron, David [0000-0002-3014-5636]When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.