Central leptin regulates heart lipid content by selectively increasing PPAR β/δ expression

in Journal of Endocrinology
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The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d) and their target genes, adipose triglyceride lipase (encoded by Pnpla2, herefater referred to as Atgl), hormone sensitive lipase (encoded by Lipe, herefater referred to as Hsl), pyruvate dehydrogenase kinase 4 (Pdk4) and acyl CoA oxidase 1 (Acox1), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 (Scd1) and diacylglycerol acyltransferase 1 (Dgat1) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight.

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  • Supplementary Table 1 - Probes used for real time PCR

 

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    Central leptin effects in the phosphorylation levels of STAT3, AMPK and ACC. (A, B, C and D) Representative microphotographs of pY-STAT3 immunohistochemistry (IHC) of hypothalamic sections from saline-infused and leptin-infused rats, out of three independent experiments. After 7-day infusion of leptin (0.2 μg/day), there is an increase in the staining of tyrosine phosphorylation of STAT3 (pY-STAT3) (see arrows) in the paraventricular nucleus (PVH) (B) and arcuate nucleus (ARC) (D) with respect to saline-infused (A and C) rats. Scale bars, 150 μm. Representative Western blot out of four and quantitative densitometric analysis showing total STAT3 and pY-STAT3 (E), and total AMPK, ACC, pT-AMPK and pS-ACC (F) in 50 µg total extracts from heart after 7 days of central saline or leptin infusion. For each analyzed protein, data were expressed as a ratio of phosphorylated to protein content. Results are the mean ± s.e.m. of 8–10 rats per group. Different letters indicate significant differences among treatments (P < 0.05, one-way ANOVA followed by Tukey test). Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    Intracerebroventricular leptin decreases cardiac triacylglyceride content and regulates the expression of genes involved in cardiac metabolism. (A) TAG content in heart after 7 days of central infusion of leptin (0.2 μg/day) (Lep), saline-infused rats fed ad libitum or saline-infused pair-fed rats. (B) mRNA levels of Atgl, Hsl in leptin or saline-infused rats. (C) mRNA levels of Cd36, Cpt1b, Mnf2, Pdk4, Ucp3, Acox1 in leptin or saline-infused rats. (D) Effect of leptin or saline infusion on mRNA levels of Acly, Scd1, Dgat1. (E) Effect of leptin or saline infusion on citrate synthase activity and relative mtDNA content. Results are the mean ± s.e.m. of 8–10 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    Effects of central leptin on cardiac expression levels of PPARs and PGC-1s. (A) mRNA levels of Ppara and Pgc1a in leptin or saline-infused rats. (B) Representative Western blot out of four and (C) quantitative densitometric analysis of PPARα and PGC-1α protein levels in 50 µg of total extracts from cardiac ventricles after central saline or leptin infusion. (D) mRNA levels of Pparb/d, Pparg and Pgc1b. (E) Representative Western blot out of four and (F) quantitative densitometric analysis of PPARβ/δ, PPARγ and PGC-1β protein levels in 50 µg of total extracts from cardiac ventricles after central saline or leptin infusion. β-Actin was used as control for protein loading and for densitometric normalization. Results are the mean ± s.e.m. of 8–10 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA folowed by Tukey test). Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    Effects of central leptin on cardiac palmitate oxidation in cardiac homogenates. (A) Complete palmitate oxidation and percent contribution of etomoxir-dependent inhibition of palmitate oxidation (CPT1-dependent) or etomoxir-independent (CPT1-independent) palmitate oxidation in saline-infused pair-fed and leptin-infused rats. (B) Effect of leptin infusion on etomoxir-dependent inhibition (CPT1-dependent) or etomoxir-independent (CPT1-independent) palmitate oxidation in saline-infused pair-fed and leptin-infused rats. (C) Representative Western blot out of four and quantitative densitometric analysis of FAT/CD36 protein levels in 15 µg of PM fraction from cardiac ventricles after 7 days of central saline or leptin infusion. Na+/K+-ATPase was used as control for PM protein loading and for densitometric normalization. Results are the mean ± s.e.m. of 8–10 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    Central leptin stimulates cardiac basal glucose uptake. (A) Cardiac ventricular explants were used for [3H]-2-deoxyglucose uptake measurements in the absence or presence of 80 nM insulin stimulation for 10 min after 7 days of central infusion of leptin or saline. Data shown are from four independent experiments per group. (B) Glut1 and Glut4 glucose transporters mRNA levels in leptin or saline-infused rats. (C) Representative Western blot out of four and quantitative densitometric analysis of GLUT1 and GLUT4 levels in 15 µg of PM fraction from cardiac ventricles after 7 days of central saline or leptin infusion. Na+/K+-ATPase was used as control for PM protein loading and for densitometric normalization. Results are the mean ± s.e.m. of 8–10 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Student’s t-test, *P ≤ 0.05 vs basal, without insulin. Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    Central leptin infusion did not alter the subcellular distribution of GLUT4 in response to insulin in heart. (A) Representative Western blot out of three and (B) quantitative densitometric analysis of GLUT4 glucose transporter in 15 µg of protein from plasma membrane (PM) and internal membrane (IM) of heart from central leptin or saline-infused rats after 7 days of treatment and 30 min of in vivo insulin (10 IU/kg body weight) stimulation. Na+/K+-ATPase and EEA1 were used as control for protein loading for PM and IM, respectively, and for densitometric normalization. (C) Representative Western blot out of three and quantitative densitometric analysis of p-S473-AKT2 and AKT2 in 50 µg of total extracts from cardiac ventricles after 7 days of central saline or leptin infusion, and 30 min of in vivo insulin (10 IU/kg body weight) stimulation. Data are expressed as ratios of phosphoserine content in AKT2 normalize to the corresponding amount of AKT2 protein. Results are the mean ± s.e.m. of 6–8 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Student’s t-test, *P ≤ 0.05 vs basal, without insulin. Lep, leptin-infused rats; PF, vehicle-infused pair-fed rats; SS, vehicle-infused rats fed ad libitum.

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    Central leptin increases cardiac protein levels of PDK4, glycogen content and activity of malic enzyme. (A) Effect of leptin infusion on protein levels of PDK4. Representative Western blot out of four and quantitative densitometric analysis of PDK4 protein levels in 50 µg of total extract from cardiac ventricles of leptin or saline-infused rats after 7 days of treatment. β-Actin was used as control for protein loading and for densitometric normalization. (B) Effect of leptin infusion on glycogen levels, mRNA levels and activity of malic enzyme. Results are the mean ± s.e.m. of 6–8 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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    The PPARβ/δ antagonist GSK0660 reduces the effects of central leptin on both, cardiac triacylglyceride content and the expression of PPARβ/δ target genes. Central saline- or leptin-infused rats were co-treated with either vehicle (0.062% DMSO) or GSK0660 (1 mg/kg per day i.p.) for 7 days. (A) TAG content in heart after 7 days of central infusion in rats, as described in Fig. 2, co-treated with either vehicle or GSK0660. (B) Effect of GSK0660 on mRNA levels of Pparb/d. (C) Effects of GSK0660 on mRNA levels of target genes of PPARβ/δ. Results are the mean ± s.e.m. of 4–6 rats per group. Different letters indicate significant differences among treatments, (P ≤ 0.05, one-way ANOVA followed by Tukey test). Student’s t-test,*P ≤ 0.05 vs leptin. Lep, leptin-infused rats; PF, saline-infused pair-fed rats; SS, saline-infused rats fed ad libitum.

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