A novel growth factor-dependent thermogenic brown adipocyte cell line from defined precursor cells
Summary (2 min read)
Introduction
- Brown adipose tissue (BAT) is a major contributor to adaptive thermogenesis in mammals including humans and rodents.
- This process involves lipid accumulation and mitochondrial biosynthesis and depends on the induction of a specific gene expression program including Ucp1 protein expression.
- Primary cells can be functionally expanded for a limited number of passages upon viral transduction with oncogenes.
- The authors sought to establish a stable brown adipocyte precursor cell line from a defined primary cell population and without the use of viral/genetic manipulation.
Results
- Establishment of a stably growing bFGF-dependent clone from interscapular brown adipose tissue Lin-Sca1+ precursor cells Efficient brown adipogenic differentiation of BATkl2 cells BATkl2 cultures were induced to differentiate with a standard adipogenic cocktail on gelatincoated dishes.
- Importantly, Ucp1 protein could be robustly detected by Western blotting in differentiated but not in undifferentiated BATkl2 cultures .
- Norepinephrine-dependent lipolysis and uncoupled mitochondrial respiration in BATkl2 adipocytes BATkl2 cells were transfected under general growth conditions with siRNA targeting the Prkaca mRNA or with non-targeting siRNA (NC) and analyzed 2 days later.
Discussion
- The novel BATkl2 cell line features a number of unique advantages compared to established human and murine brown preadipocyte cell lines.
- (1) To their knowledge it is the first BATderived cell line with defined cell type of origin, namely Lin-Sca1+ cells representing precursor cells.
- Most currently available cell lines and immortalization protocols rely on such genes (Table 1) and this is a disadvantage given the ability of oncogenes to directly and constitutively influence cell proliferation and adipogenic differentiation.
- Taken together, the BATkl2 cell line represents a novel attractive model for the investigation of brown adipocyte differentiation and thermogenic metabolism.
- The characteristics of the BATkl2 cell line may result in improved validity of conclusions related particularly to the regulation of precursor cell proliferation and differentiation.
Materials and Methods
- Tissue dissociation and cell isolation Female NMRI mice (Charles River WIGA GmbH, Sulzfeld, Germany) were housed at ambient temperature with 12-hour light-dark cycle on chow (Kliba Nafag #3437, Provimi Kliba, Kaiseraugst, Switzerland).
- Interscapular brown fat was dissected from 6 week-old mice and cleared of surrounding white-appearing fat tissue.
- Cultures were maintained at relatively low density, media were replaced every 2-3 days and passaging was performed by trypsinization.
Adipogenic differentiation
- Cells were plated on gelatin-coated wells at 1-3*104 cells per cm2 in growth medium.
- The following day media were replaced by fresh.
- 2 days after differentiation induction, media were replaced with DMEM (high glucose), 5% FCS, Pen/Strep, 1 µg/ml recombinant human insulin and 3 nM triiodothyronine (T3).
- Wherever indicated, norepinephrine was added at 500 nM. siRNA transfection FlexiTube siRNA Mm_Prkaca_1 (SI01388331, , Germany) and AllStars Negative Control siRNA were used.
- For transfection of cells after the induction of differentiation, cells were induced to differentiate for the indicated time and media were replaced with DMEM (high glucose), 5% FCS, 1 µg/ml recombinant human insulin and 3 nM triiodothyronine (T3) one day before transfection.
Plasmid DNA transfection
- For plasmid DNA transfection of undifferentiated cells, cells were plated at 4*104 cells per 24-well the day before transfection.
- The transfection mix was diluted to 500 µl DMEM supplemented with 10% FCS and used to replace the media of one 24-well.
Mitochondrial respiration assay
- Mitochondrial respiration was measured in live cells using the XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Copenhagen, Denmark) as previously described (Li et al. 2014).
- Briefly, BATkl2 cells were plated on gelatin-coated XF96 microplates and treated for adipogenic differentiation as described above (with rosiglitazone for 2 days).
- The oxygen consumption rate was calculated with XF-96 software.
- QRT-PCR mRNA expression analysis Total RNA was isolated from cultured BATkl2 cells using Qiazol according to the manufacturer’s instructions.
- Relative expression values were calculated using the ∆∆Ct method including normalization with the Tbp values.
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Cites background from "A novel growth factor-dependent the..."
...Precursors of adipocytes are able to differentiate into brown adipocytes when treated with norepinephrine, which increased lipolysis and Ucp1 mRNA expression [79]....
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References
37 citations
"A novel growth factor-dependent the..." refers background in this paper
...Thus, enhancement of BAT function has become an actively pursued approach in the development of new therapies in this area (Betz et al. 2018, Moonen et al. 2019, Sidossis et al. 2015)....
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31 citations
"A novel growth factor-dependent the..." refers background or methods or result in this paper
...As described previously, this population represents a good approximation of the Lin−CD29+CD34+Sca1+ precursor/progenitor population (Babaei et al. 2018, Bayindir et al. 2015)....
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...We have shown that Lin(TER119/CD31/Cd45)−CD29+CD34+Sca1+ cells from murine interscapular brown fat efficiently form adipocytes ex vivo with a clearly distinct brown adipocyte expression profile compared to their counterparts from white adipose tissue (Bayindir et al. 2015, Rodeheffer et al. 2008)....
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...(Bayindir et al. 2015)....
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...A single cell suspension of the stromal-vascular fraction was obtained by enzymatic tissue dissociation as previously described (Bayindir et al. 2015)....
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...Lin− Sca1+ cells were isolated by the MACS cell separation procedure described in Bayindir et al. (Bayindir et al. 2015)....
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31 citations
"A novel growth factor-dependent the..." refers background in this paper
...In this case, the proliferation of precursor cells contributes to increased capacity to form brown adipocytes (Lee et al. 2015, Nedergaard et al. 2019)....
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...TERT-hBA Human deep neck fat Telomerase (TERT) (Markussen et al. 2017)...
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