insulin

[yí dǎo sù]

Protein hormone

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This entry is made by Project of Compilation and Application of Scientific Terms in "Popular Science China" Encyclopedia to examine.

Insulin is a protein hormone secreted by islet β cells in the pancreas by stimulation of endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body that can reduce blood sugar and promote the synthesis of glycogen, fat and protein. Exogenous insulin is mainly used to treat diabetes.

Chinese name: insulin
Foreign name: Insulin^[6]
molecular weight: five thousand eight hundred and seven point six nine
Molecular formula: C two hundred and fifty-six H three hundred and eighty-one N sixty-five O seventy-six S six

Character: White or off white crystalline powder
Melting point: 233 ℃ (decomposition)
Specific curl: -64°±8°(C=2,0.003mol/L NaOH)
PH: Amphoteric, isoelectric point pI5.35-5.45
Chemical essence: protein

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Compound Introduction

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essential information

Chinese alias: ordinary insulin; Pancreatic hormone; Insulin; Regular insulin; Short acting insulin

English alias: Insulin; RI；Insulyl；Crystalline Insulin；Regular Insulin

Source content: This strain is a polypeptide with hypoglycemic effect extracted from pig pancreas. Calculated as dry product, the potency per 1mg shall not be less than 27 units. Each unit is equivalent to 0.0345 mg insulin.

Preparation requirements: This product should be extracted from frozen pancreas of quarantine qualified pigs. The production process shall meet the requirements of the current version of Good Manufacturing Practice for Drugs.

find

Islet beta cells under microscope

Insulin was introduced by F G. Banting and C H. Best was the first to discover. Since 1922, it has been used in clinical practice, which has saved the incurable diabetic patients in the past. Until the early 1980s, almost all insulin for clinical use was extracted from pig and cow pancreas. The composition of insulin in different animals is different. The structure of pig insulin is the most similar to that of human insulin, only one amino acid at the carboxyl end of the B chain is different. In the early 1980s, human insulin was successfully produced by microorganisms using genetic engineering technology and has been used in clinical practice.

In 1955, F. Sanger's group in England determined the amino acid sequence of bovine insulin, which opened the way for human beings to understand the chemical structure of protein molecules. On September 17, 1965, Chinese scientists artificially synthesized crystalline bovine insulin with full biological activity. It was the first protein artificially synthesized in the laboratory. Later, scientists in the United States and the Federal Republic of Germany also completed similar work.

In the early 1970s, British and Chinese scientists successfully measured the three-dimensional structure of pig insulin by X-ray diffraction. These work laid a foundation for further study of the relationship between insulin molecular structure and function. Analogues were prepared by chemical total synthesis and semi synthesis methods to study the effects of structural changes on biological functions; Comparative study on insulin of different species; To study abnormal insulin molecular disease, which is a molecular disease caused by the change of individual amino acids in insulin molecule due to the mutation of insulin gene. These studies also have important practical significance for clarifying the etiology of some diabetes.

First generation insulin animal insulin

In 1921, Frederick Banting cooperated with John Macleod to successfully extract insulin for the first time. The amino acid sequence and structure of insulin molecules in mammals of different races (human, cattle, sheep, pigs, etc.) are slightly different, and pig insulin is the closest to human insulin. Animal insulin is the first insulin injection preparation used for diabetes treatment. Generally, pig insulin is different from human insulin in 1 to 4 amino acids, so it is prone to immune reaction, atrophy or hyperplasia of subcutaneous fat at the injection site, insulin allergic reaction, and repeated hyperglycemia and hypoglycemia due to its high immunogenicity, It is prone to insulin resistance.

Second generation insulin human insulin

In the 1980s, people expressed highly purified synthetic human insulin through genetic engineering (recombinant DNA) transgenic yeast (brewer's yeast, Pichia pastoris or Hansen's yeast) or recombinant Chinese hamster ovary cells (CHO), and its structure was the same as the insulin secreted by human body itself.

Compared with animal insulin, human insulin has less allergic reaction or insulin resistance, so the phenomenon of subcutaneous fat atrophy is also reduced; Because human insulin antibody is less, the injection volume is 30% less than animal insulin on average; The stability of human insulin is higher than that of animal insulin. Human insulin can be stored for 4 weeks at about 25 ℃.

The physiological human insulin secretion pattern could not be simulated in the time of onset, peak time and duration of action. It needs to be injected 30 minutes before meals and has a high risk of hypoglycemia at night.

Third generation insulin insulin analogues

In the late 1990s, in the in-depth study of the structure and composition of human insulin, it was found that the peptide chain was modified: using genetic engineering technology to change the amino acid combination of some parts of the insulin peptide chain; Change isoelectric point; Increase the strength of hexamer; Replacing zinc ion with cobalt ion; Adding fatty acid chains in the molecule and increasing the combination with albumin may change its physical, chemical and biological characteristics, so as to develop insulin analogues more suitable for human physiological needs. Insulin analogues can be used immediately before meals, also known as meal insulin or quick acting insulin.

classification

Source: The control gene for insulin synthesis is on the short arm of chromosome 11. If the gene is normal, the insulin structure is normal; If the gene is mutated, the structure of insulin produced is abnormal, and it is mutated insulin. In the nucleus of β cell, the DNA of insulin gene region on the short arm of chromosome 11 is transcribed into mRNA, and the mRNA moves from the nucleus to the endoplasmic reticulum of cytoplasm, and is translated into proinsulin composed of 105 amino acid residues. Proinsulin is removed from its pro peptide by proteolysis to form a long peptide chain composed of 86 amino acids, proinsulin (45343345). Proinsulin enters the Golgi apparatus with the microbubbles in the cytoplasm. Through the action of proteolytic enzyme, the three arginine linked chains of 75, 55, and 6075 are cut off, and the broken chain generates the useless C-peptide. At the same time, insulin is generated, secreted outside the β cell, and enters the blood circulation. A small part of proinsulin without protease hydrolysis enters the blood circulation with insulin, and the biological activity of proinsulin is only 5% of insulin.

Inactivation.

The islet β cells reserve about 200U of insulin and secrete about 40U every day. On an empty stomach, the plasma insulin concentration is 5-15 μ U/mL. The plasma insulin level can increase 5-10 times after meals.

1. Animal insulin: It is extracted from the pancreas of pigs and cattle, both of which have the same effect.

1. Ultra short effect: it takes effect 15 minutes after injection, and the peak concentration is 1-2 hours (subcutaneous). (e.g. insulin aspart, insulin lispro)^[4]

2. Short acting (quick acting): it takes effect 30 minutes after injection, with peak concentration of 2-4 hours, lasting for 5-8 hours (subcutaneous, intramuscular, intravenous). (such as normal insulin and regular insulin)

3. Medium effect: it takes effect 2~4 hours after injection, the peak concentration is 6~12 hours, lasting for 24~28 hours (subcutaneous). (such as low protamine zinc insulin)

4. Long term effect: it takes effect 4-6 hours after injection and lasts for 24 to 36 hours (subcutaneous). (protamine zinc insulin)

5. Ultra long effect: it takes effect 3-6 hours after injection, and the maintenance time is 6-24 hours (subcutaneous). (insulin detemir, insulin glargine)

6. Premixing: it takes effect 0.5 hour after injection, and the maintenance time is 24 hours (subcutaneous). (Dual phase insulin)

structure

Insulin structure

The insulin function of animals of different races (human, cattle, sheep, pigs, etc.) is generally the same, but the composition is slightly different. In the figure Human insulin Chemical structure.

Insulin consists of A and B peptide chains. There are 11 kinds of 21 amino acids in the A chain and 15 kinds of 30 amino acids in the B chain of human insulin, a total of 51 amino acids. The sulfhydryl group in the four cysteine A7 (Cys) - B7 (Cys), A20 (Cys) - B19 (Cys) forms two disulfide bonds, connecting the A and B chains. In addition, there is also a disulfide bond between A6 (Cys) and A11 (Cys) in the A-chain.

secretion

The rate of insulin biosynthesis is affected by the plasma glucose concentration. When the blood glucose concentration increases, the content of proinsulin in β cells increases, and insulin synthesis accelerates.

Insulin is synthesized in islet beta cells. Insulin has a molecular weight of 5700 and is composed of two amino acid peptide chains. The A chain has 21 amino acids and the B chain has 30 amino acids. There are two disulfide bonds between A-B chains.

Insulin is secreted into the blood with the same molecule as C-peptide. In patients who are clinically treated with insulin, there is insulin antibody in the serum, which affects the radioimmunoassay of blood insulin level. In this case, the level of plasma C-peptide can be measured to understand the endogenous insulin secretion status.

influence factor

The secretion of insulin in the body is mainly affected by the following factors:

Stimulate insulin secretion

Plasma glucose concentration Plasma glucose concentration is the most important factor affecting insulin secretion. After oral or intravenous glucose injection, insulin release showed a two-phase reaction. In the early rapid phase, insulin in portal vein plasma reached the highest value within 2 minutes, and then decreased rapidly; In the slow phase of delay, the plasma insulin level gradually increased after 10 minutes, lasting for more than 1 hour. The early fast phase shows that glucose promotes the release of stored insulin, and the delayed slow phase shows the synthesis of insulin and the transformation of proinsulin.

Eating foods with more protein After eating foods with more protein, the concentration of amino acids in the blood increases, and insulin secretion also increases. Arginine, lysine, leucine and phenylalanine have strong effects on stimulating insulin secretion.

Increased gastrointestinal hormones after meals The increase of gastrointestinal hormones after meals can promote insulin secretion, such as gastrin, secretin, gastric inhibitory peptide and intestinal vasoactive peptide.

Autonomic nerve function status The vagus nerve stimulates insulin secretion; When sympathetic nerve is excited, insulin secretion is inhibited.

recipient

The biological action of insulin at the cellular level is initiated by binding to specific receptors on the target cell membrane. Insulin receptor is a specific part of the target cell membrane where insulin acts. It can only bind to insulin or proinsulin containing insulin molecules. It is highly specific and widely distributed. Receptor is a glycoprotein. Each receptor is composed of two subunits of α and β, and two subunits of each subunit form a tetrameric receptor. The alpha subunit passes through the cell membrane, and one end is exposed on the cell membrane surface, with insulin binding sites. The beta subunit extends from the cell membrane to the cytoplasm, and is a functional unit of insulin that can trigger cell membrane and intracellular effects. After insulin is combined with subunit, tyrosine kinase in β subunit is activated to phosphorylate receptor, produce mediator, regulate activity of intracellular enzyme system, and control material metabolism. The tetrameric receptor is composed of two subunits. The degree of insulin binding of each cell depends on the number and affinity of receptors, which are regulated by plasma insulin concentration. When insulin concentration increases, the number of insulin receptors tends to decrease, which is called downregulation. For example, obese patients with non-insulin-dependent diabetes are clinically insulin insensitive, called resistance, because the number of receptors on the adipocyte membrane decreases. When obese patients with non-insulin-dependent diabetes lose weight after diet control and physical exercise, the number of insulin receptors on the adipocyte membrane increases, and the binding force with insulin is strengthened, thus improving the utilization of blood sugar. This is not only the important pathogenesis of obese non-insulin-dependent diabetes, but also the theoretical basis for weight loss in treatment.

Signal path

The insulin signaling pathway requires that insulin first binds to a heterotetramer receptor on the target cell membrane. Insulin receptor is a membrane glycoprotein, which consists of two separate insulin binding domains (α subunits) and two signal transduction domains (β subunits). The binding of insulin to the receptor causes the conformational change of α subunit, so that adenosine triphosphate (ATP) can bind to the intracellular domain of β subunit. After binding to ATP, the tyrosine kinase in the β subunit is activated, which makes insulin receptor self phosphorylate. The phosphorylated insulin receptor starts from phosphorylated insulin receptor substrate (IRS) 1 and 2, and phosphorylates other protein substrates in turn. Insulin signals are further cascaded through phosphorylation networks and involve many other intracellular substances. The biochemical process of insulin action is detailed in other topics. (See "Structure and function of insulin receptor") By activating the above signal pathways, insulin can play a role as a powerful regulator of metabolic function. In addition, insulin also plays a role in growth and proliferation through insulin receptor mediated activated mitogen activated protein (MAP) kinase pathway.

metabolize

Insulin almost directly or indirectly affects the function of every tissue in the body. Among them, the metabolic effects of the three main energy storage tissues of insulin are liver, muscle and adipose tissue.^[2]

（1） Insulin and glucose metabolism - The three major sources of glucose are: intestinal absorption of food, glycogen decomposition (glycogen is the storage form of glucose), and gluconeogenesis (non sugar precursor components generated during carbohydrate, protein, and fat metabolism can synthesize glucose through gluconeogenesis).

Once transported into cells, glucose can be stored as glycogen or glycolysis into pyruvate. Pyruvate can be reduced to lactic acid, or transformed into alanine by amino transfer, or into acetyl CoA (CoA). Acetyl CoA can be oxidized into carbon dioxide and water in the tricarboxylic acid cycle, or transformed into fatty acids and stored in the form of triglycerides, or used to synthesize ketones or cholesterol.

Insulin has many functions in glucose metabolism, including inhibiting glycogen decomposition and gluconeogenesis, increasing glucose transport into fat and muscle, enhancing glycolysis in fat and muscle, and stimulating glycogen synthesis

● Glucose production: Although glycogen decomposition can occur in most tissues in the body, only the liver and kidney express glucose-6-phosphatase, which is necessary for glucose release into the blood. The liver and kidney also contain enzymes necessary for gluconeogenesis. In both organs, the liver is responsible for most of the glucose output. The tracer study found that the kidney only produces 10% - 20% glucose after overnight fasting. Therefore, the liver is the primary target organ for insulin to regulate glucose production. However, in patients with type 2 diabetes mellitus, the renal glucose output will increase to partially compensate for the decreased hepatic glucose output during the anti regulation process of hypoglycemia.

Insulin directly limits hepatic glucose output by inhibiting glycogen phosphorylase, which is essential for glycogen decomposition. Insulin also reduces hepatic gluconeogenesis through indirect action, involving the following ways: reducing the input of gluconeogenic precursors and free fatty acids into the liver; Inhibiting the secretion of glucagon, part of the mechanism is to directly inhibit the glucagon gene in islet α cells; Change the nerve signal input of the liver. After injecting insulin into the portal vein or peripheral vein of dogs, researchers found that the direct effect of insulin was still dominant in the process of hepatic glucose production, but its indirect effect was more obvious with the increase of insulin secretion.

● Utilization of glucose: insulin can stimulate skeletal muscle and fat to absorb glucose. In skeletal muscle and adipose tissue, glucose passes through the cell membrane through glucose transporter 4 (GLUT-4). GLUT-4 may exist in the cytoplasm of skeletal muscle and fat cells; The signal from insulin promotes the transfer of GLUT-4 from the cytoplasm to the cell membrane, thus assisting glucose to enter these tissues (for example, after meals). Some mouse studies have confirmed that the control of glucose homeostasis is complex, and that skeletal muscle can also ingest glucose by increasing insulin independent GLUT-4 and adenosine monophosphate activated protein kinase (AMPK) activity.

Under normal blood glucose conditions, insulin mediated glucose uptake mostly occurs in muscle tissue, and when glucose uptake increases, the additional intake of fat tissue is less than 10% of the total increase. However, adipose tissue also indirectly promotes glucose utilization through insulin mediated inhibition of lipolysis. This is due to the substrate competition mechanism, because the utilization of free fatty acids, one of the energy sources of the body, is reduced, which increases the uptake and metabolism of glucose in muscle. By affecting glycogen synthesis and glycolysis (glycolysis), insulin can also promote glucose metabolism in cells.

Insulin can enhance the activity of glycogen synthase in fat, muscle, liver and other tissues, but this effect will not lead to net glycogen synthesis unless glycogen phosphorylase is strongly inhibited. In fact, the catalytic activity of glycogen phosphorylase in human skeletal muscle is 50 times that of glycogen synthase.

Insulin can improve the rate of glycolysis in skeletal muscle and adipose tissue by enhancing the activity of hexokinase and 6-phosphate fructose kinase, two key enzymes in the glycolysis pathway.

(2) Insulin and lipid metabolism - Insulin can coordinate the use of different energy substances (glucose and free fatty acids) in the body to meet the energy needs of the body in the cycle of eating, fasting and exercise. After a meal, a large amount of glucose is available. At this time, insulin secretion increases, which can promote the storage of triglycerides to fat cells. There are many mechanisms involved:

● Insulin promotes the clearance of chyle particles rich in triglycerides (for example, chyle particles formed after mixed diet) from peripheral tissues by activating lipoprotein lipase. Lipoprotein lipase exists in the vascular endothelium of muscle and adipose tissue, which can hydrolyze triglycerides in circulating lipoproteins. The generated fatty acids are then oxidized after being ingested by muscle tissue, or stored after being ingested by adipose tissue. Insulin can also activate lipoprotein lipase in adipose tissue, but inhibit this enzyme in skeletal muscle. This tissue specific effect of insulin on lipoprotein lipase results in the transfer of triglycerides from muscle to adipose tissue for storage.

● Insulin can stimulate free fatty acids in fat cells to re esterify into triglycerides. In insulin mediated manner, more glucose is transported into adipocytes, which indirectly realizes re esterification. The increase of glycolysis activity in adipocytes leads to an increase in the level of glycolysis metabolite glycerol-3-phosphate, which is involved in the esterification of free fatty acids into triglycerides.

● Insulin inhibits the lipolysis of triglycerides in storage by inhibiting the rate limiting enzyme hormone sensitive lipase (HSL). Studies have shown that insulin can activate a protein phosphatase and inactivate HSL after dephosphorylation. Another mechanism is that insulin sensitive phosphodiesterase reduces the level of cyclic adenosine monophosphate (cAMP) in cells, thereby inhibiting the cAMP dependent protein kinase responsible for phosphorylation and activation of HSL.

The overall effect of increased triglyceride storage and reduced lipid decomposition is to reduce free fatty acids entering the liver. Although these processes are indirect, insulin may indeed play a strong regulatory role in reducing liver gluconeogenesis and glucose output.

Insulin and ketone metabolism - Under low insulin conditions such as too long fasting time or uncontrollable diabetes, fat mobilization will be significantly accelerated, leading to excessive free fatty acids entering the liver. In this case, the liver uses a large amount of acetyl CoA produced by incomplete β - oxidation of long-chain fatty acids to synthesize ketone bodies. These ketoacids (acetylacetic acid, β - hydroxybutyric acid and acetone) can be used for energy supply by extrahepatic tissues, mainly skeletal muscle and heart. In extreme cases, the brain also uses ketone bodies for energy supply.

Insulin can effectively reduce circulating ketone body concentration through various mechanisms. As mentioned above, insulin can inhibit lipid decomposition, thus reducing the supply of free fatty acids used by the liver for ketogenesis. In addition, insulin can also directly inhibit the ketogenic effect of the liver, which can also explain that in obese people and patients with type 2 diabetes, although the plasma concentration of free fatty acids is high, ketosis is not easy to occur. Finally, hyperinsulinemia is associated with increased peripheral ketone body clearance.

(3) Insulin and protein metabolism - Insulin increases nitrogen retention and protein production.

Insulin can promote the transport of amino acids into hepatocytes, skeletal muscles and fibroblasts, and can also increase the number of ribosomes and translation efficiency. In general, these effects increase protein synthesis. Insulin also inhibits protein breakdown. In human studies, using the hyperinsulinemic normoglycemic clamp technique, it was found that the physiological increase of serum insulin concentration can reduce systemic proteolysis in a dose-dependent manner [29]. This phenomenon can reduce protein hydrolysis by 40% at most, which indicates that there are other factors regulating protein hydrolysis. Insulin maintains the availability of amino acid, the substrate of protein synthesis, by inhibiting gluconeogenesis. Therefore, insulin can promote protein synthesis through direct and indirect mechanisms.

Biological function

pharmacological action

Treat diabetes and consumptive diseases. Promote glucose in blood circulation to enter hepatocytes, muscle cells, fat cells and other tissue cells to synthesize glycogen, reduce blood sugar, and promote the synthesis of fat and protein.

Physiological function

The main physiological function of insulin is to regulate the metabolic process. Glucose metabolism: promote the uptake and utilization of glucose by tissue cells, promote glycogen synthesis, inhibit gluconeogenesis, and reduce blood sugar; For fat metabolism: promote fatty acid synthesis and fat storage, reduce fat decomposition; For protein: promote amino acids to enter the cell, and promote all aspects of protein synthesis to increase protein synthesis. The overall effect is to promote anabolism. Insulin is the only hormone in the body that can reduce blood sugar, and also the only hormone that can simultaneously promote the synthesis of glycogen, fat and protein. The mechanism of action belongs to receptor tyrosine kinase mechanism.

The role of insulin in cells. Map source network

Regulate sugar metabolism

Insulin can promote the uptake and utilization of glucose by tissues and cells in the whole body, and inhibit the decomposition of glycogen and gluconeogenesis. Therefore, insulin can reduce blood sugar. When insulin is secreted excessively, blood sugar drops rapidly, brain tissue is most affected, and convulsion, coma, and even insulin shock may occur. On the contrary, insufficient insulin secretion or insulin receptor deficiency often leads to elevated blood sugar; If it exceeds the renal sugar threshold, sugar will be discharged from the urine, causing diabetes; At the same time, changes in blood composition (containing excessive glucose) also lead to hypertension, coronary heart disease, retinal vascular disease and other diseases. Insulin's hypoglycemic effect is the result of multiple actions:

(1) Promote the target cell membrane carrier in muscle, adipose tissue, etc. to transport glucose in blood into cells.

(2) Through covalent modification, the activity of phosphodiesterase is increased, the level of cAMP is reduced, and the concentration of cGMP is increased, so that the activity of glycogen synthase is increased, the activity of phosphorylase is reduced, the glycogen synthesis is accelerated, and the glycogen decomposition is inhibited.

(3) Activate pyruvate dehydrogenase by activating pyruvate dehydrogenase phosphatase, accelerate the oxidation of pyruvate to acetyl coenzyme A, and accelerate the aerobic oxidation of sugar.

(4) Inhibiting gluconeogenesis by inhibiting the synthesis of PEP carboxyl kinase and reducing the raw materials of gluconeogenesis.

(5) Inhibit hormone sensitive lipase in adipose tissue, slow down fat mobilization, and increase tissue utilization of glucose.

Regulate fat metabolism

Insulin can promote the synthesis and storage of fat, reduce free fatty acids in blood, and inhibit the decomposition and oxidation of fat. Insulin deficiency can cause disorder of fat metabolism, decrease of fat storage, enhancement of fat decomposition, and increase of blood lipids, which can cause arteriosclerosis over time, and then lead to serious cardiovascular and cerebrovascular diseases; At the same time, insulin deficiency will lead to the enhancement of body fat decomposition, the generation of a large number of ketones, and ketoacidosis.

Regulate protein metabolism

Insulin, on the one hand, promotes the uptake of amino acids and the synthesis of proteins by cells, and on the other hand, inhibits the decomposition of proteins, thus facilitating growth. The protein synthesis promoting effect of adenohypophysis growth hormone can only be shown in the presence of insulin. Therefore, insulin is also one of the indispensable hormones for growth.

Other functions

Insulin can promote potassium and magnesium ions to pass through the cell membrane and enter the cell; It can promote the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and adenosine triphosphate (ATP).

Antihormone

Glucagon

The main anti insulin hormones in the body are glucagon, epinephrine and norepinephrine, adrenocortical hormone, growth hormone, etc. They can raise blood sugar.

(1) Glucagon (glucagon). It is secreted by islet α cells and resists insulin in regulating blood glucose concentration. The main function of glucagon is to rapidly decompose glycogen in the liver and promote the production and output of glucose in the liver,

Enter the blood circulation to raise the blood sugar level. Glucagon can also enhance the intake of amino acids by hepatocytes, and strengthen gluconeogenesis by promoting lipolysis in extrahepatic tissues, increasing the input of glycerol into the liver and providing a large number of gluconeogenic materials. Glucagon and insulin coordinate the dynamic balance of blood glucose level.

When eating carbohydrates, a large amount of glucose is produced to stimulate the secretion of insulin. At the same time, the secretion of glucagon is inhibited, and the insulin/glucagon ratio increases significantly. At this time, the liver changes from a tissue that mainly produces glucose to an organ that converts glucose into glycogen and stores glycogen.

During starvation, the level of glucagon in the blood increased significantly while the level of insulin decreased. Gluconeogenesis and glycogen decomposition are accelerated, and the liver continuously transports glucose into the blood. At the same time, due to the reduced insulin level, the ability of muscle and adipose tissue to use glucose is reduced, mainly by using fatty acids, thus saving glucose to ensure that brain and other tissues have sufficient glucose supply.

(2) Adrenaline and norepinephrine. Adrenaline is secreted by the adrenal medulla, and norepinephrine is the secretion of sympathetic nerve endings. When the nervous tension or cold stimulation makes the sympathetic nerve in an excited state, the secretion of adrenaline and norepinephrine increases, which increases the output of liver glycogen decomposition, prevents glucose from entering muscle and adipose tissue cells, and increases blood sugar.

(3) Growth hormone and growth hormone inhibiting hormone.

① Growth hormone. It is secreted by the anterior lobe of the pituitary gland. It can promote human growth and regulate material metabolism in the body. Growth hormone can increase blood sugar mainly by inhibiting the utilization of glucose by muscle and adipose tissue, and promoting gluconeogenesis and glycogen decomposition in liver. Growth hormone can promote fat decomposition and increase plasma free fatty acid. When hungry, insulin secretion decreases and growth hormone secretion increases, so glucose utilization in blood decreases and fat utilization increases. At this time, glucose and free fatty acid content in plasma increases.

② Growth hormone inhibiting hormone. It is secreted by islet D cells. Growth hormone releasing inhibitory hormone not only inhibits the secretion of pituitary growth hormone, but also inhibits the secretion of insulin and glucagon under physiological conditions. However, growth hormone releasing inhibiting hormone itself has no direct effect on the production of liver glucose or the utilization of glucose in circulation.

(4) Adrenal glucocorticoid. Adrenal glucocorticoid is secreted by the adrenal cortex (mainly cortisol, namely hydrocortisone), which can promote the protein decomposition of extrahepatic tissues, increase the amount of amino acids entering the liver, and induce the synthesis of various key enzymes related to gluconeogenesis, thus promoting gluconeogenesis and raising blood sugar.

(5) Free nerve function may affect insulin secretion.

When the vagus nerve is excited, it promotes insulin secretion, while when the sympathetic nerve is excited, it inhibits insulin secretion. As we know above, the DNA in the gene region is transcribed into mRNA to generate a long peptide chain composed of 86 amino acids - Proinsulin. Proinsulin enters the Golgi apparatus with the microbubbles in the cytoplasm. Through the action of proteolytic enzyme, three arginine linked chains of 31, 32 and 60 are cut off, and the above results are obtained.

Insulin response

Systemic reaction

(1) Hypoglycemic reaction: most common. It is commonly seen in the fragile type of type I or the medium and heavy type of type II, especially in the emaciated. Generally due to too much physical activity, sometimes too little diet, reduction, lost time or excessive dosage. Symptoms include hunger, dizziness, weakness, sweating, palpitations, and even neurological symptoms, such as disorientation, irritability, incoherence, and inconstancy of crying and laughing, sometimes more serious, even fainting, tetany, epilepsy like, unconscious, and death. During the course of treatment, patients should be taught to be familiar with this reaction and be alert at any time. They should take cake and sugar or sugar water as soon as possible to relieve the symptoms. Those with severe symptoms should be immediately injected with more than 40ml of 50% glucose intravenously, followed by intravenous drip of 10% glucose water until they are awake; Sometimes glucagon can be injected first, 1mg subcutaneous or muscle each time. If the hypoglycemic reaction lasts for a long time and is serious, hydrocortisone can also be used, 100~300mg each time, and intravenous drip in 5%~10% glucose water. When hypoglycemic reaction recovers, the next dose must be carefully estimated, and the condition must be analyzed to prevent recurrence. After repeated hypoglycemia, reactive hyperglycemia (Somogyi effect) may occur due to stimulation of islet α cells and adrenal glands, which often leads to fragile type, and must be avoided as far as possible.

(2) Allergic reaction: A few patients have allergic reactions, such as urticaria, angioneurotic edema, purpura, and very few have anaphylactic shock. This reaction is mainly caused by impurities in the preparation. The mild cases can be treated with antihistamines, and the severe cases must be replaced with high-purity preparations such as single component human insulin. Because its amino acid sequence is the same as that of endogenous insulin and contains few impurities, allergy is extremely rare, or oral drugs can be used instead. If necessary, hypodermic injection of small dose of polyyi insulin can also be used for desensitization.

(3) Insulin induced edema: before diabetes mellitus is not controlled, there is often water loss and sodium loss, and glucose in cells is reduced. After control, water and sodium retention and edema may occur 4 to 6 days later, which may be related to insulin promoting renal tubular absorption of sodium, called insulin edema.

(4) Refractive disorder: during insulin treatment, sometimes the patient feels blurred vision. Due to the rapid drop of blood sugar during treatment, the osmotic pressure of the lens and vitreous body is affected, so that the water in the crystalline body escapes and the refractive rate decreases, causing hyperopia. However, this is a temporary change, which generally disappears rapidly with the recovery of blood glucose concentration, without permanent change. This kind of refractive mutation is more common in young patients with large blood sugar fluctuations.

Local reaction

(1) Skin redness, swelling, fever and subcutaneous nodules at the injection site are mostly found within weeks of the initial treatment of NPH or PZI. Due to protein and other impurities, it can disappear automatically after changing the focus, without affecting the efficacy.

(2) Subcutaneous fat atrophy or hyperplasia, fat atrophy into a concave loss of sebum, mostly seen in young women and children's thighs, abdominal wall and other injection sites; The subcutaneous tissue increases into a hard mass, which is mostly found in the injection site such as male buttocks. Sometimes it is numb and tingling, which can affect absorption. The injection site must be replaced to ensure treatment.

Drug resistance

A few patients have insulin resistance, and the daily insulin requirement exceeds 200U for more than 48 hours. At the same time, those without secondary diabetes caused by ketoacidosis and other endocrine diseases are called insulin resistance. This group does not include drug resistance caused by obesity, infection, liver disease, hemochromatosis, leukemia, rheumatoid arthritis, lipodystrophy diabetes, etc. Immune reaction: anti insulin antibodies are produced in the blood after insulin injection, which are generally IgG, especially bovine insulin. Therefore, the insulin resistance here should not be confused with insulin resistance in pathophysiology.

Solution:

① Using single component human insulin can significantly reduce antibody production and alleviate drug resistance;

② Try to use oral antidiabetic drugs and their combination;

③ In patients with significantly increased antibody concentration, try prednisone 30mg~40mg/d for three times when necessary. Most of them can also significantly reduce the insulin dose within 1~2 weeks. After taking effect, gradually reduce the dose and stop prednisone. During the course of treatment, the patient's condition and blood sugar must be closely observed to avoid repeated severe hypoglycemia when the drug resistance subsides.

Insulin treatment of nephrosis can only be said to reduce blood sugar. When patients use insulin, they must always check blood sugar. Once hypoglycemia is found, the amount of insulin should be adjusted in time.

Diabetes treatment and medication

Announce

edit

Insulin is used to treat most types of diabetes. In general, whether insulin treatment is needed depends on the degree of insulin deficiency. All patients with type 1 diabetes need insulin treatment; Many patients with type 2 diabetes will need insulin as their beta cell function gradually declines^[3] 。

indication

Patients with type 1 diabetes need insulin treatment at the onset of the disease because their pancreatic islet beta cell function is damaged and insulin secretion is absolutely insufficient, and they need lifelong insulin replacement therapy to maintain life and life. It accounts for about 5% of the total number of diabetics. On the basis of lifestyle and combined treatment of oral hypoglycemic drugs, patients with type 2 diabetes can start combined treatment of oral drugs and insulin if their blood sugar still fails to reach the control goal. Generally, when HbA1c is still greater than 7.0% after a large dose of multiple oral drugs combined treatment, insulin treatment can be considered. Emaciated diabetic patients with new onset and difficult differentiation from type 1 diabetes. In the course of diabetes (including newly diagnosed type 2 diabetic patients), when there is no obvious cause of weight loss, insulin treatment should be used as early as possible. For patients with newly diagnosed type 2 diabetes mellitus with high blood sugar, it is difficult to control blood sugar satisfactorily with oral drugs, and rapid relief of hyperglycemia toxicity can partially reduce insulin resistance and reverse beta cell function, so insulin intensive treatment can be used when newly diagnosed type 2 diabetes mellitus is accompanied by significant hyperglycemia. There are also some special circumstances that require insulin treatment: perioperative period; In case of serious acute complications or stress, temporary insulin use is required to pass the dangerous period, such as diabetic ketoacidosis, hypertonic hyperglycemia, lactic acidosis, infection, etc; Serious chronic complications, such as diabetic foot, severe diabetic nephropathy, etc; Combined with some serious diseases, such as coronary heart disease, cerebrovascular disease, blood disease, liver disease, etc; For women with gestational diabetes mellitus and diabetes mellitus complicated with pregnancy, during pregnancy, before and after delivery, and during lactation, if the blood sugar can not be controlled by diet alone to reach the required target value, insulin treatment is required, and oral hypoglycemic drugs are prohibited. Patients with secondary diabetes and specific diabetes.

Insulin preparation

（1） Human insulin

(1) Ordinary insulin ordinary insulin (short acting) is a soluble insulin zinc complex with the same amino acid sequence as endogenous human insulin. It can be used to control the increase of blood sugar after meals. After subcutaneous injection of ordinary insulin, the formed hexamer will be broken down into dimer and monomer and absorbed. This process will delay the rise of insulin concentration in the blood stream, so it is necessary to inject insulin at least 30 minutes before meals to achieve the best control effect on the rise of blood sugar after meals.

Many diabetics find it difficult to grasp the timing of this kind of meal injection. In addition, observation shows that the duration of action of ordinary insulin exceeds the duration of postprandial blood glucose rise of most meals, especially those with low carbohydrate and fat content. This can lead to hypoglycemia several hours after a meal, which can be prevented by adding some snacks.

Inpatients can also use ordinary insulin intravenously, such as treatment Diabetic ketoacidosis 。

(2) NPH insulin - NPH (medium acting insulin) is a crystalline suspension of human insulin, protamine and zinc in neutral buffer, which can delay the release of insulin into the blood. In order to achieve 24-hour basic coverage, NPH needs to be administered at least twice a day. When taking medicine in the morning, if you do not eat during the peak period of action, the risk of hypoglycemia will increase. If you are taking drugs at night or before going to bed, you may need to add some snacks before going to bed to avoid hypoglycemia at night. Subsequently, fasting blood glucose concentration may be higher than the target.

The suspension NPH should be administered at room temperature and immediately mixed before injection. The method is to roll the insulin pen or insulin bottle in your hand at least 10 times, and then turn it upside down at least 10 times.

Although NPH can be mixed with ordinary insulin or quick acting insulin in the same syringe, which may be convenient and beneficial, the two kinds of insulin should be mixed immediately before injection. Normal ("clear") insulin should be inhaled first, and then NPH ("turbid") insulin should be inhaled. The addition of protamine (NPH) into ordinary insulin bottles should be avoided, as it will change the pharmacokinetics of ordinary insulin. Inject immediately after preparation. If NPH is mixed with quick acting insulin analogue, it should be injected within a few minutes before meals.

(3) U-500 ordinary insulin - The specification of U-500 ordinary insulin is 500U/mL. It can be used to treat patients with severe insulin resistance (for example, the total daily insulin dose required is>200U). Since high concentration insulin will delay absorption, the pharmacological characteristics of U-500 ordinary insulin are most similar to NPH. However, the pharmacokinetics may vary greatly, and there may still be a risk of hypoglycemia. Higher dosage can significantly prolong the action time of insulin.

Several reports have shown that U-500 insulin is effective for obese patients, patients with immune mediated insulin resistance and patients with inherited insulin receptor abnormalities. An observational study included 11 obese patients with severe insulin resistance and uncontrolled diabetes (insulin demand>200U/d). After replacing U-100 NPH insulin with U-500 ordinary insulin, the diabetes control improved [average A1C] from 9.9% to 7.1%.

（2） Insulin analogue

Pharmacokinetics overview of currently available single insulin products

Insulin analogues are insulin analogues manufactured by recombinant DNA technology aimed at producing more physiological insulin patterns, especially for type 1 diabetes, and can reduce the risk of hypoglycemia. Fast acting insulin analogues (insulin lispro, insulin lispro aabc, insulin aspart, ultra fast acting insulin aspart and insulin glutamine lysine) have faster onset and shorter efficacy when used as pre prandial insulin; The efficacy of long-acting insulin analogues as basic insulin is more lasting, stable and predictable than that of NPH.

(1) Fast acting insulin analogue

● The insulin aspart is basically the same as that of ordinary people, except that the proline at B28 position is replaced by aspartate. This replacement reduces the formation of hexamer, so it absorbs faster, takes effect more quickly, and has a shorter effect.

● Lypro insulin is basically the same as ordinary human insulin, except that B28 and B29 are lysine and proline respectively. The other insulin lispro preparation (insulin lispro aabc) added troprostanil (a prostacyclin analog) and citric acid, so the initial absorption was slightly faster than insulin lispro, and the postprandial blood glucose control of administration at meal was slightly better.

● Glutamine B3 and B29 are lysine and glutamic acid respectively.

In terms of short-term outcomes, such as A1C and the risk of hypoglycemia, quick acting insulin may have a slight advantage over short acting (ordinary) insulin in controlling blood sugar in patients with type 1 diabetes, but not necessarily in patients with type 2 diabetes. Although quick acting insulin is more expensive than ordinary insulin, the timing of pre meal administration is more convenient for patients to master and some studies report that hypoglycemia is reduced, which has obvious advantages for patients. Individuals using the hybrid artificial pancreas system should use fast acting insulin analogues, because the algorithms that drive the automatic delivery of insulin are based on their pharmacokinetics.

Concentrated rapid acting insulin analogues U-200 lispro insulin and U-200 lispro insulin aabc, with specifications of 200U/mL instead of 100U/mL in U-100 preparations. This type of preparation is suitable for patients who need large doses of insulin during meals.

(2) Basal insulin analogue

● Insulin glargine – Insulin glargine is basically the same as human insulin, except that aspartate at A21 position of insulin molecule is replaced by glycine, and two arginine molecules are added at the amino end of B chain. After subcutaneous injection, insulin glargine precipitates in the tissue to form hexamer, thus delaying absorption and prolonging the efficacy. Insulin glargine is prepared with acid solution and cannot be mixed with quick acting insulin, which will change the pharmacokinetics of both. U-100 insulin glargine has no obvious peak effect, but the hypoglycemic effect in the first 12 hours is indeed greater than that in the following 12 hours.

The specification of the more concentrated insulin glargine preparation (insulin glargine U-300) is 300U/mL instead of 100U/mL. The efficacy of insulin glargine U-300 is more lasting and stable than that of insulin glargine U-100, lasting for more than 24 hours. It can be used for type 1 diabetes and type 2 diabetes, as well as patients with and without insulin resistance.

● Dieter insulin – Dieter insulin is an acylated insulin. Its fatty acid side chain can reversibly bind to albumin, or it can self bind in a concentration dependent manner (that is, form a dimer), thus prolonging the efficacy. The greater the dose, the longer the effect. Its potency is much weaker than that of human insulin, so it is prepared in a 4:1 molar ratio (that is, 1 unit of insulin detemir contains 4 times the molecular number of 1 unit of other insulin). Compared with insulin glargine, insulin detemir does have an obvious peak, but its efficacy rarely lasts for 24 hours.

● Degu insulin – Degu insulin is almost identical to human insulin, except that threonine at B30 is removed, and a 16 carbon fatty acid is connected to lysine at B29 through a glutamate linker, which is conducive to self binding and albumin binding. Degu insulin forms a soluble polyhexamer at the injection site, then slowly decomposes into monomer and is absorbed. This feature prolongs its action time (>40 hours), and reduces the fluctuation of plasma drug concentration with once a day administration scheme. Stable insulin concentration can be reached in 3-5 days. Different from insulin glargine and insulin detemir, the mixture of insulin degu and fast acting insulin will not significantly change the pharmacokinetics of both.

（3） Basic insulin and meal insulin

Basic insulin can inhibit the production of glucose by the liver, maintain the fasting blood glucose at a level close to normal with appropriate dosage, and pre meal insulin can meet the additional demand after food absorption, thus reducing the rise of postprandial blood glucose. After the diagnosis of type 1 diabetes mellitus, basic meal insulin therapy should be started as soon as possible to provide physiological insulin supplementation. Patients with type 2 diabetes who need insulin usually start with basic insulin treatment (except for oral drugs), but with the reduction of insulin secretion, they may need insulin at meals.

● Basic insulin – The basic insulin is generally medium or long-term insulin preparations, including NPH, NPL, insulin dete, insulin glargine or insulin degu, which are injected 1-2 times a day. The peak effect of medium effect insulin (NPH) can also partially cover breakfast and lunch, but it is not in line with the physiological model as quick acting insulin supplementation at meals.

● Meal insulin - Short acting (ordinary) or quick acting insulin (insulin lispro, insulin lispro aabc, insulin aspart, insulin aspart or insulin glutamine) is usually used as pre meal insulin to control the rise of blood sugar after food absorption. The approximate onset time, peak time and action duration of commonly used insulin are shown in the attached table.

（4） Premixed insulin

We almost never recommend the use of commercially available premixed insulin of fixed proportion for the treatment of type 1 diabetes. The strengthening program requires frequent adjustment of short-term or quick acting insulin before meals. The patients with type 1 diabetes who are unwilling or unable to follow the intensive regimen may be suitable for premixed insulin lispro/NPH.

Some patients with type 2 diabetes can use premixed preparations with appropriate effects, and premixed human insulin is generally cheaper than insulin analogues. However, if you really want to adjust the dosage of fast acting insulin before meals, it is better for patients to adjust the dosage of insulin before meals alone, rather than using a fixed proportion. However, some type 2 diabetic patients who need pre meal insulin in addition to basic insulin will prefer premixed insulin because of its convenient use and lower cost. When premixed NPH and ordinary insulin are used, the peak effect is also directly related to the proportion of ordinary insulin in premixed preparations.

（5） Inhaled insulin

In 2014, the US FDA approved another ordinary insulin dry powder preparation for oral inhalation. Some studies have shown that oral inhaled insulin causes a rapid increase in serum insulin concentration, which is faster than subcutaneous injection of ordinary insulin. However, its absorption efficiency is low, and a larger dose must be given to achieve therapeutic effect. The dose of inhaled insulin can only be adjusted by 4U, which is prohibited for patients with chronic lung disease.

（6） Basic insulin GLP-1RA injection

In October 2021, basic insulin glucagon like peptide-1 receptor agonist (GLP-1RA) injection Degu Insulin and Lilalutide Injection Approved by the National Drug Administration (NMPA) for marketing, it is used for adult patients with type 2 diabetes who have poor blood glucose control. It is combined with other oral hypoglycemic drugs on the basis of diet and exercise to improve blood glucose control^[7] 。

Degu insulin and lilalutide injection has better hypoglycemic effect than basic insulin at the same or lower dosage of insulin, and can reduce the risk of hypoglycemia and avoid adverse reactions such as weight gain caused by insulin treatment^[8] 。

Insulin administration

Subcutaneous insulin is administered by insulin pump or injection.

Insulin for injection includes prepackaged disposable insulin pen, reusable insulin pen containing disposable insulin ink cartridge or bottled preparations. The insulin pen is more convenient to use than the insulin bottle with syringe. When injecting small doses of ordinary insulin (<5U), the error of using a pen syringe to set the amount of 0 is almost 50% less than that of a traditional insulin syringe. Insulin bottles with syringes are cheaper than insulin pens. At present, there are three types of insulin injectors: 0.3mL, 0.5mL ("low dose") and 1mL. If the insulin dose does not exceed 30U, a 0.3mL syringe can be selected; 1mL syringe can be used for insulin administration up to 100U.

When using insulin pen or insulin bottle with syringe, it is recommended to use the shortest needle (such as 4mm or 5mm needle for injection pen) to avoid injection into muscle and minimize discomfort and tissue damage.

The high-pressure jet needle free syringe relies on a compression spring or a compressed gas cylinder to generate pressure, and can inject insulin under the skin without a needle. Although the device reduces the subcutaneous storage, makes the blood glucose concentration drop faster and the insulin action time shorter, it cannot improve the fluctuation of insulin absorption.

Determinants of insulin effect

Injection site of insulin

(1) Injection site: insulin injection site includes abdominal wall, thigh, arm or hip. The absorption rate of human insulin is the fastest in the abdominal wall, the slowest in the thigh and buttocks, and the arm is in the middle. These differences have clinical value

● Ordinary insulin administered before meals should be absorbed quickly, so abdominal wall injection may be preferred.

● If quick acting insulin is injected into a limb for exercise, its absorption will increase due to the increased blood flow of the active limb.

● The medium effect insulin administered before dinner should be slowly absorbed to ensure the efficacy lasts all night, so it is suitable for thigh or hip injection.

The injection site seems to have no significant effect on the absorption of long-acting basic insulin analogues insulin glargine and insulin degu.

(2) Injection method: The injection site should be clean without infection, inflammation, skin ulceration, fibrosis or fat hyperplasia. The depth of injection will affect the speed of insulin absorption. Too shallow injection may lead to intracutaneous injection, which is not only painful but also poorly absorbed. However, vertical injection at the location with less fat may cause intramuscular injection and accelerate absorption. For very thin adults, children, or anyone whose needle length is equal to or more than the distance to reach the muscle, the needle should be inserted vertically into the pinched skin fold. For many patients who use short needles (4mm), the risk of intramuscular injection is very low, and the needle can be inserted vertically without pinching the subcutaneous fat with two fingers. If a 6mm needle is used, the needle can be inserted at an angle of 45 ° without pinching the skin. Whether using insulin pen or syringe, the injection site should be rotated to avoid fat hyperplasia.

The insulin pen needs to be preassembled according to the manufacturer's instructions, and can only be injected after the needle is completely inserted (press down the piston). After injection, stay for 10 seconds before pulling out the needle to ensure complete injection and prevent leakage. When using a syringe, you should first inhale air into the syringe. The air volume should be equal to or slightly higher than the target dose. Air should be injected into the insulin bottle first, and then insulin should be extracted. Carefully remove any bubbles from the syringe before injection.

It is usually not necessary to clean the skin with an alcohol swab before injection. If alcohol is used for wiping, make sure the skin is completely dry before injecting.

(3) Subcutaneous storage: insulin injected subcutaneously will be stored subcutaneously. With the increase of subcutaneous storage, the fluctuation of absorption increased and the net absorption decreased. This may have an important impact on patients who need multiple high-dose injections every day due to insulin resistance. One of the reasons why continuous subcutaneous insulin infusion can control blood sugar smoothly is that it only uses ordinary or quick acting insulin (the latter is better), and the subcutaneous storage volume is very small, because the drugs are stored in syringes or other containers outside the body. In addition, the syringe injection has inherent variability, including the location, angle, depth and lower blood flow of each injection are different, When continuous subcutaneous infusion is performed, each catheter is fixed at the same position, which can reduce this variability.

(4) Change of subcutaneous blood flow: the degree of insulin absorption also depends on the speed of subcutaneous blood flow. Therefore, smoking can reduce insulin absorption, while exercise, sauna, hot bath, local massage and other activities that cause skin temperature rise can increase insulin absorption. The above fluctuations of ordinary and quick acting insulin are more obvious than those of long-acting insulin.

Storage method

Insulin must be stored in a refrigerator below 10 ℃, and its activity can remain unchanged for 2-3 years in a refrigerator at 2 ℃~8 ℃, even if insulin has been partially aspirated for use. When in use, the temperature can be no more than 30 ℃ or less than 2 ℃, but the sun must be avoided to prevent failure.

Insulin in use should be kept indoors in a cool place. Bottled insulin in use can be stored in the refrigerator for about 3 months. The insulin refill in use should not be returned to the refrigerator with the insulin pen, and can be kept for 4 weeks with you.

If turbid insulin is shaken for a few hours or not properly stored, it may form lumps. At this time, insulin should be discarded.

1. Insulin avoids high temperature and direct sunlight.

2. Insulin should be stored in a refrigerator at 2-8 ℃, and unopened insulin should be used before the shelf life.

3. The shelf life of opened insulin in the refrigerator is generally 1 month, indicating the opening time.

4. Remember not to put insulin on the freezing layer of the refrigerator. Iced insulin cannot be used, but can only be placed in the refrigeration room.

5. Before injection, take out insulin from the refrigerator and place it at room temperature for 20 minutes before injection.

6. The injection pen with insulin refill should not be stored in the refrigerator, but in a cool place.

7. When traveling by air, you should take insulin with you and not put it in checked baggage.

Medical examination

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Inspection name

insulin

classification

Hormone determination → pancreatic endocrine function test

Measurement principle

RIA method: non-equilibrium analysis method is adopted. First, the insulin anti antibody is fully reacted with the unlabeled insulin (i.e. the tested sample or standard) to form insulin and insulin anti antibody complex. The remaining unconjugated insulin anti antibody reacts with I-insulin to form I-insulin and insulin anti antibody complex. Separate the free and binding parts with the second antibody, and measure the radioactivity of the precipitation part (B). According to the insulin standard curve, the insulin concentration in the serum sample can be found.

reagent

(1) I - insulin: add 10ml distilled water to each bottle when using.

(2) Insulin standard: a total of 7 bottles of lyophilized powder, each bottle is fully dissolved with 1ml of distilled water, and the concentrations are 0, 5, 10, 20, 40, 80 and 160mIU/L.

(3) Anti insulin and anti anti antibody: dissolve with 10ml distilled water and mix well for use.

Operation method

Follow Table 1.

Fully mix, stand at 4 ℃ for 20min, centrifuge 2000g for 20min, discard the supernatant, and measure the precipitation radioactivity.

Normal value

5～20μU/ml。

Clinical significance of test results

(1) Most patients with type 1 diabetes mellitus are below 5 μ U/ml, and the plasma insulin level of type 2 diabetes mellitus patients can be normal, low or higher than normal. The patients with obvious increase showed hyperinsulinemia, indicating insulin resistance. The plasma insulin concentration of islets was measured at the same time as OGTT to understand the function of islet β cells in order to differentiate type 1 diabetes from type 2 diabetes. The insulin levels of type 1 diabetes mellitus patients were lower in fasting and after glucose stimulation, showing a low flat curve; Patients with type 2 diabetes mellitus may have delayed or increased peak insulin secretion, decreased or absent the first phase of insulin secretion, and insulin secretion is low compared with the blood glucose value at the same phase. Serum C-peptide can also be determined at the same time for identification.

(2) The decrease of plasma insulin in islets can also be seen in secondary diabetes caused by pheochromocytoma, somatostatin tumor, aldosteronism, primary hypoparathyroidism, and hypoglycemia caused by islet B-cell tumor, extrapancreatic tumor, adrenal hypofunction, and pituitary hypofunction.

(3) Most patients with syndrome X have obesity, hyperlipidemia, hypertension and hyperinsulinemia at the same time.

note appended

Most of the insulin secreted from pancreatic islet β cells is inactivated in the liver and kidney, of which about 40%~50% enters the liver through the portal vein to inactivate, so the liver and kidney function, especially the liver function, is an important factor affecting the insulin content in circulating blood; Diabetic patients often produce insulin anti antibody after using insulin, especially animal insulin. Because insulin and insulin anti antibody can produce a high immune response, it can affect the determination of plasma insulin in islets. In addition, the content of proinsulin and proinsulin in blood, endocrine system diseases such as anterior pituitary, adrenal cortex and hyperthyroidism, thiazide diuretics, glucocorticoids and other drugs, as well as stress states such as infection, fever and surgery are common factors affecting insulin determination.

Related diseases

Type 1 diabetes, diabetes, type 2 diabetes, pheochromocytoma, hypoparathyroidism, hypoglycemia, syndrome X, hyperlipidemia^[1]

Pharmacopoeia information

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Source content

This strain is a protein composed of 51 amino acid residues extracted from pig pancreas. According to the calculation of dried products, the insulin content (including deaminating insulin) should be 95.5%~105.0%.

Each unit is equivalent to 0.0345 mg insulin.

Preparation requirements

This product should be extracted from frozen pancreas of quarantine qualified pigs. The production process shall meet the requirements of the current version of Good Manufacturing Practice for Drugs. This product is of animal origin, and virus safety control should be carried out in the process.

character

This product is white or almost white crystalline powder.

This product is almost insoluble in water and ethanol, but soluble in inorganic acid or sodium hydroxide solution.

identify

1. In the chromatogram recorded under the content determination item, the retention time of the main peak of the test solution should be consistent with the retention time of the main peak of the reference solution.

2. Conduct the test according to the method of high performance liquid chromatography (general rule 0512).

Test solution: Take a proper amount of this product, add 0.1% trifluoroacetic acid solution to dissolve and dilute it to make a solution containing 10mg per 1mL, take 20 µ L, add 0.2mol/L trihydroxymethylaminomethane hydrochloric acid buffer solution (pH7.3) 20 µ L, 0.1% V8 enzyme solution 20 µ L and water 140 µ L, mix well, put it in a 37 ℃ water bath for 2 hours, and then add 3 µ L phosphoric acid.

Reference solution: Take an appropriate amount of insulin reference, add 0.1% trifluoroacetic acid solution to dissolve and dilute it to make a solution containing 10mg per 1mL, take 20 μ L, add 0.2mol/L trihydroxymethylaminomethane hydrochloric acid buffer solution (pH7.3) 20 µ L, 0.1% V8 enzyme solution 20 µ L and water 140 µ L, mix well, put it in a 37 ℃ water bath for 2 hours, add 3 μ L phosphoric acid.

Chromatographic conditions: octadecyl silane bonded silica gel is used as filler (5~10 µ m), 0.2mol/L sulfate buffer (28.4g anhydrous sodium sulfate is taken, dissolved with water, 2.7 mL phosphoric acid is added, the pH value is adjusted to 2.3 with ethanolamine, and 1000 mL water is added) - acetonitrile (90:10) is used as mobile phase A, and acetonitrile water (50:50) is used as mobile phase B. Gradient elution is carried out as per the following table, and the column temperature is 40 ℃, The detection wavelength is 214nm, and the injection volume is 25 µ L.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
zero	ninety	ten
sixty	fifty-five	forty-five
seventy	fifty-five	forty-five

Determination method: Precisely measure the test solution and the reference solution, respectively inject them into the liquid chromatograph, and record the chromatogram.

Result judgment: The peptide map of the test solution should be consistent with that of the control solution.

inspect

Related proteins

Determine according to the method of high performance liquid chromatography (general rule 0512), use the new preparation temporarily, and store it below 10 ℃.

Test solution: take a proper amount of this product, add 0.01mol/L hydrochloric acid solution to dissolve and dilute it to make a solution containing about 3.5mg per 1mL.

System suitability solution: see the content determination item.

Chromatographic conditions: octadecyl silane bonded silica gel is used as filler (5~10 µ m), 0.2mol/L sulfate buffer (28.4g anhydrous sodium sulfate is taken, dissolved with water, 2.7 mL phosphoric acid is added, the pH value is adjusted to 2.3 with ethanolamine, and 1000 mL water is added) - acetonitrile (82:18) is used as mobile phase A, and acetonitrile water (50:50) is used as mobile phase B. Gradient elution is performed as per the following table, and the column temperature is 40 ℃, The detection wavelength is 214 nm, and the injection volume is 20 μ L.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
zero	seventy-eight	twenty-two
thirty-six	seventy-eight	twenty-two
sixty-one	thirty-three	sixty-seven
sixty-seven	thirty-three	sixty-seven

System applicability requirements: Adjust the flow comparison to make the retention time of insulin peak about 25 minutes. See the content determination item for other requirements.

Determination method: precisely measure the test solution, inject it into the liquid chromatograph, and record the chromatogram.

Limit: calculated by peak area normalization method, A twenty-one Deamination insulin shall not be greater than 5.0%, and the sum of other related proteins shall not be greater than 5.0%.

High molecular protein

Determine according to molecular exclusion chromatography (general rule 0514).

Test solution: take a proper amount of this product, add 0.01mol/L hydrochloric acid solution to dissolve and dilute it to make a solution containing about 4mg per 1mL.

System suitability solution: take insulin monomer dimer reference substance (or take an appropriate amount of insulin and place it at 60 ℃ overnight), add 0.01mol/L hydrochloric acid solution to dissolve and dilute it to make a solution containing about 4mg per 1mL.

Chromatographic conditions: hydrophilic modified silica gel is used as filler (3~10 µ m), glacial acetic acid acetonitrile 0.1% arginine solution (15:20:65) is used as mobile phase, the flow rate is 0.5mL per minute, the detection wavelength is 276nm, and the injection volume is 100 µ L.

System suitability requirements: in the system suitability solution chromatogram, the separation of insulin monomer peak and dimer peak should meet the requirements.

Determination method: precisely measure the test solution, inject it into the liquid chromatograph, and record the chromatogram.

Limit: except for other peak areas whose retention time is greater than insulin peak, the sum of all peak areas whose retention time is less than insulin peak shall not be greater than 1.0% according to peak area normalization method.

Loss on drying

Take about 0.20g of this product, weigh it accurately, dry it at 105 ℃ to constant weight, and the weight loss shall not exceed 10.0% (general rule 0831).

zinc

Determine according to atomic absorption spectrophotometry (general rule 0406 first method).

Test solution: Take an appropriate amount of this product, weigh it accurately, add 0.01mol/L hydrochloric acid solution to dissolve it and dilute it quantitatively to make a solution containing about 0.1mg per 1mL.

Reference solution: Precisely measure an appropriate amount of zinc single element standard solution (1000 µ g per 1mL), and quantitatively dilute it with 0.01mol/L hydrochloric acid solution to prepare a solution containing 0.20 µ g, 0.40 µ g, 0.60 µ g, 0.80 µ g, 1.0 µ g and 1.2 µ g of zinc per 1mL.

Determination method: precisely measure the reference solution and test solution, measure the absorbance at the wavelength of 213.9nm, and calculate according to the standard curve method.

Limit: calculated as per dry product, the zinc content shall not exceed 1.0%.

Bacterial endotoxin

Take this product, add 0.01mol/L hydrochloric acid solution to dissolve and dilute it to make a solution containing 5mg per 1mL, check according to the law (general rule 1143), and the amount of endotoxin in every 1mg of insulin should be less than 10EU.

microbial limit

Take 0.3g of this product and check it according to the microbial count method of microbial limit test for non sterile products (general rule 1105). The total number of aerobic bacteria in 1g of test sample shall not exceed 300 cfu.

biological activity

Take an appropriate amount of this product and test it according to the insulin bioassay (general rule 1211). During the experiment, the number of experimental animals in each group can be halved. The experiment adopts a random design. The potency is calculated according to the random design method of measuring the parallel line of the volume reaction in the bioassay statistical method (general rule 1431). The potency per 1mg should not be less than 15 units.

Assay

Determine according to the method of high performance liquid chromatography (general rule 0512), use newly prepared temporarily, or store at 2~4 ℃, and use within 48 hours.

Test solution

Take an appropriate amount of this product, weigh it precisely, add 0.01mol/L hydrochloric acid solution to dissolve it and dilute it quantitatively to prepare a solution containing about 40 units per 1mL.

Reference solution

Take an appropriate amount of insulin control, weigh it precisely, add 0.01mol/L hydrochloric acid solution to dissolve it and dilute it quantitatively to prepare a solution containing about 40 units per 1mL.

System suitability solution

Take insulin control sample, add 0.01mol/L hydrochloric acid solution to dissolve and dilute it into a solution containing about 40 units per 1mL, and place it at room temperature for at least 24 hours.

Chromatographic conditions

Use octadecyl silane bonded silica gel as filler (5~10 µ m), 0.2mol/L sulfate buffer (28.4g anhydrous sodium sulfate, dissolved with water, added 2.7mL phosphoric acid, adjusted pH value to 2.3 with ethanolamine, and added water to 1000mL) - acetonitrile (74:26) as mobile phase, column temperature is 40 ℃, detection wavelength is 214nm, and injection volume is 20 µ L.

System suitability requirements

In the system suitability solution chromatogram, the insulin peak and A twenty-one The resolution between deaminating insulin peaks (relative retention time to insulin peak is about 1.2) should not be less than 1.8, and the tailing factor should not be more than 1.8.

Assay

Accurately measure the test solution and the reference solution, respectively inject them into the liquid chromatograph, and record the chromatogram. According to the external standard method, compare the insulin peak area with A twenty-one Calculate the sum of peak areas of deaminating insulin.

Storage

Shaded, sealed, stored below - 15 ℃.

preparation

1. Insulin injection.

2. Spermin zinc insulin injection.

3. Spermin zinc insulin injection (30R).^[5]

Drug description

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pharmacological action

Promote glucose in blood circulation to enter hepatocytes, muscle cells, fat cells and other tissue cells to synthesize glycogen, reduce blood sugar, and promote the synthesis of fat and protein.

indication

Mainly used for diabetes, especially insulin dependent diabetes:

1. Heavy, emaciated and malnourished;

2. Mild and medium-sized patients who failed to respond to the treatment of diet and oral hypoglycemic drugs;

3. Patients with severe metabolic disorder (such as ketoacidosis, hypertonic coma or lactic acidosis), severe infection, consumptive diseases (such as pulmonary tuberculosis and cirrhosis), progressive retinal, renal, neurological and other diseases, as well as acute myocardial infarction and cerebrovascular accident;

4. Pregnancy, delivery and major surgery are combined. It can also be used to correct intracellular potassium deficiency.

Adverse reactions

1. Allergic reaction: most of them are caused by the use of bovine insulin, which can produce corresponding antibodies such as IgE and cause allergic reaction after entering the human body as an alloprotein. The general reaction is mild and transient, occasionally causing anaphylactic shock. It can be replaced by pig insulin because it is close to human insulin.

2. Hypoglycemia: It is caused by excessive insulin. Regular insulin can rapidly reduce blood sugar, and hunger, sweating, rapid heartbeat, anxiety, tremor and other symptoms occur. In severe cases, coma, convulsion, shock, and even brain injury and death can be caused. Long acting insulin has a slow hypoglycemic effect and does not show the above symptoms, while headache, mental emotion and motor disorders are the main manifestations. In order to prevent the serious consequences of hypoglycemia, patients should be taught to be familiar with the reaction, so that they can find and take food as soon as possible, or drink sugar water. In serious cases, 50% glucose should be injected intravenously immediately. It is necessary to differentiate hypoglycemic coma from ketoacidosis coma and non ketotic diabetic coma in diabetes.

3. Insulin tolerance: acute tolerance is often caused by stress states such as infection, trauma, surgery, and emotional excitement. At this time, the amount of anti insulin substances in the blood increases, or when ketoacidosis occurs, the existence of a large number of free fatty acids and ketone bodies in the blood hinders the uptake and utilization of glucose. When acute tolerance occurs, it is necessary to increase the insulin dose to thousands of units in a short time. The causes of chronic tolerance are complex (refer to those who need more than 200U insulin per day and have no complications). It may be that anti insulin receptor antibody (AIRA) has been produced in the body, for which immunosuppressants can be used to control symptoms, so that the sensitivity of patients to insulin can return to normal; It may also be the change in the number of insulin receptors, for example, when hyperinsulinemia occurs, the number of insulin receptors on the target cell membrane decreases; It may also be that the glucose transport system on the target cell membrane is abnormal. At this time, it is often effective to switch to other animal insulin or use high-purity insulin, and adjust the dosage appropriately.

Dosage and usage

Generally, it is injected subcutaneously, 3-4 times a day. The dosage is the most before breakfast. Next before lunch, next before dinner, and least before supper. Sometimes intramuscular injection. Intravenous injection is only used in emergency (such as diabetic coma). Because the insulin requirement of patients is affected by such factors as dietary calorie and composition, severity and stability of disease, body size, physical activity intensity, number and affinity of insulin antibody and receptor, the dosage should be individualized. The dosage can be determined according to the amount of sugar in the patient's urine. Generally, one unit of sugar should be injected for every 2-4g sugar in the urine for 24 hours. For medium-sized diabetic patients, the daily requirement is about 5~40 units, which should be injected 30 minutes before each meal (to avoid hypoglycemia after administration). The dosage of heavy patients is more than 40 units. For diabetic coma, the dosage is about 100 units, which is injected intravenously with glucose (50-100g). In addition, a small amount (5~10 units) can also be used for malnutrition, emaciation, intractable pregnancy vomiting, and early cirrhosis (at the same time, glucose injection).

matters needing attention

1. Insulin excess can cause hypoglycemia. Its symptoms depend on the degree and speed of blood sugar reduction, and may include hunger, mental uneasiness, rapid pulse, dilated pupils, anxiety, dizziness, ataxia, tremor, coma, and even convulsion. The edible sugar must be given in time. When hypoglycemic shock occurs, 50 mL of 50% glucose solution is injected intravenously. If necessary, drip 5% glucose solution again. Attention must be paid to distinguish hypoglycemic coma from severe ketosis. Sometimes, rebound hyperglycemia may occur after hypoglycemia, that is, Somogyi reaction. If the urine sugar before sleep is negative, but the urine sugar in the next morning is strongly positive, refer to the insulin dosage. It should be thought that there may be hypoglycemia at night. At this time, try to reduce the insulin dosage, and do not increase the insulin dosage.

2. In order to prevent the blood sugar from suddenly dropping and losing consciousness without calling for help, each patient should be given a card with his condition and insulin use, so as to lose no time in rescue and treatment.

3. The injection site may have local reactions such as skin redness, subcutaneous nodules and subcutaneous fat atrophy. Therefore, the injection site needs to be replaced frequently.

4. A few may have urticaria, and occasionally anaphylactic shock (epinephrine can be used for rescue).

5. Very few patients can develop insulin tolerance: that is, in the absence of ketoacidosis, the daily insulin consumption is more than 200 units. The main reasons may be infection, use of corticosteroids or the presence of insulin antibodies in the body, which can bind to insulin. At this time, preparations of different animal species can be replaced or oral hypoglycemic drugs can be added.

6. Do not use it for patients with hypoglycemia, cirrhosis, hemolytic jaundice, pancreatitis, nephritis, etc.

7. The injection mostly contains preservatives, which are generally not suitable for intravenous injection. For intravenous injection, ampoule insulin preparation should be used.

Drug interaction

1. Drugs to enhance its effect: oral anticoagulants, salicylates, sulfonamides, methotrexate, etc. can compete with insulin for plasma protein, so that free insulin in the blood increases; Oral hypoglycemic drugs have synergistic effect with insulin; Protein assimilation hormone can reduce the sugar content of glucose and enhance the effect of insulin; Ethanol, chloramphenicol, etc. are said to enhance the effect of insulin.

2. Drugs that can antagonize its effects: adrenocortical hormone, thyroxine, growth hormone, etc. can increase blood sugar, and when used together, they can antagonize the hypoglycemic effect of insulin. Thiazines diuretics, oral contraceptives, and nicotinic acid derivatives are also said to reduce the hypoglycemic effect of insulin, Beta receptor blocker can block the hyperglycemic response of adrenaline and interfere with the function of regulating blood sugar of the body. When used with insulin, attention should be paid to adjusting the dosage, otherwise it is easy to cause hypoglycemia.

poisoning

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Insulin is a kind of polypeptide hormone extracted from the pancreas of livestock. According to its production source, it can be divided into four categories: animal insulin, semi synthetic adult insulin, and human insulin and insulin analogues synthesized by DNA recombination. According to the characteristics of action time, it can be divided into five categories: ultra short effect, short effect, medium effect, long effect, and ultra long effect. In addition, there are premixed insulin with ultra short effect and medium effect. It is mainly used to treat diabetes, especially insulin dependent diabetes, and also used in cases of severe metabolic disorder, pregnancy, childbirth and major surgery.

Insulin is a hormone secreted by islet β cells, which can accelerate the anaerobic fermentation and aerobic oxidation of glucose, promote glycogen synthesis, inhibit glycogen decomposition and gluconeogenesis, increase the utilization and decrease the production of glucose, and thus reduce blood sugar. Clinically, in the following cases, the injection dose is too large or the diet is disordered; The low purity insulin was changed into high purity insulin without proper reduction; Complicated with severe liver and kidney diseases, hypophysis and hypothyroidism; Combined use of anticoagulants, salicylates, sulfonamides, methotrexate and other drugs. About one month after the human body has received insulin injections for many times, insulin antibodies may appear in its circulation, which may cause local or systemic allergic reactions. If insulin is injected repeatedly at the same site for a long time, local adipose tissue may shrink or become hypertrophic. Insulin can also cause increased sodium reabsorption and retention of water and sodium, leading to edema. During the treatment of diabetes, when the blood sugar drops rapidly, the osmotic pressure in the lens and vitreous body is affected, so that the water in the crystalline body overflows and the refractive index drops, and hyperopia occurs.

clinical manifestation

1. Hypoglycemic reaction

Hyperexcitatory symptoms of sympathetic nervous system may occur, such as intense hunger, excessive sweating, trembling, palpitations, anxiety, weakness, pale face, etc; In severe cases, neurological hypoglycemia symptoms may occur. When the cerebral cortex is involved, it may be manifested as inattention, dizziness, drowsiness, dullness, visual impairment, unsteady gait, hallucination, restlessness, strange behavior and other mental disorders. When the subcortical center, midbrain, medulla oblongata and other parts are involved, it may appear confusion, dance like movements, and even tension and clonic spasms, The pyramidal sign is positive, even coma and blood pressure drop; If the disease is serious and lasts for a long time, the brain cells will be irreversibly damaged, showing some characteristics of cerebral cortex removal, and may leave behind personality changes, dementia and other symptoms. Elderly people, especially those with a long history of diabetes or insulin injection, often lack typical hypoglycemia symptoms, and once hypoglycemia occurs, it will be shown as coma. The manifestations of hypoglycemia in neonates and infants are mainly convulsions.

2. Anaphylactic reaction

Rarely seen, redness, swelling, burning, itching, etc. may occur locally, and urticaria, nausea, vomiting, diarrhea, angioneurotic edema, asthma, and occasionally anaphylactic shock may occur as systemic reactions.

3. Edema

Mild edema may occur at the initial stage of treatment, and a few may be systemic edema.

4. Skin

The skin at the injection site is red, with subcutaneous nodules and atrophy or hypertrophy of subcutaneous fat.

5. Visual impairment

Some patients have refractive errors, which may lead to blurred vision.

laboratory examination

Blood test can prove the poison. When hypoglycemia occurs, blood sugar is lower than 2.8 mmol/L.

diagnosis

The key points for diagnosis of insulin poisoning are:

1. The patient has a history of insulin injection.

2. Clinical manifestations

(1) Hypoglycemic reaction: sympathetic hyperexcitability may occur, and neurological hypoglycemia may occur in severe cases. The blood sugar was lower than 2.8 mmol/L during the attack.

(2) Allergic reaction: local reactions such as redness and swelling, and/or systemic reactions such as urticaria, angioneurotic edema, asthma, and occasionally anaphylactic shock.

(3) Edema: mild and/or systemic edema.

(4) There are subcutaneous nodules and atrophy or hypertrophy of subcutaneous fat on the skin of the injection site.

(5) Some patients have blurred vision.

3. Poisons were detected in the blood by drug analysis.

treatment

The key points for the treatment of insulin poisoning are:

1. Treatment of hypoglycemia

Light people can be relieved by eating some food such as candy and syrup. In severe cases, 60~100mL of 50% glucose injection should be injected intravenously, and then 5%~10% glucose injection should be given continuously. If the blood sugar cannot reach the standard or the patient is delirious, 0.5~1mg glucagon can be injected subcutaneously. For those who are delirious, most of them have brain edema, and mannitol and glucocorticoid can be injected intravenously. Those who inject long-acting insulin must be closely observed for several days to prevent repeated hypoglycemia.

2. Treatment of anaphylaxis

For local reactions, the vast majority do not need special treatment and can naturally subside. If it persists, you can change the type of insulin preparation, use antihistamines or glucocorticoids, and desensitization therapy. Patients with severe allergic reactions must stop or temporarily stop insulin treatment.

3. Treatment of insulin edema

The mild cases can subside naturally, and the severe cases or those with congestive heart failure must be treated symptomatically such as diuresis.

4. Local fat atrophy or hypertrophy

It is caused by insulin injection at the same site for a long time. After stopping injection at the injection site, it can recover slowly and naturally. In order to prevent it from happening, the injection site should be changed frequently. Most of the refractive errors are temporary changes, which usually recover naturally within a few weeks.

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