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

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.

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×).

Figure 2

Determination of intracellular fat content in adipocytes differentiated for 7 days with varying concentrations of rosiglitazone. (A) Oil Red O quantification according to the Materials and Methods section. (B) Fat droplet number (Spot Count/Object) and droplet intensity (Spot Avg Intensity), using the Cellomics HCS Reader. Neutral lipids were stained with BODIPY^® 493/503. Results are expressed as percentage of values obtained with 100 μM rosiglitazone (mean ± SEM, n = 8).

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.

Figure 4

Effects of varying concentrations of DHA, EPA, β-carotene, (all-E)-lycopene, resveratrol and EGCG on number and intensity of fat droplets. C3H10 T1/2 cells were differentiated in the presence of test substances for 7 days. Spot Count/Object and Spot Avg Intensity were determined, as compared to rosiglitazone control 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.

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.