Some starch‐degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley α‐amylase 1, oligosaccharide is thus bound to the ‘sugar tongs’ site. This site on the non‐catalytic domain C in the C‐terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to β‐cyclodextrin‐Sepharose, a starch‐mimic resin used for α‐amylase affinity purification. The Kd for β‐cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20–0.25 mm for the wild‐type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley α‐amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the ‘sugar tongs’ in the structure. Crystal structures of both wild‐type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the ‘sugar tongs’ site. The ‘sugar tongs’ site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg·mL−1 for the wild‐type enzyme increased to 5.9 mg·mL−1 for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild‐type activity. β‐cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild‐type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the ‘sugar tongs’ participates in multivalent binding of polysaccharide substrates.

Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/␣ collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.The insulin receptor is a disulfide linked heterodimeric protein consisting of two 135-kDa extracellular ␣-subunits and two 95-kDa transmembrane spanning ␤-subunits, containing the intracellular tyrosine kinase domain that is activated upon ligand binding (1, 2). Binding of insulin to the receptor results in a wide range of metabolic and mitogenic responses initially mediated by phosphorylation of tyrosines on several intracellular protein substrates, including insulin receptor substrates (IRS) 2 1 and 2 and Shc (3). IRS1 and IRS2 are the major insulin receptor substrates leading to glucose homeostasis and have distinct and overlapping roles in diverse organs. Furthermore alterations in both IRS1 and IRS2 have been shown to be strongly involved in the development of diabetes mellitus (4, 5). The finding of specific functions of IRS1 and IRS2 in the murine pre-muscle cell line (L6 cells) are summarized by Thirone et al.(6) but the relative contributions of IRS1 and IRS2 to insulin signaling and to the development of insulin resistance is not yet conclusive. The majority of the published literature in this field suggests that IRS1 is the major substrate leading to stimulation of glucose transport in muscle and adipose tissues, whereas in liver IRS1 and IRS2 have complementary roles in insulin signaling and metabolism. In contrast Shc does not appear to be directly involved in metabolic signaling of insulin but plays a critical role in insulin-induced mitogenesis (6, 7). Both IRS and Shc initiate the mitogenactivated pathway kinase/extracellular-regulated kinase (MAPK/ERK) pathway, including activation of ERK1/2 i...

BackgroundGene expression alterations have previously been associated with type 2 diabetes, however whether these changes are primary causes or secondary effects of type 2 diabetes is not known. As healthy first degree relatives of people with type 2 diabetes have an increased risk of developing type 2 diabetes, they provide a good model in the search for primary causes of the disease.Methods/Principal FindingsWe determined gene expression profiles in skeletal muscle biopsies from Caucasian males with type 2 diabetes, healthy first degree relatives, and healthy controls. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 Arrays covering the entire human genome. These arrays have not previously been used for this type of study. We show for the first time that genes involved in insulin signaling are significantly upregulated in first degree relatives and significantly downregulated in people with type 2 diabetes. On the individual gene level, 11 genes showed altered expression levels in first degree relatives compared to controls, among others KIF1B and GDF8 (myostatin). LDHB was found to have a decreased expression in both groups compared to controls.Conclusions/SignificanceWe hypothesize that increased expression of insulin signaling molecules in first degree relatives of people with type 2 diabetes, work in concert with increased levels of insulin as a compensatory mechanism, counter-acting otherwise reduced insulin signaling activity, protecting these individuals from severe insulin resistance. This compensation is lost in people with type 2 diabetes where expression of insulin signaling molecules is reduced.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.