β‐cell Deletion of Nr4a1 and Nr4a3 Nuclear Receptors Impedes Mitochondrial Respiration and Insulin Secretion

Abstract

Insulin secretion from β‐cells is dependent on the stimulation of mitochondrial respiration in response to an increase in glucose supply. Various studies have clearly demonstrated that the Nr4a family of orphan nuclear receptors are essential for fuel utilization and mitochondrial function in liver, muscle and fat. We have previously demonstrated that overexpression of Nr4a1 and Nr4a3 are sufficient to induce proliferation of pancreatic β‐cells. We examined the impact of nuclear receptors Nr4a1 and Nr4a3 on pancreatic β‐cell mitochondrial function and proliferation using lentivirus mediated shRNA knock‐down of Nr4a1 and Nr4a3 in INS‐1 derived 832/13 rat insulinoma cells. β‐cells deficient in Nr4a1 and Nr4a3 have a 37% reduction in proliferation rates (p<0.05). The reduction in β‐cell proliferation with Nr4a1 and Nr4a3 deficiency was accompanied by a significant decrease in glucose stimulated insulin secretion rates (GSIS) (26% decrease, p<0.05). GSIS was determined in cells preincubated in secretion assay buffer (SAB) for 1.5 hours containing 2.5 mM glucose followed by incubation in SAB with 16.7 mM glucose. High resolution respirometry (Oroboros O2k) was used to evaluate mitochondrial respiratory function and capacity with substrate‐uncoupler‐inhibitor titration protocols for both intact and permeabilized β‐cells. Permeabilized cells allow for an in depth assessment of chemiosmotic coupling within the β‐cells while the intact protocol evaluates a more physiologically relevant condition. We have shown that deletions of the Nr4a nuclear receptors clearly impact cellular respiration, lowering the oxygen consumption rate in all respiratory states of intact β‐cells between 40–65% and permeabilized β‐cells by 23–50% compared to GFP (p<0.05). Our results indicate that knockdown of Nr4a1 and Nr4a3 results in decreased expression Idh3a, Idh3g, Sdhb, and Mdh2, which would impede TCA cycle progression, and is consistent with the reduction observed in O2 consumption, ATP production and ultimately glucose stimulated insulin secretion. These data demonstrate for the first time that the Nr4a orphan nuclear receptor family members Nr4a1 and Nr4a3 are critical for basal β‐cell mitochondrial function and insulin secretion.Support or Funding InformationBrigham Young University