Apolipoprotein E null mice (apoE-/-) fed an atherogenic diet received a subcutaneous pump infusion of either EIPA (3 mg/kg/d) or the control vehicle for 4 weeks.

The nontransformed rat small intestinal epithelial IEC-18 cell line was used for activity evaluation. For the amino acid starvation-induced autophagic cell model, the IEC-18 cells were incubated in Hank’s Balanced Salt Solution (HBSS; Gibco/Invitrogen) as the culture medium for 6 h under the same atmospheric conditions. Then, alanine (1.0 mM final concentration; Sigma-Aldrich) or proline (0.5 mM final concentration, Sigma-Aldrich) with or without the NHE3 inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA; 0.3 mM final concentration; A3085, Sigma-Aldrich) was added to the culture medium and incubated with the cells for 6 h. To identify the optimal concentrations of amino acids (alanine and proline) and EIPA that elicited the best effect, various concentrations were tested. The effect of EIPA alone (without alanine or proline) was also examined in both control (DMEM cultured cells) and amino acid-starved cells. The cells were incubated for 6 h in DMEM containing 5% FBS and either 0 or 0.3 mM EIPA (labelled QNN and QNE, respectively), HBSS containing either 0 or 0.3 mM EIPA (labelled HNN and HNE, respectively), HBSS with 1.0 mM alanine (labelled HAN) or 0.5 mM proline (labelled HPN), HBSS with 1.0 mM alanine and 0.3 mM EIPA (labelled HAE), and HBSS with 0.5 mM proline and 0.3 mM EIPA (labelled HPE). To evaluate the autophagic activity, the autophagosome-lysosome fusion inhibitor chloroquine (CQ; 50 μM; Sigma-Aldrich) was used.