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CELLutions Biosystems embryonic mouse hypothalamic n38 cells
Embryonic Mouse Hypothalamic N38 Cells, supplied by CELLutions Biosystems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism
Article Snippet: Embryonic mouse hypothalamic N38 cells (Cellutions Biosystems Inc., no authentication, mycoplasma testing performed by Cellultions Biosystems Inc.) were grown in DMEM with 10% FBS at 37 °C and 5% CO 2 .

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Article Title: Compositions and methods to modulate cell activity
Article Snippet: Cell Culture and In Vitro Studies Human embryonic kidney cells (HEK 293T, (ATCC CRL-3216), mycoplasma testing and STR profiling performed by ATCC) were cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum (Gibco, Carlsbad, CA) at 37° C. and 5% CO2. .. Embryonic mouse hypothalamic N38 cells (Cellutions Biosystems Inc) were grown in Dulbecco's modified eagle medium with 10% fetal bovine serum at 37° C. and 5% CO2. .. Embryonic mouse hypothalamic N38 cells (Cellutions Biosystems Inc) were grown in Dulbecco's modified eagle medium with 10% fetal bovine serum at 37° C. and 5% CO2.

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N42 and N38 hypothalamic cells use promoter I.f to regulate aromatase mRNA expression. Esr1 mRNA and ESR1 protein expression levels in N42 hypothalamic cells are higher than N38 hypothalamic cells. A) Real-time RT-PCR was performed employing a probe complementary to the exon I.f-exon II junction to measure promoter I.f-driven aromatase mRNA expression. Relative units are shown as the ratio of aromatase mRNA:Gapdh mRNA; (presented as l.f-specific aromatase mRNA/Gapdh mRNA). Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Conventional RT-PCR (B) and IB (C) were performed to measure ESR1 expression in brain tissue (positive control) and N42 or N38 hypothalamic cells. Gapdh and actin were used as loading controls. The figures represent one of three independently performed experiments.

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: N42 and N38 hypothalamic cells use promoter I.f to regulate aromatase mRNA expression. Esr1 mRNA and ESR1 protein expression levels in N42 hypothalamic cells are higher than N38 hypothalamic cells. A) Real-time RT-PCR was performed employing a probe complementary to the exon I.f-exon II junction to measure promoter I.f-driven aromatase mRNA expression. Relative units are shown as the ratio of aromatase mRNA:Gapdh mRNA; (presented as l.f-specific aromatase mRNA/Gapdh mRNA). Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Conventional RT-PCR (B) and IB (C) were performed to measure ESR1 expression in brain tissue (positive control) and N42 or N38 hypothalamic cells. Gapdh and actin were used as loading controls. The figures represent one of three independently performed experiments.

Article Snippet: Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Positive Control

Estradiol regulates aromatase mRNA expression and enzyme activity in N42, but not N38, hypothalamic cells. A) Real-time RT-PCR was performed to measure aromatase mRNA expression after 6, 12, and 24 h of E2 (10−7 M) treatment. Aromatase mRNA levels were normalized to Gapdh mRNA levels. Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase activity assays were performed in N42 (B) and N38 (C) hypothalamic cells after 6, 12, and 24 h of E2 (10−7 M) treatment. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase enzyme activity in each neuronal line treated with the aromatase enzyme inhibitor letrozole (LET) was undetectable (data not shown).

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: Estradiol regulates aromatase mRNA expression and enzyme activity in N42, but not N38, hypothalamic cells. A) Real-time RT-PCR was performed to measure aromatase mRNA expression after 6, 12, and 24 h of E2 (10−7 M) treatment. Aromatase mRNA levels were normalized to Gapdh mRNA levels. Results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase activity assays were performed in N42 (B) and N38 (C) hypothalamic cells after 6, 12, and 24 h of E2 (10−7 M) treatment. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, paired t-test. Aromatase enzyme activity in each neuronal line treated with the aromatase enzyme inhibitor letrozole (LET) was undetectable (data not shown).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR

The E2 antagonist ICI inhibits baseline and E2-induced aromatase mRNA expression and enzyme activity in N42 hypothalamic cells. A) Real-time RT-PCR was performed after cells were incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. B) Aromatase activity assays were performed to measure enzyme activity in cells incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase activity in the N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown).

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: The E2 antagonist ICI inhibits baseline and E2-induced aromatase mRNA expression and enzyme activity in N42 hypothalamic cells. A) Real-time RT-PCR was performed after cells were incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. B) Aromatase activity assays were performed to measure enzyme activity in cells incubated in the presence or absence of 10−7 M E2 plus or minus 10−5 M ICI for 6 h. The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase activity in the N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Incubation

AP-1 cis-regulatory elements within the nt −200/−1 region of promoter I.f are essential for E2-dependent induction of aromatase expression in N42 hypothalamic cells. A) Serial deletion analysis was performed to localize the E2-responsive regions of aromatase promoter I.f. The promoter I.f-luciferase (LUC) reporter constructs were named according to the nucleotide positions of their 5′ termini. All constructs were transiently transfected into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence or absence of 10−7 M E2. Mean luciferase activity of each construct is given relative to the nt −1000/−1 promoter-luciferase reporter construct. Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, paired t-test). B) Mutational analysis of promoter I.f was performed to verify functional cis-regulatory elements. Substitution mutations were made within two putative AP-1 cis-regulatory elements in the −200/−1 promoter-luciferase reporters. Constructs were transiently cotransfected with ESR1 expression vector into N42 hypothalamic cells, and luciferase activities were measured after treatment with vehicle or 10−7 M E2. Results shown are the mean ± SEM of three independent experiments (**P < 0.01, ANOVA).

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: AP-1 cis-regulatory elements within the nt −200/−1 region of promoter I.f are essential for E2-dependent induction of aromatase expression in N42 hypothalamic cells. A) Serial deletion analysis was performed to localize the E2-responsive regions of aromatase promoter I.f. The promoter I.f-luciferase (LUC) reporter constructs were named according to the nucleotide positions of their 5′ termini. All constructs were transiently transfected into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence or absence of 10−7 M E2. Mean luciferase activity of each construct is given relative to the nt −1000/−1 promoter-luciferase reporter construct. Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, paired t-test). B) Mutational analysis of promoter I.f was performed to verify functional cis-regulatory elements. Substitution mutations were made within two putative AP-1 cis-regulatory elements in the −200/−1 promoter-luciferase reporters. Constructs were transiently cotransfected with ESR1 expression vector into N42 hypothalamic cells, and luciferase activities were measured after treatment with vehicle or 10−7 M E2. Results shown are the mean ± SEM of three independent experiments (**P < 0.01, ANOVA).

Techniques: Expressing, Luciferase, Construct, Transfection, Activity Assay, Functional Assay, Plasmid Preparation

ChIP analysis reveals ESR1 and JUN, but not FOS, bind to promoter I.f in N42 hypothalamic cells. We performed ChIP assays using N42 hypothalamic cell extracts treated with vehicle or 10−7 M E2 for 6 h. Sonicated cell supernatant was used as input DNA (positive control). Precleared chromatin was used for IP reactions with a rabbit polyclonal antibody directed against human (A) ESR1 and (B) JUN or FOS, as well as normal rabbit IgG. ESR1 was recruited to the E2-responsive nt −193/−47 but not to a more distal (nt −925/−768) region (see Fig. 4). Estradiol induced the recruitment of JUN but not FOS to the nt −193/−47 region. The figures represent one of three independently performed experiments.

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ChIP analysis reveals ESR1 and JUN, but not FOS, bind to promoter I.f in N42 hypothalamic cells. We performed ChIP assays using N42 hypothalamic cell extracts treated with vehicle or 10−7 M E2 for 6 h. Sonicated cell supernatant was used as input DNA (positive control). Precleared chromatin was used for IP reactions with a rabbit polyclonal antibody directed against human (A) ESR1 and (B) JUN or FOS, as well as normal rabbit IgG. ESR1 was recruited to the E2-responsive nt −193/−47 but not to a more distal (nt −925/−768) region (see Fig. 4). Estradiol induced the recruitment of JUN but not FOS to the nt −193/−47 region. The figures represent one of three independently performed experiments.

Techniques: Sonication, Positive Control

Putative AP-1 sites at nt −130/−124 and nt −166/−160 regions of promoter I.f are the major c-Jun-ESR1 binding sites. An EMSA was used to visualize JUN and ESR1 binding to AP-1 sites within promoter I.f. The EMSAs were performed using 5 μg of nuclear extract from N42 hypothalamic cells treated with E2 (10−7 M; 6 h) and radiolabeled oligonucleotide probes containing critical AP-1 sites and flanking sequences. A) Binding to an oligonucleotide identical to the nt −138/−114 sequence in promoter I.f and containing the nt −130/−124 AP-1 element (native; lane 2). Competition studies were performed using 100-fold excess unlabeled wild-type (consensus; lanes 3 and 4) or native oligonucleotides (lane 5) or mutant native oligonucleotides (lane 6). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 7 and 9) or 0.5 μg of anti-ESR1 (lanes 8 and 10) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 11 and 12). B) Binding to an oligonucleotide identical to the nt −175/−151 sequence in promoter I.f and containing the nt −166/−160 AP-1 element (native) (lane 2). Competition studies were performed using 100-fold excess unlabeled consensus or native probes (lanes 5 and 7) or mutant consensus or mutant native probes (lanes 6 and 8). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 10 and 12) or 0.5 μg of anti-ESR1 (lanes 9 and 11) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 13 and 14). Black arrows indicate JUN-ESR1 complexes, and white arrows indicate supershifted complexes by either c-Jun or ESR1 antibodies. The figures represent one of three independently performed experiments.

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: Putative AP-1 sites at nt −130/−124 and nt −166/−160 regions of promoter I.f are the major c-Jun-ESR1 binding sites. An EMSA was used to visualize JUN and ESR1 binding to AP-1 sites within promoter I.f. The EMSAs were performed using 5 μg of nuclear extract from N42 hypothalamic cells treated with E2 (10−7 M; 6 h) and radiolabeled oligonucleotide probes containing critical AP-1 sites and flanking sequences. A) Binding to an oligonucleotide identical to the nt −138/−114 sequence in promoter I.f and containing the nt −130/−124 AP-1 element (native; lane 2). Competition studies were performed using 100-fold excess unlabeled wild-type (consensus; lanes 3 and 4) or native oligonucleotides (lane 5) or mutant native oligonucleotides (lane 6). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 7 and 9) or 0.5 μg of anti-ESR1 (lanes 8 and 10) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 11 and 12). B) Binding to an oligonucleotide identical to the nt −175/−151 sequence in promoter I.f and containing the nt −166/−160 AP-1 element (native) (lane 2). Competition studies were performed using 100-fold excess unlabeled consensus or native probes (lanes 5 and 7) or mutant consensus or mutant native probes (lanes 6 and 8). Immunodepletion of complexes was performed using 2 μg of anti-c-Jun (lanes 10 and 12) or 0.5 μg of anti-ESR1 (lanes 9 and 11) antibody (ab). Normal rabbit IgG was added as a negative control (lanes 13 and 14). Black arrows indicate JUN-ESR1 complexes, and white arrows indicate supershifted complexes by either c-Jun or ESR1 antibodies. The figures represent one of three independently performed experiments.

Techniques: Binding Assay, Sequencing, Mutagenesis, Immunodepletion, Negative Control

The ESR1 DNA-binding domain is not required for mediating E2-dependent aromatase expression in N42 hypothalamic cells. The nt −200/−1 I.f promoter-luciferase reporter construct was transiently cotransfected with either wild-type ESR1 or ESR1 carrying a mutation at DNA-binding domain (ESR1-AA) into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence of E2 (10−7 M). Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, ANOVA).

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: The ESR1 DNA-binding domain is not required for mediating E2-dependent aromatase expression in N42 hypothalamic cells. The nt −200/−1 I.f promoter-luciferase reporter construct was transiently cotransfected with either wild-type ESR1 or ESR1 carrying a mutation at DNA-binding domain (ESR1-AA) into N42 hypothalamic cells. Luciferase assays were performed a minimum of three times in the presence of E2 (10−7 M). Summary data for three independent experiments are shown. Results are expressed as the mean ± SEM (**P < 0.01, ANOVA).

Techniques: Binding Assay, Expressing, Luciferase, Construct, Mutagenesis

ESR1 and JUN, but not FOS, form a complex in N42 hypothalamic cells. A) Immunoprecipitation with antibodies against human JUN or FOS and IB with an antibody against ESR1 (mESR1; raised in mouse, monoclonal) were performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). B) Immunoprecipitation with two different ESR1 antibodies, mESR1 or rESR1 (raised in rabbit, polyclonal), followed by IB with an antibody against JUN was performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). Immunoprecipitation with a nonspecific rabbit IgG was the negative control. The figures represent one of three independently performed experiments.

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ESR1 and JUN, but not FOS, form a complex in N42 hypothalamic cells. A) Immunoprecipitation with antibodies against human JUN or FOS and IB with an antibody against ESR1 (mESR1; raised in mouse, monoclonal) were performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). B) Immunoprecipitation with two different ESR1 antibodies, mESR1 or rESR1 (raised in rabbit, polyclonal), followed by IB with an antibody against JUN was performed in N42 hypothalamic cells treated with E2 (10−7 M; 6 h). Immunoprecipitation with a nonspecific rabbit IgG was the negative control. The figures represent one of three independently performed experiments.

Techniques: Immunoprecipitation, Negative Control

ESR1 mediates E2-stimulated aromatase mRNA expression in N42 hypothalamic cells. N42 hypothalamic cells were transfected with Esr1 siRNA or with nonspecific siRNA as a negative control (NS) and were cultured for an additional 48 h. A) Aromatase mRNA levels were analyzed by real-time RT-PCR in the presence or absence of E2 (10−7 M; 6 h). Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3; **P < 0.01, ANOVA. B) Aromatase activity assays were performed in the presence or absence of E2 (10−7 M; 6 h). The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase enzyme activity in N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown). Real-time RT-PCR (C) and IB (D) were performed to measure Esr1 mRNA and protein levels, respectively. Actin was used as a loading control. The IB figure represents one of three independently performed experiments. Real-time RT-PCR results are expressed as the mean ± SEM (n = 3), and Gapdh was used as a loading control. **P < 0.01, ANOVA. Representative results from three independent experiments are shown.

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Figure Lengend Snippet: ESR1 mediates E2-stimulated aromatase mRNA expression in N42 hypothalamic cells. N42 hypothalamic cells were transfected with Esr1 siRNA or with nonspecific siRNA as a negative control (NS) and were cultured for an additional 48 h. A) Aromatase mRNA levels were analyzed by real-time RT-PCR in the presence or absence of E2 (10−7 M; 6 h). Aromatase mRNA levels were normalized to Gapdh mRNA levels. The results are expressed as the mean ± SEM (n = 3; **P < 0.01, ANOVA. B) Aromatase activity assays were performed in the presence or absence of E2 (10−7 M; 6 h). The results are expressed as the mean ± SEM (n = 3); **P < 0.01, ANOVA. Aromatase enzyme activity in N42 neuronal line treated with the aromatase enzyme inhibitor LET was undetectable (data not shown). Real-time RT-PCR (C) and IB (D) were performed to measure Esr1 mRNA and protein levels, respectively. Actin was used as a loading control. The IB figure represents one of three independently performed experiments. Real-time RT-PCR results are expressed as the mean ± SEM (n = 3), and Gapdh was used as a loading control. **P < 0.01, ANOVA. Representative results from three independent experiments are shown.

Techniques: Expressing, Transfection, Negative Control, Cell Culture, Quantitative RT-PCR, Activity Assay, Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Article Snippet: Cell Culture Mouse embryonic hypothalamic neuronal cell lines N38 and N42 were purchased from Cellutions Biosystems Inc. (Toronto, ON, Canada) [ 29 ] and were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, New York, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Biology of Reproduction

Article Title: Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines ¹

doi: 10.1095/biolreprod.109.077206

Techniques: Expressing, Activity Assay, Quantitative RT-PCR

(A–C) Co-expression of taste signaling molecules in the embryonic mouse hypothalamic cell line N38 as assessed by immunofluorescence methods. Confocal images (×10 magnification) of N38 cells that we determine endogenously express taste-related molecules. (A) Expression of the receptor subunit T1R2; (B) expression of the receptor subunit T1R3; (C) merging, showing that these cells express both subunits of the sweet receptor T1R2/T1R3. (D–E) Relative decrease in the levels of expression of the sweet-specific receptor gene Tas1r2 in cultured N38 cells as a function of glucose levels in culture medium. The sweet receptor-specific gene Tas1r2 , but not others, was found to be expressed at significantly higher levels in N38 cells exposed to a low-glucose medium (0.1 mM) compared to cells exposed to higher glucose levels. The panels depict the results of quantifying the relative levels of expression as obtained by using real-time quantitative PCR methods. Relative gene expression levels across regions were computed using the 2 −ΔΔ C T method (see and Livak and Schmittgen, ) and were expressed as percent decrease in expression levels in cells exposed to a medium containing from 0.5 up to 10 mM glucose compared to cells exposed to a medium containing 0.1 mM glucose. Values = 100% imply that no relative differences in expression levels were detected in cells exposed to a medium containing higher glucose levels (0.5–10 mM) compared to cells exposed to a medium containing 0.1 mM glucose; values < 100% imply that significant decreases in expression levels were detected in cells exposed to a medium containing higher glucose levels compared to cells exposed to a medium containing 0.1 mM glucose. (D) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 0.5 mM glucose. (E) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 1.5 mM glucose. (F) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 2.5 mM glucose. (G) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 5 mM glucose. (H) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 10 mM glucose. (I) Decrease in Tas1r2 levels produced by adding 0.5 mM sucralose to 0.5 mM glucose. Values = 100% imply that no relative differences in expression levels were detected in cells exposed to a medium containing 0.5 mM glucose + 0.5 mM sucralose compared to cells exposed to a medium containing 0.5 mM glucose; values < 100% imply that significant decreases in expression levels were detected in cells exposed to a medium containing 0.5-mM glucose + 0.5 mM sucralose compared to cells exposed to a medium containing 0.5 mM glucose. Dashed red line depicts the 100% baseline levels. (*All p < 0.05, one-sample t -test against 100%). n.s.: p ≥ 0.05.

Journal: Frontiers in Integrative Neuroscience

Article Title: Sweet Taste Signaling Functions as a Hypothalamic Glucose Sensor

doi: 10.3389/neuro.07.012.2009

Figure Lengend Snippet: (A–C) Co-expression of taste signaling molecules in the embryonic mouse hypothalamic cell line N38 as assessed by immunofluorescence methods. Confocal images (×10 magnification) of N38 cells that we determine endogenously express taste-related molecules. (A) Expression of the receptor subunit T1R2; (B) expression of the receptor subunit T1R3; (C) merging, showing that these cells express both subunits of the sweet receptor T1R2/T1R3. (D–E) Relative decrease in the levels of expression of the sweet-specific receptor gene Tas1r2 in cultured N38 cells as a function of glucose levels in culture medium. The sweet receptor-specific gene Tas1r2 , but not others, was found to be expressed at significantly higher levels in N38 cells exposed to a low-glucose medium (0.1 mM) compared to cells exposed to higher glucose levels. The panels depict the results of quantifying the relative levels of expression as obtained by using real-time quantitative PCR methods. Relative gene expression levels across regions were computed using the 2 −ΔΔ C T method (see and Livak and Schmittgen, ) and were expressed as percent decrease in expression levels in cells exposed to a medium containing from 0.5 up to 10 mM glucose compared to cells exposed to a medium containing 0.1 mM glucose. Values = 100% imply that no relative differences in expression levels were detected in cells exposed to a medium containing higher glucose levels (0.5–10 mM) compared to cells exposed to a medium containing 0.1 mM glucose; values < 100% imply that significant decreases in expression levels were detected in cells exposed to a medium containing higher glucose levels compared to cells exposed to a medium containing 0.1 mM glucose. (D) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 0.5 mM glucose. (E) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 1.5 mM glucose. (F) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 2.5 mM glucose. (G) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 5 mM glucose. (H) Decrease in taste receptor levels produced by increasing glucose medium from 0.1 to 10 mM glucose. (I) Decrease in Tas1r2 levels produced by adding 0.5 mM sucralose to 0.5 mM glucose. Values = 100% imply that no relative differences in expression levels were detected in cells exposed to a medium containing 0.5 mM glucose + 0.5 mM sucralose compared to cells exposed to a medium containing 0.5 mM glucose; values < 100% imply that significant decreases in expression levels were detected in cells exposed to a medium containing 0.5-mM glucose + 0.5 mM sucralose compared to cells exposed to a medium containing 0.5 mM glucose. Dashed red line depicts the 100% baseline levels. (*All p < 0.05, one-sample t -test against 100%). n.s.: p ≥ 0.05.

Article Snippet: Plates containing the Embryonic Mouse Hypothalamic Cell Line N38 (Cellutions Biosystems Inc.) was grown in 1 × DMEM (high glucose, Invitrogen) with 10% fetal bovine serum, and 1% penicillin/streptomycin and maintained at 37°C under 5% CO 2 .

Techniques: Expressing, Immunofluorescence, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Produced

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