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Cushing's syndrome and glucocorticoid therapy lead to central obesity, insulin resistance, and symptoms of altered energy regulation similar to those observed in the metabolic syndrome. We hypothesized that excess glucocorticoids alter energy sensing/signaling in skeletal muscle through mediation of the LKB1/AMPK signaling pathway. To test this hypothesis, three 100 mg pellets of corticosterone were implanted subcutaneously in each of nine rats for two weeks. Responses were compared with sham operated controls fed ad libitum or food restricted to produce the body weights similar to the treatment group rats. After the treatment period, animals were anesthetized and the right gastrocnemius-plantaris and soleus were removed for analysis. After tibial nerve stimulation for 5 min, the left gastrocnemius-plantaris and soleus were also removed. We assessed AMPK activity and subunit expression, as well as several metabolic indicators including ATP, creatine phosphate, creatine, glycogen, and malonyl-CoA levels in rested and stimulated gastrocnemius-plantaris and soleus muscles. We found that high levels of glucocorticoids decreased AMPKγ3 subunit expression in the gastrocnemius-plantaris. We also observed reduced AMPKα2 activity in the stimulated gastrocnemius-plantaris, but not the soleus; and that this decreased activity corresponded to a significant reduction in phosphorylated TBC1D1, a protein involved in signaling GLUT-4 translocation. Finally, in the gastrocnemius-plantaris, we also noted an increase in glycogen stores in the hypercorticosteronemic rats. Our data suggest that altered energy sensing/signaling associated with high levels of glucocorticoids may be due in part to inhibition of AMPKα2 activity and the high energy state produced by increased glycogen stores. We also conclude that high levels of glucocorticoids decrease the levels of AMPKγ3 and diminish insulin/contraction signaling through phosphorylated TBC1D1.

Metabolic syndrome is defined as a combination of obesity, elevated triglycerides, decreased high-density lipoproteins, hypertension and insulin resistance. It is at least partially caused by sedentary life style and unhealthy dietary habits and is a major risk factor for development and progression of cardiovascular disease and type 2 diabetes. Growing medical and socioeconomic impact of the metabolic syndrome warrants further active search for novel risk markers and therapeutic targets. Recent experimental and epidemiological studies have demonstrated the multiple roles of the endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) as wells as the enzymes, which are involved in their catabolism, dimethyarginine dimethylaminohydrolases (DDAHs) and alanine:glyoxylate aminotransferase 2 (AGXT2) in the pathogenesis of metabolic syndrome and its complications. ADMA is thought to exhibit its pathological effects by inhibiting and uncoupling nitric oxide synthases (NOS), while SDMA can inhibit transport of L-arginine. DDAHs, namely DDAH1 and DDAH2, have been thought as the major enzymes metabolizing ADMA to citrulline, while being inactive towards SDMA. Experimental studies with upregulation of DDAH1 in animal models showed that lowering ADMA results in protection against endothelial dysfunction, atherosclerosis, ischemia/reperfusion injury and vascular remodeling, acceleration of angiogenesis in the settings of ischemia and improvement of insulin sensitivity. Unfortunately, all the attempts to upregulate DDAH1 using small drugs have not been successful. The data regarding the role of DDAH2 are contradictory, with some studies showing that it can metabolize ADMA under certain conditions and other studies questioning its enzymatic activity towards ADMA. AGXT2 is a mitochondrial aminotransferase, which can metabolize, among its other substrates, both ADMA and SDMA. It is a large protein with possible allosteric regulatory sites, suggesting that, in contrast to DDAH1, it could be upregulated by small molecules. The role of AGXT2 in different pathophysiological processes involving ADMA and SDMA is poorly understood. It has been recently discovered in the offspring cohort of the Framingham Heart Study participants that a composite compound, consisting of the products of metabolism of ADMA and SDMA by AGXT2 (asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV), correspondingly) is an independent biomarker of CT (computed tomography)-defined NAFLD (non-alcoholic fatty liver disease) and a predictor of future diabetes up to 12 years before disease, suggesting that AGXT2 may play a key role in development of metabolic disease and its progression. We and other have recently identified several other metabolically active substrates of AGXT2, such as a marker of cardiovascular and overall mortality homoarginine and a regulator of fatty acid oxidation and browning of adipose tissue beta-amino-isobutyric acid (BAIBA), which further supports the importance of AGXT2 in pathogenesis of cardiovascular and metabolic diseases. The data presented in the current thesis enable answering the two research aims: 1) Identification of the tissue and intracellular expression pattern of human AGXT2 and 2) Testing the hypothesis that ubiquitous transgenic overexpression of AGXT2 protects from ADMA-induced vascular damage in vivo. The first research aim provided a thorough characterization of AGXT2 expression in humans using multiple complimentary techniques and addressed the current discrepancy in the literature with previous demonstration of comparable levels of Agxt2 expression by RT-PCR and Western Blot in the kidneys and liver in mice, and previous reports on detection of predominant Agxt2 expression in the kidneys by Northern Blot and in-situ RNA-hybridization in rats. In our current study we analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. We saw the strongest expression of AGXT2 in the kidney and liver both on the mRNA and protein levels. Our immunohistochemistry stainings showed that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed the intracellular localization of AGXT2 in mitochondria. In the second research aim we investigated whether long-term upregulation of AGXT2 is safe and can protect from ADMA- mediated vascular damage in the setting of DDAH1 deficiency, which is commonly observed in cardiovascular pathologies. We generated AGXT2 transgenic (TG) mice with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of ADGV, the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. The work, included into this thesis demonstrates that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans, where the enzyme is localized in mitochondria. The expression of AGXT2 in the liver is consistent with the proposed role of AGXT2 in development and progression of NAFLD and is consistent with our previous discovery of hepatocyte nuclear factor 4 alpha (HNF4α) as the major regulator of Agxt2 expression in the mouse liver. Chronic upregulation of AGXT2 in mice lowered systemic ADMA levels without any obvious effects on viability, development, growth and fertility, suggesting potential safety of this ADMA-lowering approach. Overexpression of AGXT2 protected from ADMA-induced vascular damage in the highly clinically relevant settings of DDAH1 deficiency, suggesting that the observed vascular damage was indeed caused by ADMA itself, rather than by some ADMA-independent effects of DDAH1 deficiency. The observed protective effects of AGXT2 upregulation are especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful. The current study, therefore, provides the basis for the future screens to identify small molecules, which would upregulate AGXT2 activity.

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Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Introduction: Obesity, which is implicated in the development of the metabolic syndrome (MS) is reaching epidemic proportions worldwide. MS significantly increases the risk of developing cardiovascular disease, which includes coronary artery disease. The current absence of animal models of diet induced obesity and the MS makes the investigation of the cardiovascular consequences of MS virtually impossible. As a result the effects of the MS on cardiac function, morphology and susceptibility to ischaemia are not well understood. Aims: We set out to: 1) develop and characterize a rodent model of dietinduced obesity and the MS, 2) investigate the susceptibility of hearts from these animals to ischaemia/reperfusion induced injury and, 3) determine whether angiotensin II (Ang II) and endothelin-1 (ET-1) plays a role in cardiac remodelling and/or the severity of ischaemia and reperfusion injury in this model. Methods: Male Wistar rats were fed a standard rat chow diet or cafeteria diet (CD) for 16 weeks. After the feeding period rats were sacrificed and blood and myocardial tissue samples were collected to document biochemical changes in these animals. Hearts were perfused on the isolated working rat heart perfusion apparatus to assess myocardial mechanical function before and after ischaemia. In a separate series of experiments, hearts underwent coronary artery ligation to determine the incidence and duration of ventricular arrhythmias during ischaemia and reperfusion, using electrocardiography. To assess a possible link between myocardial remodelling and ischaemia/reperfusion injury and myocardial Ang II and ET-1 content, we also measured these peptides under basal conditions and during ischaemia. Two-dimensional targeted Mmode echocardiography was used to assess in vivo myocardial mechanical function in control and obese rats. Results: After 16 weeks on the CD, obese rats satisfied the World Health Organization (WHO) criteria for the MS by having visceral obesity, insulin resistance, dyslipidaemia and an elevated systolic blood pressure, compared to control rats. Circulating Ang II levels, but not ET-1 levels, were elevated in CD fed rats. Obese rats had cardiac hypertrophy and ex vivo basal myocardial mechanical function was depressed in the CD fed rat hearts compared to control rat hearts. CD fed rat hearts had poorer aortic output (AO) recoveries compared to hearts from control rats. These hearts also had a higher incidence and duration of reperfusion arrhythmias. No such functional differences were seen in the in vivo experiments. No differences in basal or ischaemic myocardial Ang II and ET-1 levels were seen in either group. Conclusion: We have developed and characterized a model of diet-induced obesity and the MS. Obesity is associated with cardiac hypertrophy and an increased myocardial susceptibility to ischaemia and reperfusion injury in our model. The hearts from obese rats were also more prone to reperfusion ventricular arrhythmias. As myocardial function was only poorer in the ex vivo obese animal experiments, our data suggests that the obesity associated changes in function observed in the ex vivo studies may be related to the absence of circulating substrates or factors, which are essential for their normal mechanical function.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / Insulin stimulates the production of nitric oxide (NO) in endothelial cells and cardiac myocytes by a signalling pathway that involves the insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Physiological concentrations of NO play an important part in maintaining normal vascular function. It has been suggested that nitric oxide synthase (NOS) activity and NO production are chronically impaired in diabetes mellitus by an unknown mechanism. The reninangiotensin system and subsequent production of angiotensin II (Ang II) are elevated in obesity and diabetes while antagonism of the AT1 receptor with Losartan has beneficial effects in patients with insulin resistance and type II diabetes. Aims: We therefore aimed to investigate (i) the effect of Ang II on myocardial insulin signalling with regards to key proteins (IRS-1, PKB/Akt, eNOS and p38 MAPK) in correlation with NO production, (ii) the effect of Losartan on these parameters. Methods: Hyperphagia-induced obese, insulin resistant rats (DIO=diet supplemented with sucrose and condensed milk) were compared to age-matched controls. Half the animals were treated with 10mg/kg Losartan per day for 1 week. Isolated hearts were perfused with or without 0.03 μIU/mL insulin for 15 min. Blood glucose, bodyweight, intraperitoneal fat and plasma insulin and Ang II were recorded. Proteins of interest and their phosphorylation were determined by Western blotting. NO production was flow cytometrically analyzed. ANOVA followed by the Bonferroni correction was used with a p< 0.05 considered significant. Results: DIO animals had significant elevated bodyweight, blood glucose, plasma insulin and Ang II levels. Our data showed that the hearts from the DIO animals are insulin resistant, ultimately reflected by the attenuated activation of the key proteins (IRS-1, PKB/Akt and eNOS) involved in insulin signalling as well as NO production. AT1 receptor antagonism improved NO production in isolated adult ventricular myocytes from DIO animals while concurrently enhancing expression of eNOS, PKB/Akt and p38 MAPK. In contrast, NO production as well as expression of eNOS and PKB/Akt was attenuated in control animals after Losartan treatment. Conclusion: These results suggested that Ang II via AT1 or AT2 receptors, modulates protein expression of both PKB/Akt and eNOS. This encouraged us to investigate the involvement of AT2 receptors in the observed changes. To investigate this we needed to establish a culture of neonatal rat cardiac myocytes treated with raised fatty acids and Ang II. If similar changes were induced as observed in the hearts of DIO animals, the involvement of the AT1 and AT2 receptors could be investigated using specific antagonists against these receptors. Primary cultured ventricular myocytes were isolated from 1-3 day old Wistar rat pups. They were cultured for 48 hours before the addition of palmitate and oleate at a concentration of 0.25 mM each and were treated with or without the fatty acids for a period of 4 days. After 18 hours of serum starvation, cells were stimulated with or without 10 nM insulin for 15 minutes. The effect of fatty acid treatment on cell viability and glucose uptake were assessed by trypan blue and propidium iodide staining and 2-deoxy-D-3[H] glucose uptake respectively. Protein levels and phosphorylation of key proteins (PKB/Akt, PTEN and p38 MAPK) in insulin signalling was determined by Western blotting. 0.25 mM Fatty acids did not result in the loss of cell viability. Contrary to expectation, fatty acid treatment led to enhanced basal glucose uptake but lower Glut 1 protein expression. Basal protein expression of PPARα was, however, upregulated as was the expression of the phosphatase, PTEN. The latter could explain the lower PKB/Akt phosphorylation also documented. From these results we conclude that neonatal cardiac myocytes, cultured in the presence of elevated fatty acids, did not respond in a similar manner as the intact hearts of our animals and further modifications of the system might be needed before it can be utilized as initially planned.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Genetic screening to improve obesity treatment outcomes is available despite the lack of conclusive evidence, specifically for Caucasian South Africans, in this regard. The aim of this study was to investigate the association between genotype (seven polymorphisms) and body mass index (BMI), health and lifestyle indicators in a cross-sectional sample of overweight/obese Caucasian adults (n=133), as well as the association between genotype and weight loss outcomes following an intervention (n=88) using a quasi experimental study design (time-series). The intervention consisted of a 24-week conservative weight loss programme that included dietary, physical activity and behavioural components. The primary null hypothesis for the cross-sectional sample, namely that there is no association between genotype and BMI, has not been rejected. A number of the secondary/exploratory hypotheses were rejected of which the most plausible associations (based on support by the literature and a physiological basis for the findng) are: 1) the mutant TT homozygotes of the GNB3 C825T polymorphism may have a higher risk to develop the metabolic syndrome (MetS) as they had significantly higher fasting triglyceride and glucose levels, a higher number of traits that met the diagnostic cut-off criteria for MetS and higher number of these subjects was diagnosed with MetS compared to the wild-type C-allele carriers; and 2) subjects with mutant alleles of either the FTO rs1421085 or rs17817449 polymorphisms may have poorer eating behaviours (a higher rigid control, habitual and emotional disinhibition, perceived hunger and internal locus for hunger) and higher intake of high-fat foods. The primary null hypothesis for the intervention sample, namely that there is no association between genotype and weight loss outcome, was not rejected for the FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg and GNB3 C825T polymorphisms. However, it was rejected in some instances indicating the following associations: 1) The wild-type TT homozygotes of the FTO rs17817449 polymorphism lost significantly more weight during the first two months of the program compared to the mutant allele carriers (this is a novel finding); 2) The wild-type Arg16Arg homozygotes of the ADRB2 Arg16Gly polymorphism lost significantly more weight during the first month of the program compared to the mutant allele carriers (this finding is supported by one other intervention study); 3) Subjects with a mutant C-allele of the INSIG2 rs7566605 polymorphism and a mutant Gly16-allele of the ADRB2 Arg16Gly polymorphism lost significantly less weight over the six month intervention period (this is a novel genegene interaction finding). A number of secondary/exploratory hypotheses were rejected, of which the most plausible finding include that the improvement in emotional disinhibition in the wild-type TT subjects of the FTO rs1421085 polymorphism was associated with a more pronounced decrease in BMI over the six month weight loss period. The integration of the results from this study with the literature indicates that there is insufficient evidence at this stage for genetic screening of the polymorphisms investigated in this study and the provision of evidence-based personalized recommendations for weight loss in obese individuals. It is recommended that these associations should be viewed as priority in future research. / AFRIKAANSE OPSOMMING: Genetiese sifting om die resultate van vetsug behandeling te verbeter is beskikbaar ten spyte van ‘n tekort aan genoegsame bewyse, spesifiek ten opsigte van Kaukasiërs van Suid-Afrika. Die doel van hierdie studie was om die assosiasie tussen genotipe (sewe polimorfismes) en liggaamsmassa indeks (LMI), gesondheid en lewenstyl indikatore in ‘n dwarssnit (cross-sectional) steekproef van oorgewig/vetsugtige Kaukasiër volwassenes (n=133) te ondersoek, asook die assosiasie tussen genotipe en gewigsverlies uitkomste na afloop van ‘n intervensie (n=88) in ‘n kwasi-eksperimentele studie ontwerp (tydreeks). Die intervensie het bestaan uit ‘n 24-week konserwatiewe gewigsverlies program met dieet, fisieke aktiwiteit en gedragskomponente. Die primêre nul hipotese vir die dwarsnit steekproef, naamlik dat daar geen assosiasie tussen genotipe en LMI is nie, is nie verwerp nie. ‘n Aantal sekondêre/spekulatiewe hipotesis is verwerp waarvan die mees geloofwaardige assosiasies (gebasseer op ondersteuning van die literatuur en ‘n fisiologiese basis vir die bevinding) die volgende insluit: 1) die mutante TT homosigote van die GNB3 C825T polimorfisme het moontlik ‘n hoër risiko vir die ontwikkeling van die metaboliese sindroom (MetS) aangesien hulle betekenisvolle hoër vastende trigliseriede en glukose vlakke gehad het, ‘n grooter aantal kenmerke gehad het wat aan die diagnostiese afsnykriteria vir MetS voldoen en ‘n grooter aantal van hierdie persone was met MetS gediagnoseer in vergelyking met die wilde-tipe C-alleel draers; en 2) persone met die mutante allele van die FTO rs1421085 of rs17817449 polimorfismes het moontlik ‘n swakker eetgedrag (‘n hoër rigiede kontrole, gewoonte en emosionele disinhibisie, waarneembare honger en interne lokus van honger) en ‘n hoër inname van hoë-vet voedsel. Die primêre nul hipotese vir die intervensie steekproef, naamlik dat daar geen assosiasie tussen genotipe en gewigsverlies uitkomste is nie, is nie vir die FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg en GNB3 C825T polimorfismes verwerp nie. Dit was egter in sommige gevalle vir die volgende assosiasies verwerp: 1) Die wilde-tipe TT homosigote van die FTO rs17817449 polimorfisme het betekenisvol meer gewig in die eerste twee maande van die program verloor in vergelyking met die mutante alleel draers (dit is ‘n nuwe bevinding); 2) Die wilde-tipe Arg16Arg homosigote van die ADRB2 Arg16Gly polimorfisme het betekenisvol meer gewig gedurende die eerste maand van die program verloor in vergelyking met die mutante alleel draers (hierdie bevinding word ondersteun deur een ander intervensie studie); 3) Persone met ‘n mutante C-alleel van die INSIG2 rs7566605 polimorfisme en ‘n mutante Gly16-allele van die ADRB2 Arg16Gly polimorfisme het minder gewig tydens die ses maande intervensie periode verloor (dit is ‘n nuwe geen-geen interaksie bevinding). ‘n Aantal sekondêre/ spekulatiewe hipoteses is verwerp, waarvan die mees geloofwaardigste bevinding insluit dat ‘n verbetering in emosionele disinhibisie van die wild-tipe TT persone van die FTO rs1421085 polimorfisme geassosieer was met ‘n meer prominente daling in LMI oor die ses maande gewigsverlies periode. Die integrasie van die resultate van hierdie navorsing met die literatuur dui aan dat daar op hierdie stadium onvoldoende bewyse vir genetiese sifting en die voorsiening van bewys-gebasseerde persoonlike aanbevelings vir gewigsverlies in vetsugtig individue bestaan vir die polimorfismes wat ondersoek is. Dit word aanbeveel dat hierdie assosiasies as prioriteit in toekomstige navorsing beskou moet word.