TNF and IFNγ-induced cell death requires IRF1 and ELAVL1 to promote CASP8 expression.

TNFα and IFNγ (TNF/IFNγ) synergistically induce caspase-8 activation and cancer cell death. However, the mechanism of IFNγ in promoting TNF-initiated caspase-8 activation in cancer cells is poorly understood. Here, we found that in addition to CASP8, CYLD is transcriptionally upregulated by IFNγ-induced transcription factor IRF1. IRF1-mediated CASP8 and CYLD upregulation additively mediates TNF/IFNγ-induced cancer cell death. Clinically, the expression levels of TNF, IFNγ, CYLD, and CASP8 in melanoma tumors are increased in patients responsive to immune checkpoint blockade (ICB) therapy after anti-PD-1 treatment. Accordingly, our genetic screen revealed that ELAVL1 (HuR) is required for TNF/IFNγ-induced caspase-8 activation. Mechanistically, ELAVL1 binds CASP8 mRNA and extends its stability to sustain caspase-8 expression both in IFNγ-stimulated and in basal conditions. Consequently, ELAVL1 determines death receptors-initiated caspase-8-dependent cell death triggered from stimuli including TNF and TRAIL by regulating basal/stimulated caspase-8 levels. As caspase-8 is a master regulator in cell death and inflammation, these results provide valuable clues for tumor immunotherapy and inflammatory diseases.

Progenitor cell isolation from mouse epididymal adipose tissue and sequencing library construction.

Here, we present a protocol to isolate progenitor cells from mouse epididymal visceral adipose tissue and construct bulk RNA and assay for transposase-accessible chromatin with sequencing (ATAC-seq) libraries. We describe steps for adipose tissue collection, cell isolation, and cell staining and sorting. We then detail procedures for both ATAC-seq and RNA sequencing library construction. This protocol can also be applied to other tissues and cell types directly or with minor modifications. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

Tcf21 marks visceral adipose mesenchymal progenitors and functions as a rate-limiting factor during visceral adipose tissue development.

Distinct locations of different white adipose depots suggest anatomy-specific developmental regulation, a relatively understudied concept. Here, we report a population of Tcf21 lineage cells (Tcf21 LCs) present exclusively in visceral adipose tissue (VAT) that dynamically contributes to VAT development and expansion. During development, the Tcf21 lineage gives rise to adipocytes. In adult mice, Tcf21 LCs transform into a fibrotic or quiescent state. Multiomics analyses show consistent gene expression and chromatin accessibility changes in Tcf21 LC, based on which we constructed a gene-regulatory network governing Tcf21 LC activities. Furthermore, single-cell RNA sequencing (scRNA-seq) identifies the heterogeneity of Tcf21 LCs. Loss of Tcf21 promotes the adipogenesis and developmental progress of Tcf21 LCs, leading to improved metabolic health in the context of diet-induced obesity. Mechanistic studies show that the inhibitory effect of Tcf21 on adipogenesis is at least partially mediated via Dlk1 expression accentuation.

Astragalus polysaccharide regulates brown adipogenic differentiation through miR-1258-5p-modulated cut-like homeobox 1 expression.

Astragalus polysaccharide (APS) is the major natural active component of Astragalus membranaceus, which has been recognized as one of the most popular herbal medicines worldwide. Enhancing the formation and function of brown adipose tissue increases energy expenditure and hence may potentially be used against obesity and type 2 diabetes. The aim of the present study was to explore the effect and mechanism of APS on brown adipocyte formation. Mouse C3H10T 1/2 cells were subject to APS, and both proliferation and brown adipogenic differentiation were determined. The results showed that APS exhibits a decreased proliferation ability, which is accompanied by downregulated proliferating cell nuclear antigen, cyclin D1, and cyclin-dependent kinase 4. APS promotes the differentiation of C3H10T 1/2 cells into brown adipocytes and induces the expressions of key brown adipogenic transcriptional factors, including CCAAT/enhancer-binding protein ß, uncoupling protein 1, and PR domain-containing 16. Importantly, APS enables insulin sensitization in brown adipocytes, which may proceed through activation of the canonical phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and AMP-activated protein kinase (AMPK). Furthermore, the level of cut-like homeobox 1 (CUX1) is positively related to brown adipogenic differentiation, while APS regulates Cux1 expression through interaction with miR-1258-5p. Notably, the promotional effect of APS on brown adipogenic differentiation was abolished by Cux1 knockout. Collectively, our results suggest that APS enhances the differentiation of C3H10T 1/2 cells into brown adipocytes through regulating Cux1 via miR-1258-5p.

Melatonin Regulates Differentiation of Sheep Brown Adipocyte Precursor Cells Via AMP-Activated Protein Kinase.

In sheep industry, hypothermia caused by insufficient brown adipose tissue (BAT) deposits is one of the major causes of lamb deaths. Enhancing the formation and function of BAT in neonatal lamb increases thermogenesis and hence reduces economic losses. The aim of the present study was to explore the effect and mechanism of melatonin on sheep brown adipocyte formation and function. Sheep brown adipocyte precursor cells (SBACs) isolated from perirenal BAT were treated with melatonin (1 and 10 nM). The SBACs subjected to melatonin exhibited a decreased proliferation ability, accompanied by down-regulated proliferating cell nuclear antigen, cyclin D1, and CDK4 protein contents in a melatonin dose-dependent manner. Melatonin promoted brown adipocyte formation and induced the expression of brown adipogenic markers, including uncoupling protein 1 and PR domain-containing 16 during differentiation of SBAC. Moreover, the AMP-activated protein kinase α1 (AMPKα1) activity was positively correlated with brown adipocyte formation potential. Importantly, melatonin effectively activated AMPKα1. Furthermore, promotional effects of melatonin were abolished by AMPKα1 knockout, suggesting the involvement of AMPKα1 in this process. Collectively, these results suggested that melatonin enhanced brown adipocyte formation in SBACs in vitro through activation of AMPKα1.

Effects of dietary sea buckthorn pomace supplementation on skeletal muscle mass and meat quality in lambs.

The aim of the present trial was to evaluate the effects of dietary sea buckthorn pomace (SBP) supplementation on muscle mass, meat nutritional value and quality of lambs. The results showed that dietary 16% SBP supplementation increased muscle mass and altered muscle fiber size distribution. Both nutritional compositions, including crude protein, moisture and ash, and lamb meat quality, including pH, color and cooking loss were not affected by SBP supplementation. Importantly, crude fat content was elevated, and shear force was decreased in Longissimus thoracis (LT) when lambs were fed the SBP containing diet. Moreover, the total antioxidative capacity in LT and the HDL content in serum were elevated in SBP feed lambs. Dietary SBP supplementation increased the Akt/mTOR signaling activity, and downregulated myostatin expression. Taken together, these data suggested that SBP could be used as a feed ingredient for lamb meat production by increasing muscle mass and improving tenderness, water holding capacity and antioxidative capacity of resulting meat.

Dietary Sea Buckthorn Pomace Induces Beige Adipocyte Formation in Inguinal White Adipose Tissue in Lambs.

The sea buckthorn contains substantial amounts of bioactive compounds. The objective of this study was to investigate the effects of dietary sea buckthorn pomace (SBP) on sheep beige adipocyte formation. A total of thirty lambs were equally divided into three groups and fed with diets containing different levels of SBP: 0% SBP (Control), 7.8% SBP (7.8SBP), and 16.0% SBP (16SBP). The results showed that dietary SBP affected inguinal adipocytes' size distribution, and increased both UCP1 protein content (p < 0.05) and mitochondrial numbers (p < 0.05). mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A were increased when animals were subjected to 16% SBP (p < 0.05). Supplementation with 16% SBP increased CCAAT/enhancer-binding protein ß content (p < 0.05) and PR domain containing 16 mRNA abundance (p < 0.05). Consistently, inguinal white adipose tissue (iWAT) from the 16SBP group exhibited increased insulin sensitivity, which was associated with elevated glucose transporter 4 abundance (p < 0.05). Importantly, AMP-activated protein kinase (AMPK) was activated in the 16SBP group (p < 0.05). Collectively, these results suggest that dietary SBP promotes iWAT browning in lambs, which might be through the activation of the AMPK-PGC-1α-UCP1 signaling pathway.