Insulin-like growth factor 1 alleviates high-fat diet-induced myocardial contractile dysfunction: role of insulin signaling and mitochondrial function

Abstract

Obesity is often associated with reduced plasma insulin-like growth factor 1 (IGF-1) levels, oxidative stress, mitochondrial damage, and cardiac dysfunction. This study was designed to evaluate the impact of IGF-1 on high-fat diet-induced oxidative, myocardial, geometric, and mitochondrial responses. FVB and cardiomyocyte-specific IGF-1 overexpression transgenic mice were fed a low- (10%) or high-fat (45%) diet to induce obesity. High-fat diet feeding led to glucose intolerance, elevated plasma levels of leptin, interleukin 6, insulin, and triglyceride, as well as reduced circulating IGF-1 levels. Echocardiography revealed reduced fractional shortening, increased end-systolic and end-diastolic diameter, increased wall thickness, and cardiac hypertrophy in high-fat-fed FVB mice. High-fat diet promoted reactive oxygen species generation, apoptosis, protein and mitochondrial damage, reduced ATP content, cardiomyocyte cross-sectional area, contractile and intracellular Ca(2+) dysregulation (including depressed peak shortening and maximal velocity of shortening/relengthening), prolonged duration of relengthening, and dampened intracellular Ca(2+) rise and clearance. Western blot analysis revealed disrupted phosphorylation of insulin receptor and postreceptor signaling molecules insulin receptor substrate 1 (tyrosine/serine phosphorylation), Akt, glycogen synthase kinase 3β, forkhead transcriptional factors, and mammalian target of rapamycin, as well as downregulated expression of mitochondrial proteins peroxisome proliferator-activated receptor-γ coactivator 1α and uncoupling protein 2. Intriguingly, IGF-1 mitigated high-fat-diet feeding-induced alterations in reactive oxygen species, protein and mitochondrial damage, ATP content, apoptosis, myocardial contraction, intracellular Ca(2+) handling, and insulin signaling but not whole body glucose intolerance and cardiac hypertrophy. Exogenous IGF-1 treatment also alleviated high-fat diet-induced cardiac dysfunction. Our data revealed that IGF-1 alleviates high-fat diet-induced cardiac dysfunction despite persistent cardiac remodeling, possibly because of preserved cell survival, mitochondrial function, and insulin signaling.

Fig. 1

Effect of cardiac IGF-1 overexpression on glucose tolerance, ROS production, apoptosis, ATP production, protein and mitochondrial damage following low fat (LF) or high fat (HF)-diet feeding. A: IPGTT displaying serum glucose levels following glucose challenge (2 g glucose/kg 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; and 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 = 7–10 mice/group, *p < 0.05 vs. FVB-LF group, #p < 0.05 vs. FVB-HF group, †p < 0.05 vs. IGF-LF group.

Fig. 2

Contractile properties of cardiomyocytes from LF and HF-fed FVB and 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-PS (TPS); and H: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 80 cells from 3–4 mice/group, *p < 0.05 vs. FVB-LF group, #p < 0.05 vs. FVB-HF group.

Fig. 3

Intracellular Ca²⁺ transients and stimulus frequency response in cardiomyocytes from LF and HF-fed FVB and 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; and F: Intracellular Ca²⁺ decay rate. Mean ± SEM, n = 68 cells (23–25 cells for panel A) from 3–4 mice/group, *p < 0.05 vs. FVB-LF group, #p < 0.05 vs. FVB-HF group.

Fig. 4

Effect of IGF-1 overexpression on myocardial apoptosis and hypertrophy following LF or HF feeding using TUNEL and FITC-conjugated Lectin staining, respectively. All nuclei were stained with DAPI (blue) in panels B (FVB-LF), D (FVB-HF), F (IGF-LF) and H (IGF-HF). TUNEL-positive nuclei were visualized with fluorescein (green) in panels A (FVB-LF), C (FVB-HF), E (IGF-LF) and G (IGF-HF). Original magnification = 400×. Quantified data are shown in panel I; J: FITC-conjugated Lectin immunostaining depicting transverse sections of left ventricular myocardium (×400); and K: Quantitative analysis of cardiomyocyte cross-sectional area. Mean ± SEM, n = 15 and 10 fields from 3 mice per group for panel 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.

Fig. 5

Western blot analysis of the mitochondrial proteins UCP-2 and PGC1α as well as the Ca²⁺ regulatory proteins SERCA2a, Na⁺-Ca²⁺ exchanger (NCX) and phospholamban in myocardium from LF and HF-fed FVB and 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; and F: Phospholamban. All proteins were normalized to the loading control GAPDH. Mean ± SEM, n = 8–9 mice per group, *p <0.05 vs. FVB-LF group, #p < 0.05 vs. FVB-HF group.

Fig. 6

Western blot analysis of pan and phosphorylated insulin receptor β or IGF-1 receptor β in cardiomyocytes from LF and HF-fed FVB and IGF-1 mice. A: Insulin receptor β expression; B: Basal and insulin/IGF-1-stimulated (at 100 nM for 15 min) tyrosine phosphorylation of insulin receptor β; C: IGF-1 receptor β expression; D: Basal and insulin/IGF-1-stimulated (at 100 nM for 15 min) tyrosine phosphorylation of IGF-1 receptor β; E: Tyrosine phosphorylation of IRS (Tyr1146) normalized to pan IRS; and 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. Protein expressions were normalized to that of FVB-LF group for immunoprecipitation studies. pY denotes anti-phosphotyrosine antibody; IP and IB represent immunoprecipitation and immunoblot, respectively; Mean ± SEM, n = 5–7 mice per group, *p <0.05 vs. un-stimulated FVB-LF group, †p < 0.05 vs. insulin-stimulated FVB-LF group, #p < 0.05 vs. respective FVB-HF group.

Fig. 7

Phosphorylation of Akt, GSK3β, Foxo3a and mTOR with or without insulin stimulation (100 nM for 15 min) in cardiomyocytes from LF and HF-fed FVB and IGF-1 mice. A: pAkt-to-Akt ratio; B: pGSK3β-to-GSK3β ratio; C: pFoxo3a-to-Foxo3a ratio; and D: pmTOR-to-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 = 6–9 isolations per group, *p < 0.05 vs. un-stimulated FVB-LF group, †p < 0.05 vs. insulin-stimulated FVB-LF group, #p < 0.05 vs. respective FVB-HF group.

Fig. 8

Influence of 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 medium at 37°C were exposed to palmitate (100 µM) in the absence or presence of IGF-1 (10 nM), the mitochondrial uncoupler FCCP (1 µM), or the GSK3β inhibitor SB216763 (10 µM) for 6 hrs prior to assessment of mechanical and biochemical properties A: Insulin (10 nM)-stimulated cardiomyocyte glucose uptake; B: Myocardial aconitase activity; C: Cardiomyocyte survival rate; D: Peak shortening (% of cell length); E: Maximal velocity of shortening and relengthening (± dL/dt); and 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.