Dimer (natural) Chains A and B (monomer) 11981 MW 110 a.a.  4869 b.p. Rasmol file (R click to save) Kinemage file (R click to save) Wakayama: VAL3LEU Medline Abstract

           Overview

• Role of insulin in the body
• Structure of human insulin (DNA, protein chain)
• Binding & Wakayama Mutation
• Long Term Effects - Diabetes
• Studies
• References

        Insulin is responsible for the regulation of glucose in the body.  Insulin is secreted by the pancreas and binds to the alpha receptor on the outside of the membrane receptor protein.  This causes the two beta subunits that are connected to the alpha receptors and extend into the membrane to be autophophorylated, causing a chain reaction of phosphorylation of other enzymes in the cellular bed to direct intracellular metabolic machinery to produce the desired effects.  This allows rapid entry of glucose and even amino acids and ions into the cell membrane for usual carbohydrate functions. Insulin also prevents further breakdown of glycogen to more glucose in the liver cells, and also an increased uptake of glucose and prodcution of glycogen into the liver cells; a full regulation of glucose in the body.

Structure of the Human Insulin

        Knowing the structure of insulin is important in understanding how a mutation can cause a major defect in its function and thereby lead to disease such as diabetes.
        Insulin is a protein and its production is coded by a unique sequence of DNA.The gene for insulin contains 4869 base pairs.  After the formation of  mature mRNA and then protein synthesis, an amino acid sequence for preproinsulin
is produced (110 AA).
        Preproinsulin is an inactive precursor of insulin.  It is composed of four segments:
                1.  signal sequence
                2.  B-chain
                3.  connecting peptide
                4.  A-chain
The signal sequence is responsible for "telling the cell" that preproinsulin is being produced and once it is finished, to deposit it outside the cell. The B-chain is one of the active segments of insulin. The connecting peptide has no particular function but to aid in the folding of the tertiary structure of preproinsulin. The A-chain is the second active segment of insulin.
        In the first step of insulin production, preproinsulin is converted to proinsulin via an enzyme peptsidase which cleaves off the no longer needed signal sequence.  Two disulfide bonds are formed between cysteines of the A and B chains.  Peptsidase then converts proinsulin to insulin by cleaving the connecting peptide from the A and B chains.  (This gives a good determination of how much insulin is being produced by measuring quantity of connecting peptide in blood)  An intramolecular disulfide bond is formed within the A-chain causing its a-helix to fold in a "U" shape (Both the A-chain and B-chain form a-helices in their secondary structures). This is insulin.
       There are four active binding sites for insulin to its receptor (All are found on amino acid "tyrosine").  Two on the B-chain and two and the A-chain.   There are four active binding sites for insulin to its receptor (All are found on amino acid "tyrosine").  Two on the B-chain and two and the A-chain.
 

Binding and Insulin Wakayama

Binding of the insulin to the receptor takes place at amino acid 113Y. The intra-disulfide bond (95-100) bends the insulin A chain sequence, so that site 92 lies very close to the binding site. Insulin Wakayama is the point mutation at the 1298th nucleotide position of the insulin DNA, which corresponds to the 92nd amino acid of the preproinsulin chain (3rd amino acid sequence of the A chain). It undergoes a mutation from Valine to Leucine which causes diminished binding of the insulin, although the folding of the insulin remains identical to a regular insulin molecule (mutation from a non-polar side chain to another non-polar side chain). As this mutated insulin goes undetected in the bloodstream, the overall reduced binding causes a decrease in the cell's ability to stimulate glucose uptake and oxidation in vitro. The mutation causes the insulin to retain only about 5% of its normal binding activity. Binding of the insulin and its receptor seems to be an important study in diabetes research.

Long Term Effects - Diabetes
People who do not have diabetes rely on the insulin created by the body. Insulin is a hormone that is used for the purpose of moving glucose from the blood into the body's cells. But people who have diabetes either don't produce insulin or cannot use the insulin they produce. Without insulin, the cells cannot move glucose into the cells. Glucose gathers in the blood, this alone is called hyperglycemia, which is the scientific term for too much glucose in blood. The symptoms of hyperglycemia include incredible thirst, the need to urinate frequently, unclear or blurry vision, nasea and fatigue, and much more.
        There are two different types of diabetes: Type I (insulin-dependent-diabetes) and Type II (non-insulin-dependent-diabetes). Type I diabetes is when the body no longer makes enough insulin.  This type of diabetes is shown to mostly affect children and those in their early
adult ages, and the treatment is to inject insulin every day to keep the levels of insulin safe.  Type II diabetes is different, as the body does
produce the insulin, but it cannot use that which is produced because of mutations.  This form of diabetes seems to become "active" later in one's life, that is, mainly in the middle and higher adult ages.  Type II is treated first with weight loss and exercise (as muscle activity has been
shown to actively increase the absorption of insulin); if this alone doesn't help the patient, then oral antidiabetic tablets are prescribed. According to 1995 estimates, only 800, 000 patients were diagnosed as having Type I diabetes in the United States, as compared to the 7-7.5
million patients with the Type II disease.

Studies

        Insulin Wakayama was initially discovered by K. Nanjo, et al. in 1980 when the investigators were presented with a 56 year old, non-obese Japanese woman with polyuria and weight loss.  Hyperglycemia with glucosuria and ketonuria were also present.  Initially insulin injections worked, but due to allergic reactions to the insulin at the injection site, the propositus was forced to take oral hypoglycemic agents.
        Upon performing blood work, it was discovered that the insulin countrregulatory hormone were within normal ranges, and anti-insulin,
anti-receptor, and islet cell surface antibodies were absent.  It was also noted that she did have chronic lympocytic thyroiditis, but thyroid
functions seemed normal.
       This puzzled the investigators and led them to ask the propositus if an investigation could take place.  The propositus and five of her
first degree relatives agreed to be studied, as shown in Figure A.  In this figure diagonal lines indicate members with fasting hyperinsulinemia
and asterisks indicated diabetes.
        Blood samples were taken from each of the subjects and insulin was extracted.  This insulin was then put through two tests:  High Pressure
Liquid Chromatography (HPLC), and radioimmunoassay.
      The results of the radioimmunoassay are shown in Figure 2.  The Solid Circles show the binding of radioactive iodine to rate adipose
tissue cells in the presence of an semisynthetic human insulin standard. The triangles represent insulin from a normal subject whose insulin was
extracted in the same manner as the subjects.  The Open circles are the subjects insulin.  As can be seen, when compared with the standard, the
subjects insulin only works about 19.8% as well.  This can be proven not to be an artifact of the extraction for the normal insulin follows a
similar path as the semisynthetic type.
        The HPLC analysis showed some interesting results as well as shown in Figure 3.  The first line (line S) shows the choreography of a
mixture of bovine (a), human (b), and porcine (c) insulins.  Lines 1-4 shows the results from the propositus, her brother, her sister, and her
son, respectively.  As one can see, there is a small hump where the normal human insulin is supposed to be, and a large spike near the end of the chart.  This area the end of the chart is extremely hydrophobic.  Roughly this shows that about 7.3% of the insulin of the propositus and her
relatives were similar to human, while 92.7% showed high hydrophobicity. Thus when you estimate that about 92.7% of the insulin only works about 19.8% as well as normal insulin, you in the end have about 25% of the total insulin you need to function, thus producing the effects of
diabetes.
        In conclusion it is worthy to note that upon genetic investigation, which also led to the discovery of the amino acid mutation that causes the disease, it was discovered that this was a dominant trait, unlike other diabetes which is recessive.
 

References/ Related Links

Human insulin production from a novel mini-proinsulin which has high receptor-binding activity PubMed Article
NIDDM Diabetes Info on Diabetes
Diabetes General info on Diabetes Type 2
Insulin Insulin Chain Info