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. 2020 Sep 4;9(9):2032.
doi: 10.3390/cells9092032.

NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

María-Milagros Rodríguez-Cano  1 , María-Julia González-Gómez  1 , Beatriz Sánchez-Solana  2 , Eva-María Monsalve  3 , María-José M Díaz-Guerra  3 , Jorge Laborda  1 , María-Luisa Nueda  1 , Victoriano Baladrón  3
Affiliations

NOTCH Receptors and DLK Proteins Enhance Brown Adipogenesis in Mesenchymal C3H10T1/2 Cells

María-Milagros Rodríguez-Cano et al. Cells. .

Abstract

The NOTCH family of receptors and ligands is involved in numerous cell differentiation processes, including adipogenesis. We recently showed that overexpression of each of the four NOTCH receptors in 3T3-L1 preadipocytes enhances adipogenesis and modulates the acquisition of the mature adipocyte phenotype. We also revealed that DLK proteins modulate the adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells in an opposite way, despite their function as non-canonical inhibitory ligands of NOTCH receptors. In this work, we used multipotent C3H10T1/2 cells as an adipogenic model. We used standard adipogenic procedures and analyzed different parameters by using quantitative-polymerase chain reaction (qPCR), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), luciferase, Western blot, and metabolic assays. We revealed that C3H10T1/2 multipotent cells show higher levels of NOTCH receptors expression and activity and lower Dlk gene expression levels than 3T3-L1 preadipocytes. We found that the overexpression of NOTCH receptors enhanced C3H10T1/2 adipogenesis levels, and the overexpression of NOTCH receptors and DLK (DELTA-like homolog) proteins modulated the conversion of cells towards a brown-like adipocyte phenotype. These and our prior results with 3T3-L1 preadipocytes strengthen the idea that, depending on the cellular context, a precise and highly regulated level of global NOTCH signaling is necessary to allow adipogenesis and determine the mature adipocyte phenotype.

Keywords: EGF-like proteins; adipogenic differentiation; brown-like adipocytes; mesenchymal cells; preadipocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of Notch family genes in differentiated C3H10T1/2 cells. Relative mRNA transcription levels of Notch, Hes1 (hairy and enhancer of split 1), and Hey1 (hairy and enhancer-of-split related with YRPW motif 1) (A) and Dlk (DELTA-like homologs) (B) genes in seven-day differentiated C3H10T1/2 cells. Data from qRT-PCR (quantitative reverse transcription-polymerase chain reaction) and qPCR (quantitative polymerase chain reaction) assays were normalized to P0 (ribosomal protein P0) mRNA transcription levels. The fold activation or inhibition was calculated relative to levels is non-differentiated C3H10T1/2 cells, which were set arbitrarily at 1 (horizontal black line). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 2
Figure 2
NOTCH signaling in mesenchymal C3H10T1/2 cells stably overexpressing NOTCH receptors or the HES1 protein. (A) NOTCH receptors’ transcriptional activity, as measured by luciferase assays, in each of the four stably Notch-transfected pools. The relative luciferase activities were always normalized to renilla levels. (B) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA transcription levels in each of the stably Notch-transfected pools. (C) qRT-PCR analysis of relative mRNA transcription levels of Notch genes in the stable Notch1 transfectant (N1S), the stable Notch2 transfectant (N2S), the stable Notch3 transfectant (N3S), and the stable Notch4 transfectant (N4S). (D) qRT-PCR analysis of Dlk1 (DELTA-like 1 homolog) and Dlk2 (DELTA-like 2 homolog) mRNA transcription levels in the stable Notch transfectants. (E) qRT-PCR analysis of the relative Hes1 and Dlk mRNA transcription levels in the stable Hes1 gene transfectant (H1S). All these assays were performed using non-differentiated cells. Data in qRT-PCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition in all assays was measured relative to levels in non-differentiated empty-vector-transfected cells, set arbitrarily at 1 (horizontal black line or V). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-test results is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 3
Figure 3
Expression of adipogenic markers and mitochondrial biogenesis markers in non-differentiated and differentiated C3H10T1/2 cells. (A) Relative mRNA transcription levels (1/∆ CT (cycle threshold) × 10 × E (oligonucleotide efficiency)) of the adipogenic and brown adipocyte markers aP2 (adipocyte protein 2/fatty acid binding protein 4), Pparg (peroxisome proliferator activated receptor gamma), Cebpb (CCAAT Enhancer Binding Protein Beta), Ucp2 (uncoupling protein 2), Prdm16 (PR domain-containing 16), Pgc1a (peroxisome proliferator activated receptor gamma coactivator 1-alpha), Ucp1 (mitochondrial uncoupling protein 1), Gyk (glycerol kinase), Cidea (cell death-inducing DNA fragmentation factor, alpha subunit-like effector A), and Sirt1 (Sirtuin 1) in non-differentiated C3H10T1/2 cells. (B) Relative mRNA transcription levels of the same adipogenic and brown adipocyte markers in seven-day differentiated C3H10T1/2 cells. (C) qPCR analysis of mitochondrial Cytb DNA amplification (related to genomic ApoB (apoliprotein B) DNA amplification, see Materials and Methods section) in seven-day differentiated [D] and non-differentiated [ND] C3H10T1/2 cells. Data from qRT-PCR and qPCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition values were calculated relative to those of non-differentiated C3H10T1/2 cells, which were set arbitrarily at 1 (horizontal black line or ND). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated (* p ≤ 0.05, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 4
Figure 4
Stable overexpression of Notch and Dlk genes in multipotent C3H10T1/2 cells enhanced adipogenesis levels. Representative microscopic images of adipocytes from C3H10T1/2 cells transfected with Notch (A) or Dlk genes (B) showing their adipogenic levels (50× magnification images, scale bar 200 μm) after oil red O staining, all compared with the adipogenic levels of empty-vector transfected cells (VD). D: differentiated cells. qRT-PCR analysis of indicated adipogenic marker mRNA transcription levels in the differentiated stable Notch1-overexpressing cells (N1SD) (C), the differentiated stable Notch2-overexpressing cells (N2SD) (D), the differentiated stable Notch3-overexpressing cells (N3SD) (E), the differentiated stable Notch4-overexpressing cells (N4SD) (F), the differentiated stable Dlk1-overexpressing cells (DLK1SD) (G), and the differentiated stable Dlk2-overexpressing cells (DLK2SD) (H). Data from all qRT-PCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition levels in qRT-PCR assays were calculated relative to the levels shown by seven-day differentiated empty-vector-transfected cells, which were set arbitrarily at 1 (horizontal black line). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 5
Figure 5
Stable overexpression of Notch and Dlk genes in multipotent C3H10T1/2 cells modulated brown adipogenesis. (A) Representative microscopic images of adipocytes of C3H10T1/2 cells transfected with Notch (A) or Dlk genes (B), showing the size of their lipid droplets (400× magnification images, scale bar 30 μm) after oil red O staining, all of them compared with the size of lipid droplets of empty-vector-transfected differentiated cells (VD). qRT-PCR analysis of the indicated brown marker expression levels in the differentiated stably Notch1-transfected cells (N1SD) (C), the differentiated stably Notch2-transfected cells (N2SD) (D), the differentiated stably Notch3-transfected cells (N3SD) (E), the differentiated stably Notch4-transfected cells (N4SD) (F), the differentiated stably Dlk1-transfected cells (DLK1SD) (H) and the differentiated stably Dlk2-transfected cells (DLK2SD) (I). (G) qPCR analysis of mtCytb (mitochondrial cytochrome b) DNA amplification (related to genomic ApoB DNA amplification; see Materials and Methods section) in seven-day differentiated C3H10T1/2 cells overexpressing Notch (G) and Dlk (J) genes. Data from qRT-PCR and qPCR assays were normalized to P0 mRNA transcription levels. The fold activation or inhibition levels in PCR assays was calculated relative to the levels of seven-day differentiated empty-vector-transfected cells, which were set arbitrarily at 1 (horizontal black line). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 6
Figure 6
Lipolytic potential and release of lactate to the extracellular medium in C3H10T1/2 adipocytes overexpressing Dlk or Notch genes. (A) Relative levels of glycerol released to the extracellular medium in response to isoproterenol from non-differentiated (ND) and differentiated (D) C3H10T1/2 cells. (B) Relative levels of glycerol released to the extracellular medium in response to isoproterenol from differentiated Dlk- or Notch-overexpressing cells. (C) Relative levels of lactate released into the culture media of differentiated Dlk- or Notch-overexpressing C3H10T1/2 cells. The fold activation or inhibition levels were calculated relative to the levels of non-differentiated cells (A) or empty-vector-differentiated cells (B,C), which were set arbitrarily at 1 (horizontal black line or ND cells). Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Non-statistical significance is indicated by ns.
Figure 7
Figure 7
Oxygen consumption rate (OCR) in C3H10T1/2 adipocytes overexpressing Dlk or Notch genes. (A) Relative oxygen consumption rate (OCR) in non-differentiated (C3HND) and differentiated (C3HD) C3H10T1/2 cells. Relative oxygen consumption rate (OCR) in differentiated C3H10T1/2 cells overexpressing Dlk (B), Notch1 (C), Notch2 (D), Notch3 (E), or Notch4 (F) genes. The fold activation or inhibition levels were calculated relative to time 0 of non-differentiated cells, and, additionally, to OCR of non-differentiated cells (ND) (A) or seven-day differentiated empty-vector-transfected cells (VD) (B–F), which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-test is indicated at 144 min (* p ≤ 0.05, ** p ≤ 0.01). Non-statistical significance is indicated by ns.
Figure 8
Figure 8
Outline of the roles of Notch and Dlk genes’ overexpression on C3H10T1/2 adipogenesis. The effects of adipogenic inductors at the end of the differentiation process (2 days with dexamethasone and IBMX (3-Isobutyl-1-methylxanthine) and 5 days with insulin) on C3H10T1/2 cells (wide black arrow) are compared to the effects of the same adipogenic inductors on C3H10T1/2 cells stably overexpressing each of the Notch or Dlk genes. The direction (up or down) of the variations in the expression levels of adipogenic markers, brown adipocyte markers, mitochondrial biogenesis markers, and metabolic effects are shown by grey arrows. OCR: oxygen consumption rate, ECAR: extracellular acidification rate, LP: lipolytic potential.
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