1. Insulin-Like Growth Factor 1 Alleviates High-Fat Diet–Induced Myocardial Contractile Dysfunction
by Yingmei Zhang, Ming Yuan, Katherine M. Bradley, Feng Dong, Piero Anversa, and Jun Ren
Hypertension
Volume 59(3):
February 15, 2012
Copyright © American Heart Association, Inc. All rights reserved.

2. Effect of cardiac insulin-like growth factor 1 (IGF-1) overexpression on glucose tolerance, reactive oxygen species (ROS) production, apoptosis, ATP production, protein, and mitochondrial damage after low-fat (LF) or high-fat (HF)–diet feeding.
Effect of cardiac insulin-like growth factor 1 (IGF-1) overexpression on glucose tolerance, reactive oxygen species (ROS) production, apoptosis, ATP production, protein, and mitochondrial damage after low-fat (LF) or high-fat (HF)–diet feeding. A, Intraperitoneal glucose tolerance test (IPGTT) displaying serum glucose levels after glucose challenge (2 g of glucose per kilogram of body weight). B, Area under the curve (AUC) calculated from IPGTT curves. C, Cardiomyocyte ROS production. D, Myocardial protein carbonyl formation. E, Myocyte caspase-3 activity. F, Myocardial aconitase activity. G, Myocardial ATP content. H, Mitochondrial and cytosolic cytochrome C expression from myocardium (normalized to the loading control GAPDH). Inset, Representative gel blots of mitochondrial and cytosolic cytochrome C, as well as GAPDH, using specific antibodies. Mean±SEM, n=7 to 10 mice per group. *P<0.05 vs FVB-LF group, #P<0.05 vs FVB-HF group, †P<0.05 vs IGF-LF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

3. Contractile properties of cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) transgenic mouse hearts.
Contractile properties of cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) transgenic mouse hearts. A, Representative cell shortening traces in FVB groups. B, Representative cell shortening traces in IGF groups. C, Resting cell length. D, Peak shortening (normalized to cell length). E, Maximal velocity of shortening (+dL/dt). F, Maximal velocity of relengthening (−dL/dt). G, Time to peak shortening (TPS). H, Time to 90% relengthening (TR90). Mean±SEM, n=80 cells from 3 to 4 mice per group, *P<0.05 vs FVB-LF group, #P<0.05 vs FVB-HF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

4. Intracellular Ca2+ transients and stimulus frequency response in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mouse hearts.
Intracellular Ca2+ transients and stimulus frequency response in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mouse hearts. A, Peak shortening (PS) in response to increasing stimulus frequency (0.1–5.0 Hz). Each point represents PS normalized to that of 0.1 Hz of the same cell. B, Representative intracellular Ca2+ transient traces in LF- or HF-fed groups. C, Resting fura-2 fluorescence intensity (FFI). D, Electrically stimulated rise in FFI (ΔFFI). E, Peak FFI. F, Intracellular Ca2+ decay rate. Mean±SEM, n=68 cells (23–25 cells for A) from 3 to 4 mice per group, *P<0.05 vs FVB-LF group, #P<0.05 vs FVB-HF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

5. Effect of insulin-like growth factor 1 (IGF-1) overexpression on myocardial apoptosis and hypertrophy after low-fat (LF) or high-fat (HF) feeding using TUNEL and fluorescein isothiocyanate (FITC)–conjugated lectin staining, respectively.
Effect of insulin-like growth factor 1 (IGF-1) overexpression on myocardial apoptosis and hypertrophy after low-fat (LF) or high-fat (HF) feeding using TUNEL and fluorescein isothiocyanate (FITC)–conjugated lectin staining, respectively. All of the nuclei were stained with 4′,6-diamidino-2-phenylindole (blue) in B (FVB-LF), D (FVB-HF), F (IGF-LF), and H (IGF-HF). TUNEL-positive nuclei were visualized with fluorescein (green) in A (FVB-LF), C (FVB-HF), E (IGF-LF), and G (IGF-HF). Original magnification: ×400. Quantified data are shown in I. J, FITC-conjugated lectin immunostaining depicting transverse sections of left ventricular myocardium (×400). K, Quantitative analysis of cardiomyocyte cross-sectional area. Mean±SEM, n=15 and 10 fields from 3 mice per group for I and K, respectively, *P<0.05 vs FVB-LF group; #P<0.05 vs FVB-HF group, †P<0.05 vs FVB-LF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

6. Western blot analysis of the mitochondrial proteins uncoupling protein 2 (UCP-2) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), as well as the Ca2+ regulatory proteins sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and phospholamban in myocardium from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mice.
Western blot analysis of the mitochondrial proteins uncoupling protein 2 (UCP-2) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), as well as the Ca2+ regulatory proteins sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and phospholamban in myocardium from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mice. A, Representative gel blots of UCP-2, PGC1α, SERCA2a, Na+-Ca2+ exchanger, phospholamban, and GAPDH (loading control) using specific antibodies. B, UCP-2. C, PGC1α. D, SERCA2a. E, Na+-Ca2+ exchanger. F, Phospholamban. All of the proteins were normalized to the loading control GAPDH. Mean±SEM, n=8 to 9 mice per group, *P<0.05 vs FVB-LF group, #P<0.05 vs FVB-HF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

7. Western blot analysis of pan and phosphorylated insulin receptor-β or insulin-like growth factor 1 (IGF-1) receptor-β in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and IGF-1 mice.
Western blot analysis of pan and phosphorylated insulin receptor-β or insulin-like growth factor 1 (IGF-1) receptor-β in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and IGF-1 mice. A, Insulin receptor-β expression. B, Basal and insulin/IGF-1–stimulated (at 100 nmol/L for 15 minutes) tyrosine phosphorylation of insulin receptor β. C, IGF-1 receptor-β expression. D, Basal and insulin/IGF-1–stimulated (at 100 nmol/L for 15 minutes) tyrosine phosphorylation of IGF-1 receptor-β. E, Tyrosine phosphorylation of insulin receptor substrate (IRS; Tyr1146) normalized to pan IRS. F, Serine phosphorylation of IRS (Ser307) normalized to pan IRS. Insets, Representative gel blots of pan and phosphorylated insulin receptor-β, IGF receptor-β, and IRS using respective specific antibodies. All of the protein expressions were normalized to the loading control GAPDH or FVB-LF group (for immunoprecipitation studies). pY denotes antiphosphotyrosine antibody. IP and IB represent immunoprecipitation and immunoblot, respectively; Mean±SEM, n=5 to 7 mice per group, *P<0.05 vs unstimulated FVB-LF group, †P<0.05 vs insulin-stimulated FVB-LF group, #P<0.05 vs respective FVB-HF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

8. Phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), forkhead transcriptional factors (Foxo3a), and mammalian target of rapamycin (mTOR) with or without insulin stimulation (100 nmol/L for 15 minutes) in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mice.
Phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), forkhead transcriptional factors (Foxo3a), and mammalian target of rapamycin (mTOR) with or without insulin stimulation (100 nmol/L for 15 minutes) in cardiomyocytes from low-fat (LF) and high-fat (HF)–fed FVB and insulin-like growth factor 1 (IGF-1) mice. A, phosphorylated Akt:Akt ratio. B, phosphorylated GSK3β:GSK3β ratio. C, phosphorylated Foxo3a:Foxo3a ratio. D, phosphorylated mTOR:mTOR ratio. Insets, Representative gel blots of pan and phosphorylated Akt, GSK3β, Foxo3a, and mTOR (GAPDH was used as loading control) using specific antibodies. Mean±SEM, n=6 to 9 isolations per group, *P<0.05 vs unstimulated FVB-LF group, †P<0.05 vs insulin-stimulated FVB-LF group, #P<0.05 vs respective FVB-HF group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.

9. Influence of insulin-like growth factor 1 (IGF-1) on palmitate-induced responses of glucose uptake, aconitase activity, cell survival, and cardiomyocyte contractile properties.
Influence of insulin-like growth factor 1 (IGF-1) on palmitate-induced responses of glucose uptake, aconitase activity, cell survival, and cardiomyocyte contractile properties. Cardiomyocytes from control FVB mice maintained in DEME at 37°C were exposed to palmitate (100 μmol/L) in the absence or presence of IGF-1 (10 nmol/L), the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 μmol/L), or the glycogen synthase kinase 3β (GSK3β) inhibitor SB (10 μmol/L) for 6 hours before assessment of mechanical and biochemical properties A, Insulin (10 nmol/L)-stimulated cardiomyocyte glucose uptake. B, Myocardial aconitase activity. C, Cardiomyocyte survival rate. D, Peak shortening (percentage of cell length). E, Maximal velocity of shortening and relengthening (±dL/dt). F, Time to 90% relengthening (TR90). Mean±SEM, n=5 isolations or 60 cells, *P<0.05 vs control group, #P<0.05 vs palmitate group.
Yingmei Zhang et al. Hypertension. 2012;59:
Copyright © American Heart Association, Inc. All rights reserved.