Host-agent-vector-environment measures for electronic cigarette research used in NIH grants.

OBJECTIVE: The purpose of this study is to describe the focus and comprehensiveness of domains measured in e-cigarette research. METHODS: A portfolio analysis of National Institutes of Health grants focusing on e-cigarette research and funded between the fiscal years 2007 and 2015 was conducted. Grant proposals were retrieved using a government database and coded using the Host-Agent-Vector-Environment (HAVE) model as a framework to characterise the measures proposed. Eighty-one projects met the criteria for inclusion in the analysis. RESULTS: The primary HAVE focus most commonly found was Host (73%), followed by Agent (21%), Vector (6%) and Environment (0%). Intrapersonal measures and use trajectories were the most common measures in studies that include Host measures (n=59 and n=51, respectively). Product composition was the most common area of measurement in Agent studies (n=24), whereas Marketing (n=21) was the most common (n=21) area of Vector measurement. When Environment measures were examined as secondary measures in studies, they primarily focused on measuring Peer, Occupation and Social Networks (n=18). Although all studies mentioned research on e-cigarettes, most (n=52; 64%) did not specify the type of e-cigarette device or liquid solution under study. CONCLUSIONS: This analysis revealed a heavy focus on Host measures (73%) and a lack of focus on Environment measures. The predominant focus on Host measures may have the unintended effect of limiting the evidence base for tobacco control and regulatory science. Further, a lack of specificity about the e-cigarette product under study will make comparing results across studies and using the outcomes to inform tobacco policy difficult.

Dietary supplement research portfolio at the NIH, 2009-2011.

The U.S. dietary supplement market increased by 7.5% in 2012 compared with 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine their health effects, safety, and efficacy. A portfolio analysis was performed across the NIH and the Office of Dietary Supplements (ODS) for fiscal years (FYs) 2009-2011 by using the databases Human Nutrition Research Information Management (HNRIM) and Computer Access to Research on Dietary Supplements (CARDS). The results indicated that total NIH dietary supplement-related funding for FYs 2009-2011 was $855 million ($295 million in 2009, $311 million in 2010, and $249 million in 2011). The institutes and centers with the highest investment in dietary supplement research were as follows: the National Heart, Lung, and Blood Institute ($135 million); the National Cancer Institute ($188 million); the National Center for Complementary and Alternative Medicine ($99 million); the National Institute of Diabetes and Digestive and Kidney Diseases ($68 million); the National Institute of Environmental Health Sciences ($58 million); and the ODS ($32 million). The dietary supplement ingredients receiving the most funding were botanicals (22%), vitamins (20%), lipids (14%), and minerals and trace elements (10%). The top 3 outcome research areas were cancer (61% of total dietary supplement investment), cardiovascular disease (47%), and women's reproductive health (38%). In FYs 2009, 2010, and 2011, the ODS provided 3.5%, 3.6%, and 4.1%, respectively, of the NIH investment in dietary supplement research. ODS funding focused on cellular, enzymatic, or molecular mechanisms (64% of total ODS funding). This portfolio analysis demonstrates that the NIH has committed substantial funding to dietary supplement research in an effort to expand the scientific knowledge base on the efficacy and safety of dietary supplements.

Mitochondrial isolation from skeletal muscle.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles. Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 µg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5 mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 µM) is added to start state 3. Oligomycin (1 µM), an ATPase synthase blocker, is used to estimate state. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 µM) is added to measurestate, or uncoupled respiration. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥ 4 is considered as evidence of a viable mitochondria preparation. In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

Chronic hypoxia increases insulin-stimulated glucose uptake in mouse soleus muscle.

People living at high altitude appear to have lower blood glucose levels and decreased incidence of diabetes. Faster glucose uptake and increased insulin sensitivity are likely explanations for these findings: skeletal muscle is the largest glucose sink in the body, and its adaptation to the hypoxia of altitude may influence glucose uptake and insulin sensitivity. This study tested the hypothesis that chronic normobaric hypoxia increases insulin-stimulated glucose uptake in soleus muscles and decreases plasma glucose levels. Adult male C57BL/6J mice were kept in normoxia [fraction of inspired O2 = 21% (Control)] or normobaric hypoxia [fraction of inspired O2 = 10% (Hypoxia)] for 4 wk. Then blood glucose and insulin levels, in vitro muscle glucose uptake, and indexes of insulin signaling were measured. Chronic hypoxia lowered blood glucose and plasma insulin [glucose: 14.3 ± 0.65 mM in Control vs. 9.9 ± 0.83 mM in Hypoxia (P < 0.001); insulin: 1.2 ± 0.2 ng/ml in Control vs. 0.7 ± 0.1 ng/ml in Hypoxia (P < 0.05)] and increased insulin sensitivity determined by homeostatic model assessment 2 [21.5 ± 3.8 in Control vs. 39.3 ± 5.7 in Hypoxia (P < 0.03)]. There was no significant difference in basal glucose uptake in vitro in soleus muscle (1.59 ± 0.24 and 1.71 ± 0.15 µmol·g⁻¹·h⁻¹ in Control and Hypoxia, respectively). However, insulin-stimulated glucose uptake was 30% higher in the soleus after 4 wk of hypoxia than Control (6.24 ± 0.23 vs. 4.87 ± 0.37 µmol·g⁻¹·h⁻¹, P < 0.02). Muscle glycogen content was not significantly different between the two groups. Levels of glucose transporters 4 and 1, phosphoinositide 3-kinase, glycogen synthase kinase 3, protein kinase B/Akt, and AMP-activated protein kinase were not affected by chronic hypoxia. Akt phosphorylation following insulin stimulation in soleus muscle was significantly (25%) higher in Hypoxia than Control (P < 0.05). Neither glycogen synthase kinase 3 nor AMP-activated protein kinase phosphorylation changed after 4 wk of hypoxia. These results demonstrate that the adaptation of skeletal muscles to chronic hypoxia includes increased insulin-stimulated glucose uptake.

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments.

Efficient and specific phosphorylation of PKA substrates, elicited in response to ß-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Glucose uptake in rat extraocular muscles: effect of insulin and contractile activity.

PURPOSE: Extraocular muscles show specific adaptations to fulfill the metabolic demands imposed by their constant activity. One aspect that has not been explored is the availability of substrate for energy pathways in extraocular muscles. In limb muscles, glucose enters by way of GLUT1 and GLUT4 transporters in a process regulated by insulin and contractile activity to match metabolic supply to demand. This mechanism may not apply to extraocular muscles because their constant activity may require high basal (insulin- and activity-independent) glucose uptake. The authors tested the hypothesis that glucose uptake by extraocular muscles is not regulated by insulin or contractile activity. METHODS: Extraocular muscles from adult male Sprague-Dawley rats were incubated with 100 nM insulin or were electrically stimulated to contract (activity); glucose uptake was measured with 2-deoxy-d[1,2-(3)H]glucose. The contents of GLUT1, GLUT4, total and phosphorylated protein kinase B (Akt), phosphorylated AMP-activated protein kinase (AMPK), and glycogen synthase kinase 3 (GSK3) underwent Western blot analysis. RESULTS: Insulin and activity increased glucose uptake over the basal rate to 108% and 78%, respectively. GLUT1 and GLUT4 were detectable in extraocular muscles. Phosphorylated AKT/total AKT increased by twofold after insulin stimulation, but there was no change with activity. AMPK phosphorylation increased 35% with activity. Phosphorylated-GSK3/total GSK3 did not change with insulin or activity. CONCLUSIONS: Glucose uptake in extraocular muscles is regulated by insulin and contractile activity. There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles.

The alpha1D-adrenergic receptor induces vascular smooth muscle apoptosis via a p53-dependent mechanism.

Activation of the endogenous alpha1-adrenergic receptor (AR) associated with human aortic smooth muscle cells resulted in a dose- and time-dependent increase in the levels of mitochondrial reactive oxygen species (ROS). ROS increases were apparent within 10 min and maximal after 45 min. Prolonged activation (>4 h) of the alpha1-AR resulted in smooth muscle cell apoptosis. Both the increase in ROS and apoptotic cell death were blocked by the nonselective alpha1-AR antagonist prazosin as well as the selective alpha1D-AR antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7, 9-dione (BMY 7378). Increases in ROS and apoptosis produced by alpha1-AR activation were also blocked by the p38 mitogen-activated protein kinase inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190) and the NAPDH oxidase inhibitor apocynin. The extracellular signal-regulated kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) or the c-Jun NH2-terminal kinase inhibitor 1, 9-pyrazoloanthrone anthra(1, 9-cd)pyrazol-6(2H)-one (SP 600125) was without effect on increases in ROS levels or apoptosis. Pifithrin-alpha, an inhibitor of the tumor suppressor protein p53, had no effect on ROS generation but did block alpha1D-AR-induced apoptosis. Activation of the alpha1D-AR resulted in translocation of p53 to the mitochondria. The mitochondrial translocation of p53 was blocked by prazosin, BMY 7378, apocynin, SB 202190, and pifithrin-alpha. Apoptosis was also blocked by small interfering RNA directed against p53. These data show that the alpha1D-AR is coupled to the generation of mitochondrial ROS by a pathway involving p38 and NADPH oxidase. Sustained activation of the alpha1D-AR results in smooth muscle cell apoptosis in a pathway that involves the tumor suppressor protein p53 and the mitochondrial translocation of p53. The data also provide evidence of a linkage between the alpha1D-AR and p53.

The alpha1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells.

BACKGROUND: The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. RESULTS: The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR. CONCLUSION: Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.