setup and operation of the hyperglycemic and hypoglycemic clamps in mouse for monitoring blood glucose and insulin release

小鼠高血糖和低血糖夹的手术安装

Glucose is an important source of energy for cells, and a lack of glucose can lead to a variety of symptoms and complications. In the event of low glucose (hypoglycemia, generally less than 70 mg/dL in fasting blood glucose level, but should not be determined by a single value1), the most common symptoms include weakness, confusion, sweating, and headache. It can also disrupt cerebral function and increase the risk of cardiovascular events and mortality2. Conversely, hyperglycemia is a medical condition in which the plasma glucose concentration exceeds normal levels (generally &#62; 126 mg/dL in fasting blood glucose level3). This can occur in individuals with diabetes who have either a deficit in insulin production or utilization. Hyperglycemia can lead to diabetic ketoacidosis, which occurs when the body cannot use glucose for energy but instead breaks down fatty acids for fuel. The hyperglycemic hyperosmolar state also increases mortality4. Long-term hyperglycemia can cause damage to blood vessels, nerves, and organs, leading to the development of several chronic complications such as cardiovascular disease, retinopathies, and kidney diseases. Thus, the blood glucose concentration must be maintained in a tight range between 100 mg/dL and 120 mg/dL.
Blood glucose is regulated by the balance between glucose input and output in a one-compartment model (Figure 1A). Glucose input includes absorbed glucose from food and glucose production from the liver, kidneys, and small intestine. Glucose output comprises glucose uptake in tissues and glucose disposal from the kidneys. Both the amount of glucose input and output are regulated by endocrine hormones. For example, glucagon, corticosterone, and catecholamines, known as counter-regulatory hormones, are released when blood glucose levels decrease5. They stimulate the breakdown of glycogen and the synthesis of glucose, mainly from the liver; these processes are known as glycogenolysis and gluconeogenesis, respectively. Hyperglycemia increases insulin release from pancreatic &#946;-cells and stimulates glucose uptake in the muscles, adipose tissues, and heart6,7,8,9. Exercise increases insulin-independent glucose uptake10. The sympathetic nervous system increases glucose uptake in muscles and brown adipose tissue6,11. To measure the ability to regulate glucose metabolism in peripheral tissues, researchers typically use the glucose tolerance test (GTT) and the insulin tolerance test (ITT) (Figure 1B,C). In GTT, two factors must be considered: insulin release and insulin sensitivity (Figure 1B). However, the glucose concentration curve during the 120 min test is different in each mouse, which may affect different amounts of hormone release. In ITT, blood glucose is regulated by both insulin sensitivity and the release of counter-regulatory hormones. Therefore, it is difficult to determine the precise meaning of glucose metabolism, insulin release, and insulin sensitivity in GTT and ITT, in situations where blood glucose levels are not constant.
To overcome these problems, it is desirable to keep blood glucose at a constant level (or &#34;clamp&#34;). In hyperglycemic clamp, glucose is infused into the bloodstream to raise blood glucose levels to a specific level and then maintained at that level for a period of time. The amount of infused glucose is adjusted based on measurements of blood glucose levels every 5-10 min to maintain a steady state. This technique is particularly useful for understanding the parameters of insulin secretion at a clamped glucose level. Hypoglycemic clamp is a method to maintain low blood glucose levels by infusing insulin. Glucose is infused at a variable rate to maintain a specific blood glucose level. If the mouse cannot recover from hypoglycemia, more glucose should be infused.
Although there are many advantages to performing hyperglycemic and hypoglycemic clamps, the surgical and experimental procedures are considered technically difficult. Thus, few research groups have been able to do them. We aimed to describe these methods for researchers with financial and workforce constraints to start these experiments at a lower budget.

作者聚焦：使用经济高效的高血糖和低血糖钳夹技术研究小鼠大脑中的血糖稳态

清醒小鼠的高血糖钳夹和低血糖钳夹

Our research investigates how our brain regulates the homeo status of blood glucose levels, and what kind of neurons, neuropeptides, and neuron circuits do that. Additionally, how does our memory of food, psychological stress, pain, and other somatic sensations affect it? It is difficult to assess glucose metabolism precisely.We must check in insulin sensitivity or resistance, insulin secretion and counter regulatory responses. We should also check which tissue changes glucose uptake and glucose production regarding insulin sensitivity or resistance. We believe that not only insulin sensitivity, but also insulin secretion and counter regulatory responses are all affecting normal blood glucose levels, even in healthy conditions.We are trying to understand the whole picture of glucose regulation by the brain. Our protocol is low cost and does not need a special and expensive device to create a clamp. It'll enable researchers to implement the use of the clamp easily and quickly.Because this protocol helps researchers to use the clamp very easily, we expect our protocol will encourage more research in the entire field of life sciences.

用于监测血糖和胰岛素释放的小鼠高血糖和低血糖钳的设置和操作

首先设置用于降血糖或高血糖夹的泵系统。为此，用胰岛素输注液和 30% 葡萄糖溶液填充两个汉密尔顿注射器。将填充的注射器连接到用于葡萄糖和胰岛素输注的延长管设置的两根长管上。将注射器组件连接到相应的注射泵后，将每种溶液填充到相应的延长管中。接下来，在一毫升注射器和延长管中填充生理盐水，以从动脉中抽血。接下来，将输液延长管连接到适当麻醉小鼠的预装颈静脉导管。用于将血液抽到动物颈动脉导管的延长管。使用玻璃纸胶带将管子固定在一起。将带有管子的鼠标放入带有垫料的 500 毫升空烧杯中。夹住延长管的顶端以抽血，同时将其连接到新注射器。提取 50 微升血液，将其转移到 1.5 毫升试管中并测量血糖水平。然后注入 50 微升生理盐水。要收集血液进行激素测量，请额外提取 50 微升血液并将其转移到冰上的 1.5 毫升试管中。零分钟血糖测量后，启动胰岛素注射泵注入适量的胰岛素。在 120 分钟的监测时间内，继续测量血糖水平并每 5 至 10 分钟改变一次葡萄糖输注速率，直到达到稳定状态。对于高血糖钳夹，请遵循低血糖钳夹的步骤，不包括胰岛素输注和调整后的稳态血糖水平。

setup-operation-hyperglycemic-hypoglycemic-clamps-mouse-for

Research

JoVE Journal

Medicine

Setup and Operation of the Hyperglycemic and Hypoglycemic Clamps in Mouse for Monitoring Blood Glucose and Insulin Release

107 次观看.  Kumamoto University. 首先设置用于降血糖或高血糖夹的泵系统。为此，用胰岛素输注液和 30% 葡萄糖溶液填充两个汉密尔顿注射器。将填充的注射器连接到用于葡萄糖和胰岛素输注的延长管设置的两根长管上。将注射器组件连接到相应的注射泵后，将每种溶液填充到相应的延长管中。接下来，在一毫升注射器和延长管中填充生理盐水，以从动脉中抽血。接下来，将输液延长管连接到适当麻?...

视频: 用于监测血糖和胰岛素释放的小鼠高血糖和低血糖钳的设置和操作

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively.
Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, ...