β-Adrenergic Receptors Effect On The Metabolism Of Lactate In Astrocyte And Content Of Somatostatin In Islet

The stress response of the body is a dramatic physiological change in response to difficult external environments in order to avoid injury. Under stress, the body releases numerous stress hormones for example; epinephrine, norepinephrine, glucocorticoids and angiotensin. Epinephrine and norepinephrine act mainly on adrenergic receptors and play an important role in the stress response. Adrenergic receptors are divided into two separate groups; α-AR and β-AR. The original concept of alpha and beta sympathetic receptors was put forward by Alquist in1948suggesting that beta receptors were situated mainly in the heart and alpha receptors periphery. a-AR activation increases smooth muscle contraction (resulting in increased vasoconstriction) and inhibits insulin release while β-AR controls heart rate, the regulation of glycogen synthesis and conversely, activation of p-AR results in vasodilatation. However, there are still many questions regarding regulatory mechanisms which remain to be answered. With the progress of biochemistry and molecular biology, it was discovered that a receptor can be divided into two subgroups; α1,which is Gq-coupled receptor, anda2, which is Gi-coupled receptor. Meanwhile, β-adrenergic receptor, which mediated signal transduction mainly through Gs proteins after activation, is divided into three subtypes; β1, β2and β3-AR. Since beta receptors and alpha receptor exert dierent physiological effects, the selective activation of beta-adrenergic receptor can also be generally referred to as β-aderenergic agonsim, and selective activation of a-adrenergic receptor is called the a-adrenergic agonsim.Through several selective agonists and knockout mice models, a lot of progress about physiological roles of the a-adrenergic agonsim and β-aderenergic agonsim has been achived during the past half century. However, due to widely distribution and multiple regulating environmental factors of these nine adrenergic receptors, the mechanism underlying physiologically regulation of a-adrenergic agonsim and β-aderenergic agonsim is still not completely understood. With the accelerated pace of life, human behavior changes caused by long-term pressure have attracted more and more attention recently. Therefore, physiologically regulation of a-adrenergic agonsim and P-aderenergic agonsim has long been a hot research area for a long time.(32AR is the best-characterized G protein coupled receptor in signaling and biochemical studies and this is highlighted by recent advanced work such as the crystal structure of β2AR-Gs protein complexes, multiple conformations of P2AR with different ligands and its numerous downstream signaling pathways. Generally, when stimulated by the endogenous ligand epinephrine, β2AR primarily couples to Gs proteins to mediate their physiological effect. Recently, we have known a lot about beta-aderenergic agonsim because of several interesting works. Such as, by using cell model and p2AR mutation mise, Lefkowitz group from Duke University found β2AR in kidney exerted a key role in maintaining the body’s acid-base balance by regulateing Na+/H+exchange after combination with the NHERF; through mouse model of Alzheimer disease, Professor Pei Gang found irregularly activated β2AR could promote Alzheimer disease by interaction with ghrelin to increase the y-secretase activity which resulting in accelerateing the formation of amyloid plaques; through the beta-arrestinl-/-KO mouse model, it has been found under long-term chronic stress, β2AR led to DNA damage, and further on caused genomic instability by beta-arrestinl pathway.By utilizing different β-AR agonists (Epinepherine、Isoproterenol) and antagonists (ICI118,551、Metaproterenol、Propranolol), combined with cell culture studies and the use of WT mice and β1AR-/-/β2AR-/-KO mice, we study mechanisms of the generation and transport of lactate by β-AR and the impact of astrocytes in learning and memory in mice, providing a theoretical basis of the role of lactate transport in memory loss associated with neurodegenerative diseases. We also investigated the role of P-aderenergic agonsim in astrocytes lactate production and transport and learning and memory in mice, and regulation of islets Somatostatin expreesion level.Chapter I The mechanism of metabolism lactate regulation by β-AR in astrocyte regulationβ-adrenergic receptors are typical G protein-coupled seven transmembrane receptor superfamily members. A large number of physiological experiments confirmed that activating β2-adrenergic receptor can promote long-term potentiation and enhance learning and memory in the hippocampus, amygdala and prefrontal cortex. The study found that learning and memory require energy in part from glycogenolysis in astrocytes via β2-AR activation. Astrocytes play an important role in the supply of energy in the brain; astrocytes take up glucose from the blood vessels which is then metabolized via several distinct mechanisms. Glycogen stored in astrocytes is a critical energy supply within the central nervous system during normal conditions as well as pathological conditions such as hypoxia. In astrocytes, lactate is the main product resulting from glycolysis. It is becoming clear that lactate transport from astrocytes to neurons is essential for long-term memory. Recently, an intensive study investigation the connection between astrocytic glycogenolysis and long-term memory was performed by Suzuki et al revealing lactate plays a very important role in memory formation. The lactate uptake by neuron is mainly from astrocyte glycogenolysis. The ability to shuttle lactate to various regions of the brain is dependent on monocarboxylater transporter (MCT) activity. MCT1and MCT4are expressed almost exclusively in astrocytes, whereas MCT2is strongly expressed in neurons. Decreased expression of MCT1or MCT4in astrocytes resulted in disrupted long-term memory formation which was rescued by exogenous administration of lactate. The lower expression of neuronal MCT2also resulted in loss of long-term memory, indicating the transport of lactate into neurons is required for long-term memory formation.To better understand the regulating mechanism of the role of astrocytes β-AR on lactate production and transport regulation, we used astrocytes isolated from β1AR+/+/P2AR+/+, β1AR-/-β2AR-/-mice and U251cell lines, and treated them with a variety of adrenergic agonists (eg Epinepherine, Isoproterenol, Formoterol) and antagonists (eg ICI118,551, Metaproterenol). Get the following results:1. Studies have shown that glycogen decomposition in astrocytes is mediated by P2-AR. Here we want to figure out whether β2-AR also mediates lactate generation and transport. First, we isolated β1AR+/+/β2AR+/+ and β1AR-/-/β2AR-/-astrocytes and gave them different stimulations. The results from treatment with ISO showed that lacate was not increased in β1AR-/-/β2AR-/-astrocyte but increased significantly in β1AR+/+/β2AR+/+ astrocytes. And generation of lactate could be blocked by β2-AR specific inhibitor ICI118,551but not β1-AR specific blocker Metoprolol. The similar result was also found in cell lines U251. This suggests that generation of lactate in astrocytes is mediated by β2-AR.2. To detect LDHA, MCT1and MCT4changes in mRNA and protein levels, we used RT-PCR and western blot. We found all of these genes were increased in a time-dependent manner in mRNA and protein levels after treatment with ISO(1μM) in astrocyte. But the mechanism needs to be further discussed3. Using MatInspector software from Genomatix, several potential HIF-la binding sites were predicted in LDHA and MCT4genes in the promoter sequences. We found several potential CREB binding sites in the promoter sequences of MCT1gene. Using the reporter assay with promoter of LDHA,MCT1and MCT4fused to luciferase,We showed that HIF-1α was able to activate the promoter of LDHA and MCT4and the activity was decreased after treatment with siRNA towards arrestinl. CREB was able to activate the promoter of MCTl,but can be blocked by PKA inhibitor H89.4.(32-AR mediated MCT4and LDHA’s transcriptional activation mainly through beta-arrestinl pathway in astrocytes; In this signal pathway, ERK was firstly activated and then the S506and T515sites of HIF-la was phosphorylated. HIF-la become more stabilize and it was promoted into nuclear as the transcription factor to regulate MCT4and LDHA’s transcriptional level. While β2-AR mediated MCTl’s transcriptional activation mainly through PKA pathway with phosphate-CREB in astrocytes. These results suggested that β2-AR-mediated the LDHA, MCT4, MCT1’s transcriptional activation through the different signaling pathways5. After giving β2AR’s agonist to WT mice and β1AR-/-β2AR-/-KO mice. We also detected the expression of LDHA, MCT1and MCT4which are related to lactate metabolism and also detected the activation of CREB, ERK1/2which were closely related to the memory’s formation.In summary, we found β2-AR mediated astrocytes lactic acid generation and transport by regulateing astrocytes LDHA, MCT1and MCT4transcriptional activation mainly through two signaling pathways; Gs-PKA and beta-arrestinl. We also discovered for the first time ERK activated by beta-arrestinl phosphorylated T506/S515sites of HIF-la, which regulating the expression of LDHA and MCT4. Lactic acid provided neurons energy and activates CREB and ERK1/2which are the key molecules associated with learning and memory, to enhance learning and memory in mice for certain time, but long-term β2AR agonist stimulation may produce damage to the memory. These results will be helpful to understand the role of beta-adrenergic receptors on astrocytes lactate production and transfer and their effects on learning and memory.Chapter II P-Adrenergic receptor regulates somatostatin level in islet cellsThe islets of Langerhans contain multiple types of endocrine cells, which produce glucagon (a-cells), insulin (β-cells), somatostatin (8-cells), pancreatic polypeptide (PP-cells) and ghrelin (ε-cells). The crosstalk between these cells forms a finely tuned network that controls hormone release and maintains normal growth and survival of the islets. For example, the somatostatin secreted from the islet δ cells regulates the responses of the islet β-and a-cells when faced with physiological changes. An increase in the circulating glucose concentration triggers the release of somatostatin from the pancreatic δ-cells, which exerts inhibitory effects on both insulin secretion from the β cells and glucagon secretion from the a cells. While somatostatin limits the insulin response to glucose in an inhibitory circuit, it is essential for the effects of glucose in decreasing glucagon secretion from a cells. Therefore, somatostatin is an important islet circuit hormone. An inappropriate somatostatin level may lead to disorders of islet physiology and influence islet function. Except glucose, somatostatin in the pancreatic8cells is also controlled to react to environmental changes.The adrenergic system, which is regulated by either epinephrine released from the adrenal medulla or norepinephrine released from the sympathetic nerves, is one of the most important regulators of8cells and adapts islets to increased psychological or physical demands. Previous studies have demonstrated that activation of the Adrenergic system in pancreatic8cells promotes somatostatin secretion, which is mainly through the activation of the β-adrenergic system. Somatostatin secretion is increased by agents that stimulate acetylate cyclase activity, including vasoactive intestinal peptide (VIP),glucagon, and epinephrine, suggesting that cAMP is a second messenger for somatostatin release. However, the effects of activating the adrenergic system on somatostatin in8cells and the mechanisms underlying this activation have not been investigated. Therefore, we set out to dissect the regulation of somatostatin transcription in the islets of Langerhans. We tested the effects of different adrenergic receptor agonists and antagonists on islets from wild type and β1/p2-adrenergic receptor (β1AR-/-/β2AR-/-) knockout mice and also on somatostatin secreting cell lines.1.Our results demonstrate that activation of the adrenergic system increased the production of somatostatin. EPI and ISO increased somatostatin content and transcription through the activation of β1AR/β2AR. The somatostatin content in the β1AR-/-/β2AR-/- mice was50% lower than in the WT mice.2. The GloSensor assay, qPCR, western blots and the dual luciferase assay were used to monitor the cAMP level, somatostatin expression, activations of kinases and transcriptional factors CREB and Pax6.Two parallel signaling pathways, Gs-cAMP-PKA-CREB and β-arrestinl-ERK-Pax6cooperatively regulated somatostatin transcription induced by ISO.Mechanistically, both the Gs-CREB and the β-arrestinl-Pax6pathways mediated the adrenergic effects on somatostatin transcription. These identified signaling pathways may have broader implications in maintaining celltype specificity and functions of the pancreatic8-cells.