Assignment #4 - Article Review

Assignment # 4 - Article Review

Paper: Bobes, R. J., Castro, J. I., & Miranda, C. C. (2001). Insulin modifies the proliferation and function of chicken testis cells. Poultry Science, 80(5), 637-642

Paper can be retrieved from: http://ps.fass.org/content/80/5/637.full.pdf+html

Summary

As mentioned in the previous blogs, the main role of insulin is glucose homeostasis. However, it was also mentioned that insulin has several other roles such as stimulating lipogenesis, diminishing lipolysis, modulating transcription, and stimulating growth. 

Bobes et al. (2000) conducted an experiment to investigate whether insulin plays a role in the proliferation and the androgen production of chick testis cells since insulin is already present in the chick embryo. This is the first study to examine this. To see if insulin did in fact have an effect on chick testis cell proliferation and androgen production, Bobes et al. used testes from 18 day old chick embryos or newly hatched chicks. The testes were dissociated, the cells were pre-cultured and then cell suspensions were made and each were exposed to varying concentrations of insulin and human chorionic gonadotropin (hCG) for varying amounts of time. 

                                          Figure 1:  chick embryo at 18 days of development

Bobes et al. found that when incubated with insulin for one hour, the androgen production of the chick embryo testis cells was not directly affected. However, incubating the cells in insulin for an hour did modify how the cells responded to hCG in that it resulted in a slight but significant increase in androgen production. The opposite occurred when the concentration of insulin increased.  These results are shown in figure 1 of the paper.  On the other hand, the testes cells from the newly hatched chickens showed a large increase in the production of androgens (testosterone) with the addition of increasing dosages of insulin. These results are shown in figure 4.

Bobes et al. also found that insulin enhances the proliferation of embryonic cells by observing that insulin significantly increased the uptake of H-thymidine by testes cells as shown in figure 3 of the paper. 

In conclusion, the experiment shows that insulin does effect the proliferation and the androgen production of chick testis cells and that the stage of maturity affects the cells’ response to the hormone. It also demonstrates that insulin has a slight stimulatory on hCG-dependent androgen production in embryonic chick testis cells. 

Critique

Overall, the paper is well written. It is very easy to understand and is presented in an organized manner. The experiment is explained in a way that can be easily followed by the reader.  Also, the figures are very basic but effective at displaying the results of the experiment. I like that the paper did not include an overwhelming amount of statistical analysis. 

I would have liked a better explanation in the discussion as to why the embryo testes cells did not show an increase in the androgen production in the presence of insulin but the newly hatched chick testes cells did. Saying that sensitivity to insulin depends on stage of maturity seemed like an insufficient explanation.  

A little more background information in the introduction section would have aided in the understanding of the results obtained.

The results of the experiment did seem to support to the authors’ claims and it was interesting to read how the authors’ previous experiment tied in with the one conducted in this paper.

For future experiments, it is mentioned in the paper that while the results show that insulin does influence cell proliferation and androgen production of the chick testes cells, the mechanism of how this occurs needs further investigation. It would also be interesting to conduct this same experiment on another species to see if similar results are obtained.





*References can be accessed through links on pictures and citations.*

Assignment # 3 - The Function of Insulin

The Function of Insulin

The main function of insulin is glucose homeostasis. Insulin works to lower the amount of glucose in the blood. Insulin regulates the amount of glucose in the blood by causing cells in the liver, skeletal muscles and adipose tissue to take up glucose from the blood. In most nonhepatic tissues, insulin increases glucose uptake by increasing the number of plasma membrane glucose transporters: GLUTs. Glucose uptake in the liver is the result of an increase in the activity of the enzymes glucokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK), the key regulatory enzymes of glycolysis (King, 2012). The glucose that is taken from the blood is stored as glycogen in the liver and muscles and as triglycerides in the adipose tissue.

A small amount of insulin is continuously secreted from the pancreas.  However, as blood glucose levels increase, this increase is detected by the glucose receptors located on the pancreatic beta cells which results in an increase in the amount of insulin being secreted from the pancreas.  As blood glucose levels return to normal then so does the secretion of insulin. Insulin is typically secreted immediately after eating a meal when carbohydrate levels are high. If glucose levels are low, the hormone glucagon is secreted which has opposing effects of insulin.

Figure 1: The control of glucose levels via the secretions of insulin and glucagon from the pancreas. 

While glucose homeostasis is the main function of insulin, it also has plays a role in several other processes including:

·         Stimulating lipogenesis

·         Diminishing lipolysis

·         Increasing amino acid transport into cells

·         Modulating transcription

·         Altering the cell content of numerous mRNAs

·         Stimulating growth

·         DNA synthesis

·         Cell replication

(Ramaraj, 2010) 

Insulin’s main role is to work to prevent hyperglycemia. Hyperglycemia is the medical term for high blood sugar.  Hyperglycemia affects people who have diabetes which can develop from a resistance to insulin or by the total lack of insulin secretion by the beta cells of the pancreas.

Type 1 Diabetes, also known as insulin dependent diabetes, occurs early in life.  In type 1 diabetes, the immune system attacks the insulin-producing beta cells in the pancreas and destroys them. This means the pancreas cannot secrete insulin or can only secrete insufficient amounts so the person ends up with hyperglycemia since there is not enough insulin produced to cause the uptake of glucose. Scientists do not know exactly what causes the body's immune system to attack the beta cells, but they believe that both genetic factors and viruses are involved (PHAC, 2010). While research is still ongoing, about 18 regions of the genome have been linked with influencing type 1 diabetes risk. These regions, each of which may contain several genes, have been labeled IDDM1 to IDDM18. The IDDM2 locus contains the insulin gene. Mutations of insulin gene cause a rare form of diabetes that is similar to MODY (Maturity Onset Diabetes in the Young). Other variations of the insulin gene (variable number tandem repeats and SNPs) may play a role in susceptibility to type 1 and type 2 diabetes (NCBI, 2004).

Type 2 Diabetes, also known as non-insulin dependent diabetes, is caused by the resistance of cells to the effects of insulin. Insulin can attach normally to receptors on liver and muscle cells but certain mechanisms prevent insulin from moving glucose into these cells where it can be used. Enough insulin is usually produced to overcome this resistance but eventually the pancreas will not be able produce enough to overcome the resistance. The resulting very high levels of blood glucose then beings to damage the beta cells of the pancreas and insulin secretion is halted completely (PHAC, 2010). While research is still ongoing, insulin resistance is thought to be due to the inheritance of a number of mutations in a variety of genes. Mutational analysis of the insulin signalling cascade has identified a glycine-arginine (Gly-Arg) substitution at codon 972 of the insulin receptor substrate-1 (IRS-1) gene associated with insulin resistance in obese individuals (Pedersen, 1990). 

Insulin injections are used to treat individuals with type 1 diabetes to aid in lowering blood glucose levels. Since individuals with type 2 diabetes have a resistance to insulin, treatment involves a change in diet that is low in glucose/carbohydrates. 

*References can be accessed through links on pictures and citations.*

Assignment #2 - The Structure of Insulin

Insulin Structure

Insulin is a small protein with a molecular weight of about 5800 Daltons. As mentioned in the previous blog posting, insulin consists of two peptide chains (A and B) that contain 51 amino acids in total. However, The insulin mRNA, which is coded for by the INS gene, is translated as a single chain precursor called preproinsulin which has 110 amino acids and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin (Bowan,1999). The proinsulin molecule contains 86 amino acids. Proinsulin differs from insulin in that it contains a connecting peptide, or C-peptide, of 35 amino acids (number may very between species) that connects the carboxy terminus of the B-chain to the amino terminus of the A-chain. The C-peptide has basic residues at each end, Arg-Arg at the amino terminus and Lys-Arg at the carboxy terminus. The Arg-Arg and Lys-Arg residues at either ends are the sites of enzymatic cleavage in the conversion of proinsulin to insulin. This occurs in the Golgi complex (Talwar et al., 2006).

To show how insulin can vary slightly from species to species, I prepared an alignment of preproinsulin mRNA sequences from four different species; 2 large mammals, a small mammal and a non-mammal.

Figure 1: The protein sequence alignment of preproinsulin in four different species. (Alignment produced using ClustalW2 software).

In figure 1, the preproinsulin sequence alignments of four species are compared; a *  means that the amino acids in all sequences are exact matches, : means that the sequences are very similar, .   means the sequences are slightly similar, while no symbol means the sequences are completely different. The table below summarizes the similarities between the alignments.

Table 1:  The similarity scores from comparing the alignments of the preproinsulin sequences produced in figure 1. (Table obtained from ClustalW software)

As shown in table 1, the most similar sequences are the cow and boar. This is plausible because cows and boars are both large mammals with similar lifestyles so most of the sequence is conserved. The largest differences come when the mammals are compared to the non-mammalian jungle foul. However, 65% is still a relatively high conservation. The amino acid sequence of insulin is highly conserved among vertebrates, and insulin from one mammal almost certainly is biologically active in another. Even today, many diabetic patients are treated with insulin extracted from pig pancreas (Bowan, 1999). Both the B and A-chain sequences are highly conserved in mammals and birds, with the exception of those found in the hystricomorph rodents (guinea pig, chinchilla, etc.). Even the fish insulins maintain greater than 50% homology with mammalian insulins (Permutt et al.,1984).

Relationship Between Structure and Activity

 Insulin's two chains are connected by two disulfide bridges. One bridge is found at position 7 on both chains and the other bridge is located at position 20 of chain A and position 19 of chain B. The disulfide bridges are essential for biological activity. In the three-dimensional structure of insulin the A-chain terminal residues are on the surface of the molecule. These residues are invariant and play a large role in the stability, conformation, and activity of the insulin molecule and are therefore always conserved. The C-peptide covers this region in proinsulin which explains the inactivity of proinsulin. The A₂₀ Cys and the A₂₁ Asn are always conserved as well. Any modification to these residues will result in the loss of receptor binding ability and of biological activity (Espinal, 1981). There are also several residues in the B-chain that are equally as important such as B₁ and B₂₉. The removal of eight of the C-terminal residues of the B-chain will also reduce the biological activity of insulin (Talwar et al., 2006). The biological activity of insulin is known to be closely related to these eight C-terminal residues (Shanghai Insulin Research Group, 1973).

Figure 2: The double chain structure of insulin. (Blue = A-chain, Yellow= B-chain, Grey= disulphide bonds)

The above dual chain structure and disulphide bridges are conserved in all species that have been studied which indicates that this structure is a functional requirement for the biological activity of insulin (Espinal, 1981).





*References can be accessed through links on pictures and citations.*

Assignment #1 - Insulin - Hormone Origin, Structure, and Function

Origin

Insulin is a small protein (peptide) hormone that was discovered through the efforts of several Canadian scientists; Banting, Macleod, Collip, and Best in 1921. Insulin is produced by beta cells which are located in the pancreas in clusters called islets of Langerhans. In the formation of insulin, the messenger RNA transcript is translated into an inactive protein called preproinsulin. Preproinsulin contains an amino-terminal signal sequence. This amino-terminal sequence is required for the precursor hormone to be able to pass through the endoplasmic reticulum membrane where it can undergo post-translational processing. The parts of the hormone that are not required for the biologically active hormone are clipped away during the post-translational processing. Once in the endoplasmic reticulum, the preproinsulin signal sequence is proteolytically removed to produce proinsulin. "Once the post-translational formation of three vital disulfide bonds occurs, specific peptidases cleave proinsulin. The final product of the biosynthesis is mature and active insulin. Finally, insulin is packaged and stored in secretory granules, which accumulate in the cytoplasm, until release is triggered" (Cartailler, J.P., 2012). This process is shown in figure 1.

Figure 1: The Synthesis of Insulin from Preproinsulin

 

Structure

Insulin is a 5.8 kDa peptide hormone that consists of two peptide chains (A and B) that contain 51 amino acids in total. The A chain contains 31 amino acids while the B chain contains 20 amino acids (Derewenda et al., 1989). The two peptide chains are linked by three disulphide bonds (Brange et al., 1993). The amino acid sequences as well as the placement of the disulphide bonds can be seen in Figure 2. Although insulin is active as a monomer, during its synthesis and storage it assembles into dimers and to hexamers when zinc is present (Derewenda et al, 1989). The hexamer formation is inactive but is much more stable than the active monomer form (Allison, K., 2010). Being stored as a hexamer allows large amounts of insuling to be readily available. The insulin molecule is synthesized as a single chain proinsulin molecule as mentioned previously. In the proinsulin molecule, an additional C peptide connects the carboxylic end of the B chain with the amino terminus of the A chain. The A chain forms a helical segment followed by a turn and then makes a second helical segment anti-parallel to the first one. The B chain forms a much defined alpha-helix at the N-terminal part, followed by a turn and a beta-sheet (Derewenda et al., 1989). The three dimensional structure of insulin can be seen in figures 3 and 4.

 

Figure 2: The amino acid sequence of insulin chains A and B and the placement of disulphide bonds

 

Figure 3: A structural model of an insulin monomer

 

  

 

Figure 4: A structural model of an insulin hexamer

 

Function 



 The main function of insulin is to maintain glucose homeostasis. Insulin causes muscle and fat to increase their uptake of glucose and inhibits glucose production by the liver making it the main regulator of blood glucose concentration. This is especially important after a meal. When blood glucose levels are elevated, insulin is produced and secreted by the pancreas and causes cells to take in more glucose and store it as glycogen until plasma glucose levels begin to descend. Insulin acts in a negative feed back with another hormone, glucagon. Glucagon has the opposite effects of insulin. When blood glucose levels are too low, glucagon is released from the alpha cells of the pancreas which stimulates gluconeogenesis, the break down of glycogen to glucose, and the release of glucose back into circulation resulting in a rise in blood glucose levels. Figure 5 shows how insulin and glucagon work together to maintain blood glucose levels.
 
 

 Figure 5: The negative feedback loop of  insulin and glucagon

                                                                                                                                        

 

"Insulin increases glucose uptake in cells by stimulating the translocation of the glucose transporter GLUT4 from intracellular sites to the cell surface" (Saltiel et al., 2001). Insulin action is initiated when it binds to and activates its cell-surface receptor. This cell-surface receptor consists of two α subunits and two β subunits that are linked by disulphide bonds to form an α2β2 heterotetrameric complex. Insulin binds to the extracellular α subunits, which then transmits a signal across the plasma membrane to activate the intracellular tyrosine kinase domain of the β subunit. The receptor then undergoes a series of intramolecular transphosphorylation reactions in which one β subunit phosphorylates its adjacent partner on specific tyrosine residues _{(Pessin et. al, 2000).}

"Insulin also stimulates cell growth and differentiation, and promotes the storage of substrates in fat, liver and muscle by stimulating lipogenesis, glycogen and protein synthesis, and inhibiting lipolysis, glycogenolysis and protein breakdown" (Saltiel et al., 2001).The actions of insulin are not completely understood yet and are still being studied.

 

 

 

 

Insulin is used to treat diabetes, an illness that is caused by too little insulin and/or a resistance to insulin. There are two types of diabetes. Type 1 occurs when the pancreas does not produce enough insulin or does not produce any insulin. Daily injections of insulin are required to treat type 1 diabetes. Type 1 is usually diagnosed in children and teens. The insulin can come in the form of a needle or via a pump that attaches to the patients side. Type 2 diabetes is usually caused by a poor diet resulting in high blood glucose levels. The muscle, liver, and fat cells do not respond to insulin the way they should meaning they are stimulated by insulin to uptake glucose. This is known as insulin resistance. Type 2 diabetes is usually diagnosed in adults who are overweight and is treated mostly through a change in diet and exercising to lower blood sugar levels (NCBI, 2012).

 

 

 

 

Figure 6: Healthy insulin producing beta-cells (left)  vs. damaged insulin producing beta-cells (left)

 

 

 

*References can be accessed through links on pictures and citations.*