Low Dose Metformin and Aspirin Activation of Hepatic AMP Kinase

低剂量二甲双胍和阿司匹林激活肝 AMP 激酶

• 批准号: 8781754
• 负责人: Katherine Beckwith-Fickas
• 金额: $ 5.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-18 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781754
• 关键词: 5' -AMP-activated protein kinase; Adenine Nucleotides; Adjuvant Therapy; Affect; Antibodies; Aspirin; Binding; Cells; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Diet; Dose; Drug usage; Fasting; Future; Glucagon; Hepatic; Hepatocyte; Human; Hyperglycemia; Immunoprecipitation; Impaired fasting glycaemia; Insulin Resistance; Knowledge; Liver; Measures; Mediating; Metabolic; Metformin; Modeling; Mus; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Oxidative Phosphorylation; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Prevention; Proteins; Protocols documentation; Signal Transduction; Testing; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Time; Toxic effect; Treatment Efficacy; adenylate kinase; design; diabetic patient; drug testing; glucose production; in vivo; mouse model; prevent; public health relevance; response; salicylate; sensor

DESCRIPTION (provided by applicant): This proposal will determine how metformin and salicylate activate AMP kinase (AMPK), a key step in 'turning off' hepatic glucose production. AMPK exists as a heterotrimeric complex comprised of a catalytic ? subunit and regulatory ? and ? subunits. Binding of AMP to the ? subunit causes activation of the kinase by promoting phosphorylation at T172 on the ??subunit. AMPK activation in response to pharmacological agents has considerable potential to reverse the metabolic abnormalities associated with type 2 diabetes (T2DM). Previous studies have focused on metformin and aspirin activation of AMPK for the treatment of patients with T2DM; however, both of these agents are generally tested at concentrations that are either much higher than maximally achievable therapeutic concentrations or are known to cause toxicity in humans. Therefore, this study aims to test these drugs on primary mouse hepatocytes and a mouse model of impaired fasting glucose at low, therapeutic concentrations. First, the effect of low doses of metformin and aspirin on the AMPK heterotrimeric complex will be explored by expressing a FLAG-tagged AMPK?1 subunit in primary hepatocytes and immunoprecipitating this protein (using an anti-FLAG antibody) from cell lysates after metformin or aspirin treatment. To test whether these drugs affect formation of the AMPK heterotrimeric complex directly or indirectly, FLAG-tagged AMPK subunits will be generated. Primary hepatocytes will be used to express subunits and then treated with metformin or aspirin in both a dose-dependent and time-dependent protocol. This will be followed by immunoprecipitation using an anti-?1 antibody. We hypothesize that metformin and aspirin at low concentrations will increase phosphorylation of AMPK?1 via different mechanisms, as seen in preliminary studies. These drugs will then be tested at therapeutic levels in vivo using a mouse model which disinhibits cAMP-PKA signaling in the mouse liver by ablating the hepatic PKA regulatory subunit 1A (prkar1a) yielding L-?prkar1a mice. This model has been shown to mimic the effect of increased glucagon associated with the increased insulin resistance of T2DM. Control and diet induced obesity (DIO) prkar1a and L-?prkar1a mice will be treated with metformin and aspirin alone and in combination. We anticipate the two agents, when used together, will show synergy in decreasing hepatic glucose production; salicylate by blocking cAMP phosphorylation (reversing the cAMP effect, analogous to the glucagon effect seen in T2DM) allowing metformin to promote assembly of the complex that had been blocked by cAMP stimulation. Given that diabetic patients manifest fasting hyperglycemia associated with insulin resistance, the combination of both low dose salicylate and metformin may enhance AMPK assembly, activation and subsequent treatment efficacy without producing unwanted toxicities associated with high doses of these drugs. This knowledge would be especially helpful in the design of future therapeutic interventions for the insulin resistance associated with T2DM.

描述（由申请方提供）：该提案将确定二甲双胍和水杨酸盐如何激活AMP激酶（AMPK），这是“关闭”肝脏葡萄糖生成的关键步骤。AMPK作为一种异源三聚体复合物存在，由一个催化？亚单位和调节？然后呢？亚单位。AMP与？亚基通过促进磷酸化在T172上的激酶激活？亚单位AMPK对药理学药物的响应活化具有相当大的潜力来逆转与2型糖尿病（T2 DM）相关的代谢异常。先前的研究集中在二甲双胍和阿司匹林激活AMPK治疗T2 DM患者;然而，这两种药物通常在远高于最大可达治疗浓度或已知在人体中引起毒性的浓度下进行测试。因此，本研究旨在以低治疗浓度在原代小鼠肝细胞和空腹血糖受损小鼠模型上测试这些药物。首先，低剂量的二甲双胍和阿司匹林对AMPK异源三聚体复合物的影响将通过表达FLAG标记的AMPK？1亚基，并从二甲双胍或阿司匹林处理后的细胞裂解物中免疫沉淀该蛋白（使用抗FLAG抗体）。为了测试这些药物是否直接或间接影响AMPK异源三聚体复合物的形成，将产生FLAG标记的AMPK亚基。原代肝细胞将用于表达亚单位，然后以剂量依赖性和时间依赖性方案用二甲双胍或阿司匹林处理。这将是随后的免疫沉淀使用抗？1抗体。我们假设二甲双胍和阿司匹林在低浓度下会增加AMPK的磷酸化？1通过不同的机制，如初步研究所见。然后，这些药物将在治疗水平进行测试，在体内使用的小鼠模型，解除抑制cAMP-PKA信号在小鼠肝脏消融肝PKA调节亚基1A（prkar 1a）产生L-？prkar 1a小鼠。该模型已显示模拟与T2 DM胰岛素抵抗增加相关的胰高血糖素增加的作用。控制和饮食诱导的肥胖（DIO）prkar 1a和L-？将用二甲双胍和阿司匹林单独和组合治疗PRKAR 1A小鼠。我们预计，当这两种药物一起使用时，将显示出降低肝脏葡萄糖生成的协同作用;水杨酸盐通过阻断cAMP磷酸化（逆转cAMP作用，类似于T2 DM中观察到的胰高血糖素作用），使二甲双胍促进已被cAMP刺激阻断的复合物的组装。鉴于糖尿病患者表现出与胰岛素抵抗相关的空腹高血糖症，低剂量水杨酸盐和二甲双胍两者的组合可以增强AMPK组装、活化和随后的治疗功效，而不会产生与高剂量这些药物相关的不希望的毒性。这些知识将特别有助于设计未来与T2 DM相关的胰岛素抵抗的治疗干预措施。