Single-Chain Insulins as Receptor Agonists

Abstract

Single chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. While direct linkage of insulin Band A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by radioimmunoassay with a (proinsulin insulin)-specific polyclonal antibody. While dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Argflanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling — stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes — with only limited cross-reactivity on IGF1 receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes. The MOR-1 Opioid Receptor Regulates Glucose Homeostasis by Modulating Insulin Secretion Ting Wen, Bonnie Peng, and John E. Pintar (Mol Endocrinol, published February 12, 2009, 10.1210/me.2008-0345) ABSTRACT In addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G YIn addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G Y