THE STRUCTURE OF INSULIN VARIES FROM SPECIES TO SPECIES WITHOUT CHANGING THERAPEUTIC ACTIVITY .

A major portion of the pancreas essentially comprises of glandular tissue which specially contains acinar cells that predominantly gives rise to the secretion of certain digestive enzymes. Besides, there also exist some isolated groups of pancreatic cells commonly known as the islets of Langerhans which usually made up of four cell types, each of which generates a distinct polypeptide hormone,namley : (a) Insulin in the beta () cells, (b) Glucagon in the alpha () cells, (c) Somatostatin in the delta () cells, and (d) Pancreatic polypeptide in the PP or F cell. Interestingly, the -cells made up 60-80% of the islets of Langerhans most predominantly and distinctly. Diabetes : a general term for diseases marked by excessive urination ; and is usually refers to diabetes mellitus.However, the clinical diabetes mellitus invariably occurs in two forms, associated with different causes and methods of therapy. Type 1 Diabetes : The insulin-dependent diabetes mellitus (IDDM), normally takes place when the -cells of the prevailing pancreatic islets of Langerhans are destroyed, perhaps by an autoimmune, mechanism, as a consequence of which the insulin production in vivo is overwhelmingly insufficient. Therapeutically Type-I diabetes is largely treated with insulin. Type 2 Diabetes : The noninsulin-dependent diabetes mellitus (NIDDM), i.e., type 2 diabetes, is most abundantly linked with obesity in its adult patients largely. In such a situation, the insulin levels could be either elevated or normal ; and therefore, in short, it is nothing but a disease of abnormal insulin resistance. Insulin Insulin was the first hormone identified in 1920s by Banting and Best. They discovered insulin by tying a string around the pancreatic duct of several dogs. When they examined the pancreas of these dogs several weeks later, all of the pancreatic digestive cells were died and were absorbed by the immune system and the only thing left was thousands of pancreatic islets. They then isolated the protein from these islets and discovered insulin. Sources of insulin The first successful insulin preparations came from cows (bovine)and later from pigs (porcine). Bovine and porcine insulin worked very well for the majority of patients, but some could develop an allergy or other types of reactions to the foreign protein (a foreign protein is a protein which is not native to humans). In the 1980s technology had advanced to the point where we could make human insulin. The advantage would be that human insulin would have a much lower chance of inducing a reaction because it is not a foreign protein. The technology, which made this approach possible, was the development of recombinant DNA techniques. In simple terms, the human gene, which codes for the insulin protein was cloned (copied) and then put inside of bacteria. A number of operations were performed on this gene to make the bacteria to constantly make insulin. Big vats of bacteria now make tons of human insulin. From this, one can isolate pure human insulin.

Created by fahad hussain , Noakhali Science & Technology University10.

Isolation of insulin Isolation of insulin from animal pancreas involves the following steps : (i) Mince the pancreas of slaughtered animal and extract with 80% ethanol containing small amounts of phosphoric acid (to adjust pH to 3) (ii) Centrifuge the extract to separate proteins and fats (iii) Raise the pH of extract to 8.0 by adding ammonia solution and filter (iv) Acidify and evaporate the filtrate to remove fatty material (v) Add picric acid to ethanolic solution to precipitate insulin as insulin picrate (vi) Dissolve insulin picrate in acetone and reprecipitate as hydrochloride salt (vii) Insulin is further purified by chromatography Structure of Insulin The minimum molecular weight of insulin is about 6000. Dinitro phenyl hydrazine (N-terminal amino acid determination method) showed the presence of two Nterminal amino acid residues i.e. glycine, phenylalanine. Thus insulin contains two peptide chains. Insulin was oxidized with performic acid. This produced two peptides, which were separated by electrophoresis. The two peptide chains referred to as the A chain and B chain. The peptide with N-terminal glycine residue was called the A-chain and that with the N-terminal phenylalanine residue was called B-chain. Each chain was subjected to hydrolysis (with acids or enzymes). The products of hydrolysis were separated and examined by DNP method. The chain-A contains 21 amino acid residues and the chain-B contains 30 amino acid residues. In chain-A four cysteine acid residues and chain-B two residues were present. Two disulphide bonds connecting these two chains, further chain-A contains one intra disulphide bond.

Created by fahad hussain , Noakhali Science & Technology University10.

These interactions have important clinical ramifications. Monomers and dimers readily diffuse into blood, whereas hexamers diffuse very poorly. Hence, absorption of insulin preparations containing a high proportion of hexamers is delayed and slow. This problem, among others, has stimulated development of a number of recombinant insulin analogs. The first of these molecules to be marketedcalled insulin lisprois engineered such that lysine and proline residues on the C-terminal end of the B chain are reversed; this modification does not alter receptor binding, but minimizes the tendency to form dimers and hexamers. Although the amino acid sequence of insulin varies among species, certain segments of the molecule are highly conserved, including the positions of the three disulfide bonds, both ends of the A chain and the C-terminal residues of the B chain. These similarities in the amino acid sequence of insulin lead to a three dimensional conformation of insulin that is very similar among species, and insulin from one animal is very likely biologically active in other species. Indeed, pig insulin has been widely used to treat human patients. The structure of insulin is as below :

The structure of insulin differs slightly from different sources, but all show identical hormonal activity.

Biosyntesis of insulin Insulin is synthesized in significant quantities only in cells in the pancreas. The insulin mRNA is translated as a single chain precursor called preproinsulin, and

Created by fahad hussain , Noakhali Science & Technology University10.

removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases, which excise the C peptide, thereby generating the mature form of insulin. Insulin and free C peptide are packaged in the Golgi into secretory granules, which accumulate in the cytoplasm. When the cell is appropriately stimulated, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood. C peptide is also secreted into blood, but has no known biological activity. Insulin Preparation Human insulin is absorbed more quickly than beef or pork insulin. Thus the duration of action of human insulin is shorter. If insulin preparations were administered orally, it would be degraded in gastrointestinal tract. Therefore, insulin must be administered by injection (IV or subcutaneous). The following insulin preparations are used for the treatment of diabetes. Crystalline zinc insulin. It is purified insulin crystallized as a zinc salt. It is administered subcutaneously and lowers the blood sugar within minutes. Hence it is known as rapid action insulin. Semilente insulin. It is a suspension of amorphous insulin, which is also administered subcutaneously. It is another example for rapid action insulin. Isophane insulin. It is a suspension of crystalline zinc insulin with the positively charged peptide mixture called protamine. Its duration of action is intermediate between crystalline zinc insulin and protamine zinc insulin. This is due to delayed absorption of insulin because of conjugation of insulin with protamine to form less soluble complex. Lente insulin. It is a mixture of 30% of semilente insulin and 70% ultralente insulin. It is another intermediate action insulin that is administered subcutaneously. Protamine zinc insulin. It is a prolonged action insulin preparation. It produces maximum therapeutic effect in 24 hours. It is prepared by mixing crystalline zinc insulin with protamine. Extended insulin zinc suspension. It is poorly soluble crystalline zinc insulin. This preparation has a delayed onset and prolonged duration of action. Storage Insulin in powder form should be stored in airtight containers protected from light. The injections are required to be stored in a refrigerator at 2 to 8C and not allowed to freeze.

Created by fahad hussain , Noakhali Science & Technology University10.