The Critical Role of Astragalus Polysaccharides for the Improvement of PPRAα-Mediated Lipotoxicity in Diabetic Cardiomyopathy

<div><h3>Background</h3><p>Obesity-related diabetes mellitus leads to increased myocardial uptake and oxidation of fatty acids, resulting in a form of cardiac dysfunction referred to as lipotoxic cardiomyopathy. We have shown previously that Astragalus polysaccharides (APS) administration was sufficient to improve the systemic metabolic disorder and cardiac dysfunction in diabetic models.</p> <h3>Methodology/Principal Findings</h3><p>To investigate the precise role of APS therapy in the pathogenesis of myocardial lipotoxity in diabetes, db/db diabetic mice and myosin heavy chain (MHC)- peroxisome proliferator-activated receptor (PPAR) α mice were characterized and administrated with or without APS with C57 wide- type mice as normal control. APS treatment strikingly improved the myocyte triacylglyceride accumulation and cardiac dysfunction in both db/db mice and MHC-PPARα mice, with the normalization of energy metabolic derangements in both db/db diabetic hearts and MHC-PPARα hearts. Consistently, the activation of PPARα target genes involved in myocardial fatty acid uptake and oxidation in both db/db diabetic hearts and MHC-PPARα hearts was reciprocally repressed by APS administration, while PPARα-mediated suppression of genes involved in glucose utilization of both diabetic hearts and MHC-PPARα hearts was reversed by treatment with APS.</p> <h3>Conclusions</h3><p>We conclude that APS therapy could prevent the development of diabetic cardiomyopathy through a mechanism mainly dependent on the cardiac PPARα-mediated regulatory pathways.</p> </div

Protein levels of PPARα target genes in hearts of mice at the age of 20 weeks.

<p>Cardiac tissues from db/db mice and MHC-PPARα mice with/without APS administration were detected for the protein expression at the age of 20 weeks, with C57 mice as normal control. Myocardial protein levels of PPARα target genes were determined by Western Blot analysis. (A) Representative autoradiographs of PPARα target genes and GAPDH (loading control) using specific antibodies. Protein levels of PPARα target genes in myocardium, encoding PPARα (B), M-CPF 1 (D), ACO (E), FATP 1(F) and FACS 1(G). PPARα activities in myocardial tissue were also determined at the end of the experiment by RIA (C). All groups n = 8. Bars represented means±S.E.M., and were corrected for GAPDH signal intensity and normalized to the value of C57 wide-type mice. *<i>P</i><0.05 vs. C57 mice, # <i>P</i><0.05 vs. untreated db/db mice, † <i>P</i><0.05 vs. untreated MHC-PPARα mice.</p

Serum biometric parameters of mice at different age.

<p>Serum glucose, insulin, nonesterified fatty acid (FFA) and triacylglyceride (TAG) were detected in db/db mice and MHC-PPARα mice with or without APS administration at the age of 5 weeks (A) and 20 weeks (B–E) respectively, with C57 wide-type mice as normal control. All groups n = 8. Bars represent means±S.E.M. *<i>P</i><0.05 vs. C57 mice, # <i>P</i><0.05 vs. untreated db/db mice, † <i>P</i><0.05 vs. untreated MHC-PPARα mice.</p

Echocardiographic properties and expression of cardiac dysfunction marker genes in hearts of mice.

<p>Echocardiographic studies were performed on db/db mice and MHC-PPARα mice with or without APS administration at the age of 20 weeks, with C57 wide-type mice as normal control. (A) Representative echocardiographic images; (B) fractional shortening (FS). Expression of cardiac dysfunction marker genes encoding BNP (C), Skeletal α-actin (D) and SERCA2a (E) was determined by real-time RT-PCR analysis. The relative gene expression of db/db mice and MHC-PPARα mice was normalized to the value of C57 wide-type mice, and expressed as fold change. All groups n = 8. Bars represent means±S.E.M. *<i>P</i><0.05 vs. C57 mice, # <i>P</i><0.05 vs. untreated db/db mice, † <i>P</i><0.05 vs. untreated MHC-PPARα mice. BNP, brain-type natriuretic peptide; SERCA2a, sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2a; MHC, myosin heavy chain; PPAR, peroxisome proliferator-activated receptor; APS, astragalus polysaccharides.</p

Gene expression of PPARα target genes in hearts of mice at different age.

<p>Cardiac tissues from db/db mice and MHC-PPARα mice with/without APS administration were detected for the gene expression at the age of 5 weeks (A) and 20 weeks (B-H) respectively, with C57 mice as normal control. Myocardial mRNA levels of PPARα target genes were determined by real-time RT-PCR analysis, encoding PPARα (B), M-CPF 1(C), ACO (D), FATP 1 (E), FACS 1(F), GLUT4 (G) and PDK4 (H). The relative gene expression of db/db mice and MHC-PPARα mice was normalized to the value of C57 wide-type mice, and expressed as fold change. All groups n = 6. Bars represent means±S.E.M. *<i>P</i><0.05 vs. C57 mice, # <i>P</i><0.05 vs. untreated db/db mice, † <i>P</i><0.05 vs. untreated MHC-PPARα mice. M-CPF1, muscle-typecarnitine palmitoyltransferase 1; ACO, acyl-CoA oxidase; FATP 1, fatty acid trasport protein-1; FACS 1, fatty acyl-CoA synthetase 1; GLUT4, glucose transporter 4; PDK4, pyruvate dehydrogenase kinase 4.</p

Intramyocardial lipid accumulation in mice.

<p>Myocardial triacylglyceride levels (A) in db/db mice and MHC-PPARα mice with or without APS administration at 20 week-old were determined by ESI/MS. Mean levels of TAG-associated fatty acid with chain length of 16∶0 (B), 18∶0 (C), 18∶1 (D), and 18∶2 (E) in the hearts of each group were shown respectively. (F) representative images of myocardium stained with oil red O. All groups n = 8. Bars represent means±S.E.M. *<i>P</i><0.05 vs. C57 mice, # <i>P</i><0.05 vs. untreated db/db mice, † <i>P</i><0.05 vs. untreated MHC-PPARα mice.</p

Hemodynamics and cardiac function in mice.

<p>Values are mean±S.E.M. n = 8 each group.</p>*<p>p<0.05 vs. wide type (Wt) mice;</p>#<p>p<0.05 vs. untreated db/db mice;</p>†<p>P<0.05 vs. untreated MHC-PPARα mice.</p><p>HR, heart rate; LVPWd/s, left ventricular posterior wall thickness at diastole or systole; IVSd/s, interventricular septal wall thickness at diastole or systole; LVIDd/s, left ventricular internal diameter at diastole or systole.</p