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. 2015:2015:785783.
doi: 10.1155/2015/785783. Epub 2015 Nov 16.

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

Ricardo Rodríguez-Calvo  1 , Manuel Vázquez-Carrera  2 , Luis Masana  3 , Dietbert Neumann  1
Affiliations

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

Ricardo Rodríguez-Calvo et al. PPAR Res. 2015.

Abstract

Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR), a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr(172)-AMPK and phospho-Ser(79)-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI) conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increased Cd36, Acot1, and Ucp3 mRNA levels. AICAR treatment induced the expression of Acadvl and Glut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptation via regulation of transcriptional mechanisms.

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Figures

Figure 1
Figure 1
AICAR induces AMPK activation in HL-1 cardiac cells. HL-1 cells were stimulated with AICAR for 1 h and the AMPK activation was confirmed by the protein levels of phospho-Thr172-AMPK (a) and phospho-Ser79-ACC (b). Quantifications show the ratio between phosphorylated and total forms of each protein. Data are expressed as mean ± SD of 3 different experiments. (∗ P < 0.05, ∗∗ P < 0.01 versus control nonstimulated cells).
Figure 2
Figure 2
AICAR upregulates the mRNA levels of the three PPARs in HL-1 cardiomyocytes. Analysis of the mRNA levels of Ppara (a), Ppard (b), and Pparg (c) by real-time RT-PCR in HL-1 cells stimulated by AICAR for 24 h in the presence or absence of Compound C. Data are normalized by the Cyclophilin A mRNA levels and expressed as mean ± SD of 4 different experiments. (∗ P < 0.05, ∗∗∗ P < 0.001 versus control nonstimulated cells; # P < 0.05 versus AICAR-treated cells).
Figure 3
Figure 3
Treatment with AICAR regulates the expression of PPAR-target genes. Analysis of the mRNA levels of Cd36 (a), Acot1 (b), Cpt-1b (c), Acox1 (d), Acadvl (e), Ucp3 (f), Glut4 (f), and Pdk4 (h) by real-time RT-PCR in HL-1 cells stimulated with HP/HI for 16 h in the presence or absence of AICAR (24 h). Data are normalized by the Cyclophilin A mRNA levels and expressed as mean ± SD of 4 different experiments. (∗ P < 0.05, ∗∗ P < 0.01 versus control nonstimulated cells; # P < 0.05, ## P < 0.01 versus HP/HI-stimulated cells).
Figure 4
Figure 4
AICAR prevents the HP/HI-induced intramyocellular lipid accumulation in HL-1. Lipid content was analyzed by Oil-Red-O staining in HL-1 challenged with HP/HI for 16 h in the presence or absence of AICAR (24 h). (a) Representative microphotography showing lipid droplets in cells counterstained with Haematoxylin (bar 20 μm). Squares indicate the areas shown at high magnification. (b) Quantification of stained areas relative to cell surface. Data are expressed as mean ± SD of 5 different pictures from 3 independent experiments (∗∗ P < 0.01 versus control nonstimulated cells; # P < 0.05 versus HP/HI-stimulated cells).
Figure 5
Figure 5
AICAR stimulation attenuates HP/HI-induced glucose uptake impairment in HL-1. HL-1 cells were stimulated with HP/HI for 16 h in the presence or absence of AICAR (24 h), and [3H]-deoxyglucose uptake (up) and AKT phosphorylation (down) were assessed in the presence (a) and absence (b) of insulin (200 nmol/L, 10 min). [3H]-deoxyglucose uptake (up) and AKT phosphorylation (down) determination in HL-1 cells stimulated with AICAR (24 h) in the presence and absence of insulin (200 nmol/L, 10 min) (c). Data are expressed as mean ± SD of 4 different experiments performed in duplicate. (∗ P < 0.05, ∗∗∗ P < 0.001 versus control cells without insulin stimulation; &&& P < 0.001 versus control cells stimulated with insulin; # P < 0.05 versus HP/HI-challenged cells stimulated with insulin (a) or without insulin (b); $ P < 0.05 versus AICAR-treated cells stimulated with insulin).
Figure 6
Figure 6
Schematic representation of the potential mechanisms by which AICAR regulates cardiac metabolism and protects HL-1 cardiomyocytes from the HP/HI-induced lipotoxicity and insulin resistance. HP/HI stimulation induces insulin resistance by promoting the intramyocellular lipid build-up, which in turn inhibits the insulin signalling pathway and the AKT-mediated GLUT4 membrane translocation and glucose uptake. AICAR treatment takes part in the regulation of the cardiac metabolic adaptation at several levels. First, AICAR short-term stimulation promotes the inhibition of ACC, thereby reducing the levels of the allosteric inhibitor of CPT-1 malonyl-CoA and regulating the fatty acid mitochondrial β-oxidation. In addition, AICAR-induced AMPK activation promotes GLUT4 membrane translocation through non-insulin dependent mechanisms. Finally, AICAR stimulation induces the three PPARs mRNA levels and controls the expression of some key PPAR-target genes, such as Acadvl and Glut4, involved in both glucose and fatty acid cardiac metabolism. Therefore, through these different mechanisms, AICAR is able to regulate both the acute metabolic response and the long-term metabolic adaptation in cardiac cells.
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