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 β-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.
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 > 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 β-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 "clamp"). 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.
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 °C and covered by a new lab mat for each animal. Before the surgery, the surgical area was cleaned with a betadine solution and alcohol. All surgical instruments were sterilized with an autoclave (for no more than two surgeries). Before making the incision, mice were checked to ensure they were fully anesthetized. The depth of anesthesia for each mouse was assessed prior to and during surgery by a toe pinch. The acclimatization period was no more than 5 min each time. Follow the instructions of the IACUC at the respective institution.
1. Preparation of tubings for the jugular vein and carotid artery
2. Surgery
3. Recovery
4. Set up the pump system (for hypoglycemic clamp)
5. Hypoglycemic clamp
6. Hyperglycemic clamp
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. Physiological glucose metabolism cannot be measured under poor health conditions. C57BL is more prone to clotting after surgery; mice that have a higher body weight, such as FVB or ICR mice, are less likely to lose weight due to clot washing after surgery. Thus, it's better for beginners to use FVB mice for practicing clamp procedures. C57BL mice require more intensive care. The 9 mm catheter insertion into the artery has not given us any trouble in collecting blood, even in FVB and ICR mice. Insulin was started at t = 0 min, and blood glucose levels decreased (Figure 4A). The GIR had not been stable between t = 0 min and t = 70 min. Subsequently, it became a steady state after 80 min.
The hyperglycemic clamp study was also performed in male C57BL/6N mice (8 weeks old, more than 25 g BW) 3 h fasted at the start of the experiment (Figure 4C,D). After measuring blood glucose and collecting blood samples at t = -15 and t = -5 min, glucose was infused from t = 0 min. The blood glucose level became 250 mg/dL at t = 40 min. Steady state continued from t = 30 min to the end.
Figure 1: Regulation of blood glucose levels. (A) Conceptual diagram illustrating the regulation of blood glucose in the body. (B,C) Regulation of glucose metabolism in (B) glucose tolerance test (GTT) and (C) insulin tolerance test (ITT). Several factors regulate blood glucose levels. Please click here to view a larger version of this figure.
Figure 2: Preparation of tubings and steps of the surgery. (A) Images of tubings for carotid artery and jugular vein. Tubing numbers match the protocol step numbers. (B) Method of preparing the tubings for surgery. (C, D) The numbered positions where the silk threads are ligated to insert the catheter into (C) artery and (D) vein. Positions of the silk threads in the cranial (C-(1)) and caudal side of the artery (C-(2)), and one more in the middle of them (C-(3)) (procedure 2-4). Positions of the silk threads in the cranial (D-(1)) and caudal side of the vein (D-(2)), and one more in the middle of them (D-(3)) (procedure 2-6). (E) Diagram of arterial and venous cannulas exiting the back Please click here to view a larger version of this figure.
Figure 3: Setting of the clamp. (A) Diagram showing the setup of tubings, pumps, syringes, and the mouse. (B) Sheet to record blood glucose levels and glucose infusion rate. 50 μL of blood is collected at t = -15 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min. The insulin infusion rate was 5.1 μL/min from t = 0 to 2 min and changed to 1.7 at t = 2 min. Blood glucose level was measured every 10 min. The blood glucose was measured every 5 min when its level was not stable. The glucose infusion rate was changed each time blood glucose was measured. Please click here to view a larger version of this figure.
Figure 4: Representative results of hypoglycemic clamp and hyperglycemic clamp. (A) Blood glucose levels and (B) glucose infusion rate of hypoglycemic clamp. (C) Blood glucose levels and (D) glucose infusion rate of the hyperglycemic clamp. Data represent mean ± SEM. Please click here to view a larger version of this figure.
Solution | Calculation | For 20 g mouse |
1 U/mL insulin | (2.647 x body weight) μL | 52.9 μL |
0.1% BSA saline | 300 μL – volume of insulin (μL) | 247.1 μL |
Table 1: An example calculation for preparing insulin infusate.
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 been used in mice and humans15. It is a useful method for accurately measuring glucose metabolism and is said to be the gold standard. However, it is not a common technique used by many researchers. The hyperinsulinemic-euglycemic clamp technique has been reported in mice13 and rats14, but the method of catheterization here is different, and readers can choose the easier one for their experiments. One of the purposes of this paper is to reduce the hurdle for starting an experiment. Therefore, we provided detailed information on the materials of handmade catheters, surgical procedures, and an example of experimental time course. These are informative for the researcher who attempts to perform clamp in the first time.
Insulin secretion in obesity and diabetes is stage-dependent. Many reports suggest insulin secretion is increased in obesity to reduce blood glucose in the insulin-resistant state16, but β-cell function will be damaged in Type 2 DM17,18. In fact, the number and area of pancreatic islets and insulin secretion have been reported to be increased in obesity mouse models, such as mice fed with a high-fat diet19 or leptin-deficient mice20. In these mouse models, which have an obvious phenotype, differences can be determined by examining blood insulin levels 15-30 min after glucose administration in the GTT. However, in some cases, it is not easy to determine the differences in insulin secretion. For example, if transgenic (Tg) mice have a blood glucose level of 500 mg/dL while a WT mouse has 300 mg/dL and both mice have the same blood insulin level, can we say insulin secretion decreases in Tg? In this case, we cannot compare insulin secretion ability unless blood glucose levels are the same using the method introduced here. This is one of the reasons why there is no established theory as to when β-cell function begins to deteriorate in the transition from obesity to diabetes. We can also measure insulin secretion by primary culture of the pancreas21 or ex vivo22. However, it will ruin the effect of the central nervous system on insulin release because the innervation of the vagus nerve will be removed. Post-absorptive insulin secretion is well-known, but the brain and autonomic nervous system also regulate insulin release23. The experiment to analyze the latter should be performed in an unanesthetized, unrestrained, painless blood collection. This is also why the vagus nerve has to be separated from the carotid artery in step 2.3.
Diabetes mellitus has been reported to cause hyperglycemia due to insulin resistance and increased secretions of glucagon24 and other counter-regulatory hormones25. In addition, repeated episodes of hypoglycemia in humans with diabetes due to failures of insulin dosage or other causes can lead to a condition called recurrent hypoglycemia, in which patients are prone to hypoglycemia26. It has been suggested that the rate of fall in glycemia in hypoglycemic clamp affects peripheral or central detection of the hypoglycemia27. Slow onset hypoglycemia may be appropriate to study the role of glucose sensors in portal-mesenteric veins, while a very rapid decrease in blood glucose may be for the study of brain glucose sensors27. 1 U/mL Insulin is used in lean C57BL mice. But, a higher insulin concentration will be needed in obese mice because they have insulin resistance, and 1 U/mL is not enough to decrease blood glucose levels.
In the one-compartment model of the blood glucose pool (Figure 1A), the amount of absorbed glucose may affect the rate of glucose input14. Thus, the rate of glucose production, one of the main objectives of measuring the hyperinsulinemic-euglycemic clamp, can be affected by the duration of fasting. However, long fasting time may increase the release of counter-regulatory hormones. Hence, researchers set fasting time according to their analysis purpose. Another clamp method includes blood sampling from the tail, which is simple because only an intravenous cannula needs to be inserted14. However, blood is collected in a restraint, which causes a moderate amount of restraint stress and an increase in plasma catecholamines and other stress hormones14. In addition, it is preferable to measure hormone concentrations in the blood flow in the center of the body rather than in the blood at the extremities. Therefore, blood sampling from arteries in free-moving mice is the best for measuring glucose metabolism physiologically. The mice do not move around, and swivel is not needed when they are acclimated to the experimental environment. However, it is recommended to use a swivel to prevent entangling infusion and sampling lines. Tubing1.2 and tubing set1.4 (Figure 3A) are not good for using a swivel. The system should be improved if the researcher is required to use a swivel. Reinfusion of blood cells does not influence the established steady state of blood glucose and insulin. The present method can also be applied to metabolomics studies using isotopes. For example, if 13C-glucose is continuously infused into the vein, the systemic metabolic turnover rate and intracellular intermediate metabolites can be measured28. Thus, this is a useful method to analyze glucose metabolism.
The authors have nothing to disclose.
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.
Adhesive glue | Henkel AG & Co. KGaA | LOCTITE 454 | |
ELISA kit (C-peptide) | Morinaga Institute of Bilogical Science Inc | M1304 | Mouse C-peptide ELISA Kit |
ELISA kit (insulin) | FUJIFILM Wako Pure Chemical Corporation | 633-03411 | LBIS Mouse Insulin ELISA Kit (U-type) |
Handy glucose meter | Nipro Co. | 11-777 | Free Style Freedom Lite |
Insulin (100U/ml) | Eli Lilly & Co. | 428021014 | Humulin R (100U/ml) |
Mouse | Japan SLC Inc. | C57BL/6NCrSlc | C57BL |
Suture | Natsume seisakusho | C-23S-560 No.2 | Sterilized |
Syringe Pump | Pump Systems Inc. | NE-1000 | |
Synthetic suture | VÖMEL | HR-17 | |
Tubing1 | AS ONE Corporation | 9-869-01 | LABORAN(R) Silicone Tube |
Tubing2 | Fisher Scientific | 427400 | BD Intramedic PE Tubing |
Tubing3 | IGARASHI IKA KOGYO CO., LTD. | size5 | Polyethylene tubing size5 |