The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Sprouty1 is a weight-loss target gene in human adipose stem/progenitor cells that is mandatory for the initiation of adipogenesis.

The differentiation of adipose stem/progenitor cells (ASCs) into adipocytes contributes to adipose tissue expansion in obesity. This process is regulated by numerous signalling pathways including MAPK signalling. In the present study, we show that weight loss (WL) interventions induce upregulation of Sprouty1 (SPRY1), a negative regulator of MAPK signalling, in human ASCs and elucidate the role of the Sprouty1/MAPK interaction for adipogenic differentiation. We found that the Sprouty1 protein levels are low in proliferating ASCs, increasing in density arrested ASCs at the onset of adipogenic differentiation and decreasing in the course of adipogenesis. Knock-down (KD) of Sprouty1 by RNA interference led to elevated MAPK activity and reduced expression of the early adipogenic transcription factor CCAAT/enhancer-binding protein ß (C/EBP ß), concomitant with an abrogation of adipogenesis. Intriguingly, co-treatment of Sprouty1 KD ASCs with differentiation medium and the pharmacological MEK inhibitor U0126 blunted ERK phosphorylation; however, failed to rescue adipogenic differentiation. Thus, the effects of the Sprouty1 KD are not reversed by inhibiting MAPK signalling although the inhibition of MAPK signalling by U0126 did not prevent adipogenic differentiation in wild type ASCs. In conclusion, we show that Sprouty1 is induced after WL in ASCs of formerly obese people acting as a negative regulator of MAPK signalling, which is necessary to properly trigger adipogenesis at early stages by a C/EBP ß dependent mechanism.

CD146 (MCAM) in human cs-DLK1-/cs-CD34+ adipose stromal/progenitor cells.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.

Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

The nonrecurrent laryngeal nerve: A clinical anatomic mapping with regard to intraoperative neuromonitoring.

BACKGROUND: We investigated the nonrecurrent inferior laryngeal nerve (nrILN), an important variant in the course of the inferior laryngeal nerve (ILN; 0.5-6.0%). Its importance was demonstrated in a clinical case as well as in cadaver specimens, and the pattern was identified with intraoperative neuromonitoring (IONM). METHODS: The ILN and the presence of an nrILN were investigated in 36 formaldehyde-embalmed specimens. Our anatomic findings showed differences in the anatomic course of the ILN and thus produced possible explanations for different IONM signals that would correlate with differences in the anatomic course of the ILN. Preoperative ultrasonographic evaluation of the brachiocephalic trunk and the recurrent laryngeal nerve were used for the exclusion or identification of an nrILN, respectively. RESULTS: We found 2 nrILNs (ascending, horizontal; 6%) in the anatomic specimens. These 2 specimens each showed an aberrant right subclavian artery (lusorial artery) and were, therefore, associated with the absence of a brachiocephalic trunk. The intraoperative case displayed a descending nrILN. Signals derived from the vagus nerve were positive if derived proximal to and negative if derived distal to the branching of an nrILN. By ultrasonographic identification of a normal brachiocephalic trunk, an nrILN could be excluded. CONCLUSION: Surgeons need a working knowledge about nrILNs to avoid recurrent nerve palsy and should be familiar with all the possible course variations in the ILN when IONM signals are absent with vagal stimulation. Moreover, endocrine surgeons need to be able to interpret correctly negative as well as positive signals. Preoperative ultrasonography should ideally be performed, because the presence of a normal brachiocephalic trunk is a quick method to exclude or identify a nrILN.

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue.

Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.

Novel immunohistochemical data indicate that the female foetal urethra is more than an epithelial tube.

The female urethra has often been neglected in previous studies on the development of the human urogenital system. Our aim has been to reach a consensus on the organogenesis of the female urethra and the vagina with respect to interactions between the epithelia with different evolutionary origins. Therefore we tried to clarify open questions on the spatiotemporal distribution of molecular markers raised against mesenchymal and epithelial structures within the developing human female urethra. Furthermore, we draw comparisons regarding gender-specific aspects in urethral development. To this effect, we used molecular markers such as different cytokeratins (CKs), p63, Ki67, uroplakin III, E-cadherin, vimentin, smooth muscle actin (SMA), cleaved caspase 3 and paired box gene 2 (PAX 2) to phenotype developmental changes. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay was additionally performed to reveal apoptosis. We examined different gestational stages starting from week (W) 8 until W 15. Immunohistochemistry showed a distinct staining pattern for p63 and CK17, both markers for stem cells, ensuing from the urogenital sinus (UGS) proceeding into the Muellerian duct (MD). This was observed throughout development and might be a stimulus for the formation of the vaginal anlagen that derive from the MD. In the attachment area of the MD we detected a conglomeration of cells with different embryonic origins. The epithelium of the UGS became transitional at W 9 after fertilization, and the differentiation advanced in a cranial to caudal direction. The paraurethral glands showed a slightly different staining profile than the urethral epithelium, which may be able to explain why carcinomas of these structures display various histological appearances. In addition, we could show that during the development of the female urogenital system the primary incidence is the formation of the urethra. This is followed by the establishment of the vagina, which clearly depends on the proper differentiation of the UGS/urethra.