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International Textbook of Diabetes Mellitus, 4th Ed., Excerpt #28: Insulin Gene Expression and Biosynthesis Part 4 of 6

DeFronzoCoverInsulin biosynthesis

Introduction

The previous section outlines that insulin gene transcription is a highly controlled process. The product of this process, pre-proinsulin mRNA, is unusually stable in pancreatic β cells and it is further stabilized as glucose concentrations increase [37]. As such, there is normally an abundant source of preproinsulin mRNA in the β-cell cytosol available for translation. Actually, it is the specific regulation of preproinsulin mRNA translation that is the predominant control mechanism for insulin production in the β cell under normal circumstances. This enables the β cell to rapidly replenish insulin stores back to optimal levels, after they have been depleted by stimulated insulin secretion, and is more economic energy-wise to the β cell, since translational control of insulin production bypasses the need for insulin gene transcription and preproinsulin mRNA maturation.

Structure of the insulin molecule

The primary structure A- and B-chain of insulin itself has been known for close to 50 years [88]. However, it was not until at least 10 years after this discovery that it was realized insulin is actually synthesized as a single polypeptide chain precursor molecule, preproinsulin (Figure 6.3) [88]. The N-terminal signal peptide (24 amino acids) is cleaved cotranslationally to yield proinsulin. The proinsulin molecule is a 12-kDa single chain polypeptide that encompasses the B-chain (30 amino acids) and the A-chain (21 amino acids) of insulin joined by the connecting peptide, C-peptide (Figure 6.3). Proinsulin to insulin conversion occurs by cleavage at two dibasic amino acids sequences by the B-chain/C-peptide and C-peptide/A-chain junctions to release the C-peptide moiety yielding the insulin molecule with the two independent disulphide-linked A- and B-chains correctly aligned [88].

ITDMFig6.3

Proinsulin biosynthesis: translation and translocation

Essentially, translation of preproinsulin mRNA to preproinsulin protein occurs in a fashion typical of most eukaryotic mRNAs destined to enter the cell’s secretory pathway [88–90]. During the translation process, the emerging signal sequence of pre- proinsulin binds the signal recognition particle (SRP) that then docks to the SRP-receptor, which is an integral ER membrane protein. This locates the preproinsulin mRNA/ribosomal translational complex to the ER, which is the major site of proinsulin biosynthesis in the β cell. As SRP binds to the SRP receptor, the nascent signal peptide of the newly forming preproinsulin dissociates and is transferred to another ER integral membrane protein, the signal sequence receptor (SSR). SSR is part of a “translocation pore” that facilitates transport of the newly forming preproinsulin polypeptide across the ER membrane into the ER lumen, marking the entrance of the newly synthesized preproinsulin into the β-cell’s secretory pathway. The signal peptide is cleaved by another RER protein, the signal peptidase, resulting in the nascent proinsulin molecule located to the ER lumen. There, the proinsulin molecule undergoes appropriate folding, assisted by the molecular chaperons and formation of disulfide bonds catalyzed by ER disulfide isomerase activity [88].

Proinsulin biosynthesis: effectors

and stimulus-response coupling mechanisms Proinsulin biosynthesis is translationally controlled by certain nutrients, neurotransmitters, and hormones, but glucose is the most physiologically relevant [91]. This translational control response to glucose is rapid. Significant glucose-induced proinsulin biosynthesis can be observed after a 20 – 30-min lag period that reaches a maximum rate (∼20 – 30-fold increase above basal) by 60 min [91]. Of the peptide hormones that stimulate proinsulin biosynthesis, perhaps the incretins, GLP-1 and GIP, are the most physiologically relevant. GLP-1/GIP do not increase proinsulin biosynthesis translation in their own right, but potentiate glucose-induced proinsulin biosynthesis as they do glucose-induced insulin secretion [92]. Epinepherine is also worthy of mention for specifically inhibiting glucose-induced proinsulin biosynthesis as it does glucose-induced insulin secretion [91].

Unlike that for glucose-induced insulin secretion, the secondary signals that lead to an increase in proinsulin biosynthesis are less well defined. Glucose metabolism is required for glucose-induction of both proinsulin biosynthesis and insulin secretion, but several lines of evidence indicate that the stimulus-response coupling mechanism for these β-cell functions are quite distinct [91,92]. For example, sulfonylureas stimulate and diazoxides inhibit glucose-induced insulin secretion, but these compounds have no effect on glucose-induced proinsulin biosynthesis indicating the signal transduction mechanism for regulating proinsulin biosynthesis is independent of the KATP-channel and increase in cytosolic Ca2+, unlike that for glucose-stimulated insulin secretion [91,92]. Somatostatin is a potent inhibitor of insulin secretion, but has no effect on proinsulin biosynthesis [91,92]. Long-chain fatty acids, when applied acutely, are marked potentiators of glucose-induced insulin secretion, but if anything modestly inhibit glucose-induced proinsulin biosynthesis [91,92]. It has been indicated that a secondary signal for glucose-induced proinsulin biosynthesis might be mitochondrial export of succinate and cytosolic accumulation of succinyl-CoA, but what lies downstream of succinate/ succinyl-CoA to specifically upregulate preproinsulin mRNA translational machinery requires further investigation.

Proinsulin biosynthesis: translational control mechanism

Glucose modestly increases general protein synthesis in the β cell ∼1.5–2-fold. However, the effect of glucose on proinsulin synthesis translation is much greater, and can reach ≥10-fold stimulation above basal [91,92]. This indicates a specific effect of glucose on translational control of proinsulin biosynthesis. Such specific control of glucose-induced proinsulin biosynthesis in the β cell, resides in cis-elements in the 5′- and 3′-untranslated regions (UTRs) of preproinsulin mRNA itself [92]. In the 3′ -UTR of preproinsulin mRNA, just downstream of the polyadenylation signal, there is a highly conserved primary sequence containing a UUGAA cis-element core, that has been reported to be involved in glucose-regulated preproinsulin mRNA stability in addition to the pyrimidine-rich sequence (insPRS) [37,92] (see earlier). There is some degree of cooperativity between the preproinsulin mRNA 5′ – and 3′ -UTRs for the specific glucose-induced translational control of proinsulin biosynthesis, but it appears that the 5′-UTR preproinsulin mRNA has the major influence [92]. There is also a conserved cis-element that is required for glucose-induced translational control of proinsulin biosynthesis, named ppIGE (for preproinsulin glucose element) [92]. The ppIGE has a highly conserved ppIGE palindromic core of GUCxn CUG or GUUxn UUG (where n ≤ 4 bases). A cytosolic protein trans-acting factor (ppIE-BP) binds to this translational control ppIGE cis-element of preproinsulin mRNA in a glucose-dependent manner, but the identity of the ppIGE-BP has yet to be revealed [92]. It should be noted that proinsulin is only one of a small subset of β-cell proteins (∼50 in all) [92] whose biosynthesis is regulated by glucose at the translational level. These are mostly β-granule proteins, including the proinsulin processing endopeptidases, PC2 and PC1/3 [92]. Indeed, the ppIGE is also conserved in the 5′-UTR of the majority insulin secretory granule proteins’ mRNAs, and thus, the glucose-induced specific translational control of proinsulin biosynthesis and that for insulin secretory granules can be coordinated and is the principal control mechanism for insulin secretory biogenesis in β cells [92]. The glucose-induced translational control of the proinsulin processing endopeptidases, proPC2 and proPC3, provides a means whereby proinsulin conversion is not compromised upon increased proinsulin biosynthesis [92].

 

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