2025 Theses Doctoral

The Role of Insulin Receptor Isoforms in Health and Disease

Mukhanova, Maria

The insulin receptor (IR) is a critical regulator of mammalian metabolic and mitogenic processes. It is expressed as two alternatively spliced isoforms that differ by a 12-amino-acid sequence encoded by exon 11 of the IR gene. Whether there are functional differences between the two isoforms is unknown. While early research suggests that IR isoform B (IR-B) plays a more prominent role in metabolic regulation than IR isoform A (IR-A), direct evidence remains limited due to the dearth of experimental models to assess their native properties in vivo. To address this gap in knowledge, we generated mice lacking IR-A through homologous recombination and mice lacking IR-B with CRISPR/Cas9 editing. We then characterized IR-A-/- and IR-B-/- mice in various pathophysiological conditions.

Young-adult IR-A-/- females showed lower triglyceride (TG) excursion on lipid tolerance test (LTT) and lower glycemic excursion on oral glucose tolerance test (oGTT), while their fat and lean content was preserved. Periodic acid–Schiff (PAS) staining demonstrated lower hepatic glycogen after refeeding, and qPCR analyses revealed differential expression of glycogen metabolism-related genes in liver and gastrocnemius. Hepatic IR phosphorylation was enhanced in refed females. We did not find any significant metabolic differences between male wildtype (WT) and IR-A-/- littermates across all assays.

In contrast, female and male IR-B-/- mice had higher refed plasma TG and larger LTT peaks. These mice also had greater fat mass and lower lean mass, accompanied by reduced fasting non-esterified fatty acids (NEFAs). Female, but not male IR-B-/- exhibited a trend toward lower glucose excursions on oGTT, intraperitoneal (IP) GTT (iGTT), pyruvate tolerance test (PTT), and insulin tolerance test (ITT). Radiolabeled tracer studies revealed increased 14C-oleic acid retention in liver and decreased 3H-triolein in perigonadal adipose tissue (pGAT) of male IR-B-/- mice. Hepatic IR phosphorylation was reduced during refeeding in male IR-B-/- mice. Ligand-binding assays in IR-B-/- livers showed normal insulin affinity but displayed higher IGF-2 affinity. Correspondingly, IR-B-/- females had decreased glucose levels after IP IGF-2 administration and reduced IGF-2–stimulated IR phosphorylation in the livers.

Whole-body adiposity in female and male IR-B-/- mice became markedly greater with age. Aging females also had lower fasting NEFAs and higher fasting glucose. When subjected to a high-fat diet (HFD), male IR-B-/- mice developed impaired fasting glucose and iGTT and PTT responses, with no differences in fat and lean mass between genotypes. Hepatic IR phosphorylation dropped sharply on an oGTT challenge in these mice. Calorie restriction (CR) produced the opposite phenotype: CR–IR-B-/- males showed lower refed glucose and insulin after refeeding, decreased fat and lean composition, and increased fasting NEFAs. IR phosphorylation was decreased in refed livers and gastrocnemius of these mice.

In summary, IR-A deletion impacts lipid and glucose tolerance only in females, whereas IR-B deletion raises postprandial TG under physiological conditions but drives age- and diet-sensitive shifts in glycemia and adiposity. Specifically, body-fat expansion in IR-B-/- mice increases with age, normalizes under diet-induced obesity (DIO), and reverses under CR. Collectively, the data show that IR-A and IR-B have sex- and context-dependent effects on glucose and lipid metabolism, which could, at least in part, be attributed to differential phosphorylation dynamics between the two isoforms.