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Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimulate in vivo processes) revealed (a) a rapid decline in proinsulin concommitant with formation of conversion intermediates, (b) formation of des-Arg31, Arg32-proinsulin and des-Lys64,Arg65-proinsulin in the ratio 3.3:1 at steady state, and (c) complete conversion of the precursor to insulin during extended incubation. Studies on normal human pancreas identified a similar ratio of des-Arg31,Arg32-proinsulin to des-Lys64,Arg65-proinsulin (approximately 3:1), whereas two insulinomas contained sizable amounts of des-Arg31,Arg32-proinsulin, but barely detectable amounts of des-Lys64,Arg65-proinsulin. None of the tissues contained measurable quantities of Arg32/Glu33- or Arg65/Gly66-split proinsulin. Analysis of plasma from three diabetic subjects managed by the intravenous infusion of human proinsulin revealed less than 1% processing of the circulating precursor to conversion intermediates and no processing of the precursor to human insulin. Nevertheless, analysis of plasma from the same subjects managed by the subcutaneous infusion of proinsulin revealed 4-11% processing of the precursor to intermediates that had the properties of des-Arg31,Arg32-proinsulin and Arg65/Gly66-split proinsulin. We conclude that (a) processing of proinsulin to insulin in vivo as in vitro likely occurs by preferential cleavage at the Arg32-Glu33 peptide bond in proinsulin, (b) proinsulin is inefficiently processed in the vascular compartment, and (c) subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
March 1991,
Diabetologia,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
July 1988,
The Journal of clinical endocrinology and metabolism,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
March 1996,
Clinica chimica acta; international journal of clinical chemistry,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
March 1984,
The Journal of clinical investigation,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
November 1988,
Diabetologia,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
December 1991,
Clinical biochemistry,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
July 1991,
Journal of chromatography,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
August 1997,
Pancreas,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
January 1991,
Cell biophysics,
B D Given, and
R M Cohen, and
S E Shoelson, and
B H Frank, and
A H Rubenstein, and
H S Tager
October 1988,
The Biochemical journal,
