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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

Effects of DHA, EPA, β-carotene, HT, (all-E)-lycopene, EGCG and resveratrol on lipid formation measured with the HCA assay. C3H10 T1/2 cells were treated for 7 days. Shown are three parameters (Spot Count/Object, Spot Avg Intensity and Spot Total Area/Object) that quantify lipid formation, as compared to rosiglitazone controls set as 100%. Data are shown as mean ± SEM (n = 10 - 20). Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
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Figure 3: Effects of DHA, EPA, β-carotene, HT, (all-E)-lycopene, EGCG and resveratrol on lipid formation measured with the HCA assay. C3H10 T1/2 cells were treated for 7 days. Shown are three parameters (Spot Count/Object, Spot Avg Intensity and Spot Total Area/Object) that quantify lipid formation, as compared to rosiglitazone controls set as 100%. Data are shown as mean ± SEM (n = 10 - 20). Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Mentions: Subsequently, the effects of test substances on lipid accumulation in adipocytes were investigated using the Cellomics® method. Murine pre-adipocytes were differentiated for 7 days (n = 10) in the absence or presence of ω-3 PUFAs (DHA or EPA), carotenoids, (poly)-phenols, and catechins. Incubation of rosiglitazone-treated C3H10 T1/2 cells with 25 μM DHA, 25 μM EPA, or 2 μM β-carotene, significantly decreased the number of lipid droplets (Spot Count/Object) by 56%, 42% and 41%, respectively (Figure 3). At 25 μM HT inhibited the accumulation of TGs in adipocytes by 38%, whereas (all-E)-lycopene and EGCG reduced the fat droplets by 22 and 7%, respectively. The test compounds also lowered the spot intensity (Spot Avg Intensity) and Spot Total Area/Object parameters to a similar extent, suggesting that the total fat content per cell was reduced. Compared to the PUFAs, resveratrol did not affect adipogenesis and even increased TG content.


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)

Effects of DHA, EPA, β-carotene, HT, (all-E)-lycopene, EGCG and resveratrol on lipid formation measured with the HCA assay. C3H10 T1/2 cells were treated for 7 days. Shown are three parameters (Spot Count/Object, Spot Avg Intensity and Spot Total Area/Object) that quantify lipid formation, as compared to rosiglitazone controls set as 100%. Data are shown as mean ± SEM (n = 10 - 20). Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117678&req=5

Figure 3: Effects of DHA, EPA, β-carotene, HT, (all-E)-lycopene, EGCG and resveratrol on lipid formation measured with the HCA assay. C3H10 T1/2 cells were treated for 7 days. Shown are three parameters (Spot Count/Object, Spot Avg Intensity and Spot Total Area/Object) that quantify lipid formation, as compared to rosiglitazone controls set as 100%. Data are shown as mean ± SEM (n = 10 - 20). Student's t-test: treatment versus control (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
Mentions: Subsequently, the effects of test substances on lipid accumulation in adipocytes were investigated using the Cellomics® method. Murine pre-adipocytes were differentiated for 7 days (n = 10) in the absence or presence of ω-3 PUFAs (DHA or EPA), carotenoids, (poly)-phenols, and catechins. Incubation of rosiglitazone-treated C3H10 T1/2 cells with 25 μM DHA, 25 μM EPA, or 2 μM β-carotene, significantly decreased the number of lipid droplets (Spot Count/Object) by 56%, 42% and 41%, respectively (Figure 3). At 25 μM HT inhibited the accumulation of TGs in adipocytes by 38%, whereas (all-E)-lycopene and EGCG reduced the fat droplets by 22 and 7%, respectively. The test compounds also lowered the spot intensity (Spot Avg Intensity) and Spot Total Area/Object parameters to a similar extent, suggesting that the total fat content per cell was reduced. Compared to the PUFAs, resveratrol did not affect adipogenesis and even increased TG content.

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