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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

Visualisation of lipid accumulation in differentiated adipocytes. C3H10 T1/2 cells were treated for 7 days with rosiglitazone (0.01, 0.1, 1 and 10 μM) and, after fixation with 60% isopropanol, stained with A) Oil Red O or B) Hoechst 33342 and BODIPY® 493/503. Images were acquired using A) a Nikon Coolpix 990 camera at 20× magnification or B) the Cellomics® HCS Reader camera (20×).
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Figure 1: Visualisation of lipid accumulation in differentiated adipocytes. C3H10 T1/2 cells were treated for 7 days with rosiglitazone (0.01, 0.1, 1 and 10 μM) and, after fixation with 60% isopropanol, stained with A) Oil Red O or B) Hoechst 33342 and BODIPY® 493/503. Images were acquired using A) a Nikon Coolpix 990 camera at 20× magnification or B) the Cellomics® HCS Reader camera (20×).

Mentions: Rosiglitazone dose-dependently increased the number of Oil Red O-stained lipid droplets (Figure 1A). To quantify the microscopic findings, Oil Red O was eluted from the fat droplets and measured spectro-photometrically (Figure 2A). A differentiation plateau seemed to be reached between 10 μM and 100 μM rosiglitazone, hence 100 μM values were used as references for calculations. The fluorescent stain BODIPY® likewise enabled measurement of rosiglitazone-dependent induction of TG formation (Figure 1B). Both fat droplet number (Spot Count/Object) and fat droplet intensity (Spot Avg Intensity) concentration-dependently increased with rosiglitazone (Figure 2B). Thus, our method proved valid for detecting intracellular lipid accumulation.


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)

Visualisation of lipid accumulation in differentiated adipocytes. C3H10 T1/2 cells were treated for 7 days with rosiglitazone (0.01, 0.1, 1 and 10 μM) and, after fixation with 60% isopropanol, stained with A) Oil Red O or B) Hoechst 33342 and BODIPY® 493/503. Images were acquired using A) a Nikon Coolpix 990 camera at 20× magnification or B) the Cellomics® HCS Reader camera (20×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Visualisation of lipid accumulation in differentiated adipocytes. C3H10 T1/2 cells were treated for 7 days with rosiglitazone (0.01, 0.1, 1 and 10 μM) and, after fixation with 60% isopropanol, stained with A) Oil Red O or B) Hoechst 33342 and BODIPY® 493/503. Images were acquired using A) a Nikon Coolpix 990 camera at 20× magnification or B) the Cellomics® HCS Reader camera (20×).
Mentions: Rosiglitazone dose-dependently increased the number of Oil Red O-stained lipid droplets (Figure 1A). To quantify the microscopic findings, Oil Red O was eluted from the fat droplets and measured spectro-photometrically (Figure 2A). A differentiation plateau seemed to be reached between 10 μM and 100 μM rosiglitazone, hence 100 μM values were used as references for calculations. The fluorescent stain BODIPY® likewise enabled measurement of rosiglitazone-dependent induction of TG formation (Figure 1B). Both fat droplet number (Spot Count/Object) and fat droplet intensity (Spot Avg Intensity) concentration-dependently increased with rosiglitazone (Figure 2B). Thus, our method proved valid for detecting intracellular lipid accumulation.

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