Patients suffering from hypertension often develop non-Insulin dependent diabetes mellitus (NIDDM), a condition caused by Insulin resistance. Though these patients have normal Insulin receptor (IR) and high levels of Insulin in blood, they fail to have IR-mediated signaling essential for glucose uptake and availability. NIDDM usually begins as insulin resistance, a condition in which Insulin Receptor (IR)-mediated signaling that leads to glucose uptake and glucose availability to cells is inhibited even in the presence of high levels of Insulin in blood. Mechanisms for the development of this Insulin resistance in patients suffering from hypertension are unclear. Angiotensin II (Ang II) hormone has been implicated in the pathogenesis of insulin resistance and inhibitors of Ang II receptor AT1 are shown to improve insulin sensitivity. Here we show that in the skeletal muscle tissue of SHR rats, Insulin Receptor (IR) beta- subunit forms a complex with the AT1 receptor and co-immunoprecipitates with IR-beta. Such strong AT1-IR association was not observed in normo-tensive rat tissue. To our knowledge this is the first report that shows AT1 can associate with IR-beta in mammalian tissue and that such association might play a role in the regulation of signaling by IR-beta. We further demonstrate that a 2-hour pre-incubation with Ang II (at concentrations 50pM to 1?ÝM) significantly inhibits 125I-insulin binding to IR in human cell line MCF-7. This effect was not seen when Ang II exposure was performed for shorter periods. The two-hour exposure to Ang II also led to the formation of a protein complex containing AT1 and IR-beta, similar to that seen in skeletal muscle tissue of SHR rats. Both AT1-IR association and differential tyrosine phosphorylation of IR-beta and associated proteins were inhibited when the cells were pre-treated with the AT1 antagonist losartan. These observations suggest that continuous presence of Ang II would result in sequestering IR in the AT1-IR complex and prevent IR from binding insulin. It also coincided with differential tyrosine-phosphorylation of IR beta-subunit and associated proteins, than that generated when IR was activated by insulin. Therefore, we infer that conformational alterations in IR caused by AT1-IR-beta association underlie the development of Ang II-induced insulin resistance. Based on these data we propose a model for AT1-mediated insulin resistance that involves receptor level interaction between the AT1 and the IR. Therefore, Insulin-independent, Ang II-induced tyrosine phosphorylation of IR prevents IR from binding Insulin and contributes to Insulin resistance. The observation that drugs that inhibit Angiotensin II converting enzyme (ACE), or activation of AT1 receptor, not only reduce hypertension, but also induce insulin sensitivity further supports the role for Ang II and AT1 in the development of NIDDM.

Molecular analysis of the cross talk between Angiotensin II (Ang II) and insulin signaling systems reveal that they are multifaceted and occur at cellular level and intracellular level. Experiments were carried out to evaluate the crosstalk between the Ang II receptors-AT1 and AT2 and the Insulin Receptor (IR) to understand the changes in the signaling pathway that could lead to the transition from hypertension to insulin resistance. Transient expression of rat AT2 in CHO cells induced co-immunoprecipitation of the AT2R with IRâ and inhibition of IRâ tyrosine phosphorylation. An AT2-peptide carrying the amino acids 226-363 (that spans 3rd intracellular loop (ICL) and C-terminal cytoplasmic domain) was sufficient for AT2- IRâ interaction in a yeast two-hybrid assay. An orthovanadate-insensitive AT2- IRâ association was also observed in human breast cancer cell line MCF-7. Interestingly, while AT2- IRâ complex formation was insensitive to pertussis toxin (PTX), AT2-mediated inhibition of IRâ phosphorylation was partially sensitive to PTX treatment in MCF-7. To address the mechanism behind the transition of an early hypertensive heart to an insulin resistant status, we investigated the changes that occur at post translational level in the IR and its downstream signaling molecules that modulate insulin signaling. Early hypertension was induced in 10-week old SD rats by 2% NaCl diet in combination with Ang II infusion. Enhanced serine phosphorylation of the IRâ suggestive of dysfunctional insulin signaling was observed in cardiac tissues as a result of the treatment. In addition, an enhanced association of both AT1R and AT2R with IRâ was observed in the heart tissue lysates from hypertensive rat heart. To evaluate the tissue effects of Ang II, we compared the transcriptome of hypertensive rat hearts to the controls. Analysis suggests that the Ang II induces multiple responses in heart tissue that result in changes to the gene expression pattern intended to promote insulin sensitivity and insulin resistance. Taken together our results suggest that exogenous Ang II and moderately high salt diet promote metabolic abnormalities in heart tissue that result in sequestration of IR and modulation of IR signaling, and significant changes in gene expression profile in the hypertensive heart.

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Hypertension and diabetes have long been closely linked. As such, the major player in the renin, angiotensin system, angiotensin II, has recently been investigated for its effects on metabolism and diabetes. Since skeletal muscle is one of the most metabolically active tissues, this study investigates the effects of angiotensin II specifically on skeletal muscle. In this study, L6 skeletal muscle cells were treated with angiotensin II for either 3 or 24 hours and a number of effects were investigated. Fatty acid oxidation and lipid synthesis was measured using [1-14C]-palmitate, glucose oxidation and glycogen synthesis were measured using 14C-glucose. In addition,mitochondrial oxidative capacity was measured using an XF 24 Flux Analyzer (Seahorse Bioscience) and reactive oxygen species measured using confocal microscopy. The clinical study involving the drug Benicar ® investigated the metabolic effects of blocking angiotensin II on skeletal muscle fatty acid oxidation, glucose oxidation, and oxidative and glycolytic enzyme activity. In L6 cells, angiotensin II significantly reduced fatty acid oxidation after 24 hours (p<0.01) and 3 hours (p<0.001) if angiotensin II was present during oxidation experiments. It also significantly reduced mitochondrial oxidative capacity (p<0.05) after 24 hours and significantly increased reactive oxygen species production (p<0.05) over 3 hours. The clinical study showed no significant effects of Benicar® on fatty acid or glucose oxidation or any enzyme activities. / Master of Science

Uma atividade funcional para uma via alternativa de geração de angiotensina II, como a elastase-2 foi sugerida em estudos realizados anteriormente em nosso laboratório no leito arterial mesentérico isolado de rato. No presente estudo, caracterizamos com o uso de substratos e inibidores seletivos a presença de via alternativa de geração de Ang II, independente da ECA, em carótida de ratos. Determinamos ainda a expressão do RNAm da elastase-2 nesta preparação arterial. Em anéis isolados de carótida de ratos analisamos o efeito vasoconstritor dos peptídeos Ang II, Ang I, TDP, [Pro11-D-Ala12]-Ang I (um substrato resistente a ECA) na ausência e presença de inibidores de proteases. Ang II e seus precursores produziram efeito vasoconstritor dependente da concentração em carótidas de ratos, de forma sensível ao losartan (1 M). Na presença de captopril (10 M) a resposta vasoconstritora produzida pela Ang I foi inibida, mas a resposta contrátil induzida pelo TDP e [Pro11-D-Ala12]-Ang I não foi alterada. Na presença de quimostatina (100 M) o efeito produzido pelo TDP e [Pro11-D-Ala12]-Ang I foi abolido enquanto que a curva cumulativa de Ang I foi significativamente deslocada para a direita. Inibidor Ac-AAPL-CK (seletivo para elastase-2) aboliu completamente a resposta contrátil induzida pelo PDA e não alterou o efeito vasoconstritor da Ang II. Na presença de captopril e quimostatina a resposta vasoconstritora dos peptídeos Ang I, TDP e [Pro11-D-Ala12]-Ang I foram inibidas, enquanto a resposta contrátil da Ang II não foi alterada em artéria carótida. A presença de RNAm da elastase-2 na carótida, juntamente com os dados funcionais apresentados aqui sugerem a participação desta enzima na via alternativa de geração de Ang II em carótidas de ratos. Embora a formação de Ang II a partir Ang I seja descrita como essencialmente dependente da ECA, nossos resultados sugerem a existência de vias alternativas de geração de Ang II sensível a quimostatina e Ac-AAPL-CK em artéria carótida de ratos. Muito provavelmente a elastase-2 seja a enzima responsável pela geração de Ang II nessa preparação. / We have recently described a chymostatin-sensitive elastase-2 as the major angiotensin (Ang) II-forming enzyme in the perfusate of rat mesenteric arterial bed (MAB). In the present study we investigated the role of this enzyme in generating Ang II in the isolated rat carotid artery rings by analyzing the vasoconstrictor effect of Ang II, Ang I, tetradecapetide renin-substrate (TDP), [Pro11-D-Ala12]-Ang I (an ACE-resistant substrate) in the absence and presence of proteases inhibitors. Ang II and its precursors produced a dose-dependent vasoconstrictor effect in vascular preparation that was blocked by losartan (1 M). In carotid rings, captopril (10M) abolished the responses induced by Ang I but did not affect those induced by TDP and [Pro11-D-Ala12]-Ang I. In the presence of chymostatin (100 M) alone, the effects induced by [Pro11-D-Ala12]-Ang I and TDP were abolished while the concentration-response curve to Ang I was shifted to the right. Ac-AAPL-CK (selective elastase-2 inhibitor) inhibited the responses induced by [Pro11-D-Ala12]-Ang I but did not affect Ang II-induced effects. In the presence of captopril and chymostatin, the vasoconstrictor effects of Ang I, TDP, and PDA were completely blocked while those induced by Ang II were not affected in rat artery carotid. Although Ang II formation from Ang I is essentially dependent on ACE in carotid artery, our results suggest the existence of an alternative chymostatin-sensitive pathway in rat arteries, most probably involving elastase-2.

Chan Wai-Pong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 114-135). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General review of pancreas --- p.1 / Chapter 1.2 --- The renin-angiotensin system (RAS) --- p.4 / Chapter 1.3 --- Tissue RAS --- p.12 / Chapter 1.4 --- Hypoxia and RAS --- p.21 / Chapter 1.5 --- Pancreatitis and RAS --- p.25 / Chapter 1.6 --- Aim of study --- p.27 / Chapter Chapter 2 --- Method / Chapter 2.1 --- Experimental animals and rat models --- p.30 / Chapter 2.2 --- Immunohistochemistry --- p.33 / Chapter 2.3 --- Semi-quantitative reverse transcriptase-polymase chain reaction (RT-PCR) --- p.37 / Chapter 2.4 --- Western blot analysis --- p.41 / Chapter 2.5 --- "Standard curve, quantitative competitive RT-PCR (SC-QC-RT-PCR)" --- p.45 / Chapter 2.6 --- Data analysis --- p.48 / Chapter Chapter 3 --- Result / Chapter 3.1 --- Existence of a local RAS in the rat pancreas --- p.49 / Chapter 3.2 --- Effect of chronic hypoxia on RAS expression in neonatal rat --- p.59 / Chapter 3.3 --- Effect of chronic hypoxia on RAS expression in mature rat --- p.72 / Chapter 3.4 --- Effect of experimental pancreatitis on RAS expression --- p.86 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Existence of a local RAS in the rat pancreas --- p.97 / Chapter 4.2 --- Regulation of pancreatic RAS by chronic hypoxia --- p.101 / Chapter 4.3 --- Regulation of pancreatic RAS by pancreatitis --- p.106 / Chapter 4.4 --- Conclusion --- p.111 / Chapter 4.5 --- Further work --- p.112 / Chapter Chapter 5 --- References --- p.114

Lau Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Pancreas and its functions --- p.1 / Chapter 1.1.1 --- Structure of pancreas --- p.1 / Chapter 1.1.2 --- Exocrine function --- p.4 / Chapter 1.1.3 --- Endocrine function --- p.7 / Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.7 / Chapter 1.1.3.2 --- Regulation of insulin secretion --- p.10 / Chapter 1.1.3.3 --- Mechanism for glucose-stimulated insulin release --- p.14 / Chapter 1.1.3.4 --- Bi-phase response of insulin secretion --- p.16 / Chapter 1.2 --- Pancreatic Renin-Angiotensin System --- p.19 / Chapter 1.2.1 --- Circulating RAS and local RAS --- p.19 / Chapter 1.2.2 --- RAS inhibitors --- p.25 / Chapter 1.2.2.1 --- Angiotensin converting enzyme inhibitor --- p.25 / Chapter 1.2.2.2 --- Non-specific Ang II receptor blocker --- p.28 / Chapter 1.2.2.3 --- Specific AT1 receptor antagonist --- p.29 / Chapter 1.2.2.4 --- Specific AT2 receptor antagonist --- p.30 / Chapter 1.2.3 --- RAS and Pancreas --- p.30 / Chapter 1.2.3.1 --- Expression and localization of pancreatic RAS --- p.30 / Chapter 1.2.3.2 --- Regulation of pancreatic RAS and its clinical relevance --- p.32 / Chapter 1.3 --- Islet Transplantation and RAS --- p.34 / Chapter 1.3.1 --- Whole pancreas and islet transplantation --- p.34 / Chapter 1.3.2 --- Problems encountered in islet transplantation --- p.36 / Chapter 1.3.3 --- Potential role of RAS in islet transplantation --- p.38 / Chapter 1.4 --- Diabetes Mellitus and RAS --- p.40 / Chapter 1.4.1 --- Diabetes Mellitus --- p.40 / Chapter 1.4.2 --- Type 1 diabetes and its animal model --- p.42 / Chapter 1.4.3 --- Type 2 diabetes and its animal model --- p.44 / Chapter 1.4.4 --- RAS blockade in diabetes patients --- p.46 / Chapter 1.4.5 --- Potential role of RAS in Diabetes Mellitus --- p.47 / Chapter 1.5 --- Aims of Study --- p.49 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental animals and mouse models --- p.50 / Chapter 2.1.1 --- Experimental animals for islet isolation and transplantation --- p.50 / Chapter 2.1.2 --- Mouse model for type 2 diabetes --- p.51 / Chapter 2.2 --- Islet isolation and transplantation --- p.52 / Chapter 2.2.1 --- Enzymatic islet isolation --- p.52 / Chapter 2.2.2 --- Islet transplantation --- p.53 / Chapter 2.3 --- Biological assay on islet functions --- p.53 / Chapter 2.3.1 --- Measurement of islet insulin release --- p.53 / Chapter 2.3.2 --- Measurement of islet glucose oxidation rate --- p.56 / Chapter 2.3.3 --- Measurement of islet (pro)insulin biosynthesis --- p.59 / Chapter 2.3.4 --- Measurement of islet total protein synthesis --- p.60 / Chapter 2.4 --- Chronic losartan treatment --- p.62 / Chapter 2.5 --- Perfusion experiment of transplanted islet graft --- p.62 / Chapter 2.6 --- Insulin content of the islet graft --- p.63 / Chapter 2.7 --- Islet graft (pro)insulin and total protein biosynthesis --- p.64 / Chapter 2.8 --- Real-time RT-PCR Analysis --- p.64 / Chapter 2.8.1 --- Design of primers and probes --- p.67 / Chapter 2.8.2 --- Use of internal control --- p.69 / Chapter 2.8.3 --- RT-PCR reaction --- p.69 / Chapter 2.8.4 --- Calculation using the comparative CT method --- p.70 / Chapter 2.9 --- Western Blot Analysis --- p.71 / Chapter 2.10 --- Immunocytochemistry --- p.72 / Chapter 2.11 --- Statistical data analysis --- p.73 / Chapter Chapter 3 --- Results / Chapter 3 .1 --- Effect of Angiotensin II and Losartan on islet insulin release --- p.74 / Chapter 3.1.1 --- Insulin release from normal islets --- p.74 / Chapter 3.2 --- "Effect of Angiotensin II and Losartan on islet glucose oxidation rate, (pro)insulin and total protein biosynthesis" --- p.77 / Chapter 3.2.1 --- Glucose oxidation rate of isolated normal islets --- p.77 / Chapter 3.2.2 --- (pro)insulin and total protein biosynthesis of isolated normal islets --- p.77 / Chapter 3.3 --- Regulation of RAS components in islet transplantation --- p.81 / Chapter 3.3.1 --- Expression of RAS components in endogenous islets and transplanted islets --- p.81 / Chapter 3.3.2 --- Localization of AT1-receptor in endogenous islets --- p.87 / Chapter 3.3.3 --- Expression of AT1-receptor protein in endogenous and transplanted islets --- p.89 / Chapter 3.3.4 --- Relative abundance of RAS components in kidney and liver --- p.91 / Chapter 3.3.5 --- Insulin release from perfused transplanted islet graft --- p.93 / Chapter 3.3.5 --- (pro)insulin and total protein biosynthesis of transplanted islet graft --- p.96 / Chapter 3.4 --- Effect of Angiotensin II and losartan on diabetic islets --- p.99 / Chapter 3.4.1 --- Expression of RAS components in diabetic pancreas --- p.99 / Chapter 3.4.2 --- Localization of AT1 receptors in diabetic pancreas --- p.105 / Chapter 3.4.3 --- Insulin release from islets of type 2 diabetic mice --- p.107 / Chapter 3.4.4 --- (pro)insulin and total protein biosynthesis of islets from type 2 diabetic mice --- p.112 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effect of angiotensin II and losartan on islet insulin release --- p.116 / Chapter 4.2 --- Existence of local RAS in pancreatic islets --- p.119 / Chapter 4.3 --- Regulation of islet RAS components in transplanted islets --- p.122 / Chapter 4.4 --- Clinical relevance of islet RAS in transplantation --- p.125 / Chapter 4.5 --- Regulation of islet RAS by type 2 diabetes --- p.126 / Chapter 4.6 --- Clinical relevance of islet RAS in type 2 diabetes --- p.134 / Chapter 4.7 --- Conclusion --- p.140 / Chapter 4.8 --- Further studies --- p.141 / Chapter Chapter 5 --- Bibliography --- p.142

Conclusion. These data provide a clue that AT1 receptor blocker could effectively attenuate severe form of pancreatitis and its-associated systemic inflammation in experimental models of AP. The underlying mechanisms may be involved in Ang II-induced NADPH oxidase derived oxidative stress, particularly NFkappaB and ERK1/2-dependent CREB activation. The pro-inflammatory pathways would commonly converge to transcribe an array of genes such as IL-6, thus regulating the severity of pancreatitis and the onset of its complications. All these in vivo and in vitro data provide substantial evidence that Ang II is involved in AT1 receptor-mediated signaling cascade in regulating the pathogenesis of AP. The findings provide a new insight on potential application of AT 1 receptor blockade for a therapeutic approach in the management of AP. (Abstract shortened by UMI.) / Recent advance in basic research has revealed that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of AP. In this regard, the present study aimed at investigating the effectiveness of RAS blockade in clinically relevant AP animal model and AP-associated systemic inflammation. More importantly, the underlying mechanistic pathways involved in angiotensin II (Ang II)-induced pro-inflammatory actions were elucidated using both in vivo and in vitro systems. / Results. Major components of RAS were up-regulated in obstructive pancreatitis model. Blockade of AT1 receptor attenuated pancreatic injury induced by the two models. Moreover, losartan could significantly ameliorate AP-associated systemic inflammation. Analysis of protein expression levels revealed that losartan treatment improved AP-associated elevation of NADPH oxidase p67 and p22 subunits. Double-immunostaining confirmed that expression of NADPH oxidase was localized to pancreatic acinar cells. AT1 receptor antagonism not only reduced oxidative stress but also suppressed nuclear factor kappaB (NFkappaB) activation, as evidenced by reversal effects on IkappaBbeta depletion, augmentation of phosphor NFkappaB p65, and enhanced nuclear kappaB binding activity. Blockade of AT1 receptor could also suppress the levels of kappaB-related protein expression, including intercellular adhesion molecule-1, cyclooxygenase-2, and IL-1. On the other hand, pancreatic mRNA and protein levels of IL-6 were enhanced by obstructive AP, which were antagonized by AT1 receptor blocker. Losartan treatment could reverse extracellular-regulated kinase (ERK) 1/2 and cAMP-responsive element binding protein (CREB) phosphorylation brought by obstructive AP. In vitro studies, exogenous application of Ang II induced ERK1/2 and CREB activation in AR42J cells. Concomitantly, IL-6 expression was augmented dose- and time-dependently in response to Ang II, which was reversed by treatment of AT1 receptor blocker (losartan) and ERK1/2 inhibitor (PD98059). Ang II induced NFkappaB activation was reversed by pre-treatment of AT1 receptor blocker and NADPH oxidase inhibitor but not ERK1/2 inhibitor in vitro. Moreover, Ang II-induced superoxide generation was detected. Treatment of antioxidant prevented Ang II-induced ERK1/2 activation. On top of these, in vitro experiments revealed that Ang II could sustain the activation of caerulein-induced NFkappaB and ERK1/2 in an AT1 receptor-mediated manner, but not secretagogue-induced hypersecretion. / Chan, Yuk Cheung. / Adviser: Po Siny Lzung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3246. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 228-262). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.