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Adjustment for covariates has been underused in trials intended to support approval of medical products. This underuse results partly from confusion about whether adjusting changes the thin...

The causal estimands of interest in a clinical trial are treatment effects, and a treatment effect is a comparison of the outcomes for the same patients, or for similar groups of patients, on diffe...

Clinical trials are primarily conducted to understand the average effects treatments have on patients. However, patients are heterogeneous in the severity of the condition and in ways that affect what treatment effect they can expect. It is therefore important to understand and characterize how treatment effects vary. The design and analysis of clinical studies play critical roles in evaluating and characterizing heterogeneous treatment effects. This panel discussed considerations in design and analysis under the recognition that there are heterogeneous treatment effects across subgroups of patients. Panel members discussed many questions including: What is a good estimate of the treatment effect in me, a 65-year-old, bald, Caucasian-American, male patient? What magnitude of heterogeneity of treatment effects (HTE) is sufficiently large to merit attention? What role can prior evidence about HTE play in confirmatory trial design and analysis? Is there anything described in the 21st Century Cures Act that would benefit from greater attention to HTE? An example of a Bayesian approach addressing multiplicity when testing for treatment effects in subgroups will be provided. We can do more or better at understanding heterogeneous treatment effects and providing the best information on heterogeneous treatment effects.

Advances in our understanding of the molecular underpinnings of disease have spurred the development of targeted therapies and the use of precision medicine approaches in patient care. While targeted therapies have improved our capability to provide effective treatments to patients, they also present additional challenges to drug development and benefit-risk assessment such as identifying the subset(s) of patients likely to respond to the drug, assessing heterogeneity in response across molecular subsets of a disease, and developing diagnostic tests to identify patients for treatment. These challenges are particularly difficult to address when targeted therapies are developed to treat diseases with multiple molecular subtypes that occur at low frequencies. To help address these challenges, the US Food and Drug Administration recently published a draft guidance entitled "Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease." Here we provide additional information on specific aspects of targeted therapy development in diseases with low-frequency molecular subsets.

In some randomized (drug versus placebo) clinical trials, the estimand of interest is the between-treatment difference in population means of a clinical endpoint that is free from the confounding effects of "rescue" medication (e.g., HbA1c change from baseline at 24 weeks that would be observed without rescue medication regardless of whether or when the assigned treatment was discontinued). In such settings, a missing data problem arises if some patients prematurely discontinue from the trial or initiate rescue medication while in the trial, the latter necessitating the discarding of post-rescue data. We caution that the commonly used mixed-effects model repeated measures analysis with the embedded missing at random assumption can deliver an exaggerated estimate of the aforementioned estimand of interest. This happens, in part, due to implicit imputation of an overly optimistic mean for "dropouts" (i.e., patients with missing endpoint data of interest) in the drug arm. We propose an alternative approach in which the missing mean for the drug arm dropouts is explicitly replaced with either the estimated mean of the entire endpoint distribution under placebo (primary analysis) or a sequence of increasingly more conservative means within a tipping point framework (sensitivity analysis); patient-level imputation is not required. A supplemental "dropout = failure" analysis is considered in which a common poor outcome is imputed for all dropouts followed by a between-treatment comparison using quantile regression. All analyses address the same estimand and can adjust for baseline covariates. Three examples and simulation results are used to support our recommendations.

Dropouts confound the treatment effect when the outcome and the dropout process both depend on subject characteristics. If dropout is unrelated to treatment, there is an unconfounded effect, but it...

Background This study investigated the relationship between smoking and hearing loss and deafness (HLD) and whether the relationship is modified by genetic variation. Data for these analyses was from the subset of Japanese American families collected as part of the American Diabetes Association Genetics of Non-insulin Dependent Diabetes Mellitus study. Logistic regression with generalized estimating equations assessed the relationship between HLD and smoking. Nonparametric linkage analysis identified genetic regions harboring HLD susceptibility genes and ordered subset analysis was used to identify regions showing evidence for gene-smoking interactions. Genetic variants within these candidate regions were then each tested for interaction with smoking using logistic regression models. Results After adjusting for age, sex, diabetes status and smoking duration, for each pack of cigarettes smoked per day, risk of HLD increased 4.58 times (odds ratio (OR) = 4.58; 95% Confidence Interval (CI): (1.40,15.03)), and ever smokers were over 5 times more likely than nonsmokers to report HLD (OR = 5.22; 95% CI: (1.24, 22.03)). Suggestive evidence for linkage for HLD was observed in multiple genomic regions (Chromosomes 5p15, 8p23 and 17q21), and additional suggestive regions were identified when considering interactions with smoking status (Chromosomes 7p21, 11q23, 12q32, 15q26, and 20q13) and packs-per-day (Chromosome 8q21). Conclusions To our knowledge this was the first report of possible gene-by-smoking interactions in HLD using family data. Additional work, including independent replication, is needed to understand the basis of these findings. HLD are important public health issues and understanding the contributions of genetic and environmental factors may inform public health messages and policies.

The issuance of a report in 2010 by the National Research Council (NRC) of the National Academy of Sciences entitled 'The Prevention and Treatment of Missing Data in Clinical Trials,' commissioned by the US Food and Drug Administration, had an immediate impact on the way that statisticians and clinical researchers in both industry and regulatory agencies think about the missing data problem. We believe that there is currently great potential to improve study quality and interpretability-by reducing the amount of missing data through changes in trial design and conduct and by planning and conducting analyses that better account for the missing information. Here, we describe our view on some of the recommendations in the report and suggest ways in which these recommendations can be incorporated into new or ongoing clinical trials in order to improve their chance of success. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

The National Research Council Panel on Handling Missing Data in Clinical Trials recommended that sensitivity analyses have to be part of the primary reporting of findings from clinical trials. Their specific recommendations, however, seem not to have been taken up rapidly by sponsors of regulatory submissions. The NRC report's detailed suggestions are along rather different lines than what has been called sensitivity analysis in the regulatory setting up to now. Furthermore, the role of sensitivity analysis in regulatory decision-making, although discussed briefly in the NRC report, remains unclear. This paper will examine previous ideas of sensitivity analysis with a view to explaining how the NRC panel's recommendations are different and possibly better suited to coping with present problems of missing data in the regulatory setting. It will also discuss, in more detail than the NRC report, the relevance of sensitivity analysis to decision-making, both for applicants and for regulators. Published 2015. This article is a U.S. Government work and is in the public domain in the USA

Deep inspiration-induced bronchoprotection and bronchodilation are impaired in asthma. We evaluated the effect of inhaled glucocorticosteroids on these phenomena. Two groups of subjects with asthma, 9 with moderate/severe hyperresponsiveness to methacholine, and 12 with mild/borderline hyperresponsiveness to methacholine, received inhaled fluticasone (880 microg daily) for 12 weeks. Serial bronchoprovocations were performed at Weeks 0, 6, and 12. The impact of deep inspirations on the airway response to methacholine was evaluated on the basis of inspiratory vital capacity and FEV(1). Fluticasone produced a wide spectrum of changes in the beneficial effects of deep inspiration, but the mean changes were not significant. The magnitude of the steroid-induced changes in bronchoprotection by deep inspiration correlated with baseline log PC(20) (the provocative concentration of methacholine causing a 20% fall in FEV(1); higher log PC(20) predicted improvement of the deep inspiration effect). The steroid-induced changes led to the emergence of strong positive correlations between the effects of deep inspiration and the methacholine log PC(20) that did not exist at baseline. We conclude that deep inspiration-induced bronchoprotection can be restored by inhaled glucocorticosteroids only in individuals with mild hyperresponsiveness. After steroid treatment, the beneficial effects of deep inspiration become significant determinants of the magnitude of airway hyperresponsiveness.

The effects of a deep inspiration (DI) in individuals with asthma differ from those observed in healthy subjects. It has been postulated that the beneficial effect of lung inflation is mediated by airway stretch. One hypothesis to explain the defects in the function of lung inflation in asthma is that a DI may be unable to stretch the airways. This may result from attenuation of the tethering forces between the airways and the surrounding parenchyma. In the current study, we used high-resolution computed tomography (HRCT) to examine the ability of a DI to distend the airways of subjects with asthma (n = 10) compared with healthy subjects (n = 9) at baseline and after increasing airway tone with methacholine (MCh). We found that both at baseline and after the induction of smooth muscle tone with MCh, a DI distended the airways of healthy and asthmatic subjects to a similar extent, indicating that abnormal interdependence between the lung parenchyma and the airways is unlikely to play a major role in the loss or attenuation of the beneficial effect of lung inflation that characterizes asthma. Furthermore, we observed that after constriction had already been induced by MCh, following a DI, bronchodilation occurred in the healthy subjects but further bronchoconstriction occurred in the subjects with asthma. Our findings suggest that an abnormal excitation contraction mechanism in the airway smooth muscle of subjects with mild asthma counteracts the bronchodilatory effect of a DI. Therefore, the mechanism for reduced bronchodilation after DIs in subjects with mild asthma could be intrinsic to the airway smooth muscle.

In healthy subjects, deep inspiration (DI) acts both as a bronchodilator and a bronchoprotector. The latter is impaired in asthmatics. We have now evaluated whether the lack of bronchoprotection is related to bronchial hyperresponsiveness (BHR), and whether the bronchodilatory effect is also lost in asthmatics. Ten healthy subjects (PC20 > 75 mg/ml), 12 asthmatics with moderate to severe BHR (PC20 < 1 mg/ml), 14 asthmatics with mild to borderline BHR (1 < PC20 < 25 mg/ml), and 10 rhinitics with mild to borderline BHR (1 < PC20 < 25 mg/ml) underwent single-dose methacholine provocations inducing at least 20% reduction in FEV1 after 20 min of DI inhibition. To measure the bronchodilatory effect, DIs were taken immediately after the postmethacholine spirometry, and lung function was again tested. To measure the bronchoprotective effect, DIs were taken before the administration of spasmogen. All four groups achieved the same reductions in FEV1 and FVC, in the absence of deep breaths (analysis of variance [ANOVA], p = 0.49). Only healthy subjects showed bronchoprotection (percent bronchoprotection, mean +/- SEM; healthy: 79 +/- 4.0; asthmatics with moderate to severe BHR: 12 +/- 14.5; asthmatics with mild to borderline BHR: -7 +/- 19.7; rhinitics with mild to borderline BHR: 2 +/- 14.0). In contrast, DIs were able to partially reverse bronchial obstruction in all four groups, albeit percent bronchodilation in healthy subjects was somewhat stronger. The dissociation between bronchoprotection and bronchodilation suggests that the two effects involve different mechanisms.

Lung inflation has a beneficial effect on the airways of healthy subjects. It acts as a bronchoprotector, that is to prevent bronchoconstriction, and as a bronchodilator, in that it reverses bronchial obstruction. The bronchoprotective effect of deep inspiration is more potent than the bronchodilatory one, and the two phenomena appear to advocte different mechanisms. Asthmatics and rhinitics with airways hyperresponsiveness show an impairment in bronchoprotection induced by deep breaths, whereas the bronchodilatory effect, although reduced, is still effective. The lack of the bronchoprotective effect of deep inspiration may contribute to the development of airways hyperresponsiveness. The mechanisms through which lung inflation exerts its beneficial role in healthy subjects, and the factors impairing such an effect in those with airways hyperresponsiveness, are currently under investigation.

Aim Wolfram syndrome (WFS), a rare neurodegenerative disorder, is characterized by early onset insulin-dependent diabetes mellitus, optic atrophy, deafness, diabetes insipidus, and neurological abnormalities. Although previously unreported, we hypothesized that neurological complications may be detectable in relatively early stages of the disease. As the cerebellum and brainstem seem particularly vulnerable in WFS, we focused on balance functions critically dependent on these regions. The primary goal of this investigation was to compare balance in young individuals with WFS, in relatively early stages of the disease, to an age-matched cohort using a clinically applicable test. Method Balance was assessed via the mini-BESTest in 13 children, adolescents and young adults with WFS and 30 typically developing age-matched individuals. Results A significant difference was observed between groups in balance as well as in three of four subcomponents of the mini-BESTest and in two timed tasks related to balance. Mini-BESTest scores were correlated with age among typically developing individuals. In the WFS group, mini-BESTest scores were related to overall motor dysfunction, but not age. Interpretation Impairments in balance in WFS may occur earlier in the disease process than previously recognized and appear to be related to overall neurological progression rather than chronological age. Recognizing balance impairments and understanding which balance systems contribute to balance deficits in those with WFS may allow for development of effective patient-centered treatment paradigms.

In the absence of deep inspirations, healthy individuals develop bronchoconstriction with methacholine inhalation. One hypothesis is that deep inspiration results in bronchodilation. In this study, we tested an alternative hypothesis, that deep inspiration acts as a bronchoprotector. Single-dose methacholine bronchoprovocations were performed after 20 min of deep breath inhibition, in nine healthy subjects and in eight asthmatics, to establish the dose that reduces forced expiratory volume in 1 s by >15%. The provocation was repeated with two and five deep inspirations preceding methacholine. Additional studies were carried out to assess optimization and reproducibility of the protocol and to rule out the possibility that bronchoprotection may result from changes in airway geometry or from differential spasmogen deposition. In healthy subjects, five deep inspirations conferred 85% bronchoprotection. The bronchoprotective effect was reproducible and was not attributable to increased airway caliber or to differential deposition of methacholine. Deep inspirations did not protect the bronchi of asthmatics. We demonstrated that bronchoprotection is a potent physiologic function of lung inflation and established its absence, even in mild asthma. This observation deepens our understanding of airway dysfunction in asthma.

Glycogen synthase kinase 3β (GSK-3β) is an enzyme that is suppressed by insulin and when elevated results in insulin resistance in skeletal muscle and diabetes. Its role in beta cell development and function is little known. Because of the enzyme's anti-proliferative and pro-apoptotic properties, the hypothesis to be tested here was that beta cell specific deficiency of GSK-3β in mice would result in enhanced beta cell mass and function.Mice with beta cell deficiency of GSK-3β (β-Gsk-3β [also known as Gsk3b](-/-)) were generated by breeding Gsk-3β (flox/flox) mice with mice overexpressing the Cre recombinase gene under the control of the rat insulin 2 gene promoter (RIP-Cre mice), and glucose tolerance, insulin secretion, islet mass, proliferation and apoptosis were measured. Changes in islet proteins were investigated by western blotting.On a normal diet β-Gsk-3β ( -/- ) mice were found to have mild improvement of glucose tolerance and glucose-induced insulin secretion, and increased beta cell mass accompanied by increased proliferation and decreased apoptosis. On a high-fat diet β-Gsk-3β (-/-) mice exhibited improved glucose tolerance and expanded beta cell mass with increased proliferation relative to that in control mice, resisting fat-fed diabetes. Molecular mechanisms accounting for these phenotypic changes included increased levels of islet IRS1 and IRS2 proteins and phospho-Akt, suggesting enhanced signalling through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and increased islet levels of pancreas/duodenum homeobox protein 1 (PDX1). Inhibition of GSK3 in MIN6 cells in vitro led to increased IRS1 and IRS2 protein levels through inhibition of proteosomal degradation.These results are consistent with a mechanism whereby endogenous GSK-3β activity controls islet beta cell growth by feedback inhibition of the insulin receptor/PI3K/Akt signalling pathway.

OBJECTIVE—β-Cells are particularly susceptible to fatty acid–induced apoptosis associated with decreased insulin receptor/phosphatidylinositol-3 kinase/Akt signaling and the activation of stress kinases. We examined the mechanism of fatty acid–induced apoptosis of mouse β-cells especially as related to the role played by endoplasmic reticulum (ER) stress–induced Foxo1 activation and whether decreasing Foxo1 activity could enhance cell survival.RESEARCH DESIGN AND METHODS—Mouse insulinoma (MIN6) cells were administered with fatty acids, and the role of Foxo1 in mediating effects on signaling pathways and apoptosis was examined by measuring Foxo1 activity and using dominant-negative Foxo1.RESULTS—Increasing fatty acid concentrations (100–400 μmol/l palmitate or oleate) led to early Jun NH2-terminal kinase (JNK) activation that preceded induction of ER stress markers and apoptosis. Foxo1 activity was increased with fatty acid administration and by pharmacological inducers of ER stress, and this increase was prevented by JNK inhibition. Fatty acids induced nuclear localization of Foxo1 at 4 h when Akt activity was increased, indicating that FoxO1 activation was not mediated by JNK inhibition of Akt. In contrast, fatty acid administration for 24 h was associated with decreased insulin signaling. A dominant-negative Foxo1 adenovirus (Adv-DNFoxo) conferred cells with protection from ER stress and fatty acid–mediated apoptosis. Microarray analysis revealed that fatty acid induction of gene expression was in most cases reversed by Adv-DNFoxo, including the proapoptotic transcription factor CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein).CONCLUSIONS—Early induction of JNK and Foxo1 activation plays an important role in fatty acid–induced apoptosis. Expressing a dominant-negative allele of Foxo1 reduces expression of apoptotic and ER stress markers and promotes β-cell survival from fatty acid and ER stress, identifying a potential therapeutic target for preserving β-cells in type 2 diabetes.

Glycogen synthase kinase-3 (GSK3) has been implicated in the pathophysiology of several prevalent diseases, including diabetes. However, despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, the involvement of GSK3 in islet beta cell growth and function in vivo is unknown. We therefore sought to determine whether over-activation of GSK3beta would lead to alterations in islet beta cell mass and/or function.Transgenic mice overexpressing a constitutively active form of human GSK3beta (S9A) under the control of the rat insulin promoter (RIP-GSK3betaCA) were created. Studies using mouse insulinoma cells (MIN6) were conducted to investigate the regulation of GSK3beta activity and its impact on pancreas/duodenum homeobox protein-1 (PDX-1) levels.We demonstrated that phosphorylation of GSK3beta was decreased, indicating increased GSK3beta activity in two animal models of diabetes, Lepr(-/- ) mice and Ins2 (Akita/+) mice. In MIN6 cells, the activity of GSK3beta was regulated by glucose, in a fashion largely dependent on phosphatidylinositol 3-kinase. RIP-GSK3betaCA transgenic mice showed impaired glucose tolerance after 5 months of age. Histological studies revealed that transgenic mice had decreased beta cell mass and decreased beta cell proliferation, with a 50% decrease (p0.05) in the level of PDX-1.We showed direct evidence that GSK3beta activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. These results may ultimately facilitate the development of potential therapeutic interventions targeting type 2 diabetes and/or islet transplantation.

OBJECTIVE—Single nucleotide polymorphisms (SNPs) in the P2 promoter region of HNF4A were originally shown to be associated with predisposition for type 2 diabetes in Finnish, Ashkenazi, and, more recently, Scandinavian populations, but they generated conflicting results in additional populations. We aimed to investigate whether data from a large-scale mapping approach would replicate this association in novel Ashkenazi samples and in U.K. populations and whether these data would allow us to refine the association signal. RESEARCH DESIGN AND METHODS—Using a dense linkage disequilibrium map of 20q, we selected SNPs from a 10-Mb interval centered on HNF4A. In a staged approach, we first typed 4,608 SNPs in case-control populations from four U.K. populations and an Ashkenazi population (n = 2,516). In phase 2, a subset of 763 SNPs was genotyped in 2,513 additional samples from the same populations. RESULTS—Combined analysis of both phases demonstrated association between HNF4A P2 SNPs (rs1884613 and rs2144908) and type 2 diabetes in the Ashkenazim (n = 991; P < 1.6 × 10−6). Importantly, these associations are significant in a subset of Ashkenazi samples (n = 531) not previously tested for association with P2 SNPs (odds ratio [OR] ∼1.7; P < 0.002), thus providing replication within the Ashkenazim. In the U.K. populations, this association was not significant (n = 4,022; P > 0.5), and the estimate for the OR was much smaller (OR 1.04; [95%CI 0.91–1.19]). CONCLUSIONS—These data indicate that the risk conferred by HNF4A P2 is significantly different between U.K. and Ashkenazi populations (P < 0.00007), suggesting that the underlying causal variant remains unidentified. Interactions with other genetic or environmental factors may also contribute to this difference in risk between populations.

Insulin is both a hormone regulating energy metabolism and a growth factor. We and others have shown that physiological doses of insulin initiate complex signals in primary human and mouse β-cells, but the functional significance of insulin's effects on this cell type remains unclear. In the present study, the role of insulin in β-cell apoptosis was examined. Exogenous insulin completely prevented apoptosis induced by serum withdrawal when given at picomolar or low nanomolar concentrations but not at higher concentrations, indicating that physiological concentrations of insulin are antiapoptotic and that insulin signaling is self-limiting in islets. Insulin treatment was associated with the nuclear localization of Pdx1 and the prosurvival effects of insulin were largely absent in islets 50% deficient in Pdx1, providing direct evidence that Pdx1 is a signaling target of insulin. Physiological levels of insulin did not increase Akt phosphorylation, and the protective effects of insulin were only partially altered in islets lacking 80% of normal Akt activity, suggesting the presence of additional insulin-regulated antiapoptotic pathways. Proteomic analysis of insulin-treated human islets revealed significant changes in multiple proteins. Bridge-1, a Pdx1-binding partner and regulator of β-cell survival, was increased significantly at low insulin doses. Together, these data suggest that insulin can act as a master regulator of islet survival by regulating Pdx1.

The National Research Council Panel on Handling Missing Data in Clinical Trials recommended that protocols for clinical trials ‘explicitly define... causal estimands of primary interest’. In discussions with sponsors of clinical trials since the publication of the National Research Council report, the expression causal estimands has been the subject of confusion. It may not be entirely clear what the National Research Council panel meant, and in any case, it has not been clear how this recommendation might be put in practice. This paper's purpose is to say how the working group understands it and how we think it should be put in practice. We classify possible choices of estimand according to their usefulness for regulatory purposes in various clinical settings. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

The ATP-sensitive K+ channel (KATP channel) senses metabolic changes in the pancreatic β-cell, thereby coupling metabolism to electrical activity and ultimately to insulin secretion. When KATP channels open, β-cells hyperpolarize and insulin secretion is suppressed. The prediction that KATP channel “overactivity” should cause a diabetic state due to undersecretion of insulin has been dramatically borne out by recent genetic studies implicating “activating” mutations in the Kir6.2 subunit of KATP channel as causal in human diabetes. This article summarizes the emerging picture of KATP channel as a major cause of neonatal diabetes and of a polymorphism in KATP channel (E23K) as a type 2 diabetes risk factor. The degree of KATP channel “overactivity” correlates with the severity of the diabetic phenotype. At one end of the spectrum, polymorphisms that result in a modest increase in KATP channel activity represent a risk factor for development of late-onset diabetes. At the other end, severe “activating” mutations underlie syndromic neonatal diabetes, with multiple organ involvement and complete failure of glucose-dependent insulin secretion, reflecting KATP channel “overactivity” in both pancreatic and extrapancreatic tissues.

AIMS/HYPOTHESIS: Wolfram syndrome is an autosomal recessive disorder characterised by childhood diabetes mellitus, optic atrophy and severe neurodegeneration, resulting in premature death. The aim of this study was to investigate the mechanisms responsible for the phenotype of carbohydrate intolerance and loss of pancreatic beta cells in this disorder. MATERIALS AND METHODS: To study the role of the Wolfram gene (Wfs1) in beta cells, we developed a mouse model with conditional deletion of Wfs1 in beta cells by crossing floxed Wfs1 exon 8 animals with mice expressing Cre recombinase under the control of a rat insulin promoter (RIP2-Cre). Complementary experiments using RNA interference of Wfs1 expression were performed in mouse insulinoma (MIN6) cell lines (WfsKD). RESULTS: Male knockout mice (betaWfs(-/-)) began developing variable and progressive glucose intolerance and concomitant insulin deficiency, compared with littermate controls, by 12 weeks of age. Analysis of islets from betaWfs(-/-) mice revealed a reduction in beta cell mass, enhanced apoptosis, elevation of a marker of endoplasmic reticulum stress (immunoglobulin heavy chain-binding protein [BiP]), and dilated endoplasmic reticulum with decreased secretory granules by electron microscopy. WfsKD cell lines had significantly increased apoptosis and elevated expression of the genes encoding BiP and C/EBP-homologous protein (CHOP), two markers of endoplasmic reticulum stress. CONCLUSIONS/INTERPRETATION: These results indicate that (1) lack of expression of Wfs1 in beta cells was sufficient to result in the diabetes mellitus phenotype; (2) beta cell death occurred by an accelerated process of apoptosis; and (3) lack of Wfs1 was associated with dilated endoplasmic reticulum and increased markers of endoplasmic reticulum stress, which appears to be a significant contributor to the reduction in beta cell survival.

Decreased nitric oxide (NO) is considered an important pathogenetic mechanism in pulmonary arterial hypertension (PAH), but clear evidence is lacking.We used multiple techniques to assess endogenous NO in 10 patients with untreated PAH (8 idiopathic and 2 anorexigen-associated PAH) and 12 control subjects.After a nitrite/nitrate-restricted diet, NO metabolites (NOx) were assayed in 24-hour urine collections and exhaled NO (FE(NO)) determined at multiple expiratory flows. Analysis of the relation between FE(NO) and flow allowed derivation of three flow-independent parameters: airway wall concentration (C(W)), diffusing capacity (D(NO)), and alveolar concentration (C(A)). Seven patients underwent follow-up testing after 3 months of bosentan treatment.At baseline, FE(NO) was markedly decreased at the two lowest expiratory flows in PAH: 21 +/- 4 versus 36 +/- 4 ppb at 18 ml/second and 11 +/- 2 versus 17 +/- 2 ppb at 50 ml/second, for subjects with PAH and control subjects, respectively (p0.05). C(W) was 33 +/- 11 ppb in subjects with PAH versus 104 +/- 34 in control subjects (p = 0.04). Urinary NOx was also reduced in PAH (42 +/- 6 microM NOx/mM creatinine versus 62 +/- 7 in control subjects; p = 0.04). After bosentan, FE(NO), C(W), and urine NOx increased to control values (p0.05). Exclusion of the two anorexigen cases did not alter these results.FE(NO) at low expiratory flows was decreased in PAH due to reduced C(W). Bosentan reversed these abnormalities, suggesting that suppression of NO in PAH may have been caused by endothelin.

Summary Sir2 NAD-dependent deacetylases connect transcription, metabolism, and aging. Increasing the dosage or activity of Sir2 extends life span in yeast, worms, and flies and promotes fat mobilization and glucose production in mammalian cells. Here we show that increased dosage of Sirt1, the mammalian Sir2 ortholog, in pancreatic β cells improves glucose tolerance and enhances insulin secretion in response to glucose in be ta cell-specific S ir t 1- o verexpressing (BESTO) transgenic mice. This phenotype is maintained as BESTO mice age. Pancreatic perfusion experiments further demonstrate that Sirt1 enhances insulin secretion in response to glucose and KCl. Microarray analyses of β cell lines reveal that Sirt1 regulates genes involved in insulin secretion, including uncoupling protein 2 ( Ucp2 ). Isolated BESTO islets also have reduced Ucp2, increased ATP production, and enhanced insulin secretion during glucose and KCl stimulation. These findings establish the importance of Sirt1 in β cell function in vivo and suggest therapeutic interventions for type 2 diabetes.

An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet beta-cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in beta-cell survival, we sought to determine whether this pathway is involved in ER stress-induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress-induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress-induced apoptosis and reduced levels of phospho-glycogen synthase kinase 3beta (GSK3beta). In control cells, ER stress-induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3beta. To further assess the role of GSK3beta in ER stress-induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3beta expression. These cells were found to resist ER stress-induced apoptosis. These results illustrate that ER stress-induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3beta pathway and that GSK3beta represents a novel target for agents to promote beta-cell survival.

The role of insulin signaling in pancreatic beta cells has become increasingly apparent. Stably transformed insulinoma cell lines (MIN6) were created with small interfering RNA resulting in the reduction of insulin receptor (IR) expression up to 80% (insulin receptor knockdown, IRKDDelta80). Functionally perturbed IR signaling was confirmed with the absence of insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation. Additionally, Akt phosphorylation was reduced and responded poorly to glucose stimulation. Gene expression profiling revealed that reduced IR expression was associated with alterations in expression of >1,500 genes with diverse functions. IRKD cells exhibited low rate of proliferation due to delay in transition from G0/G1 to S phase, whereas susceptibility to apoptosis did not differ from that of control cells. Insulin content was reduced in proportion to the reduction of IR. IRKD cells maintained glucose responsiveness as measured by NADPH generation, whereas Ca2+ responses and insulin secretion were enhanced. IRKDDelta80 and control cells were treated with glucose (25 mm) or insulin (100 nm) for 45 min, and gene expression profiles were assessed. Transcriptional activation of several hundred early response genes common to both glucose and insulin stimulation was observed in control cells. In IRKDDelta80 cells, insulin failed to activate any genes as anticipated. Importantly, glucose stimulation of gene expression in IRKDDelta80 cells showed that most genes previously activated by glucose were no longer activated, suggesting a major autocrine/paracrine effect of insulin on glucose-regulated gene expression. On the other hand, there were a number of glucose-regulated genes in the IRKDDelta80 cells that were not previously observed in control cells, suggesting a feedback regulation of insulin signaling on glucose-regulated gene expression. These results demonstrate important roles of the insulin receptor in islet beta cell gene expression and function and may serve to elucidate molecular defects in animal models with diminished beta cell insulin signaling.

Background Human rhinovirus (HRV) infections are associated with exacerbations of asthma, chronic obstructive pulmonary disease, and sinusitis. Nitric oxide (NO) might play an important role in host defense through its potent antiviral properties. Previous studies have shown that HRV infection in human subjects increased nasal epithelial expression of type 2 nitric oxide synthase (NOS2), an isoform of the enzyme that produces NO. Objective We sought to investigate whether increases in exhaled NO (eNO) would accompany the increased NOS2 expression and would be associated with clearance of the virus. Methods Six human subjects were infected with HRV-16 intranasally. eNO from nasal and lower airways was measured by means of direct measurement at multiple controlled flow rates. eNO was monitored at baseline (day 1) and on days 2 to 5, 8, 14, and 42 after infection. Nasal lavages were performed on days 1 to 5 and 8, and nasal scrapings were performed on days 1 to 4. NOS2 mRNA expression in nasal cells was measured by using quantitative real-time RT-PCR. Viral shedding in nasal lavage fluid was monitored by using real-time RT-PCR and bioassay. Results Peak HRV titers and symptom scores were correlated on day 3, and HRV persisted until day 5 (n=4) or day 8 (n=2). Infection was associated with transient but significant increases in lymphocytes and monocytes in nasal lavage fluid. Significant increases in both nasal and lower airway eNO concentrations accompanied HRV infection and were positively correlated. Increased nasal eNO concentrations on day 3 were associated with increased expression of NOS2 mRNA in nasal scrapings. Symptom scores on day 4 were inversely correlated with the increases in nasal eNO concentration. Conclusions We conclude that increased production of NO occurs as part of the host response to HRV infection and speculate that NO plays a beneficial role in viral clearance.

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed “PancChip 4.0,” which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.

Over the past 5 years, microarrays have greatly facilitated large-scale analysis of gene expression levels. Although these arrays were not specifically geared to represent tissues and pathways known to be affected by diabetes, they have been used in both type 1 and type 2 diabetes research. To prepare a tool that is particularly useful in the study of type 1 diabetes, we have assembled a nonredundant set of 3,400 clones representing genes expressed in the mouse pancreas or pathways known to be affected by diabetes. We have demonstrated the usefulness of this clone set by preparing a cDNA glass microarray, the PancChip, and using it to analyze pancreatic gene expression from embryonic day 14.5 through adulthood in mice. The clone set and corresponding array are useful resources for diabetes research.

According to a previously published theoretical model of emphysema, the ratio of RV to TLC (RV/TLC) reflects the size mismatch between the hyperinflated lungs in the disease and the surrounding chest. The model suggests that RV/TLC is an important predictor of improvement in FVC and that increased FVC is an important determinant of increased FEV(1) after lung volume reduction surgery (LVRS). We tested these predictions in 13 patients undergoing LVRS, in whom we made detailed measurements of lung mechanics. Using stepwise regression, we found that RV/TLC was the only preoperative independent predictor of the increase in FVC. Seventy percent of the increase in FEV(1) was attributable to increased FVC, with the remainder due to increased FEV(1)/FVC. In a separate group of 78 LVRS patients evaluated with standard preoperative pulmonary function tests, RV/TLC again was found to correlate with the increase in FVC, and changes in FEV(1) were also due largely to changes in FVC. However, RV/TLC was not predictive of the increase in FEV(1) among the group of 78 patients, because FEV(1)/FVC in patients with a low preoperative RV/TLC often increased despite little change in FVC. These findings support the proposed mechanism for increased FVC following LVRS. They also illustrate the limitations of the model, and suggest further hypotheses for selecting patients who may benefit from surgery.

Background Little is known about the perception of airflow obstruction in patients hospitalized for acute asthma. Objectives To evaluate patient perception of airflow obstruction at hospital discharge and at a 2-week follow-up visit and to determine whether symptom control and/or severity of airflow obstruction identified patients at risk for acute asthma after discharge. Methods In a prospective cohort study of inner-city adults hospitalized for acute asthma from April 1, 2001, through October 31, 2002, symptom control (Asthma Control Questionnaire) and airflow obstruction (forced expiratory volume in 1 second [FEV 1 ] percentage predicted) were evaluated at discharge and 2 weeks after discharge. We evaluated perception of airflow obstruction (symptom control vs FEV 1 percentage predicted) and perception of change in airflow obstruction (change in symptom control vs percentage change in FEV 1 ) between the 2 visits. Acute asthma after discharge was defined as an emergency department visit or hospitalization for asthma within 90 days of discharge. Results In fifty-one participants, symptom control was not significantly associated with airflow obstruction at hospital discharge ( P = .30), indicating poor perception of airflow obstruction. Among the 41 participants (80.4% of those enrolled) who completed the follow-up visit, change in symptom control was not significantly associated with change in airflow obstruction ( P = .20), indicating poor perception of change in airflow obstruction. Greater airflow obstruction at follow-up ( P = .02) and a smaller improvement in airflow obstruction ( P = .03), but not symptom control, were associated with a higher risk of acute asthma after discharge. Conclusions Patients hospitalized for acute asthma have poor perception of airflow obstruction and change in airflow obstruction. Objective measurements of lung function should guide treatment decisions after discharge in this population.

The results of the current studies define the major elements whereby glucose metabolism in islet beta-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that beta-cell depolarization and Ca(2+) influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca(2+)/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca(2+)-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser(103)-phosphorylated SRF in nuclear extracts, indicated that the SRE.SRF complexes contribute to the Ca(2+)-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

Normal subjects prevented from taking a deep breath show changes in airflow similar to those of asthmatics when challenged with methacholine (MCh). To confirm airway narrowing by MCh in this setting and to determine its location, we concurrently measured changes in airway lumenal area using high resolution computed tomography (HRCT) and airflow using partial spirometry in five normal subjects challenged with increasing doses of MCh under prohibition of deep breaths. In an attempt to improve imaging accuracy, we corrected for the changes in lung volume during bronchoprovocation. At every step of the provocation, scanning was performed at approximately the same lung volume. On the HRCT images, airway area decreased in response to the increasing doses of MCh to 91 +/- 2%, 88 +/- 2%, and 80 +/- 2% of baseline at the doses of MCh 0.25, 0.75, and 2.5 mg/ml, respectively (p0.001). Airway narrowing showed no predilection for particular airway sizes and occurred in a heterogeneous pattern. The changes in the mean airway lumenal area as measured by HRCT and the mean partial spirometric outcomes were highly correlated: FEV(1)p (r(2) = 0.46, p = 0.001), FVCp (r(2) = 0.20, p = 0.05), FEV(1)/FVCp (r(2) = 0.55, p = 0.002), MMEFp (r(2) = 0.31, p = 0.01), and taup (r(2) = 0.51, p = 0.0004). We conclude that in normal subjects who are prevented from taking a deep breath, the spirometric changes occurring with aerosol MCh challenge are associated with conducting airway narrowing.

Wolfram syndrome, originally described in 1938, is a rare, autosomal recessive disease that is characterised by young onset insulin-dependent diabetes, progressive sensorineural deafness, diabetes insipidus, autonomic nervous system dysfunction and, ultimately, brainstem atrophy and premature death [1]. The Wolfram gene (WFS1), which encodes wolframin, was mapped to chromosome 4p in families with multiple affected individuals [2], and cloned in 1998 [3]. Wolframin is a protein of 890 amino acids that is produced in a wide variety of tissues, most prominently in pancreatic beta cells and brain. Over 100 missense and non-sense mutations have been described patients. As these mutations are associated with a non-immune loss of beta cells and diabetes, the gene was subsequently evaluated in more common forms of diabetes.

The K+ inwardly rectifier channel (KIR) is one of the two sub-units of the pancreatic islet ATP-sensitive potassium channel complex (I(KATP)), which has a key role in glucose-stimulated insulin secretion and thus is a potential candidate for a genetic defect in Type II (non-insulin-dependent) diabetes mellitus. We did a molecular screening of the KIR6.2 gene by single strand conformational polymorphism (SSCP) and direct sequencing in 72 French Caucasian Type II diabetic families. We identified three nucleotide substitutions resulting in three amino acid changes (E23K, L270V and 1337V), that have also been identified in other Caucasian Type II diabetic subjects. These variants were genotyped in French cohorts of 191 unrelated Type II diabetic probands and 119 normoglycaemic control subjects and association studies were done. The genotype frequencies of the L270V and 1337V variants were not very different between Type II diabetic subjects and control groups. In contrast, analysis of the E23K variant showed that the KK homozygocity was more frequent in Type II diabetic than in control subjects (27 vs 14%, p = 0.015). Analyses in a recessive model (KK vs EK/EE) tended to show a stronger association of the K allele with diabetes (p = 0.0097, corrected p-value for multiple testing < 0.02). The data for the E23K variant obtained here and those obtained from three other Caucasian groups studied so far were combined and investigated by meta-analysis. Overall, the E23K variant was found to be significantly associated with Type II diabetes (0.001 < or = p < or = .00106, corrected p-values for multiple testing p < or = 0.01). This study shows that KIR6.2 polymorphisms are frequently associated with Type II diabetes in French Caucasians. Furthermore, a meta-analysis combining different Caucasian groups suggests an significant role of KIR6.2 in the polygenic context of Type II diabetes.

The ATP-sensitive potassium channel, K(ATP) channel, a functional complex of the sulfonylurea receptor 1, SUR1, and an inward rectifier potassium channel subunit, Kir6.2, regulates insulin secretion in the pancreas. Mutations in both the Kir6.2 and SUR1 genes are associated with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disorder of pancreatic beta-cell function characterized by excess insulin secretion and hypoglycemia. We have studied the functional properties of novel SUR1 mutations identified in PHHI patients, including H125Q, N188S, F591L, T1139M, R1215Q, G1382S, and R1394H. R1394H and deltaF1388 SUR1, a previously identified PHHI mutation, resulted in no functional channels when coexpressed with Kir6.2 in COS cells, while H125Q, N188S, F591L, T1139M, R1215Q, and G1382S SUR1 generated functional channels in the absence of ATP. With the exception of N188S and H125Q, all mutants had reduced response to stimulation by MgADP. These results indicate that lack of, or reduction of, K(ATP) channel sensitivity to MgADP is a common molecular defect associated with the disease. The mutant channels also showed varied response to activation by the potassium channel opener diazoxide. Because these mutations are distributed throughout the molecule, our data have new implications for structure-function relationships of the K(ATP) channel, suggesting that structural elements in SUR1 outside of the two nucleotide-binding folds are also important in regulating channel activity.

Spontaneous hyperinsulinemic hypoglycemia in adults is most frequently caused by sporadic, solitary pancreatic beta-cell tumors, whereas hyperinsulinemic hypoglycemia in childhood is commonly caused by generalized beta-cell dysfunction.1 Mutations in the beta-cell sulfonylurea-receptor (SUR1) gene or inward-rectifying potassium-channel (Kir6.2) gene were found in some patients.2–7 A distinct syndrome of hyperinsulinism with hyperammonemia was recently described,8,9 apparently caused by mutations in the glutamate dehydrogenase gene.10 However, many sporadic and familial cases of hyperinsulinism remain unexplained. Some may be due to somatic mutations in other genes, as suggested by reports of autosomal dominant familial hyperinsulinism that was . . .

Family studies have shown that in some populations up to 75% of the variation of body mass index can be explained by genetic factors. However, in humans, no major obesity gene has been identified to date. In contrast, there are a number of genetically well defined animal models for obesity. In two of those models (ob/ob and db/db), defects in the same pathway are responsible for obesity. Recently, some evidence has been found for the OB gene also being involved in human obesity. In this study we investigated the potential role of the OB receptor (OBR) in the etiology of massive obesity in humans using familial linkage analyses and case-control association studies. The typing of two microsatellite markers (D1S198 and D1S209), flanking the OBR gene, in 256 sib pairs showed no evidence for linkage with obesity. In order to be able to detect small gene effects, association studies with a 3'-UTR insertion/deletion polymorphism were carried out. The results of these analyses remained non-significant (chi 2 = 3.442, P = 0.18). However, subjects heterozygous for the insertion/deletion polymorphism showed a slight trend towards lower insulin values 30 min after an oral glucose load compared to homozygous individuals (P = 0.02). In summary, our results do not support a major role of the human OBR gene in the development of morbid obesity in our population.

As part of an ongoing search for susceptibility loci for NIDDM, we tested 19 genes whose products are implicated in insulin secretion or action for linkage with NIDDM. Loci included the G-protein–coupled inwardly rectifying potassium channels expressed in β-cells ( KCNJ3 and KCNJ7 ), glucagon ( GCG ), glucokinase regulatory protein ( GCKR ), glucagon-like peptide I receptor ( GLP1R ), LIM/homeodomain islet-1 ( ISL1 ), caudal-type homeodomain 3 ( CDX3 ), proprotein convertase 2 ( PCSK2 ), cholecystokinin B receptor (CCKBR}, hexokinase 1 ( HK1 ), hexokinase 2 ( HK2 ), mitochondrial FAD-glycerophosphate dehydrogenase ( GPD2 ), liver and muscle forms of pyruvate kinase ( PKL, PKM ), fatty acid–binding protein 2 ( FABP2 ), hepatic phosphofructokinase ( PFKL ), protein serine/threonine phosphatase 1 beta ( PPP1CB ), and lowdensity lipoprotein receptor ( LDLR ). Additionally, we tested the histidine-rich calcium locus ( HRC ) on chromosome 19q. All regions were tested for linkage with microsatellite markers in 751 individuals from 172 families with at least two patients with overt NIDDM (according to World Health Organization criteria) in the sibship, using nonparametric methods. These 172 families comprise 352 possible affected sib pairs with overt NIDDM or 621 possible affected sib pairs defined as having a fasting plasma glucose value of >6.1 mmol/1 or a glucose value of >7.8 mmol/1 2 h after oral glucose load. No evidence for linkage was found with any of the 19 candidate genes and NIDDM in our population by nonparametric methods, suggesting that those genes are not major contributors to the pathogenesis of NIDDM. However, some evidence for suggestive linkage was found between a more severe form of NIDDM, defined as overt NIDDM diagnosed before 45 years of age, and the CCKBR locus (11pl5.4; P = 0.004). Analyses of six additional markers spanning 27 cM on chromosome l ip confirmed the suggestive linkage in this region. Whether an NIDDM susceptibility gene lies on chromosome l ip in our population must be determined by further analyses.