This work was supported by the Leading Initiative for Excellent Young Researchers (from MEXT); a Grant-in-Aid for Scientific Research (B) (Grant Number JP21H02352); Japan Agency for Medical Research and Development (AMED-RPIME, Grant Number JP21gm6510009h0001, JP22gm6510009h9901); the Uehara Memorial Foundation; Astellas Foundation for Research on Metabolic Disorders; Suzuken Memorial Foundation, Akiyama Life Science Foundation, and Narishige Neuroscience Research Foundation. We also thank Nur Farehan Asgar, Ph.D, for editing a draft of this manuscript.

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Kumamoto University.
NOTE: For pain relief, ibuprofen was given in drinking water (0.11 mg/mL) for 48 h, and buprenorphine (0.05-0.1 mg/kg i.p.) was given 30 min before surgery. Sterile conditions include gloves, masks, and autoclaved instruments sterilized with ethylene oxide between animals. The surgery was performed on a heating pad set at 37 &#176;C and covered by a new lab mat for each animal. Befor...

Surgical Installation of Hyperglycemic and Hypoglycemic Clamps in Mice

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The method described here is a simple one that can be done with pipette tips, syringes, and other items found in ordinary laboratories. Although researchers may need to purchase additional tubes and pumps, expensive equipment is not needed. Thus, this protocol of catheterization and clamp is easier to start compared to previous reports12,13,14.
The clamp technique was developed around 1970 and has bee...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive glue</td>
			<td>Henkel AG &#38; Co. KGaA</td>
			<td>LOCTITE 454</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kit (C-peptide)</td>
			<td>Morinaga Institute of Bilogical Science Inc</td>
			<td>M1304</td>
			<td>Mouse C-peptide ELISA Kit</td>
		</tr>
		<tr>
			<td>ELISA kit (insulin)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>633-03411</td>
			<td>LBIS Mouse Insulin ELISA Kit (U-type)</td>
		</tr>
		<tr>
			<td>Handy glucose meter</td>
			<td>Nipro Co.</td>
			<td>11-777</td>
			<td>Free Style Freedom Lite</td>
		</tr>
		<tr>
			<td>Insulin (100U/ml)</td>
			<td>Eli Lilly &#38; Co.</td>
			<td>428021014</td>
			<td>Humulin R (100U/ml)</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Japan SLC Inc.</td>
			<td>C57BL/6NCrSlc</td>
			<td>C57BL</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Natsume seisakusho</td>
			<td>C-23S-560 No.2</td>
			<td>Sterilized</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Pump Systems Inc.</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic suture</td>
			<td>V&#214;MEL</td>
			<td>HR-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing1</td>
			<td>AS ONE Corporation</td>
			<td>9-869-01</td>
			<td>LABORAN(R) Silicone Tube</td>
		</tr>
		<tr>
			<td>Tubing2</td>
			<td>Fisher Scientific</td>
			<td>427400</td>
			<td>BD Intramedic PE Tubing</td>
		</tr>
		<tr>
			<td>Tubing3</td>
			<td>IGARASHI IKA KOGYO CO., LTD.</td>
			<td>size5</td>
			<td>Polyethylene tubing size5</td>
		</tr>
	</tbody>
</table>

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.

Author Spotlight: Investigating the Blood Glucose Homeostasis in Murine Brain Using a Cost-Effective Hyperglycemic And Hypoglycemic Clamp Technique

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

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.

The hypoglycemic clamp study was performed in male C57BL/6N mice (8 weeks old, more than 25 g BW) 3 h fasted at the start of the experiment (Figure 4A,B). The initial blood glucose level was 136 mg/dL (t = -15 min). If it is less than 90 mg/dL, it may be either because the surgery did not go well, or the arterial catheter was inserted too deep, or blood clots have entered the blood flow. The mouse condition after surgery affects the energy metabolism in the mouse. Physiologi...

Watch this Scientific Journal Video about Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice at JoVE.com

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, ...

hyperglycemic-clamp-and-hypoglycemic-clamp-in-conscious-mice