GLP-1 Receptor Activation Inhibits Palmitate-Induced Apoptosis via Ceramide in Human Cardiac Progenitor Cells (original) (raw)

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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Valentina Andrulli Buccheri

1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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2Department of Emergency and Organ Transplantation, Section of Cardiac Surgery, University of Bari Aldo Moro, I-70124 Bari, Italy

3Cardiac Surgery, Santa Maria Hospital, I-70124 Bari, Italy

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3Cardiac Surgery, Santa Maria Hospital, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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1Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, Andrology, and Metabolic Diseases, University of Bari Aldo Moro, I-70124 Bari, Italy

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16 August 2017

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Anna Leonardini, Rossella D’Oria, Maria Angela Incalza, Cristina Caccioppoli, Valentina Andrulli Buccheri, Angelo Cignarelli, Domenico Paparella, Vito Margari, Annalisa Natalicchio, Sebastio Perrini, Francesco Giorgino, Luigi Laviola, GLP-1 Receptor Activation Inhibits Palmitate-Induced Apoptosis via Ceramide in Human Cardiac Progenitor Cells, The Journal of Clinical Endocrinology & Metabolism, Volume 102, Issue 11, 1 November 2017, Pages 4136–4147, https://doi.org/10.1210/jc.2017-00970
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Abstract

Context

Increased apoptosis of cardiomyocytes and cardiac progenitor cells (CPCs) in response to saturated fatty acids (SFAs) can lead to myocardial damage and dysfunction. Ceramides mediate lipotoxicity-induced apoptosis. Glucagonlike peptide-1 receptor (GLP1R) agonists exert beneficial effects on cardiac cells in experimental models.

Objective

To investigate the protective effects of GLP1R activation on SFA-mediated apoptotic death of human CPCs.

Design

Human CPCs were isolated from cardiac appendages of nondiabetic donors and then exposed to palmitate with or without pretreatment with the GLP1R agonist exendin-4. Ceramide accumulation was evaluated by immunofluorescence. Expression of key enzymes in de novo ceramide biosynthesis was studied by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Apoptosis was evaluated by measuring release of oligonucleosomes, caspase-3 cleavage, caspase activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.

Results

Exposure of the CPCs to palmitate resulted in 2.3- and 1.9-fold higher expression of ceramide synthase 5 (CERS5) and ceramide desaturase-1, respectively (P < 0.05). This was associated with intracellular accumulation of ceramide and activation of c-Jun NH2-terminal protein kinase (JNK) signaling and apoptosis (P < 0.05). Both coincubation with fumonisin B1, a specific ceramide synthase inhibitor, and CERS5 knockdown prevented ceramide accumulation, JNK activation, and apoptosis in response to palmitate (P < 0.05). Exendin-4 also prevented the activation of the ceramide biosynthesis and JNK in response to palmitate, inhibiting apoptosis (P < 0.05).

Conclusions

Excess palmitate results in activation of ceramide biosynthesis, JNK signaling, and apoptosis in human CPCs. GLP1R activation counteracts this lipotoxic damage via inhibition of ceramide generation, and this may represent a cardioprotective mechanism.

The viability of progenitor cells is essential for constant tissue repair and renewal in the adult heart (1, 2), and reduced survival and dysfunction of cardiac progenitors may contribute to myocardial impairment and heart failure. Early cellular senescence, growth limitation, and enhanced apoptotic death of cardiac progenitor cells (CPCs) have been reported in human diseases (1, 2). Specifically, lipotoxicity has recently emerged as an important contributor to the development of cardiac dysfunction associated with obesity and type 2 diabetes mellitus (T2DM) (3).

Saturated fatty acids (SFAs) are known to impair metabolic pathways (3) and to increase apoptosis in cardiomyocytes (4) and H9c2 cardiac myoblasts (5). Moreover, a close relationship between cardiac lipotoxicity and impaired left ventricular function was shown in experimental models (6). Several pathways have been proposed to mediate SFA-induced apoptosis in cardiomyocytes, including activation of the stress kinase c-Jun NH2-terminal protein kinase (JNK) (7) and reactive oxygen species (ROS) production (8). De novo generation and accumulation of intracellular ceramide, resulting from the sequential action of multiple enzymes (9), are involved in lipotoxic myocardiocyte damage and apoptosis (10, 11). However, the role of ceramide and stress kinase signaling in apoptotic death of human CPCs in response to SFAs has not been investigated.

The protective role of glucagonlike peptide-1 (GLP-1) and its analogs in cardiovascular disease has been suggested by both preclinical and clinical studies (12). GLP-1 receptor (GLP1R) activation can trigger cell survival pathways in myocardial tissue, conferring protection from ischemia/reperfusion injury and amelioration of cardiac dysfunction in various animal models (13). Besides their glucoregulatory effects, GLP1R agonists have been shown to preserve endothelial function by decreasing oxidative stress (14) and inflammation (15), and improve myocardial bioenergetics (16). Moreover, recent studies have examined the cardioprotective effects of exendin-4 and liraglutide in animal models of obesity or T2DM in the context of lipotoxicity (17, 18), showing prevention of heart steatosis and inflammation in vivo. However, the underlying mechanisms are still poorly defined. We have shown that GLP-1 analogs prevent palmitate-induced apoptosis in pancreatic β cells by reducing the expression of the SFA receptor GPR40 and interfering with the activation of JNK signaling (19). Whether a similar inhibition of lipid and stress kinase signaling also occurs in human CPCs has not been investigated, to our knowledge.

Methods

Isolation and culture of human CPCs and human cardiospheres

Cardiac bioptic samples were obtained from the right atrial appendage of patients undergoing heart surgery. Samples from both sexes were used without regard to sex. The study protocol was approved by the independent Ethics Committee of the Azienda Ospedaliero-Universitaria and the University of Bari Aldo Moro and conformed with the declaration of Helsinki; informed consent to the procedure was obtained before surgery. Cardiac bioptic samples in vitro were processed to isolate cardiosphere-forming cells, ultimately leading to tridimensional multicellular structures (cardiospheres) (20) or human CPCs. The protocol to isolate and characterize human CPCs isolated from human heart biopsy specimens has been reported (21). For isolation of human cardiospheres, cardiac bioptic samples were stored on ice in cardioplegic solution and processed within 2 hours. Gross connective tissue was removed from samples, which were cut into fragments, washed, partially digested enzymatically, and cultured in dishes coated with fibronectin. After 2 weeks, a layer of cells arose from adherent explants, representing human cardiosphere-forming cells, which were seeded on poly-d-lysine–coated dishes in cardiosphere medium. Fully formed human cardiospheres were observed 12 days after collection of human cardiosphere-forming cells.

Treatments

Human cardiac bioptic samples, cardiosphere-forming cells, fully formed cardiospheres, and human CPCs, isolated from different donors, were pretreated with or without 20 nmol/L exendin-4 (Sigma-Aldrich, St. Louis, MO) for various times, as indicated, followed by stimulation with 0.1 to 0.5 mmol/L palmitate (for human CPCs) or 15 µmol/L palmitate (for cardiospheres; Sigma-Aldrich) for various times, as indicated. Palmitate was dissolved in 0.1 mol/L NaOH at 70°C for 30 minutes, and 5 mmol/L palmitate was complexed with 10% essentially fatty acid-free bovine serum albumin (BSA; fatty acid-to-BSA molar ratio, 3.3:1). Control cells received the same amount of solvent and BSA. Cells were also treated with SP600125 (10 μmol/L; Calbiochem, San Diego, CA), fumonisin B1 (FB1; 20 μmol/L; Sigma-Aldrich), exendin 9-39 (20 nmol/L; Sigma-Aldrich), H89 (5 μmol/L; Calbiochem), PKA inhibitor 14-22 amide (100 nmol/L; Merck), brefeldin-A (50 µmol/L; Sigma Aldrich), 3-isobutyl-1-methylxanthine (100 µmol/L; Sigma Aldrich) or N_-acetyl-cysteine (10 mmol/L; Sigma-Aldrich), as indicated. For studies under hypoxic conditions, human CPCs were treated with increasing concentrations of the hypoxic stressor CoCl2·6H2O (50, 100, or 150 µmol/L; Sigma-Aldrich) for 24 hours in a conventional incubator (37°C; 5% CO2) and then analyzed for HIF-1_α protein expression, using immunoblot analysis.

Gene expression by quantitative reverse-transcription polymerase chain reaction

Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), as previously described (19, 21). Total RNA (500 ng) was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Weiterstadt, Germany). Polymerase chain reactions were carried out in ABI PRISM 7500 System (Applied Biosystems) under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Relative gene expression levels were determined by analyzing the changes in SYBR green fluorescence during quantitative polymerase chain reaction using the ΔΔCt method. The mRNA level of each gene was normalized using 18S as internal control. All primers were designed using Primer Express 3.0 (Applied Biosystems; Supplemental Table 1).

Immunoblotting

Experimental cells were rapidly washed with Ca2+/Mg2+-free phosphate-buffered saline (PBS) and then mechanically detached in ice-cold lysis buffer containing 50 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 4 mmol/L EDTA, 1% Triton X-100, 10% glycerol, 50 mmol/L NaF, and 10 mmol/L NaPP, supplemented with phosphatase and protease inhibitors (Roche, Mannheim, Germany). Immunoblotting was carried out as previously described (19, 21).

Detection of apoptosis

Apoptosis was measured by multiple independent methods. Release of cytoplasmic oligonucleosomes was evaluated using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics), according to the manufacturer’s instructions.

An in vitro caspase-3 (CASP3) activity assay was performed using the Caspase-3 Colorimetric Assay Kit (Millipore, Boston, MA), according to the manufacturer’s instructions. Assays were performed in 96-well plates by incubating 80 µg of cell lysates in 100 µL of reaction buffer containing the CASP3 substrate Ac-DEVD-pNA. Lysates were incubated at 37°C for 2 hours. Absorbance at 405 nm was then measured with a microtiter plate reader (Beckman Coulter, Fullerton, CA).

Apoptosis was also assessed by the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay (Roche). Cells seeded on coverslips were treated with cold 4% paraformaldehyde for 20 minutes at room temperature, washed in PBS, and permeabilized in 0.1% Triton X-100 with 0.1% sodium citrate for 2 minutes on ice. Cells were washed in PBS and incubated with dUTP-fluorescein in the presence of working-strength terminal deoxynucleotidyl transferase in a humidified atmosphere for 60 minutes at 37°C in the dark. Fluorescein labels were detected by Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems, Heerbrugg, Switzerland).

Detection of ceramide by immunofluorescence

Cells grown to 90% confluence on coverslips were fixed in ice-cold 4% paraformaldehyde for 15 min and permeabilized in PBS/0.1% Triton X-100/0.1% sodium dodecyl sulfate for 20 minutes. Samples were incubated with a primary monoclonal antibody against ceramide (1:80; Alexis Biochemicals, San Diego, CA) in PBS/1.5% BSA overnight at 4°C, and then with a secondary Alexa (488) Fluor anti-mouse goat antibody (1:1000; Molecular Probes, Eugene, OR) for 1 hour at 25°C. Nuclei were stained with TO-PRO-3 (1:3000; Molecular Probes). Micrographs were acquired via a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems).

Detection of ROS

Intracellular ROS were detected by evaluating the oxidation of the superoxide-sensitive dye dihydroethidium (DHE). Human CPCs, cultured on glass coverslips, were fixed in ice-cold 4% paraformaldehyde for 15 minutes and permeabilized in PBS/0.1% Triton X-100 for 10 minutes. Samples were incubated with DHE at the final concentration of 15 µmol/L for 30 minutes at 37°C in the dark. Nuclei were stained with TO-PRO-3 (1:3000; Molecular Probes). Micrographs were acquired on a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems).

GLP1R and ceramide synthase 5 knockdown

After achieving 80% cell confluence, human CPCs were maintained in F-12 Nutrient Mixture medium (Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (Life Technologies) and 1% nonessential amino acids (Life Technologies), and transfected with a mixture of three GLP1R small interfering RNA (siRNA) sequences (Supplemental Table 2) at 50 nmol/L for 24 hours using 5 µL per well of Lipofectamine 2000 (Invitrogen, Carlsbad, CA). A nonsilencing fluorescently labeled siRNA was used as negative control at 33 nmol/L. The GLP1R siRNA sequences and the negative control were purchased from Invitrogen. Ceramide synthase 5 (CERS5) knockdown was obtained following the same transfection protocol and using a specific CERS5 siRNA sequence (Supplemental Table 2) at 20 nmol/L for 24 hours. The CERS5 siRNA was from Integrated DNA Technologies (Coralville, IA).

Antibodies

Polyclonal anti-SAPK/JNK, antiphospho-SAPK/JNK (Thr183/Tyr185), anti-c-Jun, antiphospho-c-Jun (Ser63), anticleaved CASP3, anti-HIF-1_α_ antibodies were obtained from Cell Signaling Technology (Danvers, MA). Monoclonal anti-_β_-actin, polyclonal anti-serine palmitoyltransferase (SPT) light chain-1, polyclonal anti-CERS5, and polyclonal anti-GLP1R antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-dihydroceramide desaturase 1 (DEGS1) antibody was from Aviva System Biology (San Diego, CA).

Statistical analyses

All data are presented as mean ± standard error of the mean of at least three independent experiments. Data are expressed as percentage of control or basal control values, as appropriate. Statistical analysis was performed by a Student t test or analysis of variance, as appropriate. Significance was assumed at P < 0.05.

Results

Exendin-4 inhibited palmitate-induced apoptosis of human CPCs and prevented the palmitate-dependent impairment of human cardiosphere isolation

Exposure of human CPCs to the SFA palmitate (0.1 to 0.5 mmol/L) resulted in a time- and dose-dependent several-fold increase of CPC apoptosis, measured by assessing oligonucleosome release (P < 0.05 vs not exposed to palmitate; Fig. 1A; Supplemental Fig. 1). Apoptosis induction by 0.25 mmol/L palmitate for 16 hours was also detected by measurements of CASP3 activity (Fig. 1B) and cleavage (Fig. 1C), as well as by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (Fig. 1D). However, preincubation with 20 nmol/L exendin-4 largely prevented the ability of palmitate to trigger apoptosis (P < 0.05 vs cells not treated with exendin-4; Fig. 1A–D). The protective action of exendin-4 against palmitate-induced apoptosis also was preserved under conditions of cellular hypoxia, achieved by exposing the human CPCs to the hypoxic stressor CoCl2·6H2O for 24 hours (P < 0.05 vs palmitate alone; Supplemental Fig. 2).

Figure 1.

Palmitate-induced apoptosis of human CPCs and protective effects of exendin-4. Human CPCs were incubated in the presence of 20 nmol/L exendin-4, or left untreated, before challenge with 0.25 mmol/L palmitate for 16 hours. (A) Quantification of cytoplasmic oligonucleosomes by enzyme-linked immunosorbent assay. The effects of exendin-4 in the presence of the GLP1R antagonist exendin 9-39 or the PKA inhibitor H89 are also shown. Cells were pretreated with 20 nmol/L exendin 9-39 for 8 hours, or 5 µmol/L H89 for 2 hours, or left untreated, and then incubated with or without 20 nmol/L exendin-4 for 8 hours before palmitate addition. (B) Quantification of CASP3 activity by Caspase-3 Colorimetric Assay Kit. (C) Evaluation of cleaved CASP3 protein levels by immunoblotting. Protein lysates (30 µg) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anticleaved CASP3 and anti-β_-actin (ACTB) antibodies, respectively. ACTB was used as a loading control. (D) Assessment of cell apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay in CPCs pretreated with 20 nmol/L exendin-4, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. Scale bar, 47.62 µm. Data are presented as mean ± standard error of the mean of at least three experiments, which were carried out using cells from different human donors; *P < 0.05 vs basal (no palmitate); †_P < 0.05 vs palmitate alone. Ex-4, exendin-4; Ex(9-39), exendin 9-39; Palm, palmitate.

We have previously demonstrated that the ability of GLP-1 to protect human CPCs from H2O2-induced apoptosis is mediated by activation of the canonical GLP1R, which signals via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/CREB pathway (21). Similarly, the ability of exendin-4 to prevent palmitate-induced apoptosis of human CPCs was abolished when cells were preincubated with the GLP1R antagonist exendin 9-39, the PKA inhibitor H89 (P < 0.05 vs cells not treated with exendin 9-39 or H89; Fig. 1A), or the PKA inhibitor 14-22 amide (P < 0.05 vs cells not treated with the PKA inhibitor 14-22 amide; Supplemental Fig. 3).

When human cardiac fragments were treated according to the cardiosphere protocol in the presence of 15 µmol/L palmitate added every 24 hours, both the number and size of the resulting cardiospheres appeared to be reduced (Supplemental Fig. 4B). However, coincubation with 20 nmol/L exendin-4 and 15 µmol/L palmitate prevented the ability of palmitate to impair cardiosphere formation (Supplemental Fig. 4D).

Exendin-4 prevented palmitate-induced accumulation of ceramide

Exposure of human CPCs to 0.25 mmol/L palmitate for 16 hours resulted in increased intracellular ceramide content, evaluated by immunofluorescence, which was almost completely abrogated in human CPCs treated with the ceramide synthase inhibitor FB1 (Fig. 2). Interestingly, ceramide accumulation was also prevented when human CPCs were treated with 20 nmol/L exendin-4 before exposure to palmitate (Fig. 2). The effects of palmitate and exendin-4 on ceramide accumulation under hypoxic conditions were difficult to interpret because the hypoxic stressor CoCl2·6H2O per se caused ceramide accumulation in the absence of palmitate (Supplemental Fig. 5). However, under hypoxia, the pattern of ceramide accumulation in the CPCs appeared to be markedly disorganized by palmitate and this was corrected after pretreatment with exendin-4 (Supplemental Fig. 5).

Figure 2.

Effects of FB1 and exendin-4 on the intracellular content of ceramide in CPCs exposed to palmitate. Human CPCs were preincubated with 20 nmol/L exendin-4 for 8 hours, or 20 µmol/L FB1 for 8 hours, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. Cells were then fixed and incubated with a ceramide antibody followed by Alexa Fluor (488) anti-rabbit antibody (green) to stain ceramide. TO-PRO-3 was used to stain nuclei (blue). Scale bar, 47.62 µm. Ex-4, exendin-4; Palm, palmitate.

Next, the mRNA and the protein levels of SPT, CERS5, and DEGS1, enzymes involved in de novo production of ceramide (Fig. 3A) (9), were evaluated. CERS5 is specific for the conversion of palmitic acid to ceramide (9). Exposure of CPCs to palmitate for different times did not affect the mRNA or protein levels of SPT (Fig. 3B). By contrast, both mRNA (after 8 hours) and protein (after 4 to 16 hours) levels of CERS5 were increased by palmitate (P < 0.05 vs cells not exposed to palmitate; Fig. 3C). Protein levels of DEGS1 were also increased (P < 0.05 vs untreated cells; Fig. 3D), albeit with a shorter time course than for CERS5, and this was not associated with changes in mRNA levels (Fig. 3D). When the CPCs were pretreated with the GLP1R agonist exendin-4, the increase of CERS5 expression, both at the mRNA and protein levels, as well as the increase in DEGS1 protein levels were prevented (P < 0.05 vs cells not treated with exendin-4; Fig. 3C and 3D). Moreover, exendin-4 reduced SPT protein levels after exposure to palmitate for 16 hours (P < 0.05 vs cells not treated with exendin-4; Fig. 3B). The ability of exendin-4 to prevent induction of CERS5 by palmitate was mimicked by the phosphodiesterase inhibitor 3-isobutyl-methylxantine, which increases cytosolic cAMP (P < 0.05 vs palmitate; Supplemental Fig. 6). The exendin-4–mediated inhibition of CERS5 induction was no longer evident (Fig. 4A) when the CPCs were incubated in the presence of three siRNA sequences specific to the human GLP1R, obtaining a 50% reduction in GLP1R mRNA (P < 0.05 vs control; Supplemental Fig. 7) and protein levels (Fig. 4A), evaluated by using an anti-GLP1R antibody previously tested for its specificity (21). In addition, the effects of exendin-4 to prevent CERS5 induction were abrogated in the presence of the PKA inhibitor 14-22 amide and brefeldin A, an inhbitor of exchange protein directly activated by cAMP (P < 0.05 vs control; Supplemental Figs. 8 and 9).

Figure 3.

Effects of exendin-4 on the regulation of the ceramide synthesis pathway by palmitate. (A) Schematic representation of the de novo synthesis of ceramide. (B–D) Human CPCs were incubated in the presence or absence of 20 nmol/L exendin-4 for 8 hours, and then exposed to 0.25 mmol/L palmitate for 16 hours. Each panel shows, for each enzyme, the mRNA levels assessed by quantitative reverse transcription PCR (left panel), a representative immunoblot (right, top panel), and the quantitation of at least three experiments (right, bottom panel). β_-actin (ACTB) was used as a loading control. (B) mRNA and protein levels of SPT. (C) mRNA and protein levels of CERS5. (D) mRNA and protein levels of DEGS1. All data are presented as mean ± standard error of the mean of at least three experiments, which were carried out using cells from different human donors. *P < 0.05 vs basal (no palmitate); †_P < 0.05 vs palmitate alone. CoA, coenzyme A; Ex-4, exendin-4; Palm, palmitate.

Figure 4.

Modulation of CERS5 expression and ceramide-associated apoptosis in human CPCs. (A) Exendin-4 reduces CERS5 protein expression through the GLP1R. Cells were transfected with a mixture of three GLP1R siRNAs (50 nmol/L) for 24 hours, then stimulated with 20 nmol/L exendin-4 for 8 hours, and finally exposed to 0.25 mmol/L palmitate for 16 hours. Protein levels of the GLP1R and CERS5 in the experimental cells are shown. Protein lysates (25 µg) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with anti-GLP1R or anti-CERS5 antibodies, respectively. β_-actin (ACTB) was used as a loading control. Data are presented as mean ± standard error of the mean (SEM) of at least three experiments, which were carried out using cells from different human donors. *P < 0.05 vs basal (Lipofectamine 2000); †_P < 0.05 vs palmitate alone; ‡P < 0.01 vs siGLP1R alone; §P < 0.05 vs siGLP1R and exendin-4. (B–E) CERS5 mediates palmitate-induced apoptosis. Human CPCs were treated with (B, C) 20 µmol/L FB1 for 8 hours or (D, E) transfected with a siRNA specific to CERS5 (20 µmol/L for 24 hours) before exposure to 0.25 mmol/L palmitate for 16 hours. Apoptosis was evaluated by (B, D) enzyme-linked immunosorbent assay for cytoplasmic oligonucleosomes and quantification of cleaved CASP3 by (C, E) immunoblotting. (C, E) White bars and black bars represent the 17-kDa and 19-kDa fragments of CASP3, respectively. ACTB was used as a loading control. Data are presented as mean ± SEM of at least three experiments, which were carried out using cells from different human donors; *P < 0.05 vs basal (no palmitate); †P < 0.05 vs palmitate alone. ‡P < 0.05 vs basal (Lipofectamine 2000); §P < 0.05 vs basal (Lipofectamine 2000) and palmitate. Ex-4, exendin-4; Palm, palmitate; siCERS5, siRNA to CERS5; siCtrl, negative control for siRNA; siGLP1R, siRNA to GLP1R.

CERS5 mediated palmitate-induced apoptosis

The role of CERS5 in SFA-induced apoptosis was investigated next. Inhibition of ceramide accumulation with FB1 prevented the apoptotic response to palmitate (P < 0.05 vs cells exposed to palmitate alone; Fig. 4B and 4C). Furthermore, confluent human CPCs transfected with a siRNA sequence targeting CERS5 and subsequently exposed to palmitate showed 70% and 35% reductions in CERS5 mRNA (P < 0.05 vs control; Supplemental Fig. 10A) and protein (P < 0.05 vs control; Supplemental Fig. 10B) levels, respectively, vs untransfected cells. Under these experimental conditions, palmitate-induced apoptosis was significantly decreased by approximately 35% to 55% (P < 0.05 vs palmitate-treated cells not transfected with CERS5 siRNA; Fig. 4D and 4E).

ROS mediated palmitate-induced apoptosis

Incubation of human CPCs with 0.25 mmol/L palmitate for 16 hours also enhanced ROS production, highlighted by increased oxidation of the intracellular dye DHE (P < 0.05 vs basal; Supplemental Fig. 11A), whereas incubation with _N_-acetyl-cysteine, a precursor compound for glutathione formation, for 2 hours before the addition of palmitate resulted in reduced ROS production (P < 0.05 vs palmitate alone; Supplemental Fig. 11A) and reduced palmitate-induced apoptosis, detected by measurement of both CASP3 cleavage (P < 0.05 vs palmitate alone; Supplemental Fig. 11B) and cytoplasmic oligonucleosomes (P < 0.05 vs palmitate alone; Supplemental Fig. 11C). Hence, ROS generation appears to contribute to palmitate-induced apoptosis.

JNK signaling pathway mediated the proapoptotic effect of palmitate, was downstream of ceramide biosynthesis, and was inhibited by exendin-4

Exposure of the human CPCs to palmitate resulted in a time-dependent phosphorylation of both JNK isoforms (i.e., JNK1 and JNK2; Supplemental Fig. 12). Phosphorylation of JNK1 and JNK2 was significantly increased beginning 8 hours after challenge with the SFA (P < 0.05 vs untreated cells; Supplemental Fig. 12A and B), in parallel with phosphorylation of the endogenous JNK substrate c-Jun (P < 0.05 vs untreated cells; Supplemental Fig. 12C). When cells were pretreated with 10 μmol/L SP600125, a specific JNK inhibitor, palmitate-induced phosphorylation of c-Jun was markedly reduced (P < 0.05 vs cells not pretreated with SP600125; Fig. 5A), and so was cellular apoptosis (P < 0.05 vs cells not pretreated with SP600125; Fig. 5B). To define the role of ceramide biosynthesis in SFA-induced activation of the JNK pathway, c-Jun phosphorylation was then evaluated in the presence of the FB1 inhibitor and CERS5 knockdown, respectively. Palmitate-induced c-Jun phosphorylation was almost completely prevented when ceramide synthases were inhibited by FB1 or after knockdown of CERS5 specifically (P < 0.05 vs cells not treated with FB1 or with siRNA to CERS5; Fig. 5C and 5D). A similar inhibition of JNK1, JNK2, and c-Jun phosphorylation was also achieved when human CPCs were preincubated with exendin-4 before challenge with the SFA palmitate (Fig. 5E).

Figure 5.

The JNK signaling pathway mediates the proapoptotic effects of palmitate, is activated by the ceramide biosynthetic pathway, and is inhibited by exendin-4. (A, B) Effects of JNK inhibition on palmitate-induced c-Jun phosphorylation and apoptosis. Human CPCs were treated with 20 µmol/L SP600125 for 1 hour and then exposed to palmitate for 16 hours. (A) Phosphorylation of c-Jun was evaluated by immunoblotting and (B) apoptosis by enzyme-linked immunosorbent assay for cytoplasmic oligonucleosomes. *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with SP600125. (C, D) Effects of ceramide synthase inhibition on palmitate-induced c-Jun phosphorylation. CPCs were pretreated with (C) 20 µmol/L FB1 for 8 hours or transfected with (D) 20 µmol/L siRNA specific to CERS5 for 24 hours, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. Representative immunoblots and quantitation of c-Jun protein content and phosphorylation from at least three independent experiments are shown. *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with FB1; ‡P < 0.05 vs cells not transfected with si_CERS5_. (E) Effects of exendin-4 on the activation of the JNK pathway by palmitate. Human CPCs were incubated in the presence of 20 nmol/L exendin-4 for 8 hours, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. The panels show the quantitation of JNK1, JNK2, and c-Jun phosphorylation. All data are presented as mean ± standard error of the mean of at least three experiments, which were carried out using cells from different human donors; *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with exendin-4. Ex-4, exendin4; p-JNK, phospho-JNK; Palm, palmitate_;_ siCERS5, siRNA to CERS5.

Discussion

Hyperglycemia and the consequent oxidative stress are enhanced in T2DM and can induce defects in both expansion and survival of the cardiac stem cell compartment, thus favoring the onset of cardiomyopathy and its progression toward heart failure (22–25). Recently, impaired function of cardiac stem cells has been reported in human diabetes (25). Moreover, hyperglycemia was found to impair the myocardial repair and proangiogenic capacity of this particular cell compartment (25). The SFA palmitate is the major constituent of circulating fatty acids in humans and has been implicated in the induction of apoptosis in rodent cardiomyoblasts (6, 26), neonatal cardiomyocytes (27), and adult cardiomyocytes (5, 28). In this study, evaluation by multiple methods showed that concentrations of palmitate in the range of 0.25 to 0.5 mmol/L can trigger an apoptotic response in the human CPCs. Unlike palmitate, treatment of human CPCs with unsaturated or polyunsaturated fatty acids, such as oleate and eicosapentaenoic acid, respectively, did not induce apoptosis (D’Oria et al., unpublished data). Therefore, apoptotic damage of the human myocardium in response to lipotoxicity can involve the CPCs pool, similarly to hyperglycemia (25). Furthermore, cardiosphere formation, a functional assay to evaluate stem cell properties (29), was impaired by palmitate (Supplemental Fig. 3B). By contrast, palmitate did not affect the differentiation of human CPCs, as assessed by determining mRNA levels of troponin-T and connexin-43 genes, which are induced upon acquisition of a mature cardiomyocyte phenotype (D’Oria et al., unpublished data).

Accumulation of ceramide as a consequence of palmitate overload can mediate cellular apoptosis and heart dysfunction both in vitro and in animal models of diabetes and obesity (10, 18, 30). Moreover, exposure of human cardiac biopsy specimens to exogenously added ceramide resulted in enhanced apoptosis of cardiomyocytes (31). In this study, exposure of the human CPCs to palmitate for several hours caused increased intracellular accumulation of ceramide (Fig. 2), and this was associated with increased expression of key enzymes in de novo ceramide synthesis, such as CERS5 and DEGS1. Ceramide synthesis occurs through _N_-acylation of a sphingoid base by one of six CERS isoforms, each mediating the synthesis of a specific subset of ceramides and possessing substrate specificity for chain-length and/or saturation of fatty acid acyl-coenzyme A (32, 33). In the myocardium, both CERS5 and CERS6 synthesize ceramide from palmitate, but the expression of CERS6 is very low (9), suggesting that CERS5 is the most important mediator of cardiac ceramide production. Induction of ceramide and its biosynthetic enzymes was found to mediate the proapoptotic response to palmitate in the human CPCs. Indeed, CERS inhibition by FB1 was sufficient to prevent intracellular accumulation of ceramide, and both FB1 and specific CERS5 silencing inhibited induction of CPC apoptosis by palmitate (Figs. 2 and 4). In line with these findings, apoptosis induced by a CERS5-dependent mechanism was also demonstrated in other cellular systems (34). However, nonceramide-dependent pathways may also be involved in palmitate-induced apoptosis in the heart (35, 36). Of note, palmitate also promoted apoptosis of CPCs via ROS generation (Supplemental Fig. 7).

The JNK signaling pathway in the heart has been implicated in various adaptive responses, including hypertrophy, and modulation of cell survival and death (8, 37). Once phosphorylated, JNK migrates into the nucleus and activates several transcription factors, such as c-Jun, which is part of the AP-1 transcription factor and regulates genes involved in apoptosis and cell proliferation (38). In this study, ceramide accumulation in response to SFAs was shown to induce JNK activation. Both blocking CERS with a specific inhibitor (FB1) and achieving knockdown of the key enzyme CERS5 reduced palmitate-induced JNK-mediated c-Jun phosphorylation. Activation of the JNK pathway mediates cellular apoptosis [Fig. 5(B)]; therefore, altogether, these data suggest that ceramide-mediated apoptosis of the human CPCs occurs through JNK signaling (Fig. 6).

Figure 6.

Agonist binding to the canonical GLP1R counteracts the increase in CERS5 expression, hence limiting the increase in intracellular ceramide content that occurs in response to palmitate overload. This results in inhibition of palmitate-induced activation of JNK signaling that triggers apoptosis of human CPCs. p-JNK, phospho-JNK.

Recently, the GLP1R agonist exendin-4 was found to revert cardiac remodeling and oxidative stress dysfunction in mice fed a high-fat diet by normalizing the imbalance of lipid metabolism (17). Moreover, another study in mice showed that short-term liraglutide treatment could ameliorate the cardiac hypertrophy and fibrosis, as well as the induction of endoplasmic reticulum stress induced by a high-fat diet (18); GLP-1 activation was shown to be cytoprotective for mouse neonatal cardiomyocytes and human coronary smooth muscle cells exposed to lipotoxic damage (18). Finally, in heart-specific diacyl-glycerol-acyl transferase knockout mice bearing lipid abnormalities similar to those observed in severe human heart failure, exendin-4 was shown to reduce the myocardial ceramide content (39). Although our results are in line with these previous findings, to our knowledge, the involvement of the ceramide biosynthetic pathway in the proapoptotic effects of palmitate in the human CPCs has not been described before.

We also show that GLP1R activation can counteract the effects of lipid overload in human CPCs, which express a functional GLP1R (21). In a previous study on human CPCs, inhibition of H2O2-induced apoptosis by GLP-1 was found to be GLP1R- and PKA-dependent (21). Similarly, in this study, the antiapoptotic action of exendin-4 in palmitate-treated CPCs was significantly reversed by the GLP1R antagonist exendin 9-39 and the PKA inhibitors H89 (Fig. 1) and 14-22 amide (Supplemental Fig. 3). Other, non-PKA–mediated signaling pathways may contribute to the GLP1R-dependent effects of exendin-4 on CERS5 in human CPCs, because the exchange protein activated by cAMP inhibitor brefeldin-A blocked the ability of exendin-4 to inhibit CERS5 induction in response to palmitate (Supplemental Fig. 8). In addition, exendin-4 was found to prevent ceramide accumulation (Fig. 2) and activation of JNK signaling (Fig. 5), and to inhibit the induction of CERS5 and DEGS1 expression (Fig. 3) in response to palmitate. Finally, exendin-4 could preserve functional properties of cardiac stem cells in vitro, because cardiosphere formation was restored when exendin-4 was coincubated with palmitate (Supplemental Fig. 3D).

In conclusion, exposure to exendin-4 prevents SFA-induced apoptosis of human CPCs. The exendin-4 prosurvival response is mediated by its ability to reduce ceramide accumulation by inhibiting key ceramide biosynthetic enzymes and prevent subsequent activation of JNK signaling (Fig. 6). Thus, GLP1R agonists may revert the abnormalities of the cardiac stem/progenitor cell compartment under conditions of lipotoxicity, and this may be relevant to the cardioprotective effects of these drugs when used in vivo in individuals with obesity and/or T2DM.

Abbreviations

• BSA
• cAMP
  cyclic adenosine monophosphate
• CASP3
• CERS5
• CPC
• DEGS1
  dihydroceramide desaturase 1
• DHE
• FB1
• GLP-1
• GLP1R
  glucagonlike peptide-1 receptor
• JNK
  c-Jun NH2-terminal protein kinase
• PBS
  phosphate-buffered saline
• PKA
• ROS
• SFA
• siRNA
• SPT
  serine palmitoyltransferase
• T2DM
  type 2 diabetes mellitus.

Acknowledgments

Financial Support: This work was supported by research grants from the Fondazione Eli Lilly Italia to A.L. and F.G. and from the Fo.Ri.SID, Italy, to L.L.; and by fellowships from the Fondazione Diabete Ricerca, Italy, and Merck Sharp & Dohme, Italy, to R.D.

Disclosure Summary: F.G. has received support from Eli Lilly (Grant 2012-2016), AstraZeneca, Sanofi, and Lifescan, and lecture fees from Eli Lilly, AstraZeneca, Sanofi, Lifescan, Novo Nordisk, Boehringer-Ingelheim, Takeda, and Janssen. L.L. has received lectures fees from Eli Lilly, AstraZeneca, Sanofi, Lifescan, Novo Nordisk, Boehringer-Ingelheim, Takeda, Janssen, Roche, and Medtronic. The remaining authors have nothing to disclose.

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Author notes

*

These authors contributed equally to this study.

Copyright © 2017 Endocrine Society

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