Abstract Insulin icodec is a once-weekly insulin analogue that has a long half-life of approximately 7 days, making it suitable for once weekly dosing. The Insulin icodec molecule was developed based ...on the hypothesis that lowering insulin receptor affinity and introducing a strong albumin-binding moiety would result in a long insulin half-life, provided that non-receptor-mediated clearance is diminished. Here, we report an insulin clearance mechanism, resulting in the splitting of insulin molecules into its A-chain and B-chain by a thiol–disulphide exchange reaction. Even though the substitutions in insulin icodec significantly stabilise insulin against such degradation, some free B-chain is observed in plasma samples from minipigs and people with type 2 diabetes. In summary, we identify thiol–disulphide exchange reactions to be an important insulin clearance mechanism and find that stabilising insulin icodec towards this reaction significantly contributes to its long pharmacokinetic/pharmacodynamic profile.
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.
Mice genetically deficient in the glucagon receptor (Gcgr(-/-)) show improved glucose tolerance, insulin sensitivity, and α-cell hyperplasia. In addition, Gcgr(-/-) mice do not develop diabetes after ...chemical destruction of β-cells. Since fibroblast growth factor 21 (FGF21) has insulin-independent glucose-lowering properties, we investigated whether FGF21 was contributing to diabetes resistance in insulin-deficient Gcgr(-/-) mice. Plasma FGF21 was 25-fold higher in Gcgr(-/-) mice than in wild-type mice. FGF21 was found to be expressed in pancreatic β- and α-cells, with high expression in the hyperplastic α-cells of Gcgr(-/-) mice. FGF21 expression was also significantly increased in liver and adipose tissue of Gcgr(-/-) mice. To investigate the potential antidiabetic actions of FGF21 in insulin-deficient Gcgr(-/-) mice, an FGF21-neutralizing antibody was administered prior to oral glucose tolerance tests (OGTTs). FGF21 neutralization caused a decline in glucose tolerance in insulin-deficient Gcgr(-/-) mice during the OGTT. Despite this decline, insulin-deficient Gcgr(-/-) mice did not develop hyperglycemia. Glucagon-like peptide 1 (GLP-1) also has insulin-independent glucose-lowering properties, and an elevated circulating level of GLP-1 is a known characteristic of Gcgr(-/-) mice. Neutralization of FGF21, while concurrently blocking the GLP-1 receptor with the antagonist Exendin 9-39 (Ex9-39), resulted in significant hyperglycemia in insulin-deficient Gcgr(-/-) mice, while blocking with Ex9-39 alone did not. In conclusion, FGF21 acts additively with GLP-1 to prevent insulinopenic diabetes in mice lacking glucagon action.
IntroductionInsulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering ...and the biological and pharmacological properties of insulin icodec.Research design and methodsA number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec. To evaluate the pharmacological properties of insulin icodec in individuals with type 2 diabetes, a randomized, double-blind, double-dummy, active-controlled, multiple-dose, dose escalation trial was conducted.ResultsThe long half-life of insulin icodec was achieved by introducing modifications to the insulin molecule aiming to obtain a safe, albumin-bound circulating depot of insulin icodec, providing protracted insulin action and clearance. Addition of a C20 fatty diacid-containing side chain imparts strong, reversible albumin binding, while three amino acid substitutions (A14E, B16H and B25H) provide molecular stability and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro cell-based studies showed that insulin icodec activates the same dose-dependent IR-mediated signaling and metabolic responses as native human insulin (HI). The affinity of insulin icodec for the insulin-like growth factor-1 receptor was proportionately lower than its binding to the IR, and the in vitro mitogenic effect of insulin icodec in various human cells was low relative to HI. The clinical pharmacology trial in people with type 2 diabetes showed that insulin icodec was well tolerated and has pharmacokinetic/pharmacodynamic properties that are suited for once-weekly dosing, with a mean half-life of 196 hours and close to even distribution of glucose-lowering effect over the entire dosing interval of 1 week.ConclusionsThe molecular modifications introduced into insulin icodec provide a novel basal insulin with biological and pharmacokinetic/pharmacodynamic properties suitable for once-weekly dosing.Trial registration numberNCT02964104.
In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is "selective", in the sense that the insulin-mediated decrease of glucose production is ...blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity.
In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated ...hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation.
In previous studies, glucagon receptor knockout mice (Gcgr(-/-)) display reduced blood glucose and increased glucose tolerance, with hyperglucagonemia and increased levels of glucagon-like peptide ...(GLP)-1. However, the role of glucagon receptor signaling for the regulation of islet function and insulin sensitivity is unknown. We therefore explored beta-cell function and insulin sensitivity in Gcgr(-/-) and wild-type mice. The steady-state glucose infusion rate during hyperinsulinemic-euglycemic clamp was elevated in Gcgr(-/-) mice, indicating enhanced insulin sensitivity. Furthermore, the acute insulin response (AIR) to intravenous glucose was higher in Gcgr(-/-) mice. The augmented AIR to glucose was blunted by the GLP-1 receptor antagonist, exendin-3. In contrast, AIR to intravenous administration of other secretagogues was either not affected (carbachol) or significantly reduced (arginine, cholecystokinin octapeptide) in Gcgr(-/-) mice. In islets isolated from Gcgr(-/-) mice, the insulin responses to glucose and several insulin secretagogues were all significantly blunted compared with wild-type mice. Furthermore, glucose oxidation was reduced in islets from Gcgr(-/-) mice. In conclusion, the present study shows that glucagon signaling is required for normal beta-cell function and that insulin action is improved when disrupting the signal. In vivo, augmented GLP-1 levels compensate for the impaired beta-cell function in Gcgr(-/-) mice.
Peptides and proteins are widely used to treat a range of medical conditions; however, they often have to be injected and their effects are short-lived. These shortcomings of the native structure can ...be addressed by molecular engineering, but this is a complex undertaking. A molecular engineering technology initially applied to insulin - and which has now been successfully applied to several biopharmaceuticals - entails the derivatization of peptides and proteins with fatty acids. Various protraction mechanisms are enabled by the specific characteristics and positions of the attached fatty acid. Furthermore, the technology can ensure a long half-life following oral administration of peptide drugs, can alter the distribution of peptides and may hold potential for tissue targeting. Due to the inherent safety and well-defined chemical nature of the fatty acids, this technology provides a versatile approach to peptide and protein drug discovery.
Summary Glucagon stimulates hepatic glucose production by activating specific glucagon receptors in the liver, which in turn increase hepatic glycogenolysis as well as gluconeogenesis and ureagenesis ...from amino acids. Conversely, glucagon secretion is regulated by concentrations of glucose and amino acids. Disruption of glucagon signaling in rodents results in grossly elevated circulating glucagon levels but no hypoglycemia. Here, we describe a patient carrying a homozygous G to A substitution in the invariant AG dinucleotide found in a 3′ mRNA splice junction of the glucagon receptor gene. Loss of the splice site acceptor consensus sequence results in the deletion of 70 nucleotides encoded by exon 9, which introduces a frame shift and an early termination signal in the receptor mRNA sequence. The mutated receptor neither bound 125I-labeled glucagon nor induced cAMP production upon stimulation with up to 1 µM glucagon. Despite the mutation, the only obvious pathophysiological trait was hyperglucagonemia, hyperaminoacidemia and massive hyperplasia of the pancreatic α-cells assessed by histology. Our case supports the notion of a hepato–pancreatic feedback system, which upon disruption leads to hyperglucagonemia and α-cell hyperplasia, as well as elevated plasma amino acid levels. Together with the glucagon-induced hypoaminoacidemia in glucagonoma patients, our case supports recent suggestions that amino acids may provide the feedback link between the liver and the pancreatic α-cells. Learning points: Loss of function of the glucagon receptor may not necessarily lead to the dysregulation of glucose homeostasis. Loss of function of the glucagon receptor causes hyperaminoacidemia, hyperglucagonemia and α-cell hyperplasia and sometimes other pancreatic abnormalities. A hepato–pancreatic feedback regulation of the α-cells, possibly involving amino acids, may exist in humans.
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