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. 2023 Apr 28;24(9):7987.
doi: 10.3390/ijms24097987.

U0126 Compound Triggers Thermogenic Differentiation in Preadipocytes via ERK-AMPK Signaling Axis

Sunday Amos Onikanni  1   2 , Cheng-Ying Yang  1 , Lloyd Noriega  1 , Chih-Hao Wang  1   3
Affiliations

U0126 Compound Triggers Thermogenic Differentiation in Preadipocytes via ERK-AMPK Signaling Axis

Sunday Amos Onikanni et al. Int J Mol Sci. .

Abstract

In recent years, thermogenic differentiation and activation in brown and white adipose tissues have been regarded as one of the major innovative and promising strategies for the treatment and amelioration of obesity. However, the pharmacological approach towards this process has had limited and insufficient commitments, which presents a greater challenge for obesity treatment. This research evaluates the effects of U0126 compound on the activation of thermogenic differentiation during adipogenesis. The results show that U0126 pretreatment primes both white and brown preadipocytes to upregulate thermogenic and mitochondrial genes as well as enhance functions during the differentiation process. We establish that U0126-mediated thermogenic differentiation induction occurs partially via AMPK activation signaling. The findings of this research suggest U0126 as a promising alternative ligand in pursuit of a pharmacological option to increase thermogenic adipocyte formation and improve energy expenditure. Thus it could pave the way for the discovery of therapeutic drugs for the treatment of obesity and its related complications.

Keywords: AMPK; MEK inhibition; U0126; adipogenesis; adipose tissue; obesity; thermogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
U0126 pretreatment upregulates thermogenic genes after the adipogenesis of human white preadipocytes. (A) The structure of U0126 compound. (B) Human white preadipocytes were pretreated with different concentrations (0, 0.1, 0.3, 1, 3, and 10 μM) of U0126 (DMSO, U-0.1, U-0.3, U-1, U-3, and U-10) for 6 days. The cell viability was measured. (C–F) After U0126 pretreatment, preadipocytes underwent adipogenic differentiation for 12 days. Thermogenic genes, UCP1 (C) and DIO2 (D), and mitochondrial biogenesis genes, PGC1α (E) and NRF1 (F), were determined. (G) The transcriptional activity of the UCP1 gene was monitored by reporter assay. The concentration of BMP7 was 3.3 nM. Unpaired Student’s t-tests were compared to the DMSO group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
U0126 pretreatment did not affect general adipogenic differentiation in human white preadipocytes. Human white preadipocytes were pretreated with different concentrations (0, 0.1, 0.3, 1, 3, and 10 μM) of U0126 (DMSO, U-0.1, U-0.3, U-1, U-3, and U-10) for 6 days and underwent adipogenic differentiation for 12 days. (A–C) Mature adipocytes were stained by Oil Red O. Gross view of wells (A), images of differentiated adipocytes (B), and the quantification of Oil Red O intensity after elution (C) were shown. Scale bar, 600 μm. (D,E) General adipogenic marker genes, AP2 (D) and FAS (E), were measured in mature white adipocytes. Unpaired Student’s t-tests were compared to the DMSO group.
Figure 3
Figure 3
U0126 pretreatment promotes thermogenic function after the adipogenesis of human white preadipocytes. Human white preadipocytes were pretreated with 3.3 nM of BMP7 or 0.3 and 3 μM of U0126 (U-0.3 or U-3) for 6 days and underwent adipogenic differentiation for 12 days. (A,B) Blot images of UCP1 protein (A) and the quantification of band intensity (B) in differentiated white adipocytes. NS, non-specific band. (C,D) Thermogenesis function of adipocytes was monitored by ERthermAC dye staining. (C) The changes of ERthermAC intensity after forskolin stimulation. (D) The quantification of area above curve in (C). Unpaired Student’s t-tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
U0126 pretreatment did not alter general adipogenic differentiation in human brown preadipocytes. (A) Human brown preadipocytes were pretreated with different concentrations (0, 0.1, 0.3, 1, 3, and 10 μM) of U0126 (DMSO, U-0.1, U-0.3, U-1, U-3, and U-10) for 6 days. The cell viability was measured. (B–D) After U0126 pretreatment, preadipocytes underwent adipogenic differentiation for 12 days. General adipogenic marker genes, AP2 (B) and FAS (C), were measured in mature brown adipocytes. (D) Images of differentiated brown adipocytes after staining with Oil Red O. Images with low (upper panel) and high (lower panel) magnifications. Scale bar, 600 μm. Unpaired Student’s t-tests compared to the DMSO group. ** p < 0.01, **** p < 0.0001.
Figure 5
Figure 5
U0126 pretreatment promotes thermogenic function after the adipogenic differentiation of human brown preadipocytes. Human brown preadipocytes were pretreated with different concentrations (0, 0.1, 0.3, 1, 3, and 10 μM) of U0126 (DMSO, U-0.1, U-0.3, U-1, U-3, and U-10) or BMP7 (3.3 nM) for 6 days and underwent adipogenic differentiation for 12 days. (A–D) Thermogenic genes, UCP1 (A) and DIO2 (B), and mitochondrial biogenesis genes, PGC1α (C) and NRF1 (D), were determined. (E,F) The oxygen consumption rate (OCR; E) of differentiated brown adipocytes was monitored while adding different drugs (OA, oligomycin A; FSK, forskolin; AA, antimycin A). (F) The quantification of maximal forskolin-induced OCR. (G,H) The thermogenesis function of adipocytes was monitored through ERthermAC dye staining. (G) The changes of ERthermAC intensity after forskolin stimulation. (H) The quantification of the area above curve in (G). Unpaired Student’s t-tests were compared to the DMSO group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 6
Figure 6
U0126-mediated AMPK activation through MEK inhibition drives the thermogenic differentiation of human white preadipocytes. (A–D) Human white preadipocytes were pretreated with 3 μM of U0126 (U-3) or without (DMSO) for 6 days. Phosphorylation and total level of ERK1/2 (A) and AMPK (C) were detected by immunoblotting. (B,D) The quantification of band intensity in (A,C), respectively. Ratios between phosphorylated and total protein (phos/total) and phosphorylated protein and β-tubulin (phos/tub), as well as total protein and β-tubulin (total/tub), were shown. (E–G) Human white preadipocytes were co-pretreated with 3 μM of U0126 (U-3) and an AMPK inhibitor, 10 μM of Compound C, for 6 days and underwent adipogenic differentiation for 12 days. The expression of UCP1 (E), DIO2 (F), and PGC1α (G) were determined. Unpaired Student’s t-tests were compared to the DMSO group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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