Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells

Abstract

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.

Glucose-Induced Expression of DAPIT in Pancreatic β-Cells

“…According this hypothetical view, the preferential rapid expression of DAPIT and its binding into the vacant sites would then switch on ATP synthesis. DAPIT would then act reciprocally to the physiological inhibitor of the ATP synthase, the ATPase inhibitory factor IF1 [ 11 , 13 ].…”

“…We found that also the inhibitory factor IF1 of the mitochondrial ATP synthase belongs to the key proteins, ensuring the physiological range of the glucose sensor [ 11 , 12 ]. The IF1 slightly inhibits synthesis of ATP, thus setting the range for elevation of phosphorylating respiration and insulin release above 3-mM glucose [ 11 , 13 ], with half-activation between 3.5 and 4-mM and saturation above 8-mM glucose [ 11 , 12 ]. When such a slight in vivo inhibition was largely cancelled using the silencing of IF1, the elevation of respiration and OXPHOS occurred at very low glucose concentration approaching to zero [ 9 ].…”

“…Simultaneously, the half-activation for the insulin release dose–response was shifted to the range of 0–2-mM glucose in INS-1E cells [ 9 ]. In contrast, overexpression of IF1 substantially blocked GSIS [ 13 ].…”

“…Similarly, Mic10 was suggested to be inserted between the neighbor dimers or tetramers of the ATP synthase [ 32 , 33 ]. Likewise, crosslinking of two F 1 moieties from the neighbor ATP synthase dimers by the un-phosphorylated dimeric inhibitory factor IF1 can stabilize the rim [ 20 ], but at the expense of ATP synthesis, since this interaction is inhibitory [ 13 ]. The inner mitochondrial membrane (IMM) bending itself may be ensured by subunits e and g [ 20 ].…”

The role of mitochondrial ATP synthase in cancer

“…These results are in line with the effects of IF1 overexpression or downregulation in cultured cells (Formentini et al 2012;Sánchez-Aragó et al 2013;Sánchez-Cenizo et al 2010). On the same line, the overexpression of IF1 in pancreatic β cells leads to a substantial decrease in ATP levels and in glucose-stimulated insulin secretion (Kahancová et al 2020), while the downregulation of the inhibitor protein in the same model causes an increase in mitochondrial respiration and in ATP levels leading to insulin secretion (Kahancová et al 2018).…”