BY: Reddy Sailaja M (MSIWM030)
Insulin is a peptide hormone that plays a critical role in human metabolism. It is synthesized and secreted by beta cells of Islets of Langerhans in the pancreas. It is the first peptide hormone to be discovered (by Frederick Banting and Charles Herbert Best, 1921). It is the first protein to be sequenced in 1951 by Frederick Sanger. Dorothy Hodgkin has determined the crystal structure of insulin in 1969. Nevertheless, it is the first hormone to be synthesized by recombinant DNA technology.
Figure 1: Structure of insulin
STRUCTURE AND FUNCTION
Human insulin is made up two polypeptide chains of 51 amino acids (A-chain- 21 amino acids and B-chain, 30 amino acids) with a molecular mass of 5808 Daltons. Insulin is an anabolic hormone that plays a crucial role in the metabolism of carbohydrates and fats by converting the free glucose available in the blood into glycogen that can be stored in the muscles.
Figure 2: Functions of insulin
Insulin is synthesized as a single polypeptide called ‘preproinsulin’ along with a 24 residue signal peptide in the pancreatic beta cells. The signal peptide guides preproinsulin to endoplasmic reticulum (ER), where the signal peptide gets separated, resulting in ‘proinsulin’ formation. In the ER, the proinsulin is further processed and folded with the formation of three disulphide bonds and gets transported to golgi complex. In golgi, the folded proinsulin is converted to ‘active insulin’ by cellular endopeptidases, namely prohormone convertases 1 & 2 and exoprotease carboxypeptidase E. These endonucleases cleave at two positions in the proinsulin, resulting in the separation of a fragment called C-peptide. The active and mature insulin now consists of two chains: A-chain (21 amino acids) and B-chain (30 amino acids), both liked to each other by two disulphide bonds.
Figure 3: Synthesis of active insulin from precursor
INSULIN MALFUNCTION AND THE ASSOCIATED DISEASES
Insulin helps maintain blood sugar level normal at all the times. When the blood sugar level is high, insulin directs liver to store glucose in the form of glycogen. In need, insulin directs the liver and muscles to release the stored glycogen in the form of glucose to boost energy to the body.
When the insulin production is less or uncontrollable, malfunction of the hormone results in the development of a condition called as diabetes mellitus (DM), where the body is unable to maintain balance between normal blood sugar levels and sysnthesis or breakdown of glycogen. Malfunction of Insulin hormone leads to two major types of diabetes milletus: Type 1 and Type 2.
Type 1 DM: It is an autoimmune disease, where one’s own immune system attacks the pancreatic cells and results in low or no insulin production. Environmental factors, genes and certain viruses trigger the immune system to damage the pancreatic cells.
Type 2 DM: The condition develops either by low insulin production by pancreatic cells or inability of the body to utilize the released insulin for glycogen synthesis. Insulin resistance is the condition developed when the major organs like muscles, body fat and liver starts ignoring the signals form insulin and fail in converting free glucose into glycogen. As more insulin is being produced, pancreatic cells get damaged and the free glucose (that was not being stored) affects the body with surge of energy. This, type 2 DM is a lifestyle disease that results majorly of over body weight, lack of exercise, smoking, lower belly fat etc.
HUMAN INSULIN PRODUCTION BY RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology is a revolutionary technique, where DNA molecules from two different organisms are joined together and inserted into a host organism in order to generate new genetic combination that adds value to varied fields like science, health care, agriculture, poultry and industry.
Human insulin is being produced by recombinant DNA technology using E.coli or Saccharomyces cerevisiae as host organisms in many ways. The popular one is the production of insulin A-chain and B-chain separately in two E.coli strains and then joined together by disulphide bonds to produce active insulin.
The mRNA sequence of A-chain (basically, mRNA is a blue print of functional protein after modifications) is fused with ß-galactosidase gene (lac Z) present in the pBR322 plasmid (now called recombinant plasmid) and inserted into E.coli by transformation process. The recombinant bacteria is allowed to grow in the presence of an antibiotic, so that only transformed E.coli with A-chain will be selected. The whole lacZ gene and the fused A-chain will synthesize ß-galactosidase enzyme and A-chain. Similarly the B-chain was also synthesized separately.
Both the chains are purified from bacteria, combined, oxidized and reduced to form disulphide bridges to produce active insulin.
Figure 4: Human insulin production using recombinant DNA technology
Recombinant human insulin was first approved in 1982 for human administration. Humulin, is the first human insulin that was released into market in 1986.
Major insulin manufacturers include: Novo Nordisk A/S (Denmark), Sanofi S.A. (France), Eli Lilly and Company (U.S.), Bioton S.A. (Poland), Wockhardt Ltd. (India) and Julphar (UAE).