Biology subjects

Brierley, G. V.

Publications and source records attributed to Brierley, G. V..

4 recordsLinked to original sources

bioRxiv2024

Deep mutational scanning of the human insulin receptor ectodomain to inform precision therapy for insulin resistance

The insulin receptor (INSR) entrains tissue growth and metabolism to nutritional conditions. Complete loss of function in humans leads to extreme insulin resistance and infantile mortality, while loss of 80-90% function permits longevity of decades. Even low-level activation of severely compromised receptors, for example by anti-receptor monoclonal antibodies, thus offers the potential for decisive clinical benefit. A barrier to genetic diagnosis and translational research is the increasing identification of INSR variants of uncertain significance. We employed saturation mutagenesis coupled to multidimensional flow-based assays to stratify approximately 14,000 INSR extracellular domain missense variants by cell surface expression, insulin binding, and insulin- or monoclonal antibody-stimulated signaling. The resulting function scores correlate strongly with clinical syndromes, offer insights into dynamics of insulin binding, and reveal novel potential gain-of-function variants. This INSR sequence-function map has high biochemical, diagnostic and translational utility, aiding rapid identification of variants amenable to activation by non-canonical INSR agonists.

genetics↗

bioRxiv2023

Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant

PIK3R1 encodes three regulatory subunits of class IA phosphoinositide 3-kinase (PI3K), each associating with any of three catalytic subunits, namely p110, p110{beta} or p110{delta}. Constitutional PIK3R1 mutations cause diseases with a genotype-phenotype relationship not yet fully explained: heterozygous loss-of-function mutations cause SHORT syndrome, featuring insulin resistance and short stature attributed to reduced p110 function, while heterozygous activating mutations cause immunodeficiency, attributed to p110{delta} activation and known as APDS2. Surprisingly, APDS2 patients do not show features of p110 hyperactivation, but do commonly have SHORT syndrome-like features, suggesting p110 hypofunction. We sought to investigate this. In dermal fibroblasts from an APDS2 patient, we found no increased PI3K signalling, with p110{delta} expression markedly reduced. In preadipocytes, the APDS2 variant was potently dominant negative, associating with Irs1 and Irs2 but failing to heterodimerise with p110. This attenuation of p110 signalling by a p110{delta}-activating PIK3R1 variant potentially explains co-incidence of gain-of-function and loss-of-function PIK3R1 phenotypes.

biochemistry↗

bioRxiv2020

Anti-Insulin receptor antibodies improve hyperglycaemia in a mouse model of human insulin receptoropathy

Loss-of-function mutations in both alleles of the human insulin receptor gene (INSR) cause extreme insulin resistance (IR) and usually death in childhood, with few therapeutic options. Bivalent anti-receptor antibodies can elicit insulin-like signaling by mutant INSR in cultured cells, but whether this translates into meaningful metabolic benefits in vivo, where dynamics of insulin signaling and receptor recycling are more complex, is unknown. To address this we adopted a strategy to model human insulin receptoropathy in mice, using Cre recombinase delivered by adeno-associated virus to knock out endogenous hepatic Insr acutely in floxed Insr mice (L- IRKO+GFP), before adenovirus-mediated add-back of wild-type (WT) or mutant human INSR. Two murine anti-INSR monoclonal antibodies, previously shown to be surrogate agonists for mutant INSR, were then tested by intraperitoneal injections. As expected, L-IRKO+GFP mice showed glucose intolerance and severe hyperinsulinemia, and this was fully corrected by add-back of WT but neither D734A nor S350L mutant INSR. Antibody injection improved glucose tolerance in D734A INSR-expressing mice and reduced hyperinsulinemia in both S350L and D734A INSR-expressing animals, and did not cause hypoglycemia in WT INSR-expressing mice. Antibody treatment also downregulated both wild-type and mutant INSR protein, attenuating its beneficial metabolic effects. Anti-INSR antibodies thus improve IR in an acute model of insulin receptoropathy, but these findings imply a narrow therapeutic window determined by competing effects of antibodies to stimulate receptors and induce their downregulation. One Sentence SummaryBivalent anti-insulin receptor antibodies improve glycaemic control, but downregulate receptor expression, in a novel mouse model of lethal human insulin receptoropathy.

biochemistry↗

bioRxiv2019

Mesenchymal Igf2 is a major paracrine regulator of pancreatic growth and function

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Here we demonstrate that many imprinted genes are highly expressed in the pancreatic mesenchyme, and explore the role of Igf2 in-vivo. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy. Surprisingly, mesenchymal mass is unaffected, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Furthermore, increased IGF2 activity specifically in the mesenchyme, through Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Ex-vivo exposure of primary acinar cells to exogenous IGF2 increases cell proliferation and amylase production through AKT signalling. We propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

developmental biology↗