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The function of the immune system is protection of the body from disease infection. Attacking the healthy cells of the body by the immune system is called an autoimmune disease.
According to the American Autoimmune Related Diseases Association, the term "autoimmune disease" refers to a varied group of more than 80 serious, chronic illnesses that involve almost every human organ system. In all of these diseases, the underlying problem is similar, the body's immune system becomes misdirected, attacking the very organs it was designed to protect."
It could be hereditary and affect many parts of the body. It is more common in women than men. Initial symptoms may include fatigue, muscle ache and low fever.
Autoimmune diseases are listed as one of the ten leading causes of death in women up to the age of 65 years. (Rose and Mackay, 2008)
Diabetes mellitus (Type 1)
Diabetes type-1 occurs due to the autoimmune destruction of the beta cells (insulin producing cells) leading to no or insufficient production of insulin (hormone regulating blood sugar) by the pancreas, which causes high blood and urine glucose levels. This leads to damage in the heart, eyes, kidney, nerves, gums and teeth. It generally occurs in young children and unless treated with insulin, it is fatal.
It is a polygenic disease (several genes contribute to its expression). The IDDM1 gene located on chromosome 6 is responsible for the histocompatibility disorder characteristic, where the beta cells render improper antigens to the T-cells which lead to the production of anti-bodies that in turn attack these beta cells. This could be due to the expression of the 'super antigen' on the beta cells, although the molecular details of this mechanism are not fully known. (Atkinson et al, 1990)
Current treatment includes islet cell transplantation, wherein islet cells on being injected in the patient's liver begins to produce insulin. However it is expensive and also leads to problems like 'hypoglycemia' (very low blood glucose level).
How many people in the UK have diabetes?
Approximately over 2.6 million people have diabetes in the UK and there up to 0.5 million people with diabetes who have the symptoms but are unaware.
[Source: Diabetes UK ââ‚¬" Core Survey Report]
0.5 % of the people did not know what type of diabetes they had. 48% of the people are found to have type-1 diabetes, closely followed by 52% having type-2.
The current treatment options are indicated below, with insulin injections being the most popular (50%).
[Source: Diabetes UK- Core Survey Report]
Recombinant-DNA Technology to produce Human Insulin
The biosynthetic 'human' insulin- HUMULIN was one of the first medicines to be developed through recombinant DNA technology and to be approved by the FDA. It was safe, effective and easy to produce. Humulin was first developed by Genentech in 1978 using the following method-
Figure 1- The production of human insulin from bacterial plasmid.
Induction of insulin secretion in engineered liver cells by nitric oxide [Muniappan L. and Ozcan S.]
In the human body, lack of insulin leads to chronic hyperglycemia along with secondary complications such as cardiovascular disease and kidney failure and thus it is very important to achieve sustained and optimal glycemic control. There is a possibility of the development of surrogate beta cells as a treatment for diabetes type 1. Usually, the beta cells in the Lengarhans islets produce and secrete insulin only when the glucose levels in the blood increase. However it is possible to achieve insulin secretion from non-beta cells independent of glucose levels. This strategy involves the production and secretion of insulin by engineering the liver cells. The stimulation of insulin secretion is achieved via the nitric oxide pathway.
Expression of either human insulin or beta specific transcription factors (PDX I, NeuroD I and Maf A) in the Hepal-6 cell line or primary liver cells via the adenoviral gene transfer, results in the production and secretion of insulin. The secretion increases three-fold when these engineered liver cells are treated with L-arginine in the presence of nitric oxide. Stimulation of insulin secretion with the help of L-arginine from surrogate beta cells via the production of nitric oxide pathway could provide a potential therapy for treatment of type 1 diabetes.
Currently, besides insulin injections, the other option is transplantation of Lengarhans islets. In gene therapy, surrogate beta cells are a potential approach in the treatment for diabetes type 1 to avoid lack of insulin production (Giannoukakis N. et al). For the diabetes Type 1 therapy to be successful, the production and secretion of mature insulin must take place from surrogate beta cells in a glucose-regulated manner. An excellent surrogate organ to produce insulin is the liver wherein successful viral gene transfer takes place and it expresses GLUT-2 and glucokinase in a manner similar to the pancreatic beta cells (Chen R. et al). However unlike the pancreatic beta cells, the hepatocytes lack insulin secretion machinery. Insulin expression in the liver is achieved by various different viral and non-viral vectors (Li S. et al, 2001).
The secretion and release of insulin in the pancreatic beta cells is directly influenced by changes in the blood glucose levels (Mulligan RC, 1993). An increase in the blood glucose levels, also leads to an alteration in the function of the beta-cell specific transcription factors (PDX I, NeuroD I and Maf A) which causes a decrease in the insulin production and hyperglycemia (Schepelmann S. et al, 2006). The production and secretion of insulin in the Hepa1-6 liver cells or the primary liver cells can be achieved through adenoviral gene transfer that leads to the expression of the human insulin or the beta-transcription factors in the liver cells. L-arginine is found to stimulate the secretion of insulin via the synthesis of nitric oxide in the fibroblasts and cervical carcinoma cells, which would make it useful in the treatment of type-1 diabetes by engineering the liver cells.
The incubation of the Hepa1-6 cell lines with an adenovirus expressing human insulin led to the production of detectable amounts of insulin as compared to the control virus. On treatment with 1mM or 25 mM glucose, the amount of insulin secreted was found to be 60 ÎÂ¼U/106 cells. The amount of insulin secreted in response to high glucose levels (25mM) was more or less the same as that secreted at low glucose levels (1mM).
Figure 2- Hepa1-6 cells expressing human insulin gene produce and secrete insulin at 1mM and 25 mM glucose concentration.
The treatment of the Hepa1-6 liver cells with 20mM L-arginine for 1 hour in the presence of 1mM glucose increased the secretion of insulin by three-fold. L-arginine acts via the nitric oxide (NO) pathway and L-NNA is the inhibitor of nitric oxide synthase (NOS) independent of L-arginine. The treatment of the Hepa1-6 cells expressing the human insulin with 100mM of L-NNA for 1 hour inhibited the secretion of basal insulin. Thus NO is responsible for a portion of the basal insulin secretion.
Figure 3- L-arginine stimulates the secretion of insulin in the Hepa 1-6 cells via the nitric oxide pathway. Cells were incubated with and without L-arginine in the absence and presence of the nitric oxide inhibitor (L-NNA).
Incubation of the following cell lines- HepG2 (human liver), NIH 3T3 (mouse fibroblast) and HeLa (human cervical carcinoma) with the human insulin adenovirus led to the production and secretion of insulin. Further incubation with 20mM of L-arginine led to an increase in the secretion of insulin by about 2-fold in the human liver cell line, less than 2-fold in the fibroblast cell line and the human cervical carcinoma. Thus, the production and secretion of insulin under the effect of L-arginine is also possible in non-beta cells and is not just restricted to only beta-cells.
Figure 4- L-arginine stimulates the secretion of insulin in various cell lines incubated with the human adenovirus insulin gene. A- Hep G2. B- NIH 3T3. C- HeLa cells.
To check for the secretion of insulin in the primary rat liver cells, they were cultured and incubated with an adenovirus containing human insulin and it was observed that insulin secretion was similar to that of the Hepa1-6 cell lines. Further incubation with 20 mM of L-arginine, the secretion increased by more than 3-fold. The secretion of insulin was abolished on treatment with 100 ÎÂ¼M L-NNA, confirming that insulin secretion is stimulated by L-arginine via the nitric oxide pathway.
Figure 5- Treatment with L-arginine induces the secretion of insulin in primary rat hepatocytes.
Advantages of using E.coli
A variety of cloning vectors can be employed with it.
The gene expression is easy to control.
The process of production is easy and cheap.
It yields large amounts of recombinant proteins.
Limitations of using E.coli
It does not perform all the post-translational modifications (eg-glycosylation).
Inclusion bodies may pose as a problem.
Secretion is poor.
If a large amount of insulin is rendered, it may lead to complications like hypoglycemia.
The wall may contain components that are toxic.
The above data indicates that the engineering of the liver cells and the non-beta cells leads to the production and secretion of insulin. The insulin secretion from these engineered cells is not very responsive towards alterations in the levels of glucose, but the secretion increases by up to 3-fold when L-arginine is added through the nitric oxide pathway.
The stimulation of insulin secretion from the surrogate beta cells through the production of nitric oxide would yield a potential therapy to treat diabetes mellitus type-1. Hypoglycemia could be avoided owing to the unstable nature of nitric oxide which leads to a transient insulin secretion. Also this therapy could replace the insulin treatments of diabetes, by further research on the molecular basis of stimulating insulin secretion by L-arginine in the surrogate beta-cells. It could also correct hyperglycemia in diabetic animals expressing insulin in liver.