Identification of Inhibitors of triacylglyceride accumulation in muscle cells: comparing HTS results from 1536-well plate-based and high-content platforms

Abstract

Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual-emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the National Institutes of Health library. The screening data from the plate reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate reader assay is a powerful high-throughout screening platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.

Keywords: 1536-well high-throughput screening; H9c2 cardiomyocytes; Nile red fluorescence; high-content analysis; human primary myocytes; lipid accumulation; phenotypic screening.

Figure 1. Assay development of 1536-well plate-reader assay

(A) The effect of increasing concentration of oleic acid on Nile red fluorescence (left Y-axis) in differentiating H9c2 myoblasts treated with (black bars) or without (white bars) 5 μM Triacsin C with no effect on cell count (striped bars) based on DAPI fluorescence (right Y-axis). (B) Cell viability was assessed by the addition of DAPI nuclear stain and measuring fluorescence. The DAPI fluorescence is plotted against the number of cells plated. A blank plate with no cells was used to obtain the background subtracted signal (open circles) from the raw signal (closed circles). This procedure was used to determine the cell count levels shown in A. (C) The effect of increasing % DMSO on Nile red fluorescence in the same cells treated with (white bars) or without (black bars) 200 μM oleic acid.

Figure 2. Assay development of 1536-well High Content assay

(A) Differentiating H9c2 myoblasts were imaged in the absence or presence of increasing concentration of oleic acid and the presence or absence of Triacsin C. Fluorescence due to excitation of Nile red at 510nm (neutral lipid) and 561nm (phospholipid) is shown in yellow and red, respectively using highlighted color scheme with equivalent settings for each image. DAPI nuclear staining is shown in blue. (B) Quantification of the Nile red fluorescence at 510nm shown in A in the presence (black bars) or absence (white bars) of 5μM Triacsin C using the Nile red – yellow gold integrated cytoplasm intensity metric.

Figure 3. Results from the pilot screen study comparing plate-reader vs. high content

(A) S/B (circles) and Z’ (squares) are shown on the left and right Y-axis respectively for each of 10 1536-well plates for data obtained from the plate-reader assay (closed symbols) using fluorescence intensity versus from the High Content imager (open symbols) using cytoplasm sum intensity algorithm (B,C) Percent inhibition and percent viability, respectively for compounds tested was correlated for data obtained by plate-reader versus high content imaging. Control for 100% inhibition (5μM Triacsin C) is shown as gray Xs. Control for 0% inhibition or 100% viability (vehicle) is shown as gray circles. Test compounds are shown as black circles. The 1:1 correlation line is drawn.

Figure 4. Results from HTS comparing plate-reader vs. high content

A cutoff criterion of ≥ 50% inhibition was applied to both the hit sets from the plate-reader and the high content imaging. (A) Hits that showed ≤ 50% viability from the DAPI fluorescence intensity in the plate-reader assay were removed and the resulting hits were correlated to their corresponding % inhibition from the high content imaging. Correlation results gave Pearson r = 0.69 and p-value (two-tailed) < 0.0001. (B) Correlation of pIC₅₀ values of confirmed hits resupplied as powders determined by plate-reader or High Content imaging. The 1:1 correlation line is drawn. Linear regression analysis gave r² = 0.74.

Figure 5. Dose-responses of lead compounds in rodent and human TAG accumulation assays

(A,B) Compounds dissolved in DMSO were added in 10-pt concentration response to oleate-exposed H9c2 myocytes as described in materials and methods. Both the lead compound, SBI-942 and its analog SBI-819 showed overlapping concentration curves for percent inhibition of Nile red fluorescence shown on the left Y-axes determined from the Envision plate-reader assay (closed circles and solid line) and the Opera high content imaging assay (triangles with dotted line). On the right Y-axes, percent viability data for each concentration tested using ATPLite is shown as open circles. (C,D) Compounds were dissolved in DMSO to 20mM stock and added in 7-pt concentration response to oleate-exposed human primary myotubes as described in materials and methods. Nile red fluorescence is shown on the left Y-axes determined from the plate-reader assay (closed circles). On the right Y-axes, percent viability data for each concentration tested using AlamarBlue is shown in open circles. Data is the average of two separate experiments performed in triplicate.

Figure 6. DGAT-1 activity of lead scaffold

SBI-942 was tested at 10μM concentration in a DGAT-1 radiolabeled assay as described in materials and methods. The commercially available DGAT-1 inhibitor A922500 was used as a positive control at 100nM. The high control wells contained DGAT-1, [¹⁴C]-decanoyl and 1,2-Dioleoyl glycerol. The low control wells contained DGAT-1 and [¹⁴C]-decanoyl only. SBI-942 was statistically significant compared to the low control by two-tailed unpaired t test (P=0.0013).