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.

G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.

Introduction: Across the world the incidence of the metabolic syndrome increases annually at an alarming rate. Two conditions associated with this are obesity and hypertension (high blood pressure). Both have negative health and lifestyle consequences, numerous studies on adrenergic receptor (AR) gene polymorphisms in various population groups have proved, although not exclusively, that these polymorphisms may be positively associated with susceptibility to and progression of obesity and hypertension. The AR encoding genes are attractive targets for such studies because the ARs, as part of the sympathetic nervous system, perform important functions like vasoconstriction, vasodilation, lipolysis and influence the heart's contraction. These functions accentuate the possible role of AR gene polymorphisms in the onset or progression of obesity and hypertension. Obesity is a health concern especially among black South African women. The prevalence of obesity (BMI > 30 kg/m2) in the North-West province of South Africa is high: 28.6%. The POWIRS (Profile of Obese Women with the Insulin Resistance syndrome) study was conducted in 2003 on 102 black South African female volunteers to search for possible associations of the p2-AR Gln27Glu and p3-AR Trp64Arg polymorphisms with parameters of the carbohydrate and lipid metabolism, the index of insulin resistance (HOMA-IR), body mass index (BMI) and body fat % (Schutte et al., 2005). To our knowledge, this was the first study of its kind in South Africa and which led to this study and dissertation. Objectives: The objectives of this study were to: • Determine the incidence of the following polymorphisms in (a) 102 black South African female volunteers and calculate the minor allele frequency: B1-AR: Ser49Gly; B2-AR: Arg16Gly; (b) 115 white South African female volunteers and calculate the minor allele frequency: B1-AR: Ser49Gly; B2-AR: Arg16Gly; Gln27Glu; B3-AR: Trp64Arg; • identify possible diplotypes and haplotypes in the study groups; • take relevant physiological parameters (measured in the POWIRS studies) into account in the search for possible associations of these polymorphisms, diplotypes and haplotypes with obesity and high blood pressure as characteristics of the metabolic syndrome; • compare the black and the white study groups with regards to the above mentioned objectives. Methods: DNA was isolated from blood spots on Guthrie cards (collected during the POWIRS studies) and the respective AR gene regions amplified by polymerase chain reaction (PCR). After restriction enzyme digestion, the DIVA fragments were separated by agarose gel electrophoresis. Genotypic findings were examined along with measured physiological parameters (measured during the POWIRS studies) and statistically processed. Area under the curve (AUC) analysis was performed on parameters measured during the oral glucose tolerance test. Diplotype and haplotype analyses were also performed on both subject groups. Results: The minor allele frequencies for both groups were calculated and compared to that reported in other published studies. For the black group, the minor allele frequencies were: 84% (B1-AR Ser49Gly), 16% (B2-AR Gln27Glu), 49% (B2-AR Arg16Gly) and 28% (B3-AR Trp64Arg) and for the white group: 94%, 46%, 50% and 7% respectively. The AUC differed in almost every instance of comparison, but was within normal ranges. Only a few significant differences were identified when the measured physiological parameters were compared between the genotypes, diplotypes and haplotypes in each group, most of which were found to be within normal ranges. When the two groups of test subjects were compared, only minimal differences were observed, most of which were still found to be well within normal ranges. Conclusions: Although no associations were identified between the separate investigated AR gene polymorphisms, diplotypes or haplotypes and obesity and hypertension or high blood pressure, indications are present that they may act as contributors to risk factors for the onset and progression of these characteristics of the metabolic syndrome. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Diabetes mellitus (DM) and hyperglycemia contribute to sympathetic nervous system (SNS) activation and cardiovascular dysfunction. SNS activation and increased norepinephrine levels downregulate cardiac β-adrenergic receptors (β-AR). The ADMIRE-HF trial identified reduced cardiac SNS innervation as an independent prognostic marker in heart failure. The β-AR antagonist [3H]-CGP12177 was used to quantify cardiac β-AR in ex vivo biodistribution studies in streptozotocin (STZ)-treated rats after 8 weeks of sustained hyperglycemia, and in the Zucker Diabetic Fatty (ZDF) rat model of type-2 diabetes at the onset of hyperglycemia (10 weeks of age) and after a sustained period of hyperglycemia (16 weeks of age). In some STZ rats, insulin was provided at the onset of hyperglycemia, or after a sustained period of hyperglycemia. Insulin treatment at both time points prevented reduced [3H]-CGP12177 binding (33-38% compared to controls) observed in STZ hyperglycemics. ZDF β-ARs were intact at 10 weeks but became reduced (16-25% relative to the Zucker leans) following 6 weeks of hyperglycemia. This work supports that cardiac β-AR are reduced in models of DM and that restoring insulin signalling to maintain glycemic control can normalize β-AR density whether provided early or after a period of sustained hyperglycemia.

Introduction: Across the world the incidence of the metabolic syndrome increases annually at an alarming rate. Two conditions associated with this are obesity and hypertension (high blood pressure). Both have negative health and lifestyle consequences, numerous studies on adrenergic receptor (AR) gene polymorphisms in various population groups have proved, although not exclusively, that these polymorphisms may be positively associated with susceptibility to and progression of obesity and hypertension. The AR encoding genes are attractive targets for such studies because the ARs, as part of the sympathetic nervous system, perform important functions like vasoconstriction, vasodilation, lipolysis and influence the heart's contraction. These functions accentuate the possible role of AR gene polymorphisms in the onset or progression of obesity and hypertension. Obesity is a health concern especially among black South African women. The prevalence of obesity (BMI > 30 kg/m2) in the North-West province of South Africa is high: 28.6%. The POWIRS (Profile of Obese Women with the Insulin Resistance syndrome) study was conducted in 2003 on 102 black South African female volunteers to search for possible associations of the p2-AR Gln27Glu and p3-AR Trp64Arg polymorphisms with parameters of the carbohydrate and lipid metabolism, the index of insulin resistance (HOMA-IR), body mass index (BMI) and body fat % (Schutte et al., 2005). To our knowledge, this was the first study of its kind in South Africa and which led to this study and dissertation. Objectives: The objectives of this study were to: • Determine the incidence of the following polymorphisms in (a) 102 black South African female volunteers and calculate the minor allele frequency: B1-AR: Ser49Gly; B2-AR: Arg16Gly; (b) 115 white South African female volunteers and calculate the minor allele frequency: B1-AR: Ser49Gly; B2-AR: Arg16Gly; Gln27Glu; B3-AR: Trp64Arg; • identify possible diplotypes and haplotypes in the study groups; • take relevant physiological parameters (measured in the POWIRS studies) into account in the search for possible associations of these polymorphisms, diplotypes and haplotypes with obesity and high blood pressure as characteristics of the metabolic syndrome; • compare the black and the white study groups with regards to the above mentioned objectives. Methods: DNA was isolated from blood spots on Guthrie cards (collected during the POWIRS studies) and the respective AR gene regions amplified by polymerase chain reaction (PCR). After restriction enzyme digestion, the DIVA fragments were separated by agarose gel electrophoresis. Genotypic findings were examined along with measured physiological parameters (measured during the POWIRS studies) and statistically processed. Area under the curve (AUC) analysis was performed on parameters measured during the oral glucose tolerance test. Diplotype and haplotype analyses were also performed on both subject groups. Results: The minor allele frequencies for both groups were calculated and compared to that reported in other published studies. For the black group, the minor allele frequencies were: 84% (B1-AR Ser49Gly), 16% (B2-AR Gln27Glu), 49% (B2-AR Arg16Gly) and 28% (B3-AR Trp64Arg) and for the white group: 94%, 46%, 50% and 7% respectively. The AUC differed in almost every instance of comparison, but was within normal ranges. Only a few significant differences were identified when the measured physiological parameters were compared between the genotypes, diplotypes and haplotypes in each group, most of which were found to be within normal ranges. When the two groups of test subjects were compared, only minimal differences were observed, most of which were still found to be well within normal ranges. Conclusions: Although no associations were identified between the separate investigated AR gene polymorphisms, diplotypes or haplotypes and obesity and hypertension or high blood pressure, indications are present that they may act as contributors to risk factors for the onset and progression of these characteristics of the metabolic syndrome. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Previous studies have suggested that sympathetic sprouting in the periphery may contribute to the development and persistence of sympathetically-maintained pain in animal models of neuropathic pain. The purpose of this thesis was to examine morphological changes in the cutaneous innervation in rats after chronic constriction injury (CCI) to the sciatic nerve. More specifically, this study addresses the question of whether sympathetic fibres sprout de novo into the upper dermis of the rat hindpaw skin after CCI of the sciatic nerve. We also determined changes in peptidergic sensory innervation following CCI. / At several periods post-injury, hind paw skin was harvested and processed using a monoclonal antibody against dopamine-beta-hydroxylase to detect sympathetic fibres and a polyclonal antibody against calcitonin gene-related peptide to identify peptidergic sensory fibres. We observed migration and branching of sympathetic fibres into the upper dermis of the hind paw skin, from where they were normally absent. This migration was first detected at 2 weeks, peaked at 4 to 6 weeks and lasted for at least 20 weeks post-lesion. At 8 weeks post-lesion, there was a dramatic increase in the density of peptidergic fibres in the upper dermis. Quantification revealed that densities of peptidergic fibres 8 weeks post-lesion were significantly above levels of sham animals. Interestingly, the ectopic sympathetic fibres did not innervate blood vessels but formed a novel association and wrapped around sprouted peptidergic nociceptive fibres. Our data show a long-term sympathetic and sensory innervation change in the rat hind paw skin after the chronic constriction injury. This novel fibre arrangement after nerve lesion may play an important role in the development and persistence of sympathetically-maintained neuropathic pain after partial nerve lesions.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.