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Prevention of diabetes

Understanding Diabetes and Synthetic Insulin

History of Insulin

During 2019, a considerable number of individuals were dealing with diabetes, a condition where the body fails to process food effectively for energy production. The creation of synthetic insulin, a fascinating biotechnological achievement, involves the use of bacteria and genetic manipulation.

Insulin as a Hormone

Insulin, a vital hormone produced by the pancreas, plays a crucial role in sugar metabolism within the body. It controls blood sugar levels and facilitates the absorption of sugar by cells for energy generation.

Production of Synthetic Insulin

Biotech techniques and bacterial resources are utilized in producing synthetic insulin, such as Humulin. This method allows for the development of various insulin types, enhancing blood sugar management capabilities.

Challenges with Insulin Prices

Biologic Drug Challenges

The production of insulin is expensive due to its biological complexity, necessitating multiple patents for different diabetes management inventions.

Market Dominance

Three major companies dominate the insulin market, leading to substantial price increases that impact diabetic patients who depend on these medications.

Lack of Generic Options

The biological features of insulin make it challenging to produce generic versions, contributing to its high cost.

Impact of High Insulin Levels

Health Risks

Elevated insulin levels can lead to diseases and insulin resistance, exacerbating health conditions.

Managing Insulin Levels

Adopting healthy lifestyle practices, including dietary choices and physical activity, can help regulate blood sugar levels and reduce the need for high insulin doses.

Recognizing Diabetes Symptoms

Recognizing Diabetes Symptoms

Early symptoms of diabetes encompass increased thirst, fatigue, and blurry vision, indicating the need for medical attention.

Synthetic Insulin and Diabetes Management

Synthetic insulin plays a key role in managing diabetes efficiently, ensuring individuals can lead healthy lives by effectively controlling their blood sugar levels.

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Insulin, a pivotal hormone in regulating blood glucose levels, facilitates the entry of glucose into cells to convert carbohydrates from the bloodstream into energy.

When the body fails to produce or utilize insulin effectively, resulting in insulin resistance, it leads to elevated blood glucose levels. Diabetic individuals may require insulin therapy based on their specific condition. Type 1 diabetics always necessitate insulin, while type 2 or gestational diabetes patients might require insulin if lifestyle changes and oral medications are insufficient.

Insulin Discovery

In 1921, experiments conducted in Toronto, Canada initiated the synthesis of commercially available insulin. Frederick Banting and Charles Best conducted experiments on diabetic dogs with limited success. A breakthrough occurred when one dog survived for 70 days with pancreatic extract injections. The first successful insulin injection to a diabetic individual took place in 1922, revolutionizing the treatment of type 1 diabetes.

Insulin Production

Insulin production has evolved since 1922, starting with animal insulin extraction. Subsequently, biosynthetic human insulin emerged in the 1980s. Later on, insulin analogues were introduced, followed by biosimilar insulin variants.

Insulin Types

There are four primary categories of insulin, each possessing distinct characteristics. Basal insulins are long-acting and administered once or twice daily, while bolus insulins are given before meals. Mixed insulins are taken at mealtimes without basal insulin. Insulins are categorized based on their effects in the body – onset, peak impact, and duration.

Received on June 29, 2014, Accepted on September 16, 2014, Collection date 2014.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction provided proper credit. The Creative Commons Public Domain Dedication waiver applies unless stated otherwise.

Abstract

The escalating number of global diabetic patients and the exploration of novel insulin delivery methods will boost the demand for recombinant insulin. Current manufacturing technologies may face challenges in meeting this rising demand. Recombinant human insulin has primarily been produced using E. coli and Saccharomyces cerevisiae for therapeutic purposes.

Introduction

The groundbreaking work of Cohen and Boyer led to genetic engineering and the formulation of recombinant proteins such as insulin and growth hormone. The first production of human insulin through recombinant DNA technology took place in 1982. Insulin production predominantly occurs in E. coli and yeast for diabetes treatment.

Figure 1.

Around the 1920s, diabetic patients were treated with insulin derived from animal pancreas. However, genetic engineering advancements enabled insulin production in E. coli and yeast for human therapeutic applications. Presently, insulin is predominantly manufactured in E. coli or Saccharomyces cerevisiae. Other expression systems like mammalian cells and transgenic plants are also utilized.

Yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris, are commonly employed for recombinant protein production. Similar to E. coli, these strains grow rapidly, are easily handled, and amenable to genetic modifications. Proteins synthesized in yeast undergo proper folding and glycosylation to a certain extent, resembling mammalian cells. Mammalian cell lines like Chinese hamster ovary (CHO) and Baby hamster kidney (BHK) cells are used to produce human therapeutic proteins, including monoclonal antibodies. Proteins expressed in mammalian cells fold correctly, undergo glycosylation, and yield functional proteins. Nevertheless, the production cost using the mammalian system is high due to pricey culture media, although the number of approved biopharmaceuticals in 2013 exceeded previous years’ averages.

Figure 2.

Figure 2.

Statistics on the approval of biopharmaceuticals in the United States and/or European Union over the past six years, indicating a trend line reflecting the mean approval rate.

Structure and function of insulin

Human insulin comprises 51 amino acids with a molecular weight of 5808 Da, playing a crucial role in metabolic regulation. Insulin is synthesized from a single polypeptide, preproinsulin, within pancreatic beta cells. Properly folded insulin forms in the trans-Golgi network aided by cellular endopeptidases. The mature insulin consists of an A-chain with 21 amino acids and a B-chain with 30 amino acids linked by disulphide bonds.

E. coli expression system for production of insulin

E. coli is the preferred host for large-scale production of recombinant proteins. However, natural post-translational modifications crucial for biological activity are lacking in E. coli. Glycosylation, phosphorylation, and disulfide bonds do not occur naturally in E. coli, but incorporating glycosylation pathways from bacteria like Campylobacter jejuni can enable glycosylated protein expression in E. coli. Codon usage impacts the expression levels of foreign proteins in E. coli. Diminished codons can be substituted to enhance expression levels. Genetically modified E. coli strains can enhance protein yield and quality. Inclusion bodies may form in E. coli, but can be managed using molecular chaperones to ensure proper protein folding and solubility.

In 1978, Genentech produced the first recombinant human insulin in E. coli using chemically synthesized cDNA encoding for the insulin A and B chains separately. Post-purification, the two chains are co-incubated under optimal conditions to generate intact and bioactive insulin through disulphide bond formation. This method led to the development of the first commercial recombinant insulin for human therapeutic use. Another approach involved expressing a single chemically synthesized cDNA encoding for human proinsulin in E. coli, followed by purification and excision of C-peptide through proteolytic digestion. This approach was more efficient for large-scale insulin production and has been commercially utilized since 1986. Eli Lilly harnessed this technology to produce Humulin, the first approved recombinant insulin in 1982. These first-generation recombinant insulins possess an identical amino acid sequence to native human insulin and are preferred over animal-derived insulin.

Advancements in genetic engineering and technological progress facilitated the creation of insulin analogues with modified amino acid sequences. The conventional insulin in commercial preparations generally exists in an oligomeric form due to high concentration, while biologically active insulin in the blood is monomeric. To develop rapid-acting insulin analogues, adjustments to critical amino acid residues involved in oligomerization were necessary. Lispro, the initial rapid-acting insulin analogue, was developed by Eli Lilly and received regulatory approval in 1996. Insulin Lispro retains a similar amino acid sequence to native insulin but with an inversion of proline-lysine at positions 28 and 29 of the B-chain, preventing dimer formation.

Yeast expression system for the production of insulin

Yeast expression system for the production of insulin

Yeast is a preferred host for expressing various foreign proteins requiring post-translational modifications. Yeast can execute post-translational changes such as phosphorylation, glycosylation, acetylation, and acylation. Producing biopharmaceuticals in yeast is cost-effective and scalable. Nonetheless, yeast’s high-mannose N-glycosylation results in the short half-life and hyper-immunogenicity of therapeutic glycoproteins. Ongoing efforts are focused on humanizing yeast N-glycosylation pathways.

Yeast is a common choice for producing therapeutic proteins such as insulin, insulin analogues, non-glycosylated somatotropin, vaccines, and growth factors. Among yeast strains, Saccharomyces cerevisiae and Pichia pastoris are frequently used for this purpose. Pichia pastoris offers benefits in terms of glycosylation and high expression levels when compared to Saccharomyces cerevisiae. When it comes to large-scale production of recombinant insulin and its analogues, Pichia pastoris shows promise as an alternative option. Bacterial systems have higher productivity rates, while yeast systems boast higher concentrations of biomass, resulting in similar overall production yields.

Table 1.

Comparison of production systems for human insulin

Origin Escherichia coli Saccharomyces cerevisiae Pichia pastoris

Since the early 1980s, Saccharomyces cerevisiae has been a popular choice for the production of recombinant human insulin, with a significant portion of commercial insulins being manufactured using this yeast system. Not only insulin, but insulin analogues like Insulin Aspart and Insulin Detemir have also been successfully made with the help of this yeast. Insulin Aspart, developed by Novo Nordisk, obtained approval from the US FDA in 2001, while Insulin Detemir, also created by Novo Nordisk, was given the green light by European regulatory authorities in 2004.
In addition to insulin analogues, Saccharomyces cerevisiae has played a role in producing more than 40 different recombinant proteins. By utilizing various leader sequences to improve protein secretion in yeast, the efficiency of production has been significantly enhanced.

Table 2.

Below are some of the biopharmaceuticals that have been successfully produced using S. cerevisiae

Category Protein Source Medical Use Initiating Segment Concentration

Transgenic plants as host for insulin production

Genetically modified plants have been used to manufacture customized proteins, offering advantages such as cost efficiency, high-quality protein production, lack of human pathogens, and the ability to modify proteins posttranslationally. The initial success was seen with human growth hormone extracted from transgenic tobacco plants, followed by other products like the Hepatitis-B-Virus surface antigen and antibodies.

For instance, recombinant human insulin has been effectively produced in the oilseeds of the plant Arabidopsis thaliana. By targeting insulin expression in the oilbodies of seeds, a high level of expression was achieved, and insulin recovery became simple. The use of genetically engineered oil seeds for targeted protein expression simplifies the purification process. Furthermore, transgenic plants have also shown potential in producing proinsulin at high levels, providing a cost-effective solution for insulin production.

Table 3.

Expression of CTB cholera toxin B subunit proinsulin in tobacco and lettuce chloroplasts

Transgenic Plant Type Tobacco Lettuce
Destination of the Product Chloroplast Chloroplast
Percentage of Leaf Protein 47% 53%
Protein yield per gram of leaf tissue 2.92/gm 3.28/gm
Reference [28]

Conclusion

Over the next two decades, the global market for insulin is expected to grow from $12 billion to $54 billion. Lifestyle changes and the increasing prevalence of diabetes worldwide are key factors driving this growth. Both Type I and Type II diabetics depend on insulin, with late-stage Type II patients requiring high doses due to insulin resistance. This rise in diabetic cases and the exploration of new insulin delivery methods like inhalation or oral routes will create a higher demand for recombinant insulin. Current production techniques may not be sufficient to meet this demand due to limited capacity and high costs. Therefore, improving the production of biologically active insulin from organisms like E. coli and Saccharomyces cerevisiae using efficient technologies is crucial. Utilizing plants for insulin production offers cost-effective solutions with high capacity, enabling stable expression and potential oral delivery options, while seeds can serve as storage for recombinant insulin.

Acknowledgments

Funded by the NSTIP strategic technologies program of Saudi Arabia (Project No. 10-BIO1257-03), this work acknowledges the support from the Science & Technology Unit, Deanship of Scientific Research, and Deanship of Graduate Studies at King Abdulaziz University, Jeddah, KSA.

Footnotes

Declaration of Interests

The authors declare no competing interests.

Contributions of the Authors

All authors contributed to the writing, design, and figures of this review and approved the final manuscript.

Contributor Information

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Here are some examples of studies that have explored different methods of producing insulin:
1. Mergulhao F, Taipa M, Cabral J, Monteiro G. conducted a study evaluating bottlenecks in proinsulin secretion by Escherichia coli.
2. Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U. utilized a simple fed-batch technique for high-level secretory production of insulin precursor using Pichia pastoris, followed by purification and conversion to human insulin.
3. Owens DR, Vora JP, Dolben J. discussed the advancements in semisynthesis and recombinant DNA technology for producing human insulin and beyond.
4. Frank B. explored the monomeric insulins produced by manipulating the position of proline in the B chain.
5. Kurtzhals P, Havelund S, Jonassen S, Markussen J. studied the effects of fatty acids and selected drugs on the albumin binding of a long-acting, acylated insulin analogue.
6. Owens DR, Zinman B, Bolli G. discussed the current status of insulins and future developments.
7. Havelund S, Plum A, Ribel U, Jonassen I, Volund A, Markussen J, Kurtzhals P. researched the mechanism of protraction of insulin detemir, a long-acting analog of human insulin.
8. Hou J, Tyo KEJ, Liu Z, Petranovic D, Nielsen J. delved into the metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.
9. Kjeldsen T, Hach M, Balschmidt P, Havelund S, Pettersson AF, Markussen J. investigated the secretory expression of prepro-leaders lacking N-linked glycosylation in Saccharomyces cerevisiae.
10. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA. achieved high-yield production of a human therapeutic protein in tobacco chloroplasts.
11. Van Rooijen GJH, Moloney MM. explored the use of plant seed oil-bodies as carriers for foreign proteins.
12. Deckers H, Moloney MM, Baum A. made a case for the recombinant production of pharmaceutical proteins in plants.
These studies provide valuable insights into different approaches for insulin production in microbial and plant systems.