Introduction

Insulin is synthesized by the b cells of the pancreatic islets as part of a single 110-amino acid precursor, preproinsulin (see Fig. 1). Processing is initiated by removal of the amino terminal, 24-amino acid signal sequence (1). The resulting 86-amino acid product folds through the formation of three disulfide bridges between Cys7-Cys72, Cys19-Cys85, and Cys71-Cys76 to produce the prohormone, proinsulin. Insulin and C-peptide are produced when endopeptidases, prohormone convertases 2 and 3 (PC2 and PC3, respectively), cleave proinsulin at two paired basic amino acid sites, Lys64-Arg65 and Arg31-Arg32 (see Fig. 1). The basic amino acid pairs are then removed from each site by carboxypeptidase H (3). Proinsulin amino acids 66-86 and 1-30 comprise the A- and B- chains, respectively, of mature insulin (see Fig. 1). "Split" proinsulin 65-66 and 32-33 are produced when cleavage is incomplete and the basic amino acid pairs are not removed from the cleavage site. "Des" proinsulin 64-65 and 31-32 are produced when cleavage is incomplete and the basic amino acid pairs are removed from the cleavage site (4). In the rat, two separate 110-amino acid preproinsulins are transcribed from two nonallelic preproinsulin genes, from which two forms of insulin and C-peptide are subsequently cleaved (1) (see Fig. 1). The mouse synthesizes two molecular forms of insulin and C-peptide, which are identical to their respective rat counterparts (5). The two rodent insulins, designated insulin I and II, are present at a ratio of1:3 in the mouse and 4: 1 in the rat (insulin I: II) (6).

B Chain

EPKPAQAFVKQHLCGPHLVEALYLVCGERGFF

A Chain

KRGIVDQCCTSICSLYQLENYCN S

69 K

KRGIVDQCCTSICSLYQLENYCN S

- Q

R

3 R

R

S A

E -

S V

V £

ELALTQLDG

A E P G G G

LELQPVQPDE

55 52

49

42 41 40 21

C Peptide

Fig. 1. Amino acid sequence of rat preproinsulin I. The superscripts indicate positions where amino acid differences exist in rat preproinsulin II and/or human preproinsulin relative to rat preproinsulin I. Mature rat insulin I and II are identical except that Ser for Pro9 and Met for Lys29 substitutions are incorporated into the B-chain of rat insulin II. Relative to rat insulin I, mature human insulin contains substitutions of Asn, Ser, Thr, and Glu for Lys3, Pro9, Ser30 and Asp69, respectively. (The rat sequence data are from ref. 1; the human data are from ref. 2.

We describe in this chapter two examples of assessment of insulin secretion in the mouse: (1) measurement of insulin in the portal vein effluent during perfusion of the mouse pancreas in situ and (2) determination of plasma insulin in mice undergoing intraperitoneal glucose tolerance testing. Each example includes details of the sample acquisition and subsequent assay of insulin in these samples.

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