ZBTB16 Overexpression Enhances White Adipogenesis and Induces Brown-Like Adipocyte Formation of Bovine White Intramuscular Preadipocytes.

BACKGROUND/AIMS: Our study aims to characterize functions of ZBTB16 gene in the process of intramuscular fat (IMF) deposition and metabolism of bovine, thereby providing insights into mechanisms for the use of ZBTB16 in fat management. METHODS: Primary preadipocytes derived from bovine IMF tissue were isolated and used as the in vitro cell model. An adenovirus Ad-ZBTB16 was transfected into bovine preadipocytes to overexpress the ZBTB16 gene. By using real-time quantitative PCR (RT-qPCR), western blotting, Oil Red-O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity assay, and cell counting kit-8 (CCK-8) test, adipogenic and proliferative signals in adipocytes were monitored to investigate effects of ZBTB16 on adipogenesis of bovine preadipocytes. RESULTS: After transfection, mRNA and protein levels of ZBTB16 gene were significantly increased. Enhanced ZBTB16 significantly promoted preadipocyte differentiation, as evidenced by accelerated lipid accumulation, enhanced GPDH activity, consistently increased mRNA expressions of adipogenic key transcription factors PPARγ, C/EBPα, FABP4, and ADIPOQ, and markedly increased protein expressions of PPARγ and FABP4. No difference was observed concerning proliferation of preadipocytes after treatment with Ad-ZBTB16. Furthermore, relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Cox8b, and PGC-1α) and beige adipocyte selective genes (CD137, TMEM26, and Tbx1) as well as UCP1 protein expression were significantly increased by Ad-ZBTB16. Meanwhile, Ad-ZBTB16 treatment remarkably induced mitochondrial biogenesis and increased relative mitochondrial DNA (mtDNA) copy number in bovine adipocytes. CONCLUSION: These results suggest that ZBTB16 overexpression can promote white adipogenesis and induce brown-like adipocyte formation for bovine white intramuscular preadipocytes.

Development of a functional beige fat cell line uncovers independent subclasses of cells expressing UCP1 and the futile creatine cycle.

Although uncoupling protein 1 (UCP1) is established as a major contributor to adipose thermogenesis, recent data have illustrated an important role for alternative pathways, particularly the futile creatine cycle (FCC). How these pathways co-exist in cells and tissues has not been explored. Beige cell adipogenesis occurs in vivo but has been difficult to model in vitro; here, we describe the development of a murine beige cell line that executes a robust respiratory response, including uncoupled respiration and the FCC. The key FCC enzyme, tissue-nonspecific alkaline phosphatase (TNAP), is localized almost exclusively to mitochondria in these cells. Surprisingly, single-cell cloning from this cell line shows that cells with the highest levels of UCP1 express little TNAP, and cells with the highest expression of TNAP express little UCP1. Immunofluorescence analysis of subcutaneous fat from cold-exposed mice confirms that the highest levels of these critical thermogenic components are expressed in distinct fat cell populations.

FABP3 and brown adipocyte-characteristic mitochondrial fatty acid oxidation enzymes are induced in beige cells in a different pathway from UCP1.

Cold exposure and ß3-adrenergic receptor agonist (CL316,243) treatment induce the production of beige cells, which express brown adipocytes(BA)-specific UCP1 protein, in white adipose tissue (WAT). It remains unclear whether the beige cells, which have different gene expression patterns from BA, express BA-characteristic fatty acid oxidation (FAO) proteins. Here we found that 5 day cold exposure and CL316,243 treatment of WAT, but not CL316,243 treatment of primary adipocytes of C57BL/6J mice, increased mRNA levels of BA-characteristic FAO proteins. These results suggest that BA-characteristic FAO proteins are induced in beige cells in a different pathway from UCP1.

Hyperoside prevents high-fat diet-induced obesity by increasing white fat browning and lipophagy via CDK6-TFEB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. (genus Hypericum, family Hypericaceae) is a flowering plant native to Europe, North Africa and Asia, which can be used in the treatment of psychiatric disorder, cardiothoracic depression and diabetes. Crataegus pinnatifida Bunge (genus Crataegus pinnatifida Bunge, family Rosaceae) was another traditional Chinese medicine for treating hyperlipidemia. Hyperoside (Hype), a major flavonoid glycoside component of Hypericum perforatum L. and Crataegus pinnatifida Bunge, possesses multiple physiological activities, such as anti-inflammatory and antioxidant effects. However, the role of Hype on obesity and related metabolic diseases still needs to be further investigated. AIM OF THE STUDY: We explored the effect of Hype on high-fat diet (HFD)-induced obesity and its metabolic regulation on white fat tissues. MATERIALS AND METHODS: In vivo four-week-old male C57BL/6J mice were randomly assigned to vehicle (0.5% methycellulose) and Hype (80 mg/kg/day by gavage) group under a normal chow diet (NCD) or HFD for 8 weeks. In vitro, 3T3-L1 preadipocyte cell line and primary stromal vascular fraction (SVF) cells from inguinal white adipose tissue (iWAT) of mice were used to investigate the molecular mechanisms of Hype regulation on adipocyte energy metabolism. RESULTS: Hype treatment in vivo promotes UCP1-dependent white to beige fat transition, increases glucose and lipid metabolism, and resists HFD-induced obesity. Meanwhile, Hype induces lipophagy, a specific autophagy that facilitates the breakdown of lipid droplets, and blocking autophagy partially reduces UCP1 expression. Mechanistically, Hype inhibited CDK6, leading to the increased nuclear translocation of TFEB, while overexpression of CDK6 partially reversed the enhancement of UCP1 by Hype. CONCLUSIONS: Hype protects mice from HFD-induced obesity by increasing energy expenditure of white fat tissue via CDK6-TFEB pathway.

Acteoside improves adipocyte browning by CDK6-mediated mTORC1-TFEB pathway.

Adipocyte browning increases energy expenditure by thermogenesis, which has been considered a potential strategy against obesity and its related metabolic diseases. Phytochemicals derived from natural products with the ability to improve adipocyte thermogenesis have aroused extensive attention. Acteoside (Act), a phenylethanoid glycoside, exists in various medicinal or edible plants and has been shown to regulate metabolic disorders. Here, the browning effect of Act was evaluated by stimulating beige cell differentiation from the stromal vascular fraction (SVF) in the inguinal white adipose tissue (iWAT) and 3T3-L1 preadipocytes, and by converting the iWAT-SVF derived mature white adipocytes. Act improves adipocyte browning by differentiation of the stem/progenitors into beige cells and by direct conversion of mature white adipocytes into beige cells. Mechanistically, Act inhibited CDK6 and mTOR, and consequently relieved phosphorylation of the transcription factor EB (TFEB) and increased its nuclear retention, leading to induction of PGC-1α, a driver of mitochondrial biogenesis, and UCP1-dependent browning. These data thus unveil a CDK6-mTORC1-TFEB pathway that regulates Act-induced adipocyte browning.

Green Tea Epigallocatechin Gallate Inhibits Preadipocyte Growth via the microRNA-let-7a/HMGA2 Signaling Pathway.

SCOPE: This study investigates the effect of epigallocatechin gallate (EGCG) on white and beige preadipocyte growth and explores the involvement of the miR-let-7a/HMGA2 pathway. METHODS AND RESULTS: 3T3-L1 and D12 cells are treated with EGCG. The effect of EGCG on cell proliferation and viability is evaluated, as well as microRNA (miRNA)-related signaling pathways. EGCG inhibits 3T3-L1 and D12 preadipocyte growth, upregulates miR-let-7a expression, and downregulates high-mobility group AT-hook 2 (HMGA2) mRNA and protein levels in a time- and dose-dependent manner. In addition, overexpression of miR-let-7a significantly inhibits the growth of 3T3-L1 and D12 cells and decreases HMGA2 mRNA and protein levels. MiR-let-7a inhibitor antagonizes the inhibitory effects of EGCG on the number and viability of 3T3-L1 and D12 cells. Furthermore, miR-let-7a inhibitor reverses the EGCG-induced increase in miR-let-7a expression levels and decrease in HMGA2 mRNA and protein levels. HMGA2 overexpression induces an increase in cell number and viability and antagonizes EGCG-suppressed cell growth and HMGA2 expression in 3T3-L1 and D12 preadipocytes. CONCLUSION: EGCG inhibits the growth of 3T3-L1 and D12 preadipocytes by modulating the miR-let-7a and HMGA2 pathways.

Perirenal Adipose Tissue from Healthy Donor: Characteristics and Promise as Potential Therapeutic Cell Source.

Perirenal adipose tissue, one of the fat masses surrounding the kidneys, can be obtained from healthy donors during a kidney transplant. Perirenal adipose tissue has only ever been known as a connective tissue to protect the kidneys and renal blood vessels from external physical stimulation. Yet, recently, as adipose tissue has begun to be considered an endocrine organ, and perirenal adipose tissue is now regarded to have a direct effect on metabolic diseases. The characteristics of perirenal adipose tissue from a healthy donor are that: (1) There are a large number of brown adipose cells (70-80% of the total), (2) Most of the brown adipose cells are inactive in the resting cell cycle, (3) Activating factors are constant low-temperature exposure, hormones, metastasis factors, and environmental factors, (4) Anatomically, a large number of brown adipose cells are distributed close to the adrenal glands, (5) Beige cells, produced by converting white adipocytes to brown-like adipocytes, are highly active, (6) Activated cells secrete BATokines, and (7) Energy consumption efficiency is high. Despite these advantages, all of the perirenal adipose tissue from a healthy donor is incinerated as medical waste. With a view to its use, this review discusses the brown adipocytes and beige cells in perirenal adipose tissue from a healthy donor, and proposes opportunities for their clinical application.

Panax notoginseng saponins modulate the gut microbiota to promote thermogenesis and beige adipocyte reconstruction via leptin-mediated AMPKα/STAT3 signaling in diet-induced obesity.

Background: Activation of the thermogenic program in white and brown adipocytes presents a promising avenue for increasing energy expenditure during the treatment of obesity. The endogenous mechanism for promoting thermogenesis in brown adipocytes or browning in white adipocytes has indicated that the gut microbiota is a crucial regulator of the host energy balance. However, whether the effects of the therapeutic intervention-induced modulation of the gut microbiota on adipocyte browning involved the regulation of leptin remains unclear. Method: The adipose features were analyzed by body composition analysis, infrared camera observations, transmission electron microscopy and H&E staining. The gene and protein expression in adipose tissue were detected by qRT-PCR, immunoblotting, immunohistochemistry and immunofluorescence staining. The gut microbiome signature was identified by 16S rRNA gene amplicon sequencing, and both mice with high-fat diet-induced obesity (DIO) and mice with antibiotics-induced microbiome depletion were subjected to fecal microbiota transplantation. Results: Treatment with Panax notoginseng saponins (PNS) shaped the murine gut microbiome by increasing the abundances of Akkermansia muciniphila and Parabacteroides distasonis, and as a result, DIO mice harbored a distal gut microbiota with a significantly increased capacity to reduce host adiposity. The PNS-induced modulation of the gut microbiota in DIO mice could increase brown adipose tissue (BAT) thermogenesis and beige adipocyte reconstruction by activating the leptin-AMPK/STAT3 signaling pathway, which results in the promotion of energy expenditure. Leptin has an essential influence on the anti-obesity effects of PNS. In cases of leptin deficiency, the PNS-induced modulation of the gut microbiota exerts negative effects on thermogenesis and browning in white adipose tissue (WAT), which indicates that PNS fail to reduce obesity in leptin gene-deficient mice. The PNS-induced modulation of the gut microbiota exerted a minimal effect on DIO mice with antibiotic-induced microbiome depletion, which confirmed the correlation between altered gut microbiota and the remodeling of adipose tissues in DIO mice. The direct influence of leptin on browning via the AMPKα/STAT3 signaling pathway in C3H101/2 cells supported our in vivo results that signalling through the leptin-AMPK/STAT3 pathway induced by the PNS-modulated gut microbiota was involved in beige adipocyte reconstruction. Conclusion: Our results revealed that leptin signaling is critical for alterations in microbiota-fat crosstalk and provide promising avenues for therapeutic intervention in the treatment of obesity.

Resveratrol liposomes and lipid nanocarriers: Comparison of characteristics and inducing browning of white adipocytes.

Trans-resveratrol (R) has a potential to increase energy expenditure via inducing browning in white adipose tissue. However, its low levels of aqueous solubility, stability, and poor bioavailability limit its application. We have successfully synthesized biocompatible, and biodegradable R encapsulated lipid nanocarriers (R-nano), and R encapsulated liposomes (R-lipo). The mean particle size of R-nano and R-lipo were 140 nm and 110 nm, respectively, and their polydispersity index values were less than 0.2. Nanoencapsulation significantly increased aqueous solubility and enhanced chemical stability of R, especially at 37 °C. R-lipo had higher physical and chemical stability than R-nano while R-nano had more prolonged release than R-lipo. Both R-nano and R-lipo increased cellular R content in 3T3-L1 cells. Both R-nano and R-lipo dose-dependently induced uncoupling protein 1 (UCP1) mRNA expression and decreased white specific marker insulin growth factor binding protein 3 expression under isoproterenol (ISO)-stimulated conditions. At the low dose (5 µM), nanoencapsulated compared to native R enhanced UCP1 and beige marker CD137 expression under ISO-stimulated conditions. Compared to R-nano, R-lipo had better biological activity, possibly due to its higher physical and chemical stability at the room and body temperature. Taken together, our study demonstrates that nanoencapsulation increased R's aqueous solubility and stability, which led to enhanced browning of white adipocytes. Even though both R-lipo and R-nano increased R's browning activities, their differential characteristics need to be considered in obesity treatment.

Effects of growth hormone on uncoupling protein 1 in white adipose tissues in obese mice.

OBJECTIVE: The transition of white adipocytes to beige cells (a phenomenon referred to as browning or beigeing) during obesity has been previously reported. Our study aimed to examine the mechanisms through which obesity induced by a high fat diet (HFD) affects uncoupling protein 1 (UCP1) expression via signal transduction and activator of transcription 5 (STAT5s). DESIGN: Seven-week-old male C57BL/6J mice were fed a normal or HFD for 11weeks. Body weight, white adipose tissue weight, and blood lipid and glucose levels were measured. To unveil the molecular mechanisms of UCP1 expression in adipose tissue, we performed further studying 3T3-L1 cells using qRT-PCR. We also measured UCP1 promoter activity in the TSA201 cell line using a dual luciferase assay. In addition, we analyzed the predicted consensus sequences for STAT5 binding in the UCP1 promoter region. RESULTS: Mice fed an HFD had higher body weight and intra-abdominal adipose tissues weight and a higher expression of UCP1, GH receptor (GHR), STATs, suppressors of cytokine signaling (SOCSs), and cytokine-inducible SH2-containing protein (CISH) compared to control mice. In 3T3-L1 cell studies, GH induced phosphorylation of the STAT5, SOCSs, CISH and UCP1 expressions. UCP1 promoter activity was associated with constitutively active STAT5 in a dose-dependent manner. We confirmed functional STAT5 binding sites at -425, -279, and -178bp of the UCP1 promoter. CONCLUSION: We suggest that endogenous GH induces UCP1 expression in adipose tissue via STAT5.

Using Thermogenic Beige Cells to Identify Biologically Active Small Molecules and Peptides.

Incorporating molecular libraries in chemical biology screenings in cultured cells has been successfully used for gene discovery in many cellular processes. It has the unique potential to uncover novel mechanisms of complex cellular biology through the screening of small molecules and protein biologics in relevant cell-based assays. Recent development in the understanding and generation of thermogenic adipocytes provides opportunities for potential anti-obesity therapeutics discovery. In this chapter, we describe screening methods using thermogenic beige cells to identify novel compounds and peptides that activate adipocyte thermogenesis.

18F-FDG PET/CT monitoring of ß3 agonist-stimulated brown adipocyte recruitment in white adipose tissue.

UNLABELLED: There is rising interest in recruitment of brown adipocytes into white adipose tissue (WAT) as a means to augment energy expenditure for weight reduction. We thus investigated the potential of (18)F-FDG uptake as an imaging biomarker that can monitor the process of WAT browning. METHODS: C57BL/6 mice were treated daily with the ß3 agonist CL316,243 (5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium salt), whereas controls received saline. (18)F-FDG small-animal PET/CT was serially performed at 1 h after CL316,243 injection. After sacrifice, interscapular brown adipose tissue (BAT) and WAT depots were extracted, weighed, and measured for (18)F-FDG uptake. Tissues underwent immunostaining, and UCP1 content was quantified by Western blotting. RESULTS: PET/CT showed low (18)F-FDG uptake in both BAT and inguinal WAT at baseline. BAT uptake was substantially increased by a single stimulation with CL316,243. Uptake in inguinal WAT was only modestly elevated by the first stimulation uptake but gradually increased to BAT level by prolonged stimulation. Ex vivo measurements recapitulated the PET findings, and measured (18)F-FDG uptake in other WAT depots was similar to inguinal WAT. WAT browning by prolonged stimulation was confirmed by a substantial increase in uncoupling protein 1 (UCP1), cytochrome-c oxidase 4 (COX4), and PR domain containing 16 (PRDM16) staining as markers of brown adipocytes. UCP1 content, which served as a measure for extent of browning, was low in baseline inguinal WAT but linearly increased over 10 d of CL316,243 injection. Finally, image-based and ex vivo-measured (18)F-FDG uptake in inguinal WAT correlated well with UCP1 content. CONCLUSION: (18)F-FDG PET/CT has the capacity to monitor brown adipocyte recruitment into WAT depots in vivo and may thus be useful for screening the efficacy of strategies to promote WAT browning.