Insulin proinsulin

Clinical Utility of Measuring Insulin, Proinsulin, C-Peptide, and Glucagon... 

Box 25-1 lists the clinical conditions in which hormones that regulate glucose, namely insulin, proinsulin, C-peptide, and glucagon, have been measured. Although there is interest in the possible clinical value of measurement of the concentrations of insulin and its precursors, the assays are useful primarily for research purposes. There is no role for routine testing for insulin, proinsulin, or C-peptide in patients with diabetes mellitus. It must be emphasized that the diagnostic criteria for diabetes mellitus do not include measurements of hormones, which remain predominantly research tools. 

A stable isotope dilution assay using mass spectrometry to measure insulin, proinsulin, and C-peptide has been developed. The difference in mass among the three analytes allows specific measurement of each protein. Comparison of patient samples revealed that most, but not all, results were higher by immunoassay than mass spectrometry. Thus immunoassays may overestimate insulin, particularly at low concentrations. The high protein concentration in the serum requires extraction of proteins (e.g., by immunoaffinity) and purification by high-performance liquid chromatography (HPLC) before quantification by mass spectrometry. This method is not suitable for routine laboratory analysis. 

Zn + is released when insulin is secreted. Conversion of proinsulin to insulin in the secretory granule is not complete and some proinsulin is also released upon secretion of insulin. Proinsulin has less than 5% of the biological activity of insulin. The C-peptide has no physiological function but assay of C-peptide helps distinguish between endogenous and exogenous sourees of insulin. 

Fig 26.10. Cleavage of proinsulin to insulin. Proinsulin is converted to insulin by proteolytic cleavage, which removes the C-peptide and a few additional amino acid residues. Cleavage occurs at the arrows. From Murray RK, et al. Harper s Biochemistry, 23rd Ed. Stanford, CT Appleton Lange, 1993 560. 

CK8 +, CK18 +, CK19 +, Chromogranin +, NSE +, PGP9.5 +, Synaptophysin +, Leu7 +, SlOO +/—, CK5/6 -, CK7 -, CK20 -Proliferation index [Ki-67 (MIB-1) index] in well differentiated neuroendocrine tumor <2% Proliferation index [Ki-67 (MIB-1) index] in well differentiated neuroendocrine carcinoma >2% Proliferation index ] Ki-67 (MIB-1) index] in poorly differentiated neuroendocrine carcinoma >30% Insulin +, Proinsulin +... 

Figure 7 shows a schematic comparison of insulin, proinsulin, IGF, and relaxin. 

Desmethylcyproheptadine 10,11-epoxide, a biotransformation product of the antihistaminic and antiserotoninergic drug, cyproheptadine inhibited proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets (Chow et al. 1989). Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of H-leucine showed that desmethylcyproheptadine epoxide, desmethylcyproheptadine and cyproheptadine epoxide were 22,10, and 4 times, respectively, more potent than cyproheptadine in inhibiting hormone synthesis. In man, there was no evidence for metabolic alteration at the tricyclic ethylene bridge (C-10, C-11), whereas dog, cat, and rat all metabolise the drug, at least in part, at this site (Porter et al. 1974). A minor N-oxide conjugate as a metabolite of cyproheptadine has been found in man (Johnson et al. 1962). 

Recovery nd Purifica.tion. The production of EH Lilly s human insulin requires 31 principal processing steps of which 27 are associated with product recovery and purification (13). The production process for human insulin, based on a fermentation which yields proinsulin, provides an instmctive case study on the range of unit operations which must be considered in the recovery and purification of a recombinant product from a bacterial fermentation. Whereas the exact sequence has not been pubUshed, the principle steps in the purification scheme are outlined in Figure la. 

Insulin and Amylin. Insulin is a member of a family of related peptides, the insulin-like growth factors (IGFs), including IGF-I and IGF-II (60) and amylin (75), a 37-amino acid peptide that mimics the secretory pattern of insulin. Amylin is deficient ia type 1 diabetes meUitus but is elevated ia hyperinsulinemic states such as insulin resistance, mild glucose iatolerance, and hypertension (33). Insulin is synthesized ia pancreatic P cells from proinsulin, giving rise to the two peptide chains, 4. and B, of the insulin molecule. IGF-I and IGF-II have stmctures that are homologous to that of proinsulin (see INSULIN AND OTHER ANTIDIABETIC DRUGS). 

FIGURE 15.3 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges. 

INSULIN. Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin, an important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin (Figure 15.3). 

Figure 42-12. Structure of human proinsulin. Insulin and C-peptide molecules are connected at two sites by dipeptide links. An initial cleavage by a trypsin-like enzyme (open arrows) followed by several cleavages by a car-boxypeptidase-like enzyme (solid arrows) results in the production of the heterodimeric (AB) insulin molecule (light blue) and the C-peptide.

A small number of proteins, and again insulin is an example, are synthesized as pro-proteins with an additional amino acid sequence which dictates the final three-dimensional structure. In the case of proinsulin, proteolytic attack cleaves out a stretch of 35 amino acids in the middle of the molecule to generate insulin. The peptide that is removed is known as the C chain. The other chains, A and B, remain crosslinked and thus locked in a stable tertiary stiucture by the disulphide bridges formed when the molecule originally folded as proinsulin. Bacteria have no mechanism for specifically cutting out the folding sequences from pro-hormones and the way of solving this problem is described in a later section. 

The IGFs (also termed somatomedins ), constitute a family of two closely related (small) polypeptides IGF-I and IGF-II. As the names suggest, these growth factors bear a strong structural resemblance to insulin (or, more accurately, proinsulin). Infusion of IGF-I decreases circulating... 

Proinsulin is proteolytically processed in the coated secretory granules, yielding mature insulin and a 34-amino acid connecting peptide (C peptide, Figure 11.1). The C peptide is further proteolytically modified by removal of a dipeptide from each of its ends. The secretory granules thus contain low levels of proinsulin, C peptide and proteases, in addition to insulin itself. The insulin is stored in the form of a characteristic zinc-insulin hexamer, consisting of six molecules of insulin stabilized by two zinc atoms. 

Figure 11.1 Proteolytic processing of proinsulin, yielding mature insulin, as occurs within the coated secretory granules...

Although a high degree of homology is evident between insulins from various species, the same is not true for proinsulins, as the C peptide sequence can vary considerably. This has therapeutic implications, as the presence of proinsulin in animal-derived insulin preparations can potentially elicit an immune response in humans. 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

FIGURE 18-12 The/af//af mutation in carboxypeptidase E (CPE) leads to secretion of proinsulin, not mature insulin, and results in diabetes. The S202P mutation within CPE results in degradation of the enzyme and defective insulin processing in the fat/fat heterozygous mouse. LDCV, large dense-core vesicle. 

P cells of the pancreatic islets in combination with atoms of zinc, but when required to regulate blood glucose concentration, the prohormone is cleaved and functional insulin is released into the circulation along with the C-peptide. This example of post-translational processing is mediated by peptidases which are contained in the vesicles along with the proinsulin. The fusion of the secretory vesicles with the cell membrane and activation of the peptidase prior to exocytosis of the insulin are prompted by an influx of calcium ions into the P-cell in response to the appropriate stimulus. Similarly, catecholamines are synthesized and held within the cell by attachment to proteins called chromogranins. 

Structurally insulin is a small peptide, with a molecular mass of around 5500 and composed of two subunits, denoted a and (3 chains. Insulin is synthesized as a single peptide, Proinsulin and stored within the pancreatic p-cells. At the moment of secretion, pro-insulin is cleaved, releasing C-peptide and functional insulin in to the blood circulation (Figure 4.22). 

Yamamoto A, Hayakawa E, Lee VH (1990) Insulin and proinsulin proteolysis in mucosal homogenates of the albino rabbit Implications in peptide delivery from nonoral routes. Life Sci 47 2465-2474... 

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