A cellular model for the investigation of depot specific human adipocyte biology

Todorčević, Marijana; Hilton, Catriona; McNeil, Catriona; Christodoulides, Constantinos; Hodson, Leanne; Karpe, Fredrik; Pinnick, Katherine E.

doi:10.1080/21623945.2016.1277052

Cited by 21 publications

(45 citation statements)

References 62 publications

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“…We investigated the role of TCF7L2 in AP biology using de-differentiated fat (DFAT) cells generated from immortalised abdominal adipocytes (22). These retain their depot-specific gene expression signature (Fig.…”

Section: Resultsmentioning

confidence: 99%

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TCF7L2 plays a complex role in human adipose progenitor biology which may contribute to genetic susceptibility to type 2 diabetes

Verma

Loh

Vasan

et al. 2019

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Non-coding genetic variation at TCF7L2 is the strongest genetic determinant of type 2 diabetes (T2D) risk in humans. TCF7L2 encodes a transcription factor mediating the nuclear effects of WNT signalling in adipose tissue (AT). Here we mapped the expression of TCF7L2 in human AT and investigated its role in adipose progenitor (AP) biology. APs exhibited the highest TCF7L2 mRNA abundance compared to mature adipocytes and adipose-derived endothelial cells. Obesity was associated with reduced TCF7L2 transcript levels in subcutaneous abdominal AT but increased expression in APs. In functional studies, TCF7L2 knockdown (KD) in APs led to dose-dependent activation of WNT/β-catenin signaling, impaired proliferation and dose-dependent effects on adipogenesis. Whilst partial KD enhanced adipocyte differentiation, complete KD impaired lipid accumulation and adipogenic gene expression. Overexpression of TCF7L2 accelerated adipogenesis.Transcriptome-wide profiling revealed that TCF7L2 can modulate multiple aspects of AP biology including extracellular matrix secretion, immune signalling and apoptosis. The T2D-risk allele at rs7903146 was associated with reduced AP TCF7L2 expression and enhanced AT insulin sensitivity.Our study highlights a complex role for TCF7L2 in AP biology and suggests that in addition to regulating pancreatic insulin secretion, genetic variation at TCF7L2 may also influence T2D risk by modulating AP function. 51Cell culture: Primary APs (derived from AT biopsies) or AP lines were cultured and differentiated 52 as described (21,22). Primary endothelial cells were isolated using a CD31 MicroBead Kit (Miltenyi 53 Biotec). Quantification of intracellular lipid was undertaken using AdipoRed lipid stain (Lonza) and 54 multi-well plate reader (PerSeptive Biosystems, Perkin Elmer). 55 56 Generation of de-differentiated fat (DFAT) cells: DFAT cells were generated by selection and de-57 differentiation of lipid-laden, in vitro differentiated immortalised APs (23) with modifications (See 58 supplemental information). 59 60 Lentiviral constructs and generation of stable AP lines: TCF7L2 (sh843, TRCN0000262843; 61 sh897, TRCN0000061897) and control (scrambled) shRNA plasmid vectors were purchased (Sigma-62 Aldrich). The TOPflash reporter vector (24) was a gift from Roel Nusse (Addgene #24307). Lentiviral 63 particles were produced in HEK293 cells using MISSION ® (Sigma-Aldrich) packaging mix. Stable 64 AP lines were generated by transduction of cells with lentiviral particles and selected using (2µg/ml) 65 puromycin. 66 67 Doxycycline-inducible AP lines: The TCF7L2 sequence (from TCF4E pcDNA3, a kind gift from 68 Frank McCormick, Addgene #32738) (25) was cloned into the tet-pLKO-puro doxycycline-inducible 69 expression lentiviral vector (gift of Dmitri Wiederschain, Addgene #21915) (26). Stable doxycycline-70 inducible AP lines were generated by transduction of cells with lentiviral particles and selected using 71 (2µg/ml) puromycin. 72 73 Proliferation assays: Equal number of APs were seeded, trypsinised and counted...

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Section: Resultsmentioning

confidence: 99%

“…Primary APs (derived from AT biopsies) or AP lines were cultured and differentiated as described (21,22). Primary endothelial cells were isolated using a CD31 MicroBead Kit (Miltenyi Biotec).…”

Section: Methodsmentioning

confidence: 99%

TCF7L2 plays a complex role in human adipose progenitor biology which may contribute to genetic susceptibility to type 2 diabetes

Verma

Loh

Vasan

et al. 2019

Preprint

Self Cite

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“…cDNA expression of CCDC92 ( A ), ZNF664 ( B ), SPATA20 ( C ), UQCC1 ( D ) and GDF5 ( E ) was measured over a 14-day adipogenic differentiation time-course using primary preadipocytes from abdominal subcutaneous (ASAT) and gluteal subcutaneous (GSAT) fat depots[37]. Data are shown as DDCt values (normalized to PPIA and PGK1; n=6, mean ± SEM).…”

Section: Resultsmentioning

confidence: 99%

“…For cell-cultured human primary preadipocytes, of both abdominal subcutaneous fat (ASAT) and gluteofemoral fat (GSAT) origin, a differentiation time course (n=6) was performed as described in Todorčević, et al . [37]. All biopsies and cells were homogenized in Tri-reagent (cat.…”

Section: Methodsmentioning

confidence: 99%

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Regional fat depot masses are influenced by protein-coding gene variants

Neville

Wittemans

Pinnick

et al. 2019

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23 With the identification of a large number of genetic loci associated with human fat 24 distribution and its importance for metabolic health, the question arises as to what 25 the genetic drivers for discrete fat depot expansion might be. To date most studies 26 have focussed on conventional anthropometric measures such as waist-to-hip ratio 27 (WHR) adjusted for body mass index. We searched for genetic loci determining 28 discrete fat depots mass size using an exome-wide approach in 3 large cohorts.29 Here we report an exome-wide analysis of non-synonymous genetic variants in 30 17,212 participants in which regional fat masses were quantified using dual-energy 31 X-ray absorptiometry. The missense variant CCDC92 S70C, previously associated with 32 WHR, is associated specifically with reduced visceral and increased leg fat masses.33 Allele-specific expression analysis shows that the deleterious minor allele carrying 34 transcript also has a constitutively higher expression. In addition, we identify two 35 variants associated with the transcriptionally distinct fat depot arm fat (SPATA20 K422R 36 and UQCC1 R51Q ). SPATA20 K422R , a rare novel locus with a large effect size specific 37 to arm, and UQCC1 R51Q , a common variant exome-wide significant in arm but 38 showing similar trends in other subcutaneous fat depots. In terms of the 39 understanding of human fat distribution, these findings suggest distinct regulation of 40 discrete fat depot expansion. 42Author summary 43 Human fat storing tissues are heterogeneous and comprise functionally and 44 structurally distinct regional fat depots, the relative size of which appear to have 45 significant implications for health. Whilst it is known that inter-individual differences in 46 fat distribution have genetic drivers, studies to date have focussed on crude 3 47 anthropometric approximations of region fat masses rather than precise measures.48 Here we describe an exome-wide analysis of a large collection of men and women 49 who have undergone body scanning using dual-energy X-ray absorptiometry (DXA) 50 to better define regional fat masses and identify new genetic drivers for human fat 51 distribution. With this approach we identify three gene regions associated with 52 distinct fat depots which can help to explain the variation in fat distribution between 53 people and may lead to a better understanding of the depot specific fat 54 tissue expansion.4 56 57 58 Beyond associations with chronic disease and overall obesity, as defined by body-59 mass index (BMI), it is becoming increasingly apparent that there is an even stronger 60 relationship between body fat distribution and cardio-metabolic disease [1, 2]. For 61 example, Yusuf et al. [1] showed that waist-to-hip ratio (WHR) is a stronger predict of 62 myocardial infarction than BMI. To date, the overwhelming majority genome and 63 exome-wide association studies on fat distribution have focussed on waist and hip 64 circumference and WHR [3, 4]. While these measures are easy and cheap to obtain 65 on a large sca...

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A cellular model for the investigation of depot specific human adipocyte biology

Cited by 21 publications

References 62 publications

TCF7L2 plays a complex role in human adipose progenitor biology which may contribute to genetic susceptibility to type 2 diabetes

TCF7L2 plays a complex role in human adipose progenitor biology which may contribute to genetic susceptibility to type 2 diabetes

Regional fat depot masses are influenced by protein-coding gene variants

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution

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