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Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets.

Warnke I, Goralczyk R, Fuhrer E, Schwager J - Nutr Metab (Lond) (2011)

Bottom Line: Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation.The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: DSM Nutritional Products Ltd,; Department of Human Nutrition and Health, CH-4002, Basel, Switzerland. ines.warnke@dsm.com.

ABSTRACT

Background: Adipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation.

Methods: C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.

Results: The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β.

Conclusion: This in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

No MeSH data available.


Related in: MedlinePlus

Summary of overall effects of the dietary ingredients DHA, β-carotene and resveratrol on lipid accumulation and on gene expression clusters. mRNA levels of enzymes involved in glucose and fat metabolism (Glut4, LPL, FAS, ACC1 and CPT-1β) and of adipocyte differentiation markers (PPARγ, C/EBPα, PPARα, aP2 and adiponectin) were determined in maturing C3H10 T1/2 cells. Depicted are the dose-dependent effects of DHA and β-carotene and the impact of resveratrol on lipid accumulation (after 7 days treatment, top of the table) and on gene expression (after 4 days treatment) relative to rosiglitazone control cells. Data are shown as % of positive control ± SEM (fat accumulation parameters) and fold change ± error (based on SEM, n = 6 - 15) for gene expression levels, respectively. Exemplarily, the fold changes of the genes FAS and PPARγ are shown as illustration. Fat droplet number equates to Spot Count/Object; fat droplet intensity equates to Spot Avg Intensity and fat droplet area equates to Spot Total Area/Object. Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. The average coefficient of variation (CV) for dCT values was less than 5% for all analysed genes at all concentration of the investigated compounds.
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Figure 5: Summary of overall effects of the dietary ingredients DHA, β-carotene and resveratrol on lipid accumulation and on gene expression clusters. mRNA levels of enzymes involved in glucose and fat metabolism (Glut4, LPL, FAS, ACC1 and CPT-1β) and of adipocyte differentiation markers (PPARγ, C/EBPα, PPARα, aP2 and adiponectin) were determined in maturing C3H10 T1/2 cells. Depicted are the dose-dependent effects of DHA and β-carotene and the impact of resveratrol on lipid accumulation (after 7 days treatment, top of the table) and on gene expression (after 4 days treatment) relative to rosiglitazone control cells. Data are shown as % of positive control ± SEM (fat accumulation parameters) and fold change ± error (based on SEM, n = 6 - 15) for gene expression levels, respectively. Exemplarily, the fold changes of the genes FAS and PPARγ are shown as illustration. Fat droplet number equates to Spot Count/Object; fat droplet intensity equates to Spot Avg Intensity and fat droplet area equates to Spot Total Area/Object. Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. The average coefficient of variation (CV) for dCT values was less than 5% for all analysed genes at all concentration of the investigated compounds.

Mentions: Treatment with DHA (25 μM) reduced the expression of aP2 and adiponectin, to 48% and 59% respectively (Table 3). The expression of aP2 and adiponectin after treatment with 2 μM β-carotene was also significantly decreased to 31% and 39%, and at 25 μM HT to 47% and 69%, respectively (Figure 5, Table 3). The mRNA levels of the adipogenesis-associated transcription factors, PPARγ, PPARα and C/EBPα were strongly suppressed by PUFAs, β-carotene and HT at the highest concentration (up to 85%; Table 3). Although PPARγ and C/EBPα were suppressed by β-carotene, as by DHA and EPA, not exactly the same down-stream genes were affected. Interestingly 1 μM DHA led to a significant increase in the mRNA level of C/EBPα.


Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets.

Warnke I, Goralczyk R, Fuhrer E, Schwager J - Nutr Metab (Lond) (2011)

Summary of overall effects of the dietary ingredients DHA, β-carotene and resveratrol on lipid accumulation and on gene expression clusters. mRNA levels of enzymes involved in glucose and fat metabolism (Glut4, LPL, FAS, ACC1 and CPT-1β) and of adipocyte differentiation markers (PPARγ, C/EBPα, PPARα, aP2 and adiponectin) were determined in maturing C3H10 T1/2 cells. Depicted are the dose-dependent effects of DHA and β-carotene and the impact of resveratrol on lipid accumulation (after 7 days treatment, top of the table) and on gene expression (after 4 days treatment) relative to rosiglitazone control cells. Data are shown as % of positive control ± SEM (fat accumulation parameters) and fold change ± error (based on SEM, n = 6 - 15) for gene expression levels, respectively. Exemplarily, the fold changes of the genes FAS and PPARγ are shown as illustration. Fat droplet number equates to Spot Count/Object; fat droplet intensity equates to Spot Avg Intensity and fat droplet area equates to Spot Total Area/Object. Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. The average coefficient of variation (CV) for dCT values was less than 5% for all analysed genes at all concentration of the investigated compounds.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117678&req=5

Figure 5: Summary of overall effects of the dietary ingredients DHA, β-carotene and resveratrol on lipid accumulation and on gene expression clusters. mRNA levels of enzymes involved in glucose and fat metabolism (Glut4, LPL, FAS, ACC1 and CPT-1β) and of adipocyte differentiation markers (PPARγ, C/EBPα, PPARα, aP2 and adiponectin) were determined in maturing C3H10 T1/2 cells. Depicted are the dose-dependent effects of DHA and β-carotene and the impact of resveratrol on lipid accumulation (after 7 days treatment, top of the table) and on gene expression (after 4 days treatment) relative to rosiglitazone control cells. Data are shown as % of positive control ± SEM (fat accumulation parameters) and fold change ± error (based on SEM, n = 6 - 15) for gene expression levels, respectively. Exemplarily, the fold changes of the genes FAS and PPARγ are shown as illustration. Fat droplet number equates to Spot Count/Object; fat droplet intensity equates to Spot Avg Intensity and fat droplet area equates to Spot Total Area/Object. Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. The average coefficient of variation (CV) for dCT values was less than 5% for all analysed genes at all concentration of the investigated compounds.
Mentions: Treatment with DHA (25 μM) reduced the expression of aP2 and adiponectin, to 48% and 59% respectively (Table 3). The expression of aP2 and adiponectin after treatment with 2 μM β-carotene was also significantly decreased to 31% and 39%, and at 25 μM HT to 47% and 69%, respectively (Figure 5, Table 3). The mRNA levels of the adipogenesis-associated transcription factors, PPARγ, PPARα and C/EBPα were strongly suppressed by PUFAs, β-carotene and HT at the highest concentration (up to 85%; Table 3). Although PPARγ and C/EBPα were suppressed by β-carotene, as by DHA and EPA, not exactly the same down-stream genes were affected. Interestingly 1 μM DHA led to a significant increase in the mRNA level of C/EBPα.

Bottom Line: Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation.The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: DSM Nutritional Products Ltd,; Department of Human Nutrition and Health, CH-4002, Basel, Switzerland. ines.warnke@dsm.com.

ABSTRACT

Background: Adipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation.

Methods: C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.

Results: The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β.

Conclusion: This in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

No MeSH data available.


Related in: MedlinePlus