Insulin is a protein hormone that is critical for reducing the level of glucose

Question

Insulin is a protein hormone that is critical for reducing the level of glucose in the blood. Insulin is produced by beta-cells in the pancreas and the expression of the insulin gene is controlled by three regulatory transcription factors that are specific to the insulin gene. In addition, high levels of glucose in the blood increase the production of pre-mRNA molecules from the insulin gene.

a. Explain how the three regulatory transcription factors might be involved in regulating expression of the insulin gene only in pancreatic beta-cells.

b. Do you expect that glucose is acting as an enhancer, a transcription factor, or an effector molecule in this gene regulation system? Explain your answer.

3. In a healthy person, protein M is produced at moderate levels. But when a person has NON-M disease they produce lower levels of protein M. Is it the regulatory sequence or protein coding sequence of the gene that is most likely to be defective in this person? Explain your answer briefly.

Explanation / Answer

2 a.Insulin is a 51 amino acid protein that is secreted by the beta cells of Islets of Langerhans in pancreas. It is synthesized as a 110 amino acid preproprotein that is translocated to ER. IN ER, the signal peptide is cleaved. The proprotein is then folded and enters the Golgi, where it is cleaved to form insulin. Insulin is then secreted by packaging in secretory vesicles that fuse with plasma membrane. Insulin has three enhancer elements, A, C, E present in its core binding motifs.

MafA, PDX-1, and NeuroD are three transcription factors that regulate glucose dependent insulin secretion. Duodenal homeobox-1(PDX-1) interacts with insulin gene through a central domain recognition motif (TAAT core in A3 element). This factor is specific for insulin A element. As a result, the PDX-1 transcription factor binds to A3 element enhancer region and will enhance transcription of insulin.

The Insulin promoter element 3b1 (IPE3b1) and 3b2 (IPE3b2) form DNA binding complexes with C1 element present in the insulin promoter. IPE3b1 is also known as MafA in rat that is expressed only in insulin beta cells. Thus, MafA enhances transcription of insulin gene.

E elements (5-GCCATCTG-3) are two separated mini-enhancer units within the insulin enhancer. A helix-loop-helix binding domain in BETA2/NeuroD allows it to bind the E element and thereby increase transcription.

All these transcription factors bind the insulin promoter elements when glucose is high in blood.

2b. Insulin mRNA stability is maintained by glucose.

Glucose promotes the binding of PDX-1 to the A3 site as well as increases PDX-1 trans-activating potency. Glucose promotes translocation and modification of a cytoplasmic, inactive PDX-1 to the nuclear, active PDX-1, possibly via posttranslational phosphorylation and modification by O-linked N-acetylglucosamine or small ubiquitin-related modifier 1. Binding and expression of MafA and NeuroD is enhanced by glucose. Glucose mediates translocation of Neuro D1 by O-linked glycosylation. Glucose stimulates transcription and translation of MafA protein. These three factors act synergistically in response to glucose stimulation.

3. The non-M disease person is producing lower levels of M protein. The protein is still being produced, albeit at lower levels. If the mutation or defect were in the protein-coding region, no protein or a mutant protein that was shorter in length would have been synthesized. As functional protein is still being synthesized, the defect should be in the regulatory sequence. Most likely the regulatory sequence is modified such that enhances can no longer bind to it or a negative regulator binds more efficiently to affect protein levels.

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