Biometric and echocardiographic parameters of rats fed the control or HS diet.

<p>Data are the means ± SD.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p><p>SBP, systolic blood pressure; DBP, diastolic blood pressure; IVSd, Interventricular septal end-diastolic dimension; LVEdD or LVEsD, left ventricular end-diastolic or systolic diameter; LVPWd, left ventricular posterior wall thickness in diastole; EF, ejection fraction; FS, fractional shortening; E/A, transmitral flow ratio.</p

FA and glucose oxidations in SCD1-overexpressed cardiac myocytes.

<p>(A): FA oxidation was significantly increased in rat neonatal cardiac myocytes treated with 100 µM palmitic acid (PA). (B): On the other hand, glucose oxidation was significantly reduced in cardiac myocytes treated with PA (100 µM). (C): PA (50 to 100 µM) induced excessive FA oxidation was attenuated in cardiac myocytes transduced with a MOI of 20 of Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). (D): PA (50 to 100 µM) -induced suppression of glucose oxidation recovered in cardiac myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). BSA was used as a vehicle. Each sample was counted in a scintillation counter (cpm) and data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA concentration. (E); Phosphorylation levels of AMPK and ACC decreased in cardiac myocytes transduced with an MOI of 20 of Ad-SCD1 in comparison with control cells transduced with Ad-LacZ. Oligomycin (1 µM) was used as an AMPK and ACC activator.</p

Expression of SCD1 in HS diet rat hearts and in obese and diabetes patients' hearts.

<p>(A) and (B): Semi-quantitative RT-PCR (A) and real-time RT-PCR (B) showed the increased expression of SCD1 after 3 months in HS diet rat hearts in comparison with normal fed rat hearts. GAPDH was measured as an internal control. mRNA of the normal fed group was normalized to a value of 1, and the mRNA level in the HS-fed group is shown relative to the control level. Values are reported as the means ± SD. N = 6–8, **p<0.01 vs. control. (C): Semi-quantitative RT-PCR of SCD1, lipoprotein lipase (LPL), fatty acid synthase (FAS), acyl-CoA synthase (ACS), elongation of long chain fatty acid member 6 (Elovl6), peroxisome proliferator coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor α (PPARα), pyruvate dehydrogenase kinase isozyme 1 and 4 (PDK1, PDK4) in HS- or normal fed rat hearts after 3 months. Expression levels of other lipid biosynthesis genes or FA oxidative genes were similar between two groups at the same time point. (D) to (G): Pathology of SCD1 in HS- or normal fed rat heart. HE-stained sections (D) and (E) and Masson's trichrome staining (F) and (G) of rat myocardium after 3 months of feeding. (D), (F): Normal fed; (E), (G): High-sucrose fed. (H) and (I): Immunohistochemical staining showed increased expression of SCD1 after 3 months in HS-fed rat hearts (I), while minimal SCD1 staining was observed in control hearts (H). (J) and (K): Although SCD1 was not detectable in cardiac myocytes of normal healthy subject (J) using immunohistochemical staining, SCD1 expression was increased in obese and diabetes patients' hearts (K). Counterstaining was performed with 2% methyl green. Scale bar = 200 µm.</p

Effect of several humoral factors on SCD1 mRNA expression in neonatal rat cardiac myocytes.

<p>Real-time RT-PCR of SCD1 expressions in neonatal rat cardiac myocytes stimulated with (A) angiotensin II (0.1 to 1 µM), endothelin-1 (0.01 to 0.1 µM), norepinephrine (1 to 10 µM), lipopolysaccharide (10 to 50 ng/ml) for 24 h, (B) hypoxia (3 to 9 h) and (C) Hydrogen peroxide (H₂O₂: 0.1 to 50 µM) for 24 h. High dose (10–50 µM) of hydrogen peroxide significantly inhibited SCD1 mRNA expression in neonatal rat cardiac myocytes. Each SCD1 expression is presented relative to the gene expression of GAPDH and control is normalized to a value of 1. Values are reported as the means ± SD. **p<0.01 vs. control.</p

Saturated FA-induced apoptosis, DAG and ceramide level in SCD1-overexpressed cardiac myocytes.

<p>Cardiac myocytes were infected with Ad-LacZ as a control or Ad-SCD1 at an MOI of 20, and simultaneously treated with palmitic acid (PA) or stearic acid (SA). (A): Cleaved-caspase 3 and caspase 3 protein levels were analyzed by Western blotting. 250 µM of PA or SA induced cleaved-caspase 3 levels, and these inductions were attenuated in myocytes transduced with Ad-SCD1. (B): Caspase 3 and 7 activities were increased with PA or SA (100 to 250 µM) in cardiac myocytes, and inhibited in cells transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ(□). Values are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA or SA concentration. (C): To detect cell death via DNA fragmentation, left panels illustrate TUNEL fluorescent staining (green) and right panel illustrates nuclei (DAPI: blue). Scale bar = 200 µm. 250 µM of PA or SA induced TUNEL-positive cells in neonatal rat cardiac myocytes transduced with Ad-LacZ, and these TUNEL-positive cells were reduced by Ad-SCD1 transduction. (D) and (E): Both (D) DAG and (E) ceramide levels in lipid extracts were significantly increased by the addition of PA or SA (250 µM), and these increases of DAG and ceramide were attenuated in myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). Each sample was counted in a scintillation counter (cpm). An arbitrary value of 1.0 was assigned to cells transduced with Ad-LacZ by the addition of BSA. Data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA or SA concentration.</p

Heart tissue fatty acid composition of rats fed the control or HS diet for 3 months.

<p>Data are the means ±SD, and are presented as percent of total fatty acids.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p

Plasma fatty acid composition of rats fed the control or HS diet for 3 months.

<p>Data are the means ±SD, and are presented as percent of total fatty acids.</p>*<p>P<0.05, **P<0.01 vs. control fed group.</p

SCD1 modulated saturated FA-induced ROS levels in cardiac myocytes.

<p>Cardiac myocytes were (A) infected with Ad-LacZ as a control or Ad-SCD1 at an MOI of 20 or (B) transfected with siGFP as a control or siSCD1 at 20 µM, and simultaneously treated with palmitic acid (PA, 250 µM) or stearic acid (SA, 250 µM). Intracellular ROS levels were detected with CM-H₂DCFDA. PA or SA-induced ROS levels in neonatal rat cardiac myocytes transduced with Ad-LacZ, and these ROS levels were reduced by Ad-SCD1 transduction. In contrast, knockdown of SCD1 by siRNA increased basal ROS levels, and enhanced PA or SA-induced ROS levels. Scale bar = 200 µm.</p

Effect of glucose, insulin and FFAs on SCD1 mRNA expression in neonatal rat cardiac myocytes.

<p>(A) to (C): Real-time RT-PCR showed that SCD1 expressions were increased in neonatal rat cardiac myocytes which treated with (A) glucose (100 to 450 mg/dl), (B) insulin (1 to 10 µM), and (C) saturated FAs, palmitic acid (PA) or stearic acid (SA) for 24 h. On the other hand, unsaturated FAs, oleic acid (OA) or linoleic acid (LA) didn't affect the expression of SCD1.</p

Saturated FA-induced apoptosis in cardiac myocytes with loss of SCD1 function using siRNA transfection.

<p>Cardiac myocytes were transfected with siGFP as a control or siSCD1 at 20 µM, and simultaneously treated with palmitic acid (PA) or stearic acid (SA). (A): Loss of SCD1 function (siSCD1) augmented cleaved-caspase 3 levels induced by 250 µM of PA or SA. (B): Caspase 3 and 7 activities induced by PA or SA (100 to 250 µM) in cardiac myocytes were also augmented by the loss of SCD1 function (siSCD1: ▪) in comparison with control cells transduced with siGFP (□). Values are shown as the mean ± SD. *P<0.05 or **P<0.01 vs. siGFP in each PA or SA concentration. (C): 250 µM of PA or SA induced TUNEL-positive cells in neonatal rat cardiac myocytes transduced with siGFP, and these TUNEL-positive cells increased by the loss of SCD1 function (siSCD1). Scale bar = 200 µm.</p