The transport of glucose across the plasma membrane is a fundamental mechanism to provide cells with its basic requirements for energy yielding processes. It is also vital for clearing glucose from blood into tissues, a process normally stimulated by the hormone insulin in mammals. The sympathetic nervous system, normally activated during stress, also regulates glucose transport. The sympathetic neurotransmitter noradrenaline, acts on the family of adrenergic receptors (ARs). An important subtype of the AR family is the β-AR, which is subdivided into the β1, β2, and β3-AR. Glucose is transported across the plasma membrane by the family of glucose transporters (GLUT1-12, and HMIT). In this thesis, I have investigated the β-AR regulation of GLUT1 and 4, and glucose uptake, in skeletal muscle cells and brown adipocytes in culture, model systems which correspond to metabolically active, sympathetically innervated and insulin-sensitive tissues. In brown adipocytes, activation of the β3-ARs induced the expression of GLUT1, resulting in a large increase of glucose uptake. In skeletal myotubes, we postulate there is a possible mechanism where β2-ARs can regulate the intrinsic activity of GLUT1. We found that insulin signaling, but not β-adrenergic signaling, mediated glucose uptake through class I phosphatidylinositol 3-kinase (PI3K). The β-adrenergic signaling to glucose uptake appeared to involve a PI3K related kinase (PIKK), in both skeletal myotubes and brown adipocytes. Furthermore, the increase of glucose uptake by β-ARs in brown adipocytes is partially mediated by AMP-activated protein kinase (AMPK). However, in an artificially constructed system, with cells expressing GLUT4 and β2-ARs, both insulin and β-adrenergic activation translocated GLUT4 and increased glucose uptake. These results show that β-adrenergic signaling increase glucose uptake by regulating glucose transporters through distinct pathways, in skeletal myotubes and brown adipocytes.

No description available.

Noradrenergic signaling has important functions in the central nervous system (CNS) with respect to emotion, learning and memory. Activation of β- adrenergic receptors (β ARs) stimulates protein kinase A via Gs-protein, adenylyl cyclase, and cAMP. Synaptic β←2 -adrenergic receptors, targets of the neurotransmitter norephinephrin, are associated with the GluA1 subunit of AMPA-type glutamate receptors, which mediate most excitatory synaptic transmission in mammalian CNS. PKA-mediated phosphorylation of GluA1 on Ser845 is important for GluA1 surface expression, activity induced postsynaptic accumulation, and synaptic plasticity. Postsynaptic localization of PKA is mediated by a major scaffolding protein `A kinase anchor protein 5 (AKAP5)'. AKAP5 associates with AMPA receptors via SAP97 and PSD95. We have two strains of AKAP5 mutant mice: AKAP5 knockout and AKAP5 D36. AKAP5 KO mice have a complete loss of AKAP5 gene expression. D36 mice miss the last 36 residues (PKA binding site) of AKAP5 but without affecting other interactions. These mutant mice provide us with appropriate in vivo models for studying the functional roles of AKAP5. We compared the functional and physical association of β2AR and AMPA receptors among wild type, AKAP5 KO, and AKAP5 D36 mice. Although AKAP5 was not necessary for the assembly of the β2AR / GluA1 complex, we found that AKAP5 anchored PKA activity was required for full β2AR stimulation-induced GluA1 Ser845 phosphorylation. Recording and analysis of field EPSPs (fEPSPs) of CA1 pyramidal neurons with brief bath perfusion of the β2AR agonist isoproterenol indicated a role of AKAP5 anchored PKA in the regulation of postsynaptic AMPAR responses by norephinephrin. Moreover, we observed a delayed extinction of contextual fear memory in AKAP5 D36 mice, which suggests the involvement of AKAP5 anchored PKA in memory formation and modification.

<p>Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit.</p>

Both obesity and type 2 diabetes are significant health burdens in our society. The prevention of these conditions is vital to individual health and to the health care system, which is inordinately stressed by these chronic diseases. Due to variations in individual response to interventions, prevention strategies may require some tailoring based on heritable traits.The objective of this study was to determine whether insulin sensitivity could be altered by resistance training, and further if body composition or insulin sensitivity response to resistance training in postmenopausal women may be influenced by adrenergic receptor genetic variants and gene-gene interactions.Completers of a 12-month randomized controlled trial of resistance training in sedentary post-menopausal (PM) women, using or not using hormone therapy, were measured for fasting plasma glucose, insulin, and non-esterified fatty acids (NEFA) at baseline and one year. These biomarkers were used to compute models of insulin sensitivity. Body composition was measured by dual x-ray absorptiometry. Subjects were also re-consented for genotyping of adrenergic receptor (ADR) gene variants, ADRA2B Glu9/12, ADRB3 Trp64Arg, ADRB2 Gln27Glu.The resistance training intervention did not have an overall effect on insulin sensitivity in the largest sample and change in insulin sensitivity was largely dependent body composition. There were small favorable effects of genotype on initial measures of both body composition and insulin sensitivity in the ADRA2B Glu9+ carriers versus non-carriers. The effects of ADRA2B alone were no longer present following intervention, but ADRB3 Arg64+ and ADRB2 Glu27+ contribute to improved insulin sensitivity with exercise, when accounting for body composition. ADRB2 Glu27+ was the key to improved biomarkers of insulin sensitivity when in combination with ADRA2B Glu9+ or ADRB3 Arg64+ and a model of insulin sensitivity was most improved by the combination ADRB3 Arg64+ by ADRB2 Glu27+, compared to other ADRB3 by ADRB2 combinations.This is the first trial of ADRA2B, ADRB3, and ADRB2 genetic variation combinations and resistance training in postmenopausal women relative to body composition and insulin sensitivity. Some specific genotypes were identified as responders and non-responders to exercise. These data support independent associations between body composition and insulin sensitivity and the ADR gene variants.

We have shown that the stimulation of β-adrenergic receptors (β-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of αβ-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases β1 integrins expression in ARVMs via the stimulation of α1-AR, not β-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited α1-AR-stimulated increases in β1 integrins expression. Activation of β1 integrins signaling pathway using laminin (LN) inhibited β-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of β1 integrins with anti-β1 integrin antibodies prevented β-AR-stimulated apoptosis. Treatment of cells using LN or anti-β1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of α1-AR regulates β1 integrins expression via the activation of ERK1/2, (2) β1 integrins signaling protects ARVMs from β-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of β1-integrin signaling. These data suggest that β1 integrin signaling protects ARVMs against β-AR-stimulated apoptosis possibly via the involvement of ERK1/2.

Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Introdução: A influência de variantes genéticas dos genes da visfatina e dos recep- tores ?2-adrenérgicos em resposta ao treinamento físico é ainda inconclusiva. Obje- tivos: Verificar os efeitos do treinamento com danças afro-brasileiras por 13 sema- nas sobre parâmetros de saúde de crianças com sobrepeso ou obesidade e a in- fluência das variantes genéticas acima descritas na magnitude de resposta a este treinamento. Materiais e métodos: 30 crianças (9 ± 1,1 anos) realizaram um trei- namento que consistia em 5 minutos de aquecimento a 60% da FCmáx, quatro mo- mentos dez minutos de 70% a 80% da FCmáx intercalados com cinco momentos de dois minutos de recuperação ativa a 60% da FCmáx, 3x/sem, 60 min por sessão. Antes e após o treinamento realizou-se avaliação da composição corporal, índice de massa corporal (IMC), z- score do IMC, circunferência da cintura (CC), relação cintu- ra/estatura (RCE), pressão arterial sistólica (PAS) e diastólica (PAD), capacidade aeróbia e analise da variabilidade da frequência cardíaca (VFC). Análises sanguí- neas foram realizadas para genotipagem, avaliação do perfil lipídico, glicemia, insu- lina e HOMA-IR. A análise estatística foi realizada utilizando modelo de regressão de efeitos mistos. Resultados: Após o treinamento com dança afro-brasileira houve redução significativa (p<0,05) no z-score do IMC (-6,6%), na relação cintura/estatura (-4,8%) e consumo calórico (-15,3%). Considerando os genótipos dos polimorfismos estudados houve uma diminuição significativa no valor do z-score do IMC (-10%) para o genótipo AG do gene do receptor ?2-adrenérgico Arg16Gly, mas nenhuma alteração significativa para os genótipos relacionados aos genes da visfatina e Gln27Glu do receptor ?2-adrenérgico. Conclusão: O treinamento com dança afro- brasileira foi uma estratégia com efeito positivo sobre o score-z do IMC e relação cintura/estatura em crianças com sobrepeso e obesidade. E o genótipo AG do poli- morfismo do receptor ?2-adrenérgico Arg16Gly apresentou melhor resposta ao trei- namento no z-score do IMC, o que pode indicar uma influência genética à resposta ao treinamento. / Introduction: The influences of NAMPT and ?2-adrenergic receptors polymorphisms in response to dance training remain unclear. Objectives: To verify the effects of dance training on health parameters of overweight or obese children and to verify the influence of the genetic variants previously mentioned in response to 13 weeks of training with African-Brazilian dance. Methods: Thirty children (9 ± 1.1 years) per- formed a training that consisted of 10 minutes at 60% of HRmax, four moments from 70% to 80 % of HRmax interspersed with five minutes of active recovery at 60% of HRmax, in total of 60 minutes of training session. Before and after the training body composition, body mass index (BMI), waist circumference (WC), systolic and diastolic blood pressure (DBP), physical fitness and heart rate variability (HRV) were evaluat- ed. Blood analyzes were performed for genotyping and evaluation of the lipid profile, glucose, insulin and HOMA-IR. Statistical analysis was performed using a general linear mixed effects model. Results: The African-Brazilian dance training resulted in a significant reduction (p<0.05) in BMI z-score (-6.6%), waist-to-height ratio (-4.8%) and caloric intake (-15.3%). In the analysis of each polymorphism, it was possible to observe a significant decrease in the z-score BMI (-10%) for the AG genotype of the Arg16Gly polymorphism in the ?2-adrenergic receptor gene, but there is no signifi- cant difference for the visfatin and Gln27Glu of the ?2-adrenergic receptor polymor- phisms. Conclusion: Afro-Brazilian dance training was a strategy with a positive ef- fect on BMI-z score and waist-to-height ratio in overweight and obese children. In addition, the AG genotype of the ?2-adrenergic receptor Arg16Gly polymorphism presented a better response to training on the BMI z-score, which can suggest a ge- netic influence on the training response.