Carbohydrates are enzymaticaliy digested into monosaccharides in the alimentary canal of animals

Carbohydrates are enzymaticaliy digested into monosaccharides in the alimentary canal of animals. These monosaccharides are absorbed into the blood, which carries them to the cells of the tissues for oxidation and release of metabolic energy (ATP), carbon dioxide and water.

Glucose is the commonest monosaccharide digestion product among all and frequently used as a metabolic fuel.

Cells pick up their required amount of glucose from the blood by a process known as assimilation. A fixed concentration of glucose is maintained in the blood plasma. Any excess is transported to the liver and muscle for storage in the form of insoluble glycogen by a process known as glycogen sis. When the blood glucose concentration falls below normal, a precise quantity of glycogen, mainly of the liver, is broken down into glucose by a process known as glycogenolysis and this glucose is released into the blood to make up for the shortfall. Thus there is a homeostasis of glucose concentration in the blood plasma.

This state is maintained by the action of several hormones secreted from the endocrine glands. Insulin is one among these hour manes. Any excess of glucose in the blood is converted into glycogen for storage. Thus insulin lowers blood glucose level i.e. it favours glycogens.

It is a hypoglycemic (blood glucose lowering) agent. It comes into action, when the blood glucose level increases (hyperglycemia). It is secreted from the islet of Langerhans cells of the pancreas. Other hormones, like glucagon from the pancreas, epinephrine from the adrenal medulla and glucocorticoids from the adrenal cortex increase plasma glucose concentration by glycogenolysis and gluconeogenesis.

These are hyperglycemic (blood glucose elevat­ing) agents. Insulin alone counters the joint hyperglycemic actions of all these hormones. This is the main reason for insulin being the most important hormone for maintaining a homeostasis of glucose in the blood plasma.

In some persons, the islet cells fail to secrete the required amount of insulin, which leads to an elevated blood glucose level or hyperglycemia.

This increase, when becomes acute, leads to a pathological condition known as diabetes mellitus. This state is known as insulin dependent diabetes mellitus (IDMM) and occurs in only 10% of the diabetic population. In 90% of this population, required amount of insulin is secreted from the pancreas.

However, the hepatocytes of the liver fail to respond to the insulin. This state is known as non-insulin dependent diabetes mellitus (NIDDM). Persons, suffering from IDDM need exogenous insulin to maintain a constant glucose level in the blood plasma.

World Health Organization (WHO) estimates that India has the largest population of diabetics (35 millions) followed by China and USA. By the turn of 2025, this figure may touch 57 millions and by 2030, 79.4 millions. At present, the world figure is esti­mated at 140 million and is projected at 300 million by the turn of 2025.

Nearly 90% of the insulin comes from slaughtered cows and pigs. Insulin derived from these animals has minor variations in the amino acid sequence and hence are immu­nogenic, i.e. produce antibodies in the blood of the recipients. Secondly, the production cost is very high due to complex purification procedure and finally, this insulin bears the risk of many infectious viral diseases, such as bovine spongiform encephalitis, transmis­sible spongiform encephalitis, Creutzteldt-Jacob disease and other neurological disorders.

In the present day, these problems have been circumvented by producing human insulin in bacterial or yeast expression hosts by recombinant DNA technology. This insulin has been referred to as recombinant insulin.

Structure of insulin

Insulin is a heterodimeric protein (two different polypeptides connected together) consisting of two polypeptides, A and B, joined by two inter-chain disulfide bridges. Polypeptide A consists of 21 amino acid residues, while B, 30. It is synthesized as pre-pro-insulin on the ribosomes of islet cells.

The stretch of 23 amino acid residues from the amino terminal end directs the molecule into the endoplasmic reticulum lumen, where it is removed. This stretch is known as the pre or leader sequence. The resultant polypeptide is a pro-insulin molecule.

The B-chain starts at the amino-terminal end followed by a C (connecting) peptide and then the A-chain. This molecule undergoes two interchange disulfide bridge formation and a series of site specific peptide cleavages, resulting in a mature insulin molecule and a C-peptide

Structure of human mature insulin two polypeptides (A and B) are con­nected by two inter-chain disulfide bridges.

Insulin gene

The human insulin gene is located on the short arm of the chromosome 11. The gene has three untranslated regions, two intervening sequences and five reading frames (translated regions). The reading frames are organized into a leader, a B- chain, two C-chain and A-chain sequences. The two C-chain sequences are separated from each other by an intervening sequence.

There are two approaches for alleviating the problems of diabetics: (1) tissue engi­neering and (2) recombinant DNA.

Tissue engineering approach

Prior to the advent of the recombinant DNA technology, tissue engineering method was the practice for curing the diabetics. In this technique, islet cells from another mammal, such as cow or pig are transplanted into the intra-peritoneal cavity of a diabetic.

This method encountered a serious problem. The grafted islet cells were considered foreign and were rejected by the immune system of the host. Therefore, the islet cells were grafted in an encapsulated form, such that the antibodies, gen­erated due to an immune response, could not move across the capsule to damage the tissue.

The islet cells were first immobilized by the polysaccharide, alginate and then encapsulated in hollow spheres made up of fibers. However, following the synthesis of recombinant insulin, this practice has become obsolete.

Recombinant DNA approach

The problem of exogenous [bovine (cow) or orcine (pig)] insulin and / or trans­planted islet cells being foreign, is overcome by the recombinant DNA technol­ogy. The insulin produced by this. Technology is known as recombinant insulin.

The recombinant insulin is as pure as the insulin synthesized by the islet cells in vivo. In the first approach, the human insulin gene is inserted into a cloning-ex­pression vector and then cloned and expressed in a transformed Escherichia coli cell. Following its synthesis, it is purified, enzymatic ally cleaved and refolded into its active tertiary structure by down-stream processing.

A second approach is to use yeast (Saccharomyces cerevisiae) host for the expression of the insulin gene. This approach has an advantage over the E.coli host in that S. cerevisisae, being a eukaryotic host, expresses and refolds the insulin polypeptide correctly. Despite these advantages, most of the insulin manufacturing biotech companies prefers to use E.coli as the cloning-expression host.

Synthetic oligonucleotide approach

The initial approach was to construct synthetic genes from oligonucleotides that encoded for A and B chains separately. These were then inserted separately into E. coli cloning-expression plasmids carrying p -galactosidase gene, such that the inserted A or B genes followed the (3 -galactosidase gene, also engineered into the plasmid.

A codon for the aminoacid, methionone was also engineered into the plasmid between the p -galactosidase and A and B genes. The expression prod­ucts were large fusion proteins consisting of the polypeptide p -galactosidase and A or B polypeptide of the insulin molecule.

These fusion proteins were isolated from the host. Cyanogen bromide was used to clip the mature A and B chains from the p -galactosidase. The A and B chains were purified and reconstituted into ma true insulin molecule. This approach was refined by cloning a single P -galactosi-

Dase-insulin fusion protein gene. The fusion protein synthesized could be cleaved in a single step to release the insulin molecule. Following its synthesis, it is re folded into its active tertiary structure by treating it in a refolding vessel with buffers of various concentrations.

Recombinant Insulin Manufacturing Companies

Genentech is the first biotech company to manufacture recombinant insulin in 1978 by using bacteriophages as vectors and E. coli cells as host cells for cloning and expression. It licenced the human insulin technology to Eli Lilly Corporation of USA, where it was named “HUMULIN” or Recombinant Human Insulin.

In 1982, it became the first recombinant drug approved by the Food and Drug Administration (FDA) of USA. Several other companies, world over, are manufacturing human insulin to meet the insulin demand of diabetics. Among these, the front runners are Novo Nordisk, Denmark, Hoechst, Germany; A vends Germany and Pfizer, USA.

Wockhardt Ltd has launched its first insulin manufacturing unit of Asia in Aurangabad, Maharastra, India. It manufactures human insulin under the trade name of WOSULIN. It has joined the band of a few human insulin manufacturing com­panies of the world. Following its establishment, all the companies have cut their insulin prices in India by 35-40%.