Losartan counteracts the effects of cardiomyocyte swelling on glucose uptake and insulin receptor substrate-1 levels

Abstract

A growing body of evidence demonstrates an association between Angiotensin II (Ang II) receptor blockers (ARBs) and enhanced glucose metabolism during ischemic heart disease. Despite these encouraging results, the mechanisms responsible for these effects during ischemia remain poorly understood. In this study we investigated the influence of losartan, an AT1 receptor blocker, and secreted Ang II (sAng II) on glucose uptake and insulin receptor substrate (IRS-1) levels during cardiomyocyte swelling. H9c2 cells were differentiated to cardiac muscle and the levels of myogenin, Myosin Light Chain (MLC), and membrane AT1 receptors were measured using flow cytometry. Intracellular Ang II (iAng II) was overexpressed in differentiated cardiomyocytes and swelling was induced after incubation with hypotonic solution for 40min. Glucose uptake and IRS-1 levels were monitored by flow cytometry using 2-NBDG fluorescent glucose (10μM) or an anti-IRS-1 monoclonal antibody in the presence or absence of losartan (10^-7M). Secreted Angiotensin II was quantified from the medium using a specific Ang II-EIA kit. To evaluate the relationship between sAng II and losartan effects on glucose uptake, transfected cells were pretreated with the drug for 24h and then exposed to hypotonic solution in the presence or absence of the secreted peptide. The results indicate that: (1) swelling of transfected cardiomyocytes decreased glucose uptake and induced the secretion of Ang II to the extracellular medium; (2) losartan antagonized the effects of swelling on glucose uptake and IRS-1 levels in transfected cardiomyocytes; (3) the effects of losartan on glucose uptake were observed during swelling only in the presence of sAng II in the culture medium. Our study demonstrates that both losartan and sAng II have essential roles in glucose metabolism during cardiomyocyte swelling.

Keywords: Cardiomyocytes swelling; Glucose uptake; Insulin receptor substrate-1; Losartan; Secreted Angiotensin II.

Figure 1. Myogenin and Myosin Light Chain (MLC) in differentiated H9c2 cells and overexpression of intracellular Angiotensin II (iAng II)

H9c2 cells were differentiated to cardiac phenotype and intracellular levels of myogenin and MLC were quantified by flow cytometry using specific monoclonal antibodies. In addition, differentiated cardiomyocytes were transfected with a plasmid containing the entire Ang II sequence and the levels of the intracellular peptide were quantified using the Ang-II EIA kit. (A) Intracellular MLC levels significantly increased after differentiation to cardiac phenotype (black bar) when compared to undifferentiated (open bar). No significant differences were observed in the levels of myogenin after differentiation. (B) AT1 receptor levels significantly increased after differentiation to cardiac phenotype (black bar) when compared to undifferentiated (open bar). (C) iAng II levels significantly increased in cardiomyocytes after transfection (black bar) when compared to untransfected cells (open bar). Each bar is the average of six experiments. Vertical lines at each bar represent SD (**p<0.01, ****p<0.0001).

Figure 2. Swelling of transfected cardiomyocytes and losartan effect on glucose uptake and IRS-1 levels

Transfected cardiomyocytes were incubated with hypotonic solution and changes in cell size changes were monitored by flow cytometry. For analysis of losartan effects, cells were incubated with the drug prior to swelling. (A) Representative flow cytometric dot plot of cells prior to swelling. (B) Representative dot plot of cells after swelling. (C) Cell size increased significantly after incubation with hypotonic solution (black bar) when compared to untreated (open bar). (D, E) Glucose uptake significantly decreased in cardiomyocytes exposed only to hypotonic solution (gray bar) when compared to untreated (open bar). However, the pretreatment with losartan significantly increased glucose uptake during swelling (black bar) when compared to both, cells exposed to hypotonic solution only (gray bar) and untreated (open bar). Each bar is the average of six experiments. Vertical lines at each bar represent SD (*p<0.05, **p<0.01, ****p<0.0001). SSC=Side Scatter, FSC= Forward Scatter, MFI= Mean Fluorescence Intensity.

Figure 3. Secreted Ang II (sAng II) from transfected cardiomyocytes after incubation with hypotonic solution

Transfected cardiomyocytes were exposed to hypotonic solution for 1 minute or 40 minutes and the levels of sAng II in the cultured medium were quantified by ELISA. Ang II secretion significantly increased from cells exposed to hypotonic solution for 40 min (black bar) when compared to cells in solution for 1 min (gray bar) and to Ang II baseline levels (open bar). Each bar is the average of six experiments. Vertical lines at each bar represent SD (**p<0.01).

Figure 4. Effect of losartan and secreted Ang II (sAng II) on glucose uptake during cardiomyocyte swelling

Transfected cardiomyocytes were exposed to hypotonic solution in the presence or absence of sAng II and losartan. Glucose uptake significantly decreased when the cells were maintained in hypotonic solution containing sAng II for 40 minutes (light gray bar) when compared to cells exposed to solution without sAng II and losartan (open bar). However, when the cells were maintained in hypotonic solution with losartan glucose uptake significantly increased (black bar) when compared to cells in solution with sAng II only (light gray bar). No significant differences were observed on glucose uptake between cardiomyocytes incubated with hypotonic solution replaced every 5 minutes (no sAng II nor losartan; open bar) and cells in solution containing only losartan (no sAng II; dark gray bar). Each bar is the average of six experiments. Vertical lines at each bar represent SD (*p<0.05, ***p<0.001, ****p<0.0001). MFI= Mean Fluorescence Intensity.