Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation.

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-ß and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-ß, TNF-α, TGFß-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

Increased hippocampal excitability in miR-324-null mice.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3'UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

Growth Hormone-Releasing Hormone in Lung Physiology and Pulmonary Disease.

Growth hormone-releasing hormone (GHRH) is secreted primarily from the hypothalamus, but other tissues, including the lungs, produce it locally. GHRH stimulates the release and secretion of growth hormone (GH) by the pituitary and regulates the production of GH and hepatic insulin-like growth factor-1 (IGF-1). Pituitary-type GHRH-receptors (GHRH-R) are expressed in human lungs, indicating that GHRH or GH could participate in lung development, growth, and repair. GHRH-R antagonists (i.e., synthetic peptides), which we have tested in various models, exert growth-inhibitory effects in lung cancer cells in vitro and in vivo in addition to having anti-inflammatory, anti-oxidative, and pro-apoptotic effects. One antagonist of the GHRH-R used in recent studies reviewed here, MIA-602, lessens both inflammation and fibrosis in a mouse model of bleomycin lung injury. GHRH and its peptide agonists regulate the proliferation of fibroblasts through the modulation of extracellular signal-regulated kinase (ERK) and Akt pathways. In addition to downregulating GH and IGF-1, GHRH-R antagonist MIA-602 inhibits signaling pathways relevant to inflammation, including p21-activated kinase 1-signal transducer and activator of transcription 3/nuclear factor-kappa B (PAK1-STAT3/NF-κB and ERK). MIA-602 induces fibroblast apoptosis in a dose-dependent manner, which is an effect that is likely important in antifibrotic actions. Taken together, the novel data reviewed here show that GHRH is an important peptide that participates in lung homeostasis, inflammation, wound healing, and cancer; and GHRH-R antagonists may have therapeutic potential in lung diseases.

microRNA-seq of cartilage reveals an overabundance of miR-140-3p which contains functional isomiRs.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

CRELD2 Is a Novel LRP1 Chaperone That Regulates Noncanonical WNT Signaling in Skeletal Development.

Cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER)-resident chaperone highly activated under ER stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage-specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whereas deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating noncanonical WNT4 signaling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor low-density lipoprotein receptor-related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF-ß1 signaling. Therefore, our data provide a novel link between an ER-resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT-responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Effects of 100 % oxygen during exercise in patients with interstitial lung disease.

PURPOSE: Hypoxemia limits exercise in some patients with interstitial lung disease (ILD). High levels of supplemental oxygen during exercise might allow physical training at a higher level and more effective pulmonary rehabilitation (PR). Our goals were to use graded cardiopulmonary exercise testing (CPET) to determine whether hyperoxia (FIO2≈1.0) increased exercise tolerance in patients with mild to moderate ILD. METHODS: We studied 6 patients with ILD, including idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP). The study population included 3 females and 3 males (age 69 ± 5 [SD] years; FVC 61 ± 14 %; absolute DLCO 53 ± 19 %). Subjects underwent 2 ramped (15 W/min) CPET protocols on a cycle ergometer (Jaeger Oxycon Pro™, CareFusion Respiratory Care) breathing either air or oxygen (FIO2≈1.0) from a Douglas bag in random order. RESULTS: Minute ventilation (VE) increased significantly during CPET breathing air (pre CPET, 18 ± 2 [SEM] L/min; post CPET, 47 ± 6; P = 0.01), but it did not increase significantly breathing oxygen (pre CPET, 15 ± 3 [SEM]; post CPET, 29 ± 9; P = 0.06). Likewise, carbon dioxide production (VCO2) increased significantly during CPET breathing air (pre CPET, 450 ± 93 [SEM] mL/min; post CPET, 1311 ± 200; P = 0.01), but it did not increase significantly breathing oxygen (pre CPET, 369 ± 129; post CPET, 847 ± 832; P = 0.09). Exercise time during CPET did not differ significantly (P = 0.34) in air (5.6 ± 0.9 [SEM] min) or oxygen (7.0 ± 1.8). Increases in heart rate (HR) and Borg dyspnea index (BDI) after CPET were not affected by breathing oxygen. CONCLUSION: Exercise-induced increases in VE and VCO2 were prevented by breathing pure oxygen during CPET, demonstrating both decreased ventilatory drive and more efficient exercise at achieved workloads. Hyperoxia could enhance the ability of patients with ILD to train at higher workloads, resulting in more effective rehabilitation.

Weight Loss Results in Increased Expression of Anti-Inflammatory Protein CRISPLD2 in Mouse Adipose Tissue.

OBJECTIVE: Obesity is a major risk factor for cardiovascular disease, metabolic syndrome, and type 2 diabetes mellitus, whereas weight loss is associated with improved health outcomes. It is therefore important to learn how adipose contraction during weight loss contributes to improved health. It was hypothesized that adipose tissue undergoing weight loss would have a unique transcriptomic profile, expressing specific genes that might improve health. METHODS: This study conducted an RNA-sequencing analysis of the epididymal adipose tissue of mice fed either a high-fat diet (HFD) or a regular rodent chow diet (RD) ad libitum for 10 weeks versus a cohort of mice fed HFD for the first 5 weeks before being swapped to an RD for the remainder of the study (swapped diet [SWAP]). RESULTS: The swapped diet resulted in weight loss, with a parallel improvement in insulin sensitivity. RNA sequencing revealed several transcriptomic signatures distinct to adipose tissue in SWAP mice, distinguished from both RD and HFD adipose tissue. The analysis found a unique upregulated mRNA that encodes a secreted lipopolysaccharide-binding glycoprotein (CRISPLD2) in adipose tissue. Whereas cellular CRISPLD2 protein levels were unchanged, plasma CRIPSLD2 levels increased in SWAP mice following weight loss and could correlate with insulin sensitivity. CONCLUSIONS: Taken together, these data demonstrate that CRISPLD2 is a circulating adipokine that may regulate adipocyte remodeling during weight loss.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. METHODS: We tested whether MIA-602 (5 µg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. RESULTS: Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. CONCLUSIONS: MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

XBP1 signalling is essential for alleviating mutant protein aggregation in ER-stress related skeletal disease.

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific 'disease signature' of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation.

Nuclear Respiratory Factor 1 Acting as an Oncoprotein Drives Estrogen-Induced Breast Carcinogenesis.

We have previously shown nuclear respiratory factor 1 (NRF1)-mediated transcriptional programming of mitobiogenesis contributes to estrogen-induced breast cancer through modulating cell cycle progression. In this study, we report a new role of NRF1 that goes beyond that of programming mitobiogenesis. Specifically, we report a novel oncogenic function of NRF1 supporting its causative role in breast cancer development and progression. The gain of NRF1 and/or treatment with 17ß-estradiol (E2) produced heterogeneous breast cancer stem cell (BCSC)-like subsets composed of more than 10 distinct cell sub-populations. Flow sorting combined with confocal imaging of markers for pluripotency, epithelial mesenchymal transition (EMT), and BCSCs phenotypically confirmed that the BCSC-like subset arise from cell re-programming. Thus, we determined the molecular actions of NRF1 on its target gene CXCR4 because of its known role in the acquisition of the BCSC-like subset through EMT. CXCR4 was activated by NRF1 in a redox-dependent manner during malignant transformation. An NRF1-induced BCSC-like subset was able to form xenograft tumors in vivo, while inhibiting transcription of CXCR4 prevented xenograft tumor growth. Consistent with our observation of NRF1-driven breast tumorigenesis in the experimental model, higher protein levels of NRF1 were also found in human breast cancer tissue specimens. This highly novel role of NRF1 in the stochastic acquisition of BCSC-like subsets and their progression to a malignant phenotype may open an entirely new research direction targeting NRF1 signaling in invasive breast cancer. Our discovery of targeting transcriptional activation of CXCR4 to inhibit NRF1-induced oncogenic transformation provides a mechanistic explanation for estrogen-dependent breast carcinogenesis and opens new avenues in strategic therapeutics to fight breast cancer.

Translocation and Redistribution of GLUT4 Using a Dual-Labeled Reporter Assay.

It is crucial to determine the regulation of GLUT4 translocation and redistribution to the plasma membrane. The HA-GLUT4-GFP dual-reporter construct has become an important tool in the assessment of GLUT4 recycling in cultured adipocytes and myocytes. Through the use of light microscopy, this reporter construct allows for visualization of GLUT4 specifically at the cell surface or GLUT4 that has recycled from the cell surface while simultaneously marking the total GLUT4 pool. Here, we discuss and outline the general application of this reporter construct and its use in evaluating GLUT4 translocation within cultured adipocytes.

Glucose availability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of nicotinamide metabolism.

Expansion of adipose tissue in response to a positive energy balance underlies obesity and occurs through both hypertrophy of existing cells and increased differentiation of adipocyte precursors (hyperplasia). To better understand the nutrient signals that promote adipocyte differentiation, we investigated the role of glucose availability in regulating adipocyte differentiation and maturation. 3T3-L1 preadipocytes were grown and differentiated in medium containing a standard differentiation hormone mixture and either 4 or 25 mm glucose. Adipocyte maturation at day 9 post-differentiation was determined by key adipocyte markers, including glucose transporter 4 (GLUT4) and adiponectin expression and Oil Red O staining of neutral lipids. We found that adipocyte differentiation and maturation required a pulse of 25 mm glucose only during the first 3 days of differentiation. Importantly, fatty acids were unable to substitute for the 25 mm glucose pulse during this period. The 25 mm glucose pulse increased adiponectin and GLUT4 expression and accumulation of neutral lipids via distinct mechanisms. Adiponectin expression and other early markers of differentiation required an increase in the intracellular pool of total NAD/P. In contrast, GLUT4 protein expression was only partially restored by increased NAD/P levels. Furthermore, GLUT4 mRNA expression was mediated by glucose-dependent activation of GLUT4 gene transcription through the cis-acting GLUT4-liver X receptor element (LXRE) promoter element. In summary, this study supports the conclusion that high glucose promotes adipocyte differentiation via distinct metabolic pathways and independently of fatty acids. This may partly explain the mechanism underlying adipocyte hyperplasia that occurs much later than adipocyte hypertrophy in the development of obesity.

Enhanced GLUT4-Dependent Glucose Transport Relieves Nutrient Stress in Obese Mice Through Changes in Lipid and Amino Acid Metabolism.

Impaired GLUT4-dependent glucose uptake is a contributing factor in the development of whole-body insulin resistance in obese patients and obese animal models. Previously, we demonstrated that transgenic mice engineered to express the human GLUT4 gene under the control of the human GLUT4 promoter (i.e., transgenic [TG] mice) are resistant to obesity-induced insulin resistance. A likely mechanism underlying increased insulin sensitivity is increased glucose uptake in skeletal muscle. The purpose of this study was to investigate the broader metabolic consequences of enhanced glucose uptake into muscle. We observed that the expression of several nuclear and mitochondrially encoded mitochondrial enzymes was decreased in TG mice but that mitochondrial number, size, and fatty acid respiration rates were unchanged. Interestingly, both pyruvate and glutamate respiration rates were decreased in TG mice. Metabolomics analyses of skeletal muscle samples revealed that increased GLUT4 transgene expression was associated with decreased levels of some tricarboxylic acid intermediates and amino acids, whereas the levels of several glucogenic amino acids were elevated. Furthermore, fasting acyl carnitines in obese TG mice were decreased, indicating that increased GLUT4-dependent glucose flux decreases nutrient stress by altering lipid and amino acid metabolism in skeletal muscle.

Physical activity and quality of life improvements of patients with idiopathic pulmonary fibrosis completing a pulmonary rehabilitation program.

INTRODUCTION: Pulmonary rehabilitation is effective for patients with COPD, but its benefit is less clearly established in idiopathic pulmonary fibrosis (IPF), especially in regard to levels of physical activity and health-related quality of life. The objectives were to determine whether pulmonary rehabilitation increased physical activity as assessed by the International Physical Activity Questionnaire (IPAQ), and improved quality of life and symptoms as assessed by the St George respiratory questionnaire for IPF (SGRQ-I) and the Borg dyspnea index (BDI). METHODS: Subjects who met current criteria for IPF were randomized to a 3-month pulmonary rehabilitation program (n = 11) or to a control group (n = 10). The rehabilitation group participated in twice-weekly, 90-min exercise sessions (24 total sessions). The control group maintained its preceding, normal physical activity. All subjects underwent 6-min walk tests to assess the postexertion BDI. The SGRQ-I and a 5-point self-assessment of health were completed at baseline, after 3 months of intervention or observation, and after 3-month follow-up. All subjects completed the IPAQ weekly. RESULTS: Subjects in the rehabilitation group maintained significantly higher levels of physical activity throughout the 3-month rehabilitation program (rehabilitation: 51,364 ± 57,713 [mean ± SD] metabolic equivalent of task-minutes; control: 20,832 ± 37,155, P = .027 by 2-tailed Mann-Whitney test). SGRQ-I symptom domain scores improved considerably by -9 ± 22 in the rehabilitation group, whereas in the control group they worsened (16 ± 12 rehabilitation compared with control, P = .013 by 2-tailed Mann-Whitney test). During the 3-month follow-up, self-reported physical activity levels in the rehabilitation group were 14,428 ± 8,884 metabolic equivalent of task-minutes and in the control group 16,923 ± 32,620 (P = .17 by 2-tailed Mann-Whitney test), demonstrating substantial reversal of activity in the rehabilitation group. BDI scores after 6-min walk tests did not change significantly. CONCLUSIONS: A 3-month rehabilitation program significantly improved symptoms (SGRQ-I) and physical activity levels (IPAQ) in subjects with IPF while they participated actively in the program. (ClinicalTrials.gov registration NCT01118221.).

Rab5 activity regulates GLUT4 sorting into insulin-responsive and non-insulin-responsive endosomal compartments: a potential mechanism for development of insulin resistance.

Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.

Exercise limitation in IPF patients: a randomized trial of pulmonary rehabilitation.

BACKGROUND: Patients with idiopathic pulmonary fibrosis (IPF) have severely limited exercise capacity due to dyspnea, hypoxemia, and abnormal lung mechanics. This pilot study was designed to determine whether pulmonary rehabilitation were efficacious in improving the 6-min walk test (6-MWT) distance, exercise oxygen uptake, respiratory muscle strength [maximum inspiratory pressure (MIP)], and dyspnea in patients with IPF. Underlying physiological mechanisms and effects of the intervention were investigated. METHODS: Subjects were randomly assigned to a 3-month pulmonary rehabilitation program (n = 11) or to a control group (n = 10). All subjects initially underwent the 6-MWT and constant load exercise gas exchange studies. RESULTS: Subjects in the rehabilitation group increased treadmill exercise [metabolic equivalent of task-minutes] over the first 14 sessions. Beneficial effects on physical function resulted in those who completed rehabilitation. Subjects who completed the program increased cycle ergometer time and maintained exercise oxygen consumption (exercise VO(2)) at the baseline level over 3 months, while the control group suffered a significant decrease in exercise VO(2). Rehabilitation subjects also increased their MIP. Plasma lactate doubled and brain natriuretic peptide levels increased significantly after exercise, as did the plasma amino acids glutamic acid, arginine, histidine, and methionine. These changes were associated with significant decreases in arterial oxygen saturation and increases in 15-F(2t)-isoprostanes after exercise. CONCLUSIONS: Pulmonary rehabilitation effectively maintained exercise oxygen uptake over 3 months and lengthened constant load exercise time in patients with moderately severe IPF. Exercise endurance on cycle ergometry testing was limited by dyspnea and severe hypoxemia associated with systemic oxidant stress.

A novel androgen-regulated isoform of the TSC2 tumour suppressor gene increases cell proliferation.

TSC2 (Tuberous sclerosis complex 2) is an important tumour suppressor gene, mutations within which are linked to the development of tuberous sclerosis and implicated in multiple tumour types. TSC2 protein complexes with TSC1 and blocks the ability of the Rheb (Ras homolog enriched in brain) GTPase to activate mTOR (mammalian target of rapamycin), a crucial signal transducer which regulates protein synthesis and cell growth. Here, we report the characterisation of a novel isoform of TSC2 which is under direct control of the ligand-activated androgen receptor. TSC2 isoform A (TSC2A) is derived from an internal androgen-regulated alternative promoter and encodes a 508-amino acid cytoplasmic protein corresponding to the C-terminal region of full-length TSC2, lacking the interaction domain for TSC1 and containing an incomplete interaction domain required for Rheb inactivation. Expression of TSC2A is induced in response to androgens and full-length TSC2 is co-ordinately down-regulated, indicating an androgen-driven switch in TSC2 protein isoforms. In contrast to the well-characterised suppressive effect on cell proliferation of full-length TSC2 protein, both LNCaP and HEK293 cells over-expressing TSC2 isoform A proliferate more rapidly (measured by MTT assays) and have increased levels of cells in S-phase (measured by both Edu staining and FACS analysis). Our work indicates, for the first time, a novel role for this well-known tumour suppressor gene, which encodes an activator of cell proliferation in response to androgen stimulation.