Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

Abstract

Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na⁺/H⁺-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm

“…We first probed the role of Na + /H + exchange in the ZP-induced alkalization using Na + substitution and pharmacology. Indeed, the ZP-induced pH i response was abolished by substitution of extracellular Na + by NMDG (N-methyl-D-glucamine) or addition of EIPA, a commonly used non-selective inhibitor of Na + /H + exchangers (Shi et al, 2017). The NH 4 Cl-induced pH i response was, however, similar in the absence or presence of Na + or EIPA ( Figures 1I-K).…”

SGLT2 Inhibitors Produce Cardiorenal Benefits by Promoting Adaptive Cellular Reprogramming to Induce a State of Fasting Mimicry: A Paradigm Shift in Understanding Their Mechanism of Action

“…An increase in AMPK and SIRT1 may also directly enhance tubuloglomerular feedback, thereby linking changes in the activity of these energy sensors to the action of SGLT2 inhibitors to ameliorate glomerular hyperfiltration (35,36). Finally, activation of AMPK/SIRT1 and autophagic flux has been linked to downregulation of ion exchangers that have been implicated in the pathogenesis of diabetic cardiac and renal disease (37,38).…”

Autophagy stimulation and intracellular sodium reduction as mediators of the cardioprotective effect of sodium–glucose cotransporter 2 inhibitors

“…There may be an interplay between autophagy and the activity of ion exchangers, thus potentially linking the two broad mechanistic frameworks that may underlie the benefits of SGLT2 inhibitors. Experimentally, an increase in AMPK may lead to a decrease in the expression of NHE‐1 and other transporters that mediate sodium influx into cells, and conversely, suppression of NHE‐3 activity has been reported to be followed by an increase in autophagic flux . Increased Akt phosphorylation following SGLT2 inhibition may also contribute to inhibition of NHE‐1 in cardiomyocytes …”

“…Experimentally, an increase in AMPK may lead to a decrease in the expression of NHE-1 and other transporters that mediate sodium influx into cells, 149 and conversely, suppression of NHE-3 activity has been reported to be followed by an increase in autophagic flux. 164 Increased Akt phosphorylation following SGLT2 inhibition may also contribute to inhibition of NHE-1 in cardiomyocytes. 84,165 It is noteworthy that metformin also stimulates autophagy, 135,[166][167][168][169] primarily by activating AMPK and SIRT1.…”