Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic ␣ cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr ؊/؊ ). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr ؊/؊ mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to ␣ cell, and to a lesser extent, ␦ cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3-to 10-fold increase in circulating GLP-1 amide. Gcgr ؊/؊ mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and ␣ and ␦ cell numbers. Furthermore, the lean phenotype of Gcgr ؊/؊ mice suggests glucagon action may be involved in the regulation of whole body composition.

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.

Here, we describe the molecular engineering of insulin icodec to achieve a plasma half-life of 196 h in humans, suitable for once-weekly subcutaneously administration. Insulin icodec is based on re-engineering of the ultra-long oral basal insulin OI338 with a plasma half-life of 70 h in humans. This systematic re-engineering was accomplished by (1) further increasing the albumin binding by changing the fatty diacid from a 1,18-octadecanedioic acid (C18) to a 1,20-icosanedioic acid (C20) and (2) further reducing the insulin receptor affinity by the B16Tyr → His substitution. Insulin icodec was selected by screening for long intravenous plasma half-life in dogs while ensuring glucose-lowering potency following subcutaneous administration in rats. The ensuing structure–activity relationship resulted in insulin icodec. In phase-2 clinical trial, once-weekly insulin icodec provided safe and efficacious glycemic control comparable to once-daily insulin glargine in type 2 diabetes patients. The structure–activity relationship study leading to insulin icodec is presented here.

We have studied the distribution of activin receptor gene expression in the brain, pituitary, ovary, and testis of the adult rat by in situ hybridization, using probes complementary to the mRNAs encoding the mouse activin receptor subtypes II and IIB (ActRII and ActRIIB). Throughout the brain, ActRII mRNA expression was stronger than that of ActRIIB, and the patterns of expression were similar, although not identical. The most intense sites of activin receptor gene expression were the hippocampal formation, especially the dentate gyrus (ActRII), taenia tecta, and induseum griseum; the amygdala, particularly the amygdaloid-hippocampal transition zone; and throughout the cortical mantle, including the primary olfactory cortex (piriform cortex and olfactory tubercle); other regions of the cortex showing lesser degrees of hybridization included the cingulate cortex, claustrum, entorhinal cortex, and subiculum. In addition, moderate levels of expression were observed in several hypothalamic areas involved in neuroendocrine regulation, such as the suprachiasmatic, supraoptic, paraventricular, and arcuate nuclei. Moreover, activin receptors were also expressed in regions with inputs to the hypothalamus, both in the forebrain (bed nucleus of the stria terminalis and medial preoptic area) and within the brainstem (nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, and mesencephalic raphé system). ActRII mRNA was observed in the intermediate lobe of the pituitary and, less prominently, in the anterior lobe, whereas ActRIIB appeared to be weakly expressed throughout all three pituitary divisions. In both male and female gonads, activin receptor message was clearly present in germ cells, and ActRII was the predominant form. In the ovary, in addition to an intense signal in the oocyte, activin receptor was expressed in corpus luteum and granulosa cells during diestrous day 1. In the testis, there was a strong ActRII signal in rounded spermatids, and a moderate signal in pachytene spermatocytes. In contrast, ActRIIB was absent within tubules, but weakly expressed in interstitial and Leydig cells. This is the first report of the distribution of activin receptor message in adult mammalian tissues. Although consistent with some previously suggested functional associations of activin-containing pathways in the brain, this pattern of expression suggests a greater role for activin than was previously appreciated in cortical, limbic, and somatosensory pathways and in the maturation of germ cells in the gonads of both male and female rats.