HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling

Aims/hypothesis Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. Methods Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. Results We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR–HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. Conclusions/interpretation In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. Data availability Array data have been submitted to the GEO database at NCBI (GSE148699). Graphical abstract Supplementary Information The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-021-05470-y.

for 10 minutes at 800 g. The supernatant was discarded, and the pellet resuspended in DMEM 10% v/v Serum and filtered through a 70 µm nylon mesh and centrifuged for 10 min at 800 g. Once again, the pellet was resuspended in DMEM 10% v/v serum and filtered through a 48 µm nylon mesh. The cells were then diluted in DMEM supplemented with 10% v/v FBS, 10 mmol/l HEPES, 50 mg/ml penicillin, 50 mg/ml streptomycin and 50mg/ml gentamycin. At day 0, the differentiation was induced using DMEM 10% v/v serum supplemented with 200 nmol/l insulin, 1 µmol/l rosiglitazone, 0.2 nmol/l triiodothyronine, 1 mmol/l DEX and 1mmol/l IBMX. From day 2 to day 8, the medium was changed every other day with DMEM 10% v/v serum supplemented with 200 nmol/l insulin.
Human SVF (hSVF) was isolated from scWAT, collected from healthy patients (abdominoplasty) and differentiated by administering the same adipogenic cocktail as for hMADS cells [3]. The study was approved by the University Ulm ethical committee (vote no. 300/16) and all patients gave written informed consent.
For microarray expression profiling, total RNA was isolated employing the RNAeasy kit (Qiagen) using the small and large RNA protocols for animal tissue and cultured cells, including on-column DNase digestion. RNA quality was measured with an Agilent 2100 Bioanalyzer and only high-quality RNA (RIN>7) was used for microarray analysis. Total RNA (150 ng) was amplified using the WT PLUS Reagent Kit (Thermo Fisher Scientific Inc., Waltham, USA) and the resulting cDNA was hybridized on Human Clariom S arrays (Thermo Fisher Scientific). Staining and scanning (Gene Chip Scanner 3000 7G) was done according to manufacturer`s instructions.
Transcriptome Analysis Console (TAC;version 4.0.0.25;Thermo Fisher Scientific) was used for quality control and to obtain annotated normalized SST-RMA genelevels. Statistical analyses were performed by utilizing the statistical programming environment R [6,7]. Gene-wise testing for differential expression was done with the limma t-test including batch correction and Benjamini-Hochberg (BH) multiple testing correction (FDR < 10%). To reduce background, gene sets were filtered for TAC Data Above Background p-values<0.05. Heat maps were generated in R. Pathway analyses were generated through the use of Ingenuity Pathway Analysis software (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity) using Fisher`s Exact Test p-values (Diseases & Functions), BH-corrected p-values (Canonical Pathways), or zscores (Upstream Regulators). Array data have been submitted to the GEO database at NCBI (GSE148699).
For RNAseq analysis, library preparation and sequencing were performed by Novogene. An amount of 800 ng of total RNA was used and controlled quantitatively and qualitatively with Agilent Bioanalyzer 2100 system. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. Prepared PCR products were purified and size-selected on AMPure XP beads (Beckman #A63881). The prepared libraries were sequenced (paired-end) on an Illumina platform. Read counts post processing, statistical analysis and differential gene expression analysis were performed using DESeq2 [8] in R [7]. In order to observe the overall effect of experimental covariates a two-dimensional PCA plot was employed. GO gene enrichment analysis of differentially expressed genes was performed using ClusterProfiler [9]. ChIP-qPCR was performed as previously described [10]. . 20 million cells per biological replicate were crosslinked with formaldehyde solution (1% v/v in PBS) for 15 min. Chromatin was isolated using a lysis buffer (150 mmol/l NaCl; 5 mmol/l EDTA; 5 mmol/l Tris; 1% Triton; 0.5% NP40), and then sonicated in the shearing buffer (1% SDS; 10 µmol/l EDTA; 0.05 mmol/l Tris). Immunoprecipitations were performed by adding 3 µg of the following antibodies: αIgG (Cell Signaling Technology #2729); αGlucocorticoid Receptor (ProteinTech, Germany #24050-1-AP), αFLAG (Sigma, Germany #F1804) and sepharose protein A/G beads (Biomol, Germany #PAG50-00-0002) in a dilution buffer (0.1 g/L SDS, 1.10 g/L Triton, 1.2 µmol/l EDTA, 16.7 µmol/l Tris, 0.167 mol/l NaCl). DNA was eluted and qPCR was performed to validate the binding using the following primers (Supplementary table 1). All the experiments were performed at least 3 times.

Mouse experiments:
All animal studies were conducted in accordance with German animal welfare legislation and protocols were approved by the state ethics committee and government of Upper Bavaria (nos. ROB-55.2-2532.Vet_02-16-117, ROB-55.2-2532.Vet_02-17-125, ROB-55.2-2532. All mice were group-housed maintained in a climate-controlled environment at 22 °C with a 12-h dark-light cycle under specific pathogen-free conditions in the animal facility of the Helmholtz Center Munich. db/db Mice (JAX Mice Strain) were purchased from Charles River (https://www.criver.com/products-services/find-model/jax-dbdb-mice?region=23).
Adipocyte-specific Hand2 knockout mice (Hand2 AdipoqCre ) were generated by crossing AdipoqCRE mice (Jackson laboratory, stock number 028020; C57BL/6J) with Hand2 floxflox mice (NMRI strain) kindly provided by R. Zeller [11]. Hand2 AdipoqCre (CRE+) and wild type littermates (CRE-) were used for all experiments. Animals were fed a high-fat diet (HFD) 60 kcal % fat (D12492, Research Diets Inc., New Brunswick, NJ, USA) ad libitum from the age of 6 weeks for 12 weeks after which glucose and insulin tolerance tests was performed. Animals were fasted at 8 am for 4 to 6 hours and subsequently injected intraperitoneally (i.p.) with glucose at 2 g/kg or insulin 0.8 U/kg (HUMINSULIN Normal 100, Lilly Germany #PA257FSGE05). Blood samples were taken from the tail vein before and 15, 60, 90 and 120 min after the i.p. injection to assess glucose levels. (AccuChek Performa glucose meter, Roche Diabetes Care, Mannheim, Germany). Assessment of the fat mass vs lean mass was performed using whole-body magnetic resonance analysis (EchoMRI).
Histological analysis of the samples has been performed as following: The samples were fixed in 4% w/v neutrally buffered formalin and subsequently embedded in paraffin. 3 µm thick sections were stained with hematoxylin and eosin (HE), using a HistoCore SPECTRA ST automated slide stainer (Leica, Germany) with prefabricated staining reagents (Histocore Spectra H&E Stain System S1, Leica, Germany), according to the manufacturer's instructions. Stained tissue sections were scanned with an AxioScan. Z1 digital slide scanner (Zeiss, Jena, Germany) equipped with a 20x magnification objective. Quantification of lipid amount was morphometrically determined by automatic digital image analysis using the commercially available software Definiens Developer XD 2 (Definiens AG, Germany). The calculated parameter for BAT was the ratio of total area of lipid droplets per whole tissue section and for WAT the mean size of lipid droplets per whole tissue section. The experiment has been performed on 3 different cohorts of littermates with a total of 20 to 28 animals per group.
Hand2 expression in wildtype mice versus diet-induced obese mice was measured in samples from 18 weeks old (10 animals per group) C57BL/6J males (C57BL/6J (JAX Mice Strain), Charles River https://www.criver.com/products-services/find-model/jax-c57bl6j-mice) fed chow or HFD. To correlate Hand2 mRNA expression to body weight, https://www.criver.com/products-services/find-model/jax-c57bl6j-mice) and NMRI (Hand2 AdipoqCre (CRE-) male mice (18 to 24 weeks old) fed ad libitum with regular rodent chow were used. At termination of all experiments, the animals were killed by cervical dislocation and organs were collected and immediately frozen in liquid nitrogen and stored at -80 °C for further processing. The expression of Hand2 (and other genes) was analyzed using SYBR-based qPCR (Thermo Fisher, Germany #A25741) (primers listed in ESM Table 1).

Human studies:
Human BAT was collected from FDG-PET-positive scan sites in the supraclavicular localization and subcutaneous WAT was taken from the same incision. The Study protocol was approved by the ethics committee of the Hospital District of Southwestern Finland, and participants provided written informed consent following the committee's instructions. The study was conducted according to the principles of the Declaration of HelsinkiparticipantsThe metabolic status of all potential participants who donated BAT was assessed, and only those with normal glucose tolerance and normal cardiovascular status (as assessed on the basis of electrocardiograms and measured blood pressure) were included. The age range of the participants was 23-49 years.
We studied a group of 7 healthy volunteers (2 men and 5 women). 1) men or premenopausal women, 2) age >18years, 3) stable body weight, defined as the absence of fluctuations of >3% of body weight for ≥3 months before blood tests.
In addition, the following exclusion criteria have been defined: 1) any acute or chronic inflammatory disease or symptoms of infection; 2) clinical evidence of either cardiovascular or peripheral artery disease; 3) smoking; 4) LDL-cholesterol > 4mmol/l; 5) any type of malignant disease; 6) thyroid dysfunction; 7) Cushing's disease or hypercortisolism; 8) alcohol or drug abuse; 9) pregnancy. The study was approved by Ethics committee of the University of Leipzig (approval number: 159-12-21052012) and all participants gave written informed consent before taking part in the study.
To analyze HAND2 mRNA expression in human adipose tissue, RNA from visWAT and scWAT was extracted by using the RNeasy Lipid tissue Mini Kit (Qiagen, Hilden, Germany (Hs01003267_m1) span exon-exon boundaries to improve the specificity of the qPCR.

Statistics:
Data presented as bar chart were expressed as mean ± standard error of the mean (s.e.m.). Data presented as box and whiskers plot were shown as median with upper and lower quartile as well as maximum and minimum. Two-tailed, unpaired t-Test was used when comparing two conditions. 1-way ANOVA and 2-ANOVA with Tukey test were used when comparing three or more groups as reported in the figure legends.
Analysis was performed using GraphPad Prism. P < 0.05 was considered significant as indicated by asterisks in the figure's legends. Mice experiments involving    Data are presented as fold change compared to the condition siCtr/1h (a-c). Data are presented as arbitrary unit representing copy number normalized on TBP (d-p).
Statistics: two-way ANOVA with Tukey test (a-c). one-way ANOVA with Tukey test (dp), mean ± SEM. Statistical significance is indicated by *p<0.05. ChIP-qPCR analysis of selected GR or HAND2 binding sites was performed on 3T3L1 transfected with pcDNA-3XFlag-Hand2 vector (n=3 replicates) (b, d, f) and scWAT SVF from Hand2 3XFlag mice (n=2-4 replicates) (c, e, g). ChIP-qPCR was used to amplify chromatin derived from immunoprecipitations with anti-IgG antibody, used as control (b, c), anti-GR antibody (d, e) and anti-Flag antibody (f, g). Data are presented as fold change compared to the condition CRE-(a). Statistics: two-way ANOVA with Tukey test (a, b, d, f), one-way ANOVA with Tukey test (c, e, g); median with upper and lower quartile ± maximum and minimum (a), mean ± SEM (bg). Statistical significance is indicated by *p<0.05.