Molecular engineering of safe and efficacious oral basal insulin.

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.

Cross-species reactive monoclonal antibodies against the extracellular domains of the insulin receptor and IGF1 receptor.

Translation across species of immunoassay results is often challenging due to the lack of cross-species reactivity of antibodies. In order to investigate the biology of insulin and IGF1 receptors, we generated new versatile monoclonal assay antibodies using the extracellular domain of the insulin/IGF1 hybrid receptor as the bait protein in the Adimab yeast antibody discovery platform and as the antigen in a rabbit monoclonal antibody platform. The resulting antibody clones were screened for receptor specificity as well as cross-species reactivity to both tissue and cell line derived samples. Using these strategies, we were able to identify highly specific insulin receptor monoclonal antibodies that lack cross-reactivity to the IGF1 receptor using the Adimab platform and a highly specific IGF1 receptor monoclonal antibody that lacks cross-reactivity to the insulin receptor using the rabbit antibody platform. Unlike earlier monoclonal antibodies reported in the literature, these antibodies show cross-species reactivity to the extracellular domains of mouse, rat, pig, and human receptors, indicating that they bind conserved epitopes. Furthermore, the antibodies work well in several different assay formats, including ELISA, flow cytometry, and immunoprecipitation, and therefore provide new tools to study insulin and IGF1 receptor biology with translation across several species and experimental model systems.

Elucidating the Biological Roles of Insulin and Its Receptor in Murine Intestinal Growth and Function.

The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models.

Quantitative Proteomics of Intestinal Mucosa From Male Mice Lacking Intestinal Epithelial Insulin Receptors.

The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by liquid chromatography mass spectrometry was applied to jejunal and colonic mucosa from mice fed a normal chow diet and mice fed a Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell lineages: Paneth, goblet, absorptive, and enteroendocrine cells. Only goblet and absorptive cells were affected in the colon. Also, a marked effect of WD consumption was found on the gut proteome. A substantial reduction was detected in Paneth cell proteins with antimicrobial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence 1C-3 and -2, α-defensin 17, and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase, apolipoprotein A-II, and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice compared with controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and the expression of proteins of the P-type adenosine triphosphatases and the solute carrier-transporter family was reduced in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell specificity in the intestinal epithelium.

Treatment with insulin analog X10 and IGF-1 increases growth of colon cancer allografts.

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.

Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation.

Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr³°8 on Akt (p-Akt-Thr³°8), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr³°8 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser47³ or IRS-1-PI3K activity. Furthermore, p-Akt-Thr³°8 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for "fine-tuning" insulin-mediated GS activation in humans.

Stimulation of MC38 tumor growth by insulin analog X10 involves the serine synthesis pathway.

Recent evidence suggests that type II diabetes is associated with increased risk and/or aggressive behavior of several cancers, including those arising from the colon. Concerns have been raised that endogenous hyperinsulinemia and/or exogenous insulin and insulin analogs might stimulate proliferation of neoplastic cells. However, the mechanisms underlying possible growth-promoting effects of insulin and insulin analogs in cancer cells in vivo, such as changes in gene expression, are incompletely described. We observed that administration of the insulin analog X10 significantly increased tumor growth and proliferation in a murine colon cancer model (MC38 cell allografts). Insulin and X10 altered gene expression in MC38 tumors in a similar fashion, but X10 was more potent in terms of the number of genes influenced and the magnitude of changes in gene expression. Many of the affected genes were annotated to metabolism, nutrient uptake, and protein synthesis. Strikingly, expression of genes encoding enzymes in the serine synthesis pathway, recently shown to be critical for neoplastic proliferation, was increased following treatment with insulin and X10. Using stable isotopic tracers and mass spectrometry, we confirmed that insulin and X10 increased glucose contribution to serine synthesis in MC38 cells. The data demonstrate that the tumor growth-promoting effects of insulin and X10 are associated with changes in expression of genes involved in cellular energy metabolism and reveal previously unrecognized effects of insulin and X10 on serine synthesis.

Agonism and antagonism at the insulin receptor.

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B'29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1-10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints ((3)H-thymidine incorporation), and not on metabolic endpoints ((14)C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity ((3)H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B'29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.

Insulin therapy in diabetes and cancer risk: current understanding and implications for future study: proceedings from a meeting of a European Insulin Safety Consensus Panel, convened and sponsored by Novo Nordisk, held Tuesday October 5, 2010 at The Radisson Edwardian Heathrow Hotel, Hayes, Middlesex, UK.

INTRODUCTION: Interest in the possibility of certain insulin treatments having the potential to modify cancer development and prognosis was reawakened in 2009, following publication of several epidemiological studies addressing this issue. This interest extends to how diabetes itself and cancer might be linked, and makes desirable an exchange of expert views and knowledge to enhance understanding in this subject among those treating diabetes and cancer, or those developing diabetes therapies. METHODS: A European meeting was convened with participants invited based on known relevant interests in endocrinology, oncology, epidemiology, and insulin analog design and investigation. Experts in these fields were invited to present on relevant topics, with open discussions held after each presentation. RESULTS: Concern over the potential mitogenic properties of certain insulin analogs has arisen from some (but not all) epidemiological studies, although confounding factors render interpretation controversial. Future epidemiological studies are likely to strengthen confidence in drawing conclusions. Meanwhile, pharmacological studies, and a consideration of cancer pathophysiology, implicate increased insulin-like growth factor-1 receptor affinity, and/or deranged insulin receptor interaction/signaling properties as possible a priori causes for concern with some insulin analogs. Again, interpretation of the body of pharmacological evidence is confounded by the array of test systems and methodologies used, and by studies frequently succumbing to methodological pitfalls. Reassuringly, most available insulin analogs do not differ in their receptor interaction response profile to human insulin, and for those that do there are reasons to question any potential clinical relevance. Nevertheless, it is desirable that new experimental models are devised that can better determine the likely clinical consequences of any variance in receptor response profile versus human insulin. CONCLUSION: More data are required to increase our understanding of this issue. To facilitate and disseminate such understanding, close cooperation and communication between diabetologists, epidemiologists, oncologists, and insulin engineers will be essential.

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.

Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin.

Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by (3)H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as (3)H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing.

Impaired insulin activation and dephosphorylation of glycogen synthase in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment.

CONTEXT: Insulin resistance is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). The molecular mechanisms underlying reduced insulin-mediated glycogen synthesis in skeletal muscle of patients with PCOS have not been established. SUBJECTS AND METHODS: We investigated protein content, activity, and phosphorylation of glycogen synthase (GS) and its major upstream inhibitor, GS kinase (GSK)-3 in skeletal muscle biopsies from 24 PCOS patients (before treatment) and 14 matched control subjects and 10 PCOS patients after 16 wk treatment with pioglitazone. All were metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry. RESULTS: Reduced insulin-mediated glucose disposal (P < 0.05) was associated with a lower insulin-stimulated GS activity in PCOS patients (P < 0.05), compared with controls. This was, in part, explained by absent insulin-mediated dephosphorylation of GS at the NH2-terminal sites 2+2a, whereas dephosphorylation at the COOH-terminal sites 3a+3b was intact in PCOS subjects (P < 0.05). Consistently, multiple linear regression analysis showed that insulin activation of GS was dependent on dephosphorylation of sites 3a+3b in women with PCOS. No significant abnormalities in GSK-3alpha or -3beta were found in PCOS subjects. Pioglitazone treatment improved insulin-stimulated glucose metabolism and GS activity in PCOS (all P < 0.05) and restored the ability of insulin to dephosphorylate GS at sites 2 and 2a. CONCLUSIONS: Impaired insulin activation of GS including absent dephosphorylation at sites 2+2a contributes to insulin resistance in skeletal muscle in PCOS. The ability of pioglitazone to enhance insulin sensitivity, in part, involves improved insulin action on GS activity and dephosphorylation at NH2-terminal sites.

L-glutamate and glutamine improve haemodynamic function and restore myocardial glycogen content during postischaemic reperfusion: A radioactive tracer study in the rat isolated heart.

1. L-Glutamate and glutamine have been suggested to have cardioprotective effects. However, the issue is controversial and the metabolic mechanisms underlying a beneficial effect are not well understood. 2. In the present study we investigated the effects of L-glutamate and glutamine on haemodynamic recovery, the rate of de novo glycogen synthesis and myocardial glucose uptake during postischaemic reperfusion. 3. Hearts from male Wistar rats (250-300 g) were divided into three groups as follows: (i) control (n = 12); (ii) L-glutamate (n = 12); and (iii) glutamine (n = 12). Hearts were mounted in a Langendorff preparation and perfused with oxygenated Krebs'-Henseleit solution at 80 mmHg and 37C. Global ischaemia for 20 min was followed by 15 min reperfusion, during which L-glutamate (50 mmol/L) or glutamine (20 mmol/L) were administered. Left ventricular developed pressure (LVDP), de novo synthesis of glycogen using [14C]-glucose and myocardial glucose uptake using D-[2-3H]-glucose were measured. 4. L-Glutamate and glutamine increased postischaemic LVDP (P < 0.01 vs control hearts for both). L-Glutamate and glutamine increased de novo glycogen synthesis by 78% (P < 0.001) and 55% (P < 0.01), respectively. At the end of reperfusion, total myocardial glycogen content was increased by both L-glutamate and glutamine (5.7 +/- 0.3 and 6.2 +/- 0.7 micromol/g wet weight, respectively; P < 0.05 and 0.01, respectively) compared with that in control hearts (3.6 +/- 0.4 micromol/g wet weight). Neither L-glutamate nor glutamine affected myocardial glucose uptake during reperfusion. 5. Improved postischaemic haemodynamic recovery after L-glutamate and glutamine supplementation during reperfusion is associated with increased de novo glycogen synthesis, suggesting a favourable modulation of intracellular myocardial carbohydrate metabolism.

Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes.

In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites, but the mechanism involved in human muscle and the defect in type 2 diabetes remain unclear. We studied the effect of insulin at physiological concentrations on glucose metabolism, insulin signaling and phosphorylation of GS in skeletal muscle from type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact. Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 + 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase in the phosphorylation of sites 2 + 2a. This phosphorylation abnormality likely caused the impaired GS activation and glucose storage, thereby contributing to skeletal muscle insulin resistance, and may therefore play a pathophysiological role in type 2 diabetes.