Loss of Stearoyl-CoA Desaturase-1 Activity Induced Leptin Resistance in Neuronal Cells

Abstract

The lack of response to leptin’s actions in the brain, “leptin resistance,” is one of the main causes of the pathogenesis of obesity. However, although high-fat diets affect sensitivity to leptin, the underlying mechanisms of leptin resistance are still an enigma. Here we examined the effect of excess saturated fatty acids (SFAs) on leptin signaling in human neuronal cells. Palmitate, the principle source of SFAs in diet, induced leptin resistance in a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb). We next investigated the function of stearoyl-CoA desaturase-1 (SCD1), an enzyme which converts SFAs into monounsaturated fatty acids (MUFAs), on leptin-induced signaling. We found that reduction of SCD1 activity, through SCD1 inhibition and knockdown, impairs leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human neuronal cells. Our findings suggested that SCD1 plays a key role in the pathophysiology of leptin resistance in neuronal cells associated with obesity.

A lack of energy balance likely contributes to overweight and obesity. The hypothalamus plays a vital role in body weight maintenance.¹⁾ Leptin, a hormone secreted from adipocytes, centrally regulates energy homeostasis by suppressing food intake and by accelerating energy expenditure.^2,3) There are six different isoforms of leptin receptors, ObRa-f. These isoforms are classified as short (ObRa, ObRc, OBRd, and ObRf), long (ObRb), and secreted (ObRe) isoforms.^4,5) Leptin’s major anti-obesity signal transduction pathway is mediated by the binding of leptin to its long isoform receptor ObRb which then mediates the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) signals.⁶⁾ Although leptin is considered a potential anti-obesity hormone, most obese subjects are leptin resistant.⁷⁾ Over the last few decades, searching for the related factors and underlying mechanisms of leptin resistance has drawn interest.

Excessive consumption of saturated fats is a risk factor for metabolic syndrome.⁸⁾ The most common saturated fatty acid (SFA) in the human diet, palmitate, was shown to impair leptin’s effect on anorexia.^9,10) Furthermore, obese subjects display increased levels of plasma free fatty acids,¹¹⁾ which has been associated with lipotoxicity.¹²⁾ Lipotoxicity is associated with endoplasmic reticulum (ER) stress,¹³⁾ one of the mechanisms of the pathogenesis of leptin resistance.^14–16)

Exogenous fatty acids from the diet influence the fatty acid composition of the membrane phospholipids.¹⁷⁾ The degree of fatty acid unsaturation affects the biophysical properties of the membrane.¹⁸⁾ Stearoyl-CoA desaturase-1 (SCD1), an enzyme that converts SFAs into monounsaturated fatty acids (MUFAs), plays a critical role in the regulation of the ratio of SFAs to MUFAs.¹⁹⁾ Knocking down SCD1 results in loss of fatty acid desaturation, thereby increasing SFAs.²⁰⁾ An alteration of the ratio between SFAs and MUFAs is linked to disease states such as cardiovascular diseases, hypertension, diabetes, obesity, and neurological disorders.²¹⁾

Loss of the SCD1 ability to desaturate fatty acids increases SFA accumulation. Increase in SFAs may be detrimental to the anti-obesity action of leptin.^9,10) Global SCD1 deficiency was shown to produce a lean and obesity-resistant phenotype,²²⁾ while the role of SCD1 in central nervous system (CNS) function has not yet been elucidated. Therefore, the aim of the present study is to examine the function of SCD1 on leptin signaling in human neuronal cells.

MATERIALS AND METHODS

Materials and Reagents

Human leptin was received from Enzo Life Science (NY, U.S.A.). Sodium palmitate was purchased from Tokyo Chemical Industry (Japan). BSA was provided by Sigma (MO, U.S.A.). SCD1 inhibitor (CAY10566) was obtained from Cayman Chemical (MI, U.S.A.).

Cell Culture

Human neuroblastoma (SH-SY5Y) cell lines stably transfected with Ob-Rb long isoform of leptin receptor (SH-SY5Y-ObRb)²³⁾ were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C in 5% CO₂/95% air. We did not add G418 in the culture medium.

Confluent SH-SY5Y-ObRb cells, which were plated at a density of approximately 7×10⁵ cells per 60 mm culture dishes were used for stimulation.

Palmitate Preparation

Palmitate was prepared based on previous reports with minor modifications.²⁴⁾ Briefly, 100-mM palmitate was prepared as follows: 27.8 mg sodium palmitate was dissolved in 1 mL sterile water by alternating heating and vortexing. Palmitate was completely dissolved when it reaches 70°C. Then, 200 µL of the 100 mM palmitate solution was immediately added to 3.8 mL of 0.1% non-esterified fatty acid (NEFA)-free BSA, obtaining a 5 mM palmitate solution. 0.1% NEFA-free BSA was prepared in serum-free DMEM. The 5 mM palmitate solution was shaken at 140 rpm at 40°C for 1 h and was then immediately used to treat the cells. Serum-free DMEM containing 0.1% NEFA-free BSA was used as the vehicle control.

RNA Interference (RNAi) Experiment

The transient transfection of small interfering RNAs (siRNAs) was conducted in SH-SY5Y-ObRb cells. At a time of transfection, cells were seeded 5×10⁵ cells per 35 mm culture dishes. Lipofectamine RNAiMAX (Life Technologies, U.S.A.) was used to transfect (siRNA) according to the manufacturer’s instruction. Opti-MEM medium was used for transfections. Single sequence of Silencer^® Select Pre-designed (Inventoried) siRNA (Life Technologies, siRNA ID for SCD1: s12503) and Silencer^® Select Negative Control siRNA #1 (Life Technologies) were used. The final concentrations of siRNAs were 5 nM. Cells were harvested 72 h after the transfections.

Western Blotting Analysis

Western blotting was performed as described previously.²³⁾ Briefly, cells were washed with ice-cold phosphate-buffered saline and lysed in a buffer containing 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)–NaOH, pH 7.5, 150 mM NaCl, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA), 1 mM Na₃VO₄, 10 mM NaF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40 for 20 min. The supernatants were collected after centrifuging at 15000 rpm for 20 min at 4°C. The samples were boiled with Laemmli buffer for 3 min, fractionated via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred at 4°C to nitrocellulose membranes. The membranes were incubated with rabbit anti-phospho-STAT3 (Tyr705) (#9145S, Cell Signaling, U.S.A.), rabbit anti-STAT3 (#9132, Cell Signaling), rabbit anti-SCD1 (#2438, Cell Signaling), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ACR001P, Acris, U.S.A.) antibodies followed by incubation with anti-horseradish peroxidase-linked antibody (Medical & Biological Laboratories Co., Ltd., Japan). Peroxidase was detected by chemiluminescence using an enhanced chemiluminescence using an ECL system (Thermo Scientific, U.S.A.). Band densities were measured by Image J software (Wayne Rasband, NIH).

Statistics

One-way ANOVA was used with Bonferroni’s post hoc analysis for statistical analysis. A p value <0.05 was defined to be statistical significant.

RESULTS

Palmitate Attenuated Leptin Signaling in Neuronal Cells

A major anti-obesity action of leptin is known to dominantly occur through the JAK2-STAT3 pathway. To evaluate the involvement of palmitate in leptin signaling, STAT3 phosphorylation levels were analyzed. In the current study, we used the SH-SY5Y human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb).²³⁾ In line with previous observations,^9,10) palmitate (100 µM, 2 h) significantly inhibited leptin-induced STAT3 phosphorylation (Fig. 1).

Fig. 1. Palmitate Attenuated Leptin Signaling in Neuronal Cells

A. SH-SY5Y-ObRb cells were treated with 0.1% BSA vehicle or 100 µM palmitate for 2 h followed by leptin (0.03 µg/mL, 15 min). Phospho(Tyr705)-STAT3 and STAT3 levels were analyzed by Western blotting. B. A densitometric analysis of phospho(Tyr705)-STAT3 was performed using image analysis software. Data are expressed as the mean±S.E. of 4 independent experiments (n=4). ** p<0.01.

Inhibition of SCD1 Induced Leptin Resistance

Due to the harmful effect of SFAs on leptin-induced signaling, we reasoned that inhibition of SCD1 would increase SFA levels and thereby induce leptin resistance. To test this hypothesis, we used an SCD1 inhibitor, CAY10566 (CAY). CAY 10 µM was shown to sufficiently increased an accumulation of SFAs.²⁵⁾ SH-SY5Y-ObRb cells were treated with CAY (10 µM, 2, 4 and 8 h), followed by leptin (0.03 µg/mL, 15 min) stimulation. Proteins were then isolated and Western blotting analysis was performed. It was found that CAY significantly inhibited leptin-induced STAT3 phosphorylation in a time-dependent manner (Fig. 2).

Fig. 2. SCD1 Inhibitor Attenuated Leptin Signaling in Neuronal Cells

A. SH-SY5Y-ObRb cells were treated with CAY10566 (CAY) 10 µM for 2, 4, and 8 h. Leptin (0.03 µg/mL) stimulation was done for another 15 min. Phospho(Tyr705)-STAT3 and STAT3 levels were analyzed by Western blotting. B. A densitometric analysis of phospho(Tyr705)-STAT3 was performed using image analysis software. Data are expressed as the mean±S.E. of 4 independent experiments (n=4). ** p<0.01.

We further analyzed the involvement of SCD1 in leptin resistance by knocking down SCD1. SH-SY5Y-ObRb cells were transfected with SCD1 siRNA (5 nM) for 72 h. The expression level of SCD1 was analyzed by Western blotting to check the knockdown efficacy. We showed that SCD1-specific siRNA inhibited its expression in SH-SY5Y-ObRb cells (Figs. 3A, B). Under these conditions, we analyzed leptin-induced signaling. As expected, the SCD1 knockdown significantly inhibited the leptin-induced STAT3 phosphorylation in SH-SY5Y-ObRb cells (Figs. 3C, D).

Fig. 3. Leptin Signaling Was Attenuated in SCD1-Knocked Down Neuronal Cells

SH-SY5Y-ObRb cells were transfected with control or SCD1 siRNAs (5 nM) for 72 h. Seventy-two hours after the transfection, cells were treated with leptin (0.03 µg/mL, 15 min). Phospho(Tyr705)-STAT3, STAT3, SCD1, and GAPDH levels were analyzed by Western blotting. A. SCD1 was efficiently knocked down by its specific siRNA. B. A densitometric analysis of SCD1 was performed using image analysis software. C. Leptin-induced STAT3 phosphorylation was attenuated in SCD1-knocked down cells. D. A densitometric analysis of phospho(Tyr705)-STAT3 was performed using image analysis software. Data are expressed as the mean±S.E. of 4 independent experiments (n=4). ** p<0.01.

DISCUSSION

A diet high in saturated fats is a critical cause of leptin resistance in obesity.²⁶⁾ Consistent with previous observations,^9,10) we observed that palmitate, a principal source of SFAs in the diet, induced leptin resistance. We also demonstrated that inhibition of SCD1, an enzyme that synthesizes MUFAs from SFAs, caused leptin resistance in neuronal cells.

Saturated fatty acids such as palmitate are known to have a strong lipotoxic effect in inducing inflammation and ER stress.^27,28) Accordingly, overnutrition activates pro-inflammatory pathway IKKβ/nuclear factor-kappa B (NF-κB) in the hypothalamus, which in turn disrupts leptin signaling.²⁹⁾ ER stress also plays a key role in the pathophysiology of leptin resistance.^14–16)

The activation of unfolded protein response (UPR) by membrane lipid saturation mediates different mechanism from those activated by ER stress.³⁰⁾ In the presence of unfolded or misfolded proteins in the ER, cells activates an adaptive response UPR by sensing a luminal stress signals to activate the luminal stress-sensing domain of the UPR-related proteins, inositol-requiring enzyme 1α (IRE1α) and protein kinase R-like endoplasmic reticulum-resident kinase (PERK).³¹⁾ In contrast, perturbation of membrane lipid unsaturation activated IRE1α and PERK arms of UPR although the luminal stress-sensing domain was deleted.³⁰⁾ Therefore, it would be interesting to assess whether the loss of SCD1 activity activates UPR genes in this manner thereby contributing to leptin resistance.

SCD1 plays a vital role in modulating the toxic effects of SFAs.^32,33) Knocking down SCD1 successfully reduces membrane lipid unsaturation and increases 18 : 0 of phospholipid levels approximately 1.5-fold in Hela cells.²⁰⁾ Consistent with the effects of excess SFAs, we revealed that the loss of SCD1 activity impaired leptin-induced signaling, as signified by the inhibition of leptin-induced STAT3 in SCD1 inhibitor-treated cells and SCD1-knockdown cells. These results suggested that SCD1 might play an important role in the regulation of lipid saturation levels and thereby prevent lipotoxicity. Similarly, elevated SCD1 had a protective effect against fatty acid-induced insulin resistance in rat muscle cells.²⁹⁾ In the MIN6 pancreatic β-cell line, upregulation of SCD1 expression results in resistance to SFA-induced cell death.³⁴⁾ However, discrepancies in the role of SCD1 have been reported. Evidence suggested that SCD1 expression levels are strongly upregulated in the livers of obese mice.³⁵⁾ The inhibition of SCD1 activity in mice, as signified by the use of SCD1-antisense oligonucleotide inhibitors (ASOs) and deletion of SCD1 (SCD1−/−), prevented diet-induced obesity.^36,22) Since SCD1 is thought to mediate fat storage in adipose tissue, attenuation of SCD1 activity might inhibit body weight by blocking fat storage.³⁷⁾ These controversies in the biological outcome of SCD1 may be explained by distinction in each specific organ’s SCD1 activity and functions. Of note, in the present study, we used a human neuronal cell model. It is possible that the role of neuronal SCD1 is different from that of peripheral SCD1. Thus, leptin-induced STAT3 activation should be further examined in SCD1-overexpressing neuronal cells and SCD1-neuron specific knockout mouse models.

In the present study, we found an interesting function of SCD1 in regulating neuronal action; i.e., leptin-induced STAT3 activation. Pharmacological intervention that can regulate SCD1 activity in the CNS would be useful for the treatment of obesity caused by leptin resistance. Further studies may be required to conduct these possibilities.

Acknowledgments

This research was supported by the Kobayashi International Scholarship Foundation, JSPS KAKENHI, and Takeda Science Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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