Jinlida Granules Reduce Obesity in db/db Mice by Activating Beige Adipocytes.

Recent studies indicate existence of beige adipocytes in adults. Upon activation, beige adipocytes burn energy for thermogenesis and contribute to regulation of energy balance. In this study, we have analyzed whether Jinlida granules (JLD) could activate beige adipocytes. JLD suspended in 0.5% carboxymethyl cellulose (CMC) was gavage fed to db/db mice at a daily dose of 3.8 g/kg. After 10 weeks, body weight, biochemical, and histological analyses were performed. In situ hybridization, immunofluorescence, and western blotting were conducted to test beige adipocyte activation in mice. X9 cells were induced with induction medium and maintenance medium containing 400 µg/mL of JLD. After completion of induction, cells were analyzed by Nile red staining, time polymerase chain reaction (PCR), western blotting, and immunofluorescence to understand the effect of JLD on the activation of beige adipocytes. A molecular docking method was used to preliminarily identify compounds in JLD, which hold the potential activation effect on uncoupling protein 1 (UCP1). JLD treatment significantly improved obesity in db/db mice. Biochemical results showed that JLD reduced blood glucose (GLU), triglyceride (TG), and low-density lipoprotein cholesterol (LDL) levels as well as liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice. Hematoxylin and eosin staining (H&E) showed that JLD reduced hepatocyte ballooning changes in the liver. Immunofluorescence showed that JLD increased the expression of the thermogenic protein, UCP1, in the beige adipose tissue of mice. JLD also increased the expression of UCP1 and inhibited the expression of miR-27a in X9 cells. Molecular docking results showed that epmedin B, epmedin C, icariin, puerarin, and salvianolic acid B had potential activation effects on UCP1. The results suggest that JLD may activate beige adipocytes by inhibiting miR-27a expression, thereby promoting thermogenesis in beige adipocytes. This study provides a new pharmacological basis for the clinical use of JLD.

The natural compound rutaecarpine promotes white adipocyte browning through activation of the AMPK-PRDM16 axis.

The prevalence of obesity is increasing globally and is associated with many metabolic disorders, such as type 2 diabetes and cardiovascular diseases. In recent years, a number of studies suggest that promotion of white adipose browning represents a promising strategy to combat obesity and its related metabolic disorders. The aim of this study was to identify compounds that induce adipocyte browning and elucidate their mechanism of action. Among the 500 natural compounds screened, a small molecule named Rutaecarpine, was identified as a positive regulator of adipocyte browning both in vitro and in vivo. KEGG pathway analysis from RNA-seq data suggested that the AMPK signaling pathway was regulated by Rutaecarpine, which was validated by Western blot analysis. Furthermore, inhibition of AMPK signaling mitigated the browning effect of Rutaecaripine. The effect of Rutaecaripine on adipocyte browning was also abolished upon deletion of Prdm16, a downstream target of AMPK pathway. In collusion, Rutaecarpine is a potent chemical agent to induce adipocyte browning and may serve as a potential drug candidate to treat obesity.

The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes.

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect ß-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

Targeting Glutamine Synthesis Inhibits Stem Cell Adipogenesis in Vitro.

BACKGROUND/AIMS: Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. METHODS: Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. CONCLUSION: These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.

Vitamin D ameliorates adipose browning in chronic kidney disease cachexia.

Patients with chronic kidney disease (CKD) are often 25(OH)D3 and 1,25(OH)2D3 insufficient. We studied whether vitamin D repletion could correct aberrant adipose tissue and muscle metabolism in a mouse model of CKD-associated cachexia. Intraperitoneal administration of 25(OH)D3 and 1,25(OH)2D3 (75 µg/kg/day and 60 ng/kg/day respectively for 6 weeks) normalized serum concentrations of 25(OH)D3 and 1,25(OH)2D3 in CKD mice. Vitamin D repletion stimulated appetite, normalized weight gain, and improved fat and lean mass content in CKD mice. Vitamin D supplementation attenuated expression of key molecules involved in adipose tissue browning and ameliorated expression of thermogenic genes in adipose tissue and skeletal muscle in CKD mice. Furthermore, repletion of vitamin D improved skeletal muscle fiber size and in vivo muscle function, normalized muscle collagen content and attenuated muscle fat infiltration as well as pathogenetic molecular pathways related to muscle mass regulation in CKD mice. RNAseq analysis was performed on the gastrocnemius muscle. Ingenuity Pathway Analysis revealed that the top 12 differentially expressed genes in CKD were correlated with impaired muscle and neuron regeneration, enhanced muscle thermogenesis and fibrosis. Importantly, vitamin D repletion normalized the expression of those 12 genes in CKD mice. Vitamin D repletion may be an effective therapeutic strategy for adipose tissue browning and muscle wasting in CKD patients.

Human beige adipocytes for drug discovery and cell therapy in metabolic diseases.

Human beige adipocytes (BAs) have potential utility for the development of therapeutics to treat diabetes and obesity-associated diseases. Although several reports have described the generation of beige adipocytes in vitro, their potential utility in cell therapy and drug discovery has not been reported. Here, we describe the generation of BAs from human adipose-derived stem/stromal cells (ADSCs) in serum-free medium with efficiencies >90%. Molecular profiling of beige adipocytes shows them to be similar to primary BAs isolated from human tissue. In vitro, beige adipocytes exhibit uncoupled mitochondrial respiration and cAMP-induced lipolytic activity. Following transplantation, BAs increase whole-body energy expenditure and oxygen consumption, while reducing body-weight in recipient mice. Finally, we show the therapeutic utility of BAs in a platform for high-throughput drug screening (HTS). These findings demonstrate the potential utility of BAs as a cell therapeutic and as a tool for the identification of drugs to treat metabolic diseases.

Adipogenic commitment induced by green tea polyphenols remodel adipocytes to a thermogenic phenotype.

The potential contribution of green tea (GT) to the development of thermogenic/beige cells have been scarcely investigated. Here we investigated if the beneficial effects of GT in the induction of thermogenic/beige adipocytes results from an initial cell commitment during adipogenesis. Male C57Bl/6 mice (3 months) were divided into 3 groups: Control (chow diet), Obese (cafeteria diet), and Obese + GT. Mice received GT gavage (500 mg/kg of BW) over 12 weeks (5 days/week), after 4 weeks of diet, totalizing 16 weeks of experimentation. GT treatment increased energy expenditure (EE) in mice fed with cafeteria-diet leading to reduced BW gain, decreased adiposity, reduced inflammation, and improving insulin sensitivity. Those phenotypes were associated with enhanced expression of oxidative, thermogenic and beige genes. GT induced a futile cycle through de novo lipogenesis activating the thermogenic pathway. Induction of beige phenotype occurs autonomously in adipocytes and involves the PPARγ/FGF21/AMPK/UCP1 pathway. Our study identified that metabolic changes caused by GT may involve the temporal expression of PPARγ promoting the induction of thermogenic cells by reprogramming initial steps of adipocyte commitment.

Dietary apple polyphenols promote fat browning in high-fat diet-induced obese mice through activation of adenosine monophosphate-activated protein kinase α.

BACKGROUND: Promoting brown and beige adipogenesis contributes to adaptive thermogenesis, which provides a defense against obesity and related disorders. Apple polyphenols (APs) play a significant role in treating variety of metabolic diseases. This study was conducted to determine the effects of APs on the development of brown and beige adipocytes and thermogenesis and investigate whether these effects are mediated by adenosine monophosphate-activated protein kinase (AMPK). High-fat diet (HFD)-induced obese mice and differentiated 3T3-L1 adipocytes were subjected to APs treatment. The thermogenic program and associated regulatory factors, and the involvement of AMPKα was assessed. RESULTS: Dietary APs supplementation reduced adiposity and improved insulin sensitivity in HFD-induced obese mice. Moreover, APs increased the oxygen consumption and heat production and decreased respiratory exchange ratio, which were accompanied by the upregulation of thermogenic genes expression and the activation of AMPKα in brown fat and inguinal white fat. Further, APs treatment directly increased expression of brown adipogenic markers and induced phosphorylation of AMPKα in differentiated 3T3-L1 adipocytes, whereas the beneficial effects of APs were reversed by AMPK inhibition. CONCLUSION: Our results provide new insights into the function of APs in regulating brown/beige adipogenesis and adaptive thermogenesis and suggest the potential application of APs in the prevention and therapeutics of obesity and associated metabolic diseases. © 2020 Society of Chemical Industry.

Scd1 controls de novo beige fat biogenesis through succinate-dependent regulation of mitochondrial complex II.

Preadipocytes can give rise to either white adipocytes or beige adipocytes. Owing to their distinct abilities in nutrient storage and energy expenditure, strategies that specifically promote "beiging" of adipocytes hold great promise for counterbalancing obesity and metabolic diseases. Yet, factors dictating the differentiation fate of adipocyte progenitors remain to be elucidated. We found that stearoyl-coenzyme A desaturase 1 (Scd1)-deficient mice, which resist metabolic stress, possess augmentation in beige adipocytes under basal conditions. Deletion of Scd1 in mature adipocytes expressing Fabp4 or Ucp1 did not affect thermogenesis in mice. Rather, Scd1 deficiency shifted the differentiation fate of preadipocytes from white adipogenesis to beige adipogenesis. Such effects are dependent on succinate accumulation in adipocyte progenitors, which fuels mitochondrial complex II activity. Suppression of mitochondrial complex II by Atpenin A5 or oxaloacetic acid reverted the differentiation potential of Scd1-deficient preadipocytes to white adipocytes. Furthermore, supplementation of succinate was found to increase beige adipocyte differentiation both in vitro and in vivo. Our data reveal an unappreciated role of Scd1 in determining the cell fate of adipocyte progenitors through succinate-dependent regulation of mitochondrial complex II.

Black Raspberry (Rubus coreanus Miquel) Promotes Browning of Preadipocytes and Inguinal White Adipose Tissue in Cold-Induced Mice.

The alteration of white adipose tissue (WAT) "browning", a change of white into beige fat, has been considered as a new therapeutic strategy to treat obesity. In this study, we investigated the browning effect of black raspberry (Rubus coreanus Miquel) using in vitro and in vivo models. Black raspberry water extract (BRWE) treatment inhibited lipid accumulation in human mesenchymal stem cells (hMSCs) and zebrafish. To evaluate the thermogenic activity, BRWE was orally administered for 2 weeks, and then, the mice were placed in a 4 °C environment. As a result, BRWE treatment increased rectal temperature and inguinal WAT (iWAT) thermogenesis by inducing the expression of beige fat specific markers such as PR domain zinc-finger protein 16 (PRDM16), uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), and t-box protein 1 (TBX1) in cold-exposed mice. Furthermore, ellagic acid (EA), a constituent of BRWE, markedly promoted beige specific markers: UCP1, PGC1α, TBX1, and nuclear respiratory factor 1 in beige differentiation media (DM)-induced 3T3-L1 adipocytes. Our findings indicate that BRWE can promote beige differentiation/activation, and EA is the active compound responsible for such effect. Thus, we suggest the nature-derived agents BRWE and EA as potential agents for obesity treatment.

Immature Citrus reticulata Extract Promotes Browning of Beige Adipocytes in High-Fat Diet-Induced C57BL/6 Mice.

Obesity has become a global public health issue. Promoting browning of white adipose tissue (WAT) helps to maintain energy homeostasis. Previous studies have found that citrus fruit exhibits a number of biological activities. Although most citrus fruit drop has been considered agricultural waste, the ability to use it may be desirable. In this study, we investigate the antiobesity effects of immature citrus fruits in high-fat diet (HFD)-fed mice. The main phytochemical components of immature Citrus reticulata in water extraction analyzed by HPLC are synephrine, narirutin, hesperidin, nobiletin, and tangeretin (16.0 ± 1.08, 4.52 ± 0.31, 9.14 ± 0.32, 2.54 ± 0.07, 1.67 ± 0.05 mg/g, respectively). Oral administration of 1% immature Citrus reticulata extract (ICRE) for 11 weeks markedly reduced body weight gain, epididymal fat weight, fasting blood glucose, serum triglyceride, and total cholesterol ( P < 0.05 for all). In addition, histological analysis revealed that dietary ICRE decreased adipocyte size and hepatic steatosis compared to the HFD group ( P < 0.05 for both). Furthermore, we found that mice treated with ICRE have improved cold tolerance during acute cold challenge. These effects were associated with increased expression of uncoupling protein 1 (UCP1) and thermogenic genes in inguinal WAT. Taken together, these results suggest that ICRE can prevent obesity and lipid accumulation through induction of brown-like adipocyte formation.

Phytol stimulates the browning of white adipocytes through the activation of AMP-activated protein kinase (AMPK) α in mice fed high-fat diet.

Stimulating the browning of white adipocytes contributes to the restriction of obesity and related metabolic disorders. This study aimed to investigate the browning effects of phytol on mice inguinal subcutaneous white adipose tissue (iWAT) and explore the underlying mechanisms. Our results demonstrated that phytol administration decreased body weight gain and iWAT index, and stimulated the browning of mice iWAT, with the increased expression of brown adipocyte marker genes (UCP1, PRDM16, PGC1α, PDH, and Cyto C). In addition, phytol treatment activated the AMPKα signaling pathway in mice iWAT. In good agreement with the in vivo findings, the in vitro results showed that 100 µM phytol stimulated brown adipogenic differentiation and formation of brown-like adipocytes in the differentiated 3T3-L1 by increasing the mitochondria content and oxygen consumption, and promoting mRNA and/or protein expression of brown adipocyte markers (UCP1, PRDM16, PGC1α, PDH, Cyto C, Cidea and Elovl3) and beige adipocyte markers (CD137 and TMEM26). Meanwhile, phytol activated the AMPKα signaling pathway in the differentiated 3T3-L1. However, the inhibition of AMPKα with Compound C totally abolished phytol-stimulated brown adipogenic differentiation and formation of brown-like adipocytes. In conclusion, these results showed that phytol stimulated the browning of mice iWAT, which was coincident with the increased formation of brown-like adipocytes in the differentiated 3T3-L1, and appeared to be primarily mediated by the AMPKα signaling pathway. These data provided new insight into the role of phytol in regulating the browning of WAT and suggested the potential application of phytol as a nutritional intervention for the restriction of obesity and related metabolic disorders.

Moderate alcohol intake induces thermogenic brown/beige adipocyte formation via elevating retinoic acid signaling.

Clinically, low and moderate alcohol intake improves human health with protection against metabolic syndromes, including type 2 diabetes; however, mechanisms that are associated with these effects remain to be elucidated. The aims of this study were to investigate the effects of moderate alcohol intake on thermogenic brown/beige adipocyte formation and glucose and lipid homeostasis, as well as the involvement of retinoic acid (RA) signaling in the entire process. C57BL6 male mice were supplemented with 8% (w/v) alcohol in water for 1 or 4 mo. Alcohol intake prevented body weight gain, induced the formation of uncoupling protein 1-positive beige adipocytes in white adipose tissue, and increased thermogenesis in mice, which is associated with decreased serum glucose and triacylglycerol levels. Mechanistically, alcohol intake increased RA levels in serum and adipose tissue, which was associated with increased expression of aldehyde dehydrogenase family 1 subfamily A1 (Aldh1a1). When RA receptor-α signaling was conditionally blocked in platelet-derived growth factor receptor-α-positive adipose progenitors, the effects of alcohol on beige adipogenesis were largely abolished. Finally, moderate alcohol prevented high-fat diet-induced obesity and metabolic dysfunction. In conclusion, moderate alcohol intake induces thermogenic brown/beige adipocyte formation and promotes glucose and lipid oxidation via elevation of RA signaling.-Wang, B., Wang, Z., de Avila, J. M., Zhu, M.-J., Zhang, F., Gomez, N. A., Zhao, L., Tian, Q., Zhao, J., Maricelli, J., Zhang, H., Rodgers, B. D., Du, M. Moderate alcohol intake induces thermogenic brown/beige adipocyte formation via elevating retinoic acid signaling.

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.