Journal: International Journal of Molecular Sciences

Article Title: Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

doi: 10.3390/ijms23105331

Figure Lengend Snippet: Compounds 5f and 4d attenuate inflammation in vitro. Effects of compounds (10 µM) on the protein levels of STAT3, IκB, and NF-κB in AML-12 cells. ( a ) Expression levels of inflammation related-proteins in vitro. ( b – d ) Protein expression levels were normal-ized against the indicated protein. * p < 0.05 compared with the group treated with the PBS (vehicle). + p < 0.05 compared with the group treated with the LPS. Data are pre-sented as the mean ± SD.

Article Snippet: The human keratinocytes HaCaT or the alpha mouse liver 12 (AML-12) cells were obtained from Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: In Vitro, Expressing

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 1. Palmitate upregulates NNMT expression in he- patocytes. A: NNMT catalyzes SAM-dependent methyla- tion/degradation of nicotinamide, which serves as a predominant endogenous precursor for NAD þ biosyn- thesis via the salvage pathway. B: AML12 cells were treated with the indicated concentrations of palmitate for 16 h. NNMT gene expression was quantified using real time-qPCR. Data are expressed as means ± SD, n = 4 separate experiments. Differences between the 2 groups were determined using Student’s t test (P < 0.05; P < 0.001 vs. control). C: AML12 cells were treated with the 0.4 mM palmitate for 16 h. Protein abun- dance of NNMT was detected by Western blotting. The signal of NNMT protein band was measured by densi- tometry and then divided by the signal of its correspond- ing actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). D: AML12 cells were treated with the 0.4 mM palmitate for 16 h. Intracellular total NAD (tNAD) concentrations were measured. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Expressing, Gene Expression, Control, Western Blot

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 3. mTORC1 activation upon palmitate exposure contributes to NNMT upregulation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) expo- sure for 16 h. Total protein was extracted. Protein abun- dance of phosphorylated (p-)S6, total-S6, and actin were detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B and C: AML12 cells were pretreated with or without Torin1 (0.25 mM) (B) or rapamycin (Rapa at 50 nM) (C) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. NNMT gene expressions were detected by real time-qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001 vs. control). NNMT, nicotina- mide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 4. mTORC1 activation is an upstream event in palmitate induced ATF4 activation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total protein was extracted. Protein abundance of ATF4 and actin were detected by Western blotting. The signal of ATF4 protein band was measured by densitometry and then divided by the sig- nal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B: AML12 were pretreated with Torin1 (0.25 mM) for 2 h before a 16-h palmitate (0.4 mM) exposure. Total RNA was extracted. ATF4 mRNA levels were detected by real time-qPCR. Data are expressed as means ± SD, n = 4 sep- arated experiments. Differences between the two groups were deter- mined using Student’s t test (P < 0.001 vs. control). ATF, activating transcription factor.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Quantitative Proteomics, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 5. mTORC1 activation contributes to palmi- tate-induced ER stress and NNMT upregulation. A and B: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before the palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. The gene expres- sions of Xbp1, Xbp1s, and Xbp1u were quantified by real time-qPCR and Xbp1s/Xbp1u ratio calculated. Data are expressed as means ± SD, n = 4 different experiments. Differences between the two groups were determined using Student’s t test (P < 0.001vs. control). C: AML12 cells were pretreated with Tornin1 for 2 h before tunicamycin (10 μm) treat- ment for 16 h. Protein abundance of p-S6 and actin was detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 5 separate experiments. Student’s t test was used for statistical evaluation (P < 0.01; P < 0.0001 vs. control). D: Ten- week-old male C57BL/6N mice were injected with tunicamycin (2 mg/kg body wt ip) or isovolumic vehi- cle (150 mM dextrose) and 16 h later livers were har- vested. Protein abundance of ATF4, p-S6 and actin was detected by Western blotting. E: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before tunicamycin (10 μm) treatment for 16 h. Protein abun- dance of ATF4 was detected by Western blotting. F: AML12 cells were pretreated with Torin1 for 2 h before tunicamycin (10 μm) treatment for 16 h. Total RNA was extracted and NNMT gene expression quantified by real time-qPCR. All data were expressed as means ± SD, n = 4 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.0001 vs. control). NNMT, nicotinamide N-methyl- transferase; XBP1, X-box binding protein 1.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Control, Quantitative Proteomics, Western Blot, Injection, Gene Expression, Binding Assay

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 6. NNMT inhibition protects against palmitate-induced cell death. A: AML12 cells were pretreated with either JBSNF-000088 (20 mM) or II399 (20 mM) at the indicated concentrations for 4 h before palmitate (0.4 mM) exposure for 16 h. Cell viability was determined by LDH release mea- surement. Data are expressed as mean ± SD, n = 3 separated experi- ments. Bars with different character differ significantly (P < 0.05). B: AML12 cells were transfected with either scramble siRNA or NNMT siRNA for 24 h and treated with palmitate at 0.4 mM for 16 h. Cell death was determined by LDH release measurement. Data are expressed as means ± SD, n = 3 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.001 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Transfection, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 7. Protein kinase A (PKA) inhibition compro- mises the protective effect of NNMT inhibition against palmitate-induced cell death. A and B: AML12 cells were treated with either JBSNF-000088 (25 mM) or II399 (25 mM) for 6 h. Total proteins were isolated and PKA substrates detected by Western blotting. The signal of PKS substrates was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05; P < 0.01 vs. untreated cells). C: AML12 cells were pretreated with either JBSNF-000088 (25 mM) or II399 (25 mM) at the pres- ence/absence of PKA inhibitor, either SQ22536 (200 mM) or H89 (10 mM) for 4 h before palmitate exposure for 16 h. Cell death was determined by LDH release. All data are expressed as means ± SD, n = 3 sepa- rated experiments. Differences between the two groups were determined using Student’s t test (P < 0.05; P < 0.01; P < 0.001 vs control). D: sche- matic illustration of the role and mechanism of NNMT upregulation in palmitate-induced hepatocyte lipotox- icity. The mTORC1-ATF4 pathway activation contrib- utes to palmitate-elicited NNMT upregulation and protein kinase A (PKA) activation contributes to NNMT inhibition-conferred protection against hepatolipotox- icity. NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Isolation, Western Blot, Control, Activation Assay

Journal: Adipocyte

Article Title: Adipocytes as lipid sensors of oleic acid transport through a functional Caco-2/HT29-MTX intestinal barrier

doi: 10.1080/21623945.2019.1580842

Figure Lengend Snippet: Dose-response of hepatocytes AML12 to oleic acid (OA) after 24 h. (a) Real-time analysis of OA uptake by enterocytes AML12 (left panel) and corresponding slopes and IC50 (right panel). Data are presented as mean values ± SEM (8 replicates). (b) OA-induced TG accumulation monitored by cytometry analysis of AdipoRed fluorescence. Mean AdipoRed-H was analyzed on living cell populations in duplicates (right panel). (c) Merged images of AdipoRed (green) and Hoechst 33258 (red) acquired with identical parameters with objective x20. Significant Student t-test-p-values are reported for p < 0.05, compared to control.

Article Snippet: Mouse hepatocyte cell line AML12 (LGC Standards) were grown in 1:1 mixture of DMEM and Ham’s F12 medium with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium 40 ng/mL dexamethasone (Sigma Aldrich), antibiotics and 10% FCS during one week after confluency before experiments.

Techniques: Cytometry, Fluorescence, Control

Journal: Adipocyte

Article Title: Adipocytes as lipid sensors of oleic acid transport through a functional Caco-2/HT29-MTX intestinal barrier

doi: 10.1080/21623945.2019.1580842

Figure Lengend Snippet: Caco-2/HT29-MTX intestinal barriers pre-treated with oleic acid (OA) in co-culture induce TG accumulation in AML12 hepatocytes. ( a) Intestinal barriers were pre-treated with either 10 or 20 μM OA during 2 h then OA was removed by replacing media and intestinal barrier 96-wells inserts were loaded onto AML12 hepatocytes during 24 h in HG media. TG accumulation was assessed using AdipoRed fluorescent intensities normalized to time of pre-treated intestinal barrier before loading onto AML12 cells in either Caco-2/HT29-MTX (T0) or 24 h after co-culture (A) and AML12 cells. Corresponding images are presented as merged AdipoRed (green)/Hoechst 33258 (red) images at magnification x4 (96-wells inserts) or x20 (AML12). Dot line at 1 represents the value for control at T = 0. (b) AML12 sensing of intestinal barriers pre-treated with OA (10 µM) in co-culture media containing either low (LG) or high glucose (HG) with or without insulin (INS 0,05 mU/mL) after 24 h. Data are presented as mean values ± SEM (n = 8) with significant Student t-test p-values <0.05 compared to T0.

Article Snippet: Mouse hepatocyte cell line AML12 (LGC Standards) were grown in 1:1 mixture of DMEM and Ham’s F12 medium with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium 40 ng/mL dexamethasone (Sigma Aldrich), antibiotics and 10% FCS during one week after confluency before experiments.

Techniques: Co-Culture Assay, Control