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. 2021 May 19;22(10):5357. doi: 10.3390/ijms22105357

Table 2.

Characteristics and analysis of outcomes for in vitro studies.

Outcomes Type
Authors Design Treatment Sphingolipid Extraction/Analysis Method Sphingolipid Levels Inflammatory Parameters Neuropeptide Levels
Sortino et al., 1999 Hypothalamic GT1-7 neuronal cells.

  1. TNF-α (20 ng/mL) for 15, 30 and 60 min.

  2. D609 (5 mg/mL) for 1 h.

 Diacylglycerol kinase assay
(commercially available kit)		 ↑ Cer-1-P (accumulation of ceramide) in GT1-7 cells stimulated with TNF-α.
↓ Cer-1-P in GT1-7 cells after pretreatment with D609.		 ↑ TNFR1 and TNFR2 in GT1-7 cells after TNF-α exposure Uninformed
Davis et al., 2006 Wild-type mixed AH cultures
(containing neurons and glia).

  1. IL-1b (10–12 nM) for 30 s, 1, 2,5 and 10 min.

  2. C2-ceramide (5–10 lM) for 2,5,10 and 15 min.

 Uninformed ↑ Src phosphorylation in the AH cultures after treatment with C2-Ceramide or IL-1β (concentration and time-dependent) Uninformed Uninformed
McFadden et al., 2014 PHN, N38HN, or R7HN cells.

  1. Palmitate (PA) (5200 mM).

  2. C75 (570 mM).

  3. FSG67 (5160 mM).

  4. C89b (540 mM).

  5. TG (5300 nM).

  6. For 18 or 24 h.

 Lipidomics ↑ Ceramide levels in PHD exposed to palmitate.
↓ Ceramide levels in PDH exposed to C75		 ↑ TNF-α, IL-1β, and IL-6 mRNA levels in PHN after exposed to C16:0 for 18 h.
↓ TNF-α, IL-1β mRNA levels in PHN exposed to C75 alone or in the presence of C16:0, FSG67, and C89b for 18 h.		 Uninformed
Morselli et al., 2014a N43 cells and BV2 cells.

  1. Pretreated for the indicated time with 10−8 M E2 conjugated with fatty-acid-free BSA (albumin).

  2. 10−8 M E2 or 100 μM PA conjugated with BSA alone or in combination for 8 h.

  3. Cells were transfected with siRNAs targeting murine ERa or murine PGC-1a or with an unrelated control siRNA.

  4. N43 cells were infected with the FLAG-ERa (AdERa) and GFP adenoviral. A total of 4 h after virus exposure and 48 h later, cells were treated as indicated.

 Liquid chromatography/electrospray ionization/tandem mass spectrometry ↑ Ceramide levels in N43 cells exposed to PA. ↑ TNF-α and IL-6 mRNA expression in N43 and BV2 cells exposed to PA for 8 h.
↓ TNF-α and IL-6 in N43 cells transfected with siRNA for PGC-1a, followed by treatment with PA.
↑ TNF-α and IL-6 in N43 cells transfected with siRNA for PGC-1a, followed by infection with AdGFP-Era followed by infection with AdGFP-ERa, with or without exposed to PA; 		Uninformed
Morselli et al., 2014b N43 hypothalamic cell line and primary neuronal cell cultures.

  1. Palmitate.

  2. The steroid hormone 17-β estradiol (E2).

 Mass spectrometry Uninformed ↑ Inflammation in N43 cell line and primary neurons exposed to PA.
↓ Inflammation in N43 cell line and primary neurons were pretreatment with E2.		 Uninformed
Silva et al., 2014 GT1-7 cells.

  1. Leptin (40 µmol) for 12 h.

  2. Transfection with siRNA targeted to STAT3 or scrambled control siRNA.

 Western blotting ↑ S1PR1 protein levels in GT1-7 cells treated with leptin, in a time-dependent manner.
↓ S1PR1 protein levels in GT1-7 cells transfected with STAT3 siRNA.		 Uninformed Uninformed
Campana et al.,2017 Hypothalamic GT1-7 neuronal cells.

  1. Palmitate (1 mM) for 24 h. After, cells were stimulated with insulin (100 nM) for 5 min.

 Liquid chromatography coupled with high-resolution mass spectrometry. ↑ Ceramide levels (C:16, C:18, C22, C24) in GT1-7 cells after treatment with palmitate.
↓ SPT2 blocked ceramide accumulation induced by palmitate in GT1-7 cells.		 Uninformed Uninformed
Dusaban et al., 2017 Astrocytes were isolated from P1-P3 postnatal WT and S1P3 KO mice.

  1. siRNA for S1P1, S1P2, S1P3, and control siRNA.

  2. S1P2 antagonist JT-013 (1 μM).

  3. S1P treatment (0,5 μM, 6 h).

  4. S1p3 antagonist SPM-354 (5 μM, 15 min).

FTY720 (100 nM, 6 h). 		Quantitative-PCR ↑ S1P3 in WT astrocytes after scratch injury. ↑ COX2 and IL-6 levels in S1P3 KO astrocytes treated with S1P. Uninformed
Tse, E. and Belsham, D., 2018 mHypoA-POMC/GFP-1, -2, -3, and -4 neurons.

  1. 10, 50, or 100 mM palmitate for 8 and 24 h.

  2. 50 mM methylpalmitate for 8 h.

  3. 10 nM insulin for 15 min.

  4. 1mM C16-ceramide for 8 h.

  5. 50 mM oleate for 8 h or cotreated with 50 μM palmitate and 50 μM oleate for 8 h.

  6. 100 mM myriocin or 50 mM L-cycloserine for 1 h.

  7. Inhibitors treatment for 1 h, [TAK-242, JNK (SP 600125), p38 MAP kinase (SB, 202190), or MEK1/2 (ERK1/2; PD 0325901) and PS- 1145 (10 mM)].

 Uninformed Uninformed ↑ mRNA levels of IL-6, IL-1B, TLR4, TNF-α, and NFkB after treatment with 50 mM palmitate for 8 h.
↓ IL6 mRNA expression after inhibition of TLR4 and pretreatment with PS 1145.
↑ IL-6 mRNA expression after pretreatment with SP 600125 and SB 202190.
↓ IL-6 mRNA expression after Oleate co-treatment.
↓ JNK phosphorylation in mHypoAPOMC/GFP-2 neurons pretreated with 50 mM palmitate and insulin stimulation.		 ↑ POMC mRNA expression in mHypoA-POMC/GFP-2 neurons with 50 mM palmitate for 8 h or 1 mM C16-ceramide for 8 h.
↓ POMC mRNA expression after pretreatments with 50mM SP 600125 (JNK inhibitor) or 10-mM PD 0325901 (ERK inhibitor).
↑ POMC mRNA expression in mHypoA-POMC/GFP-2 neurons with pretreatment with 100 mM myriocin or 50 mM L-cycloserine for 1 h.
Cotreatment rescue PA induced POMC mRNA.
Sergi et al., 2018 mHypoE-N42 hypothalamic cell line.
Primary hypothalamic culture from Sprague Dawley rats.

  1. Palmitate (PA- 200 μM).

  2. Lauric acid (LA- 200 μM).

  3. Oleanolic acid (OA-200 or 125 μM).

  4. Eicosapentaenoic acid (EPA 200 or 125 μM).

  5. LPS 100 ng/mL.

  6. Synthesis, L-cycloserine (inhibitor of ceramide) 250 μM.

  7. TLR4 inhibitor (CLI-095) 1 μM.

All for 6 h. 		LC- ESI-MS/MS ↑ C16 ceramide after PA treatment.
↓ C16 ceramide when L-cycloserine was added in combination with PA.
↓ C16 ceramide in N42 neurons were treated with PA in the presence of OA or EPA.		 ↑ IL-6 and TNF-α expression in N42, N49 cells and hypothalamic cultures, after treatment with PA or LPS.
↓ IL-6 e TNF-α expression after treatment with L-cycloserine, in N42 cells compared with LPS group.
↓ IL-6 expression after treatment with LA in N42 cells.
↓ IL-6 and TNF-α expression after cotreatment with OA and EPA in N42 cells.
↓ IL-6 expression after treatment with PA combined with cycloserine compared to PA alone in N42 cells		 Uninformed
Maldonado-Ruiz et al., 2019 Microglia primary culture from Wistar rats.

  1. 100 uM palmitic acid.

  2. 100 uM palmitoleic acid.

  3. 100 uM stearic acid.

  4. 100 uM linoleic acid.

  5. 25 uM N-hexanoyl-D-sphingosine (C6).

  6. 0.1 ug/mL LPS.

All for 24 h. 		Uninformed Uninformed ↑ TNF-α, IL-6 and IL-1 after treatment with palmitate.
↑ TNF-α after treatment with stearic acid.
↑ IL-6 after treatment with palmitoleic acid.
↓ IL-6 after treatment with C6.		 Uninformed