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We constructed a recombinant oral GLP-1 analogue in Lactococcus lactis (L. lactis) and evaluated its physiological functions.In silico docking suggested the alanine at position 8 substituted with serine (A8SGLP-1) reduced binding of DPP4, which translated to reduced cleavage by DPP4 with minimal changes in stability. This was further confirmed by an in vitro enzymatic assay which showed that A8SGLP-1 significantly increased half-life upon DPP4 treatment. In addition, recombinant L. lactis (LL-A8SGLP-1) demonstrated reduced fat mass with no changes in body weight, significant improvement of random glycemic control and reduced systemic inflammation compared with WT GLP-1 in db/db mice.LL-A8SGLP-1 adopted in live biotherapeutic products reduce blood glucose in db/db mice without affecting its function.

Cold-activated thermogenesis of brown adipose tissues (BAT) is vital for the survival of animals under cold stress and also inhibits the development of tumours. The development of small-molecule tools that target thermogenesis pathways could lead to novel therapies against cold, obesity, and even cancer. Here, we identify a chemical signal that is produced in beetles in the winter to activate fat thermogenesis. This hormone elevates the basal body temperature by increasing cellular mitochondrial density and uncoupling in order to promote beetle survival. We demonstrate that this hormone activates UCP4-mediated uncoupled respiration through adipokinetic hormone receptor (AKHR). This signal serves as a novel fat-burning activator that utilizes a conserved mechanism to promote thermogenesis not only in beetles, nematode and flies, but also in mice, protecting the mice against cold and tumor growth. This hormone represents a new strategy to manipulate fat thermogenesis.

Liver fibrosis is caused by the accumulation of extracellular matrix proteins on the surface of hepatocytes and results from chronic liver injury. TGFβ1 is one of the most important promoters of hepatic fibrosis, which accelerates the transformation of hepatic stellate cells to myofibroblasts and collagen expression. It is well-known that TGFβ1 binds to TGFβR2 to mediate its downstream signal cascades to regulate target gene transcription. Therefore, the TGFβR2 blocker might be a prominent drug candidate. We constructed TGFβR2 extracellular domain into living biotherapeutics Lactococcus lactis to reduce hepatic fibrosis in CCl4 treated mice in the present study. We found that the culture supernatant of the recombinant bacteria can inhibit the TGFβ1-induced collagen synthesis in the hepatic stellate cells at the cellular level. In addition, results of in vivo study showed that the recombinant bacteria significantly reduced the degree of liver fibrosis in CCl4-treated mice. Furthermore, flow cytometry results indicated that the recombinant bacteria treatment significantly reduced the CD11b+ Kupffer cells compared with the empty vector bacteria group. Consistently, fibrosis-related gene and protein expression were significantly reduced upon recombinant bacteria treatment. Finally, the subchronic toxicity test results showed that this bacteria strain did not have any significant side effects. In conclusion, our recombinant Lactococcus lactis shows tremendous therapeutic potential in liver fibrosis. KEY POINTS: • The supernatant of L. lactis expressing TGFβR2 inhibits the activation of myofibroblast. • The oral recombinant strain reduced the degree of liver fibrosis and inflammation in mice. • The recombinant strain was safe in subchronic toxicity test in mice.

Brown adipose tissue (BAT) is the primary site of adaptive thermogenesis, which is involved in energy expenditure and has received much attention in the field of obesity treatment. By screening a small-molecule compound library of drugs approved by the Food and Drug Administration, pantothenic acid was identified as being able to significantly upregulate the expression of uncoupling protein 1 (UCP1), a key thermogenic protein found in BAT. Pantothenate (PA) treatment decreased adiposity, reversed hepatic steatosis, and improved glucose homeostasis by increasing energy expenditure in C57BL/6J mice fed a high-fat diet. PA also significantly increased BAT activity and induced beige adipocytes formation. Mechanistically, the beneficial effects were mediated by UCP1 because PA treatment was unable to ameliorate obesity in UCP1 knockout mice. In conclusion, we identified PA as an effective BAT activator that can prevent obesity and may represent a promising strategy for the clinical treatment of obesity and related metabolic diseases.

Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disease affecting women of reproductive age. Due to its complex aetiology, there is no currently effective cure for PCOS. Brown adipose tissue (BAT) activity is significantly decreased in PCOS patients, and BAT activation has beneficial effects in animal models of PCOS. Here, we investigated the effect of ginsenoside compound K (CK) in an animal model of PCOS and its mechanism of BAT activation.Primary brown adipocytes, Db/Db mice and dehydroepiandrosterone (DHEA)-induced PCOS rats were used. The core body temperature, oxygen consumption, energy metabolism related gene and protein expression were assessed to identify the effect of CK on overall energy metabolism. Oestrous cycle, serum sex hormone, ovarian steroidogenic enzyme gene expression and ovarian morphology were also evaluated following CK treatment.Our results indicated that CK treatment could significantly protect against body weight gain in Db/Db mice via BAT activation. Furthermore, we found that CK treatment could normalize hyperandrogenism, oestrous cyclicity, normalize steroidogenic enzyme expression and decrease the number of cystic follicles in PCOS rats. Interestingly, as a potential endocrine intermediate, C-X-C motif chemokine ligand-14 protein (CXCL14) was significantly up-regulated following CK administration. In addition, exogenous CXC14 supplementation was found to reverse DHEA-induced PCOS in a phenotypically similar manner to CK treatment.In summary, CK treatment significantly activates BAT, increases CXCL14 expression and ameliorates PCOS. These findings suggest that CK might be a potential drug candidate for PCOS treatment.

Sperm quality is an important indicator of male fertility, and a suitable biomarker enables the selection of high-quality spermatozoa. We previously found that L-amino acid oxidase encoded by the L-amino acid oxidase 1 (Lao1) gene exerts biological roles in the mammary gland and brain by converting specific L-amino acids into keto acids, ammonia, and hydrogen peroxide (H2O2). Here, we describe the role of Lao1 in male reproduction. Lao1-deficient (Lao1–/–) male mice generated fewer pregnant embryos and pups as well as lower ratios of fertilized oocytes and even ovulated number was not different, suggesting that male subfertility caused the smaller litters. We found that LAO1 expressed in acrosomes is associated with high malformation ratios and low viability of Lao1–/– sperm. Wild-type (WT) sperm produced more H2O2 than Lao1–/– sperm, and 10 µM H2O2 restored knockout (KO) sperm viability in vitro. In addition, the sperm ratio of induced acrosome reaction was higher in WT than in Lao1–/– sperm incubated with the calcium ionophore A23187. Moreover, LAO1 expression was abundant in bovine sperm with high fertilization ratios. We concluded that LAO1 localized in the sperm acrosome influences sperm viability and morphology as well as the acrosome reaction, and that LAO1-deficient sperm might cause male subfertility. Thus, LAO1 might serve as a novel marker for selecting high-quality spermatozoa, especially for livestock reproduction.Summary sentenceLAO1 localized in the sperm acrosome may influence sperm viability and acrosome reaction by metabolizing L-amino acid to produce H2O2.Graphical Abstract

White adipose tissue (WAT) browning may have beneficial effects for treating metabolic syndrome. miRNA are important regulators of the differentiation, development, and function of brown and beige adipocytes. Here, we found that the cold-inducible miRNA17-92 cluster is enriched in brown adipose tissue (BAT) compared with WAT. Overexpression of the miR17-92 cluster in C3H10T1/2 cells, a mouse mesenchymal stem cell line, enhanced the thermogenic capacity of adipocytes. Furthermore, we observed a significant reduction in adiposity in adipose tissue-specific miR17-92 cluster transgenic (TG) mice. This finding is partly explained by dramatic increases in white fat browning and energy expenditure. Interestingly, the miR17-92 cluster stimulated WAT browning without altering BAT activity in mice. In addition, when we removed the intrascapular BAT (iBAT), the TG mice could maintain their body temperature well under cold exposure. At the molecular level, we found that the miR17-92 cluster targets Rb1, a beige cell repressor in WAT. The present study reveals a critical role for the miR17-92 cluster in regulating WAT browning. These results may be helpful for better understanding the function of beige fat, which could compensate for the lack of BAT in humans, and may open new avenues for combatting metabolic syndrome.

Identification of key regulators of energy homeostasis holds important therapeutic promise for metabolic disorders, such as obesity and diabetes. ACE2 cleaves angiotensin II (Ang II) to generate Ang-(1-7) which acts mainly through the Mas1 receptor. Here, we identify ACE2 pathway as a critical regulator in the maintenance of thermogenesis and energy expenditure. We found that ACE2 is highly expressed in brown adipose tissue (BAT) and that cold stimulation increases ACE2 and Ang-(1-7) levels in BAT and serum. Ace2 knockout mice (Ace2-/y) and Mas1 knockout mice (Mas1-/-) displayed impaired thermogenesis. Mice transplanted with brown adipose tissue from Mas1-/- display metabolic abnormalities consistent with those seen in the Ace2 and Mas1 knockout mice. In contrast, impaired thermogenesis of Leprdb/db obese diabetic mice and high-fat diet-induced obese mice were ameliorated by overexpression of Ace2 or continuous infusion of Ang-(1-7). Activation of ACE2 pathway was associated with improvement of metabolic parameters, including blood glucose, lipids, and energy expenditure in multiple animal models. Consistently, ACE2 pathway remarkably enhanced the browning of white adipose tissue. Mechanistically, we showed that ACE2 pathway activated Akt/FoxO1 and PKA pathway, leading to induction of UCP1 and activation of mitochondrial function. Our data propose that adaptive thermogenesis requires regulation of ACE2 pathway and highlight novel potential therapeutic targets for the treatment of metabolic disorders.

RNF20, an E3 ligase critical for monoubiquitination of histone H2B at lysine 120 (H2Bub), has been implicated in the regulation of various cellar processes; however, its physiological roles in adipocytes remain poorly characterized. Here, we report that the adipocyte-specific knockout of Rnf20 (ASKO) in mice led to progressive fat loss, organomegaly and hyperinsulinemia. Despite signs of hyperinsulinemia, normal insulin sensitivity and improved glucose tolerance were observed in the young and aged CD-fed ASKO mice. In addition, high-fat diet-fed ASKO mice developed severe liver steatosis. Moreover, we observed that the ASKO mice were extremely sensitive to a cold environment due to decreased expression levels of brown adipose tissue (BAT) selective genes, including uncoupling protein 1 (Ucp1), and impaired mitochondrial functions. Significantly decreased levels of peroxisome proliferator-activated receptor gamma (Pparγ) were observed in the gonadal white adipose tissues (gWAT) from the ASKO mice, suggesting that Rnf20 regulates adipogenesis, at least in part, through Pparγ. Rosiglitazone-treated ASKO mice exhibited increased fat mass compared to that of the non-treated ASKO mice. Collectively, our results illustrate the critical role of RNF20 in control of white and brown adipose tissue development and physiological function.

The prevention of diseases through health control is essential at zoos. Here, we investigated the gut microbiota formation during infancy in an Asian elephant and compared the composition between infant and mother. Besides, we analyzed the components of breast milk and examined the correlation with the infant gut microbiota. Analysis revealed the gut microbiota of the infant contained high amount of Lactobacillales and its diversity was relatively low compared to that of the mother. We found several milk components, showed a positive correlation with the change of Lactobacillales. The present study revealed the mechanism of gut microbiota formation during infancy in an Asian elephant and provides important insights into the health control of Asian elephants in zoos.

Type 2 diabetes is currently experiencing an outbreak worldwide. GLP-1 effectively lowers blood glucose level as an emerging target for the treatment of type 2 diabetes. However, the application of GLP-1 is limited by short half-life and too expensive cost in clinic. In this study we employed the food-grade probiotics as delivery system to express human GLP-1 and its analogue. Recombinant lactococcus lactis could express GLP-1 and analogue in vitro and modified GLP-1 analogue was more resistant to DPP-4 degradation. Oral administration of GLP-1 analogue could reduce the fat mass. More importantly, GLP-1 analogue improved hyperglycemia and insulin resistance in Db/Db mouse although the insulin secretion is not observed in vitro. Our study demonstrates that lactococcus lactis genetically modified with single amino acid mutation could prolong half-life of GLP-1 and increase insulin sensitivity in Db/Db mouse model as an oral drug delivery system driving the development and innovation of drug therapy for type 2 diabetes.

TGFβ1 signaling pathway is associated with many diseases, which can induce the activation of hepatic stellate cells (HSCs) and induce liver fibrosis. Studies have shown that 20S-protopanaxadiol (PPD) has a therapeutic effect on liver fibrosis, but the target is unknown. In this study, we confirmed that PPD reduced the mRNA expression of downstream genes of the TGFβ1 pathway, which suggesting PPD is associated with the TGFβ1 pathway. The protein dissociation temperature and dissociation constant (Kd) of PPD on TGFβR1 and TGFβR2 were determined, which showed that PPD combined with TGFβR1 (Kd = 1.54 μM). The docking and simulation methods were used to find their binding sites. Site mutations, protein expression and in vitro binding experiments were performed to demonstrated these sites. In particular, these sites of TGFβR1 were also the active sites of TGFβR2. Therefore, we speculated that PPD blocked the combination of TGFβR1 and TGFβR2 by binding to the D57, R58, P59, and N78 of the TGFβR1 extracellular domain. Thus, PPD could block the transmission of TGFβ1 pathway and inhibit the activation of HSCs, and treating fibrosis. Our studies showed that PPD has the potential to treat diseases related to the TGFβ1 pathway and broadens its clinical application.

Environmental temperature serves a major driver for adaptive changes in wild organisms, however, its role in domestication has been less characterized. To uncover the mechanisms of cold tolerance in domestic animals, we sequenced genomes of 28 cattle at median coverage from warm and cold areas across China. By characterizing the population structure and demographic history, we identified two genetic clusters, i.e., northern and southern cattle groups, and a common historic population peak at 30 kilo years ago. Genome scan of cold tolerant breeds revealed genes that under selection sweeps enriched in thermogenesis related pathways. Specifically, we determined a substitution of PRDM16 (p.P779L) in north cattle, which maintains the formation of brown adipocytes through boosting expression of thermogenic related genes, indicating a vital role of this gene in cold tolerance. The findings provide a basis of genetic variations in domestic cattle that shaped by temperature environments and highlight a role of reverse mutation in livestock species.

Background& AimsThe Coronavirus Disease 2019 (COVID-19) has become a global epidemic and has caused a lasting and huge loss of life security, economic development and social stability in more than 180 countries around the world. Unfortunately, there is still no specific treatment for COVID-19 till now, therefore, at this point, all potential therapies need to be critically considered. LL-37 is one of the best-studied human antimicrobial peptide (AMPs) that has a broad-spectrum activity against bacteria and viruses. The use of living, genetically modified organisms (GMOs) is an effective approach for delivery of therapeutic proteins. The aim of this study was to determine the safety and efficacy of the Lactococcus lactis which has been genetically modified to produce the therapeutic human antimicrobial peptide LL-37 (herein after referred to cas001) in the patients of COVID-19.MethodsFirstly we constructed genetically modified food-grade probiotic, Lactococcus lactis, with sequence of seven tandem repeats of mature human LL-37 under control of the nisin-inducible nisA promoter to produce the cas001. A total of 20 healthy SD rats, half male and half female (There were five male and five female in the control group, the same in treatment group) were used to observe the acute toxic reaction and death after daily administration of cas001 for three weeks, which helps to provide necessary reference basis for clinical dose selection, verificaition of toxic reaction and possible target organs. According to the estimated clinical dosage of 1 × 108CFU /kg/day, considering the conversion of body surface area, the dose for rats should be multiplied by 6.17 to 6 × 108 CFU/kg/day. We administrated 100 times higher dose at 6 × 1010 CFU/ /kg/day to rats. In order to investigate the pharmacokinetics of cas001, male SD rats (body weight 250-300g, 1 × 1010 /animal, n=3) were given oral administration of LL-37 bacteria powder. The concentration of LL-37 in the blood before and after gavage was detected by ELISA kit (Hycult biotechnology Cat# HK321).Human clinical study was approved by Ethics committee of Chinese PLA General Hospital (S2020-074-04) and a total of 11 patients with mild symptoms were enrolled in Wuhan hankou hospital and Huoshenshan hospital. They were enrolled voluntarily and all patients signed informed consent. Among them, there were 5 males and 6 females, aged 55 ± 12 (36-70) years old, and the duration from onset to medication enrollment was 35 ± 19 (5-68) days. 6 patients were nucleic acid positive and 5 patients were nucleic acid negative when they were enrolled. All patients received the oral drug cas001 treatment according to requirement(1 × 109 CFU/capsule, 3 capsules/time, three times a day for 3weeks), with an average follow-up time of 33 ± 15 days (see table 1 for the results).FindingsWestern blot analysis shows that reasonable amount of LL-37 were induced by different concentrations of nisin, which means we have successfully constructed cas001. In the pre-clinical safety evaluation test, after three weeks administration of cas001, no adverse effects were observed on the rat’s body weight, food and water intake, hematological or serum biochemical parameters. The results showed that the LD50 of cas001 was higher than that of the 100 times of the expected clinical dose of 6 × 1010 CFU/day. These results showed that cas001 could be safe in animal experiments. In addition, rat pharmacokinetics results showed that the serum concentration of LL-37 reached peak 2 hours after gavage of cas001 and returned to basal level 6 hours after gavage. During study period, the volunteers did not feel any discomfort while taking the cas001 capsules, and two hours after oral administration, the concentration of LL-37 were increased in healthy volunteers.cas001 shows definite effect in the improvement of gastrointestinal symptoms and is possible to have effects in improving the systemic symptoms and respiratory symptoms and may play a role in the improvement of results of nucleic acid test and lung CT test. 11 patients enrolled showed good compliance, tolerance, subjective feeling and actively interacted with the doctors. None of the patients had any adverse reactions.ConclusionsBased on above observations, we conclude here that as an oral anti-viral agent, cas001 displayed good safety profiles. It is very hard to reach conclusion of clinical outcomes related to the cas001, although changes of several symptoms indicate encouraging findings.

The strong effects of classic brown adipose tissue (BAT) and recruited beige adipocytes in treatment of obesity and metabolic syndrome have been attracting increasing research interest. Cold treatment is an effective, convenient approach to stimulate BAT activity and induce white adipose tissue (WAT) browning. Here, we utilized prolonged cold exposure (from 2 h to 2 weeks in a 4° cold chamber) to elucidate dynamic changes in BAT and in WAT browning during acute and chronic cold exposure in mice. BAT mass decreased quickly, with reduced lipid droplet sizes within 8 h of cold exposure owing to the utilization of BAT pre-storage triglycerides, and subsequently increased during prolonged cold exposure. These dynamic morphological changes in BAT were confirmed by gene expression changes in ADRB3 and PGC1α, while UCP1 and ELOVL3 expression was continuously up-regulated throughout the entire cold exposure period. Additionally, cold treatment increased BAT secretion of FGF21, which has been reported to activate beige adipocyte formation. Thus, to illustrate potential crosstalk between secreted BAT proteins (so-called BATokines) and beige adipogenesis during cold stress, we performed an interscapular BAT (iBAT) removal experiment in mice. Surprisingly, loss of classic iBAT enhanced WAT browning due to compensatorily increased sympathetic WAT input. Unexpectedly, we observed significantly reduced adiposity in the iBAT removal group compared with the control group. These results further suggest that WAT browning plays an important role in whole-body energy metabolism during cold acclimation, even without iBAT. Furthermore, our data imply that enhanced WAT browning may be an efficient therapeutic tool to combat obesity and related syndromes.

Objective Previous work has suggested that white adipocytes may also show a mammary luminal secretory cell phenotype during lactation. The capacity of brown and beige/brite adipocytes to display a mammary cell phenotype and the levels at which they demonstrate such phenotypes in vivo is currently unknown. Methods To investigate the putative adipocyte origin of mammary gland cells, we performed genetic lineage-labeling experiments in BAT and the mammary glands. Results These studies indicated that the classic brown adipocytes (Ucp1+) and subcutaneous beige/brite adipocytes (Ucp1−/+) were found in the mammary gland during lactation, when they exhibited a mammary myoepithelial phenotype. Up to 2.5% of the anterior dorsal interscapular mammary myoepithelial cell population had a brown adipocyte origin with an adipose and myoepithelial gene signature during lactation. Eliminating these cells, along with all the brown adipocytes, significantly slowed offspring growth, potentially demonstrating their functional importance. Additionally, we showed mammary epithelial lineage Mmtv+ and Krt14+ cells expressed brown adipocyte markers after weaning, demonstrating that mammary gland cells can display an adipose phenotype. Conclusions The identification of a brown adipocyte origin of mammary myoepithelial cells provides a novel perspective on the interrelationships between adipocytes and mammary cells with implications for our understanding of obesity and breast cancer., Highlights • Brown adipocytes can show a mammary myoepithelial cell phenotype in vivo. • Myf5+/Ucp1+ myoepithelial cells express an adipose and myoepithelial signature. • Mammary-derived epithelial cells can display adipose features after weaning.

Intermittent fasting (IF), as a dietary intervention for weight loss, takes effects primarily through increasing energy expenditure. However, whether inter‐organ systems play a key role in IF remains unclear. Here, a novel hepatokine, pregnancy zone protein (PZP) is identified, which has significant induction during the refeeding stage of IF. Further, loss of function studies and protein therapeutic experiment in mice revealed that PZP promotes diet‐induced thermogenesis through activating brown adipose tissue (BAT). Mechanistically, circulating PZP can bind to cell surface glucose‐regulated protein of 78 kDa (GRP78) to promote uncoupling protein 1 (UCP1) expression via a p38 MAPK‐ATF2 signaling pathway in BAT. These studies illuminate a systemic regulation in which the IF promotes BAT thermogenesis through the endocrinal system and provide a novel potential target for treating obesity and related disorders., This paper reports a novel hepatokine, pregnancy zone protein (PZP), which is significant induction by refeeding. Loss of function studies and protein therapeutic experiment in mice reveal that PZP promotes diet‐induced thermogenesis through activating brown adipose tissue (BAT) via p38 MAPK‐ATF2‐UCP1 signaling pathway in BAT. These studies provide a novel potential target for treating obesity and related disorders.

Promoting development and function of brown and beige fat may reduce obesity. Here, we show that fat SIRT6 expression is markedly induced by cold exposure and a β-adrenergic agonist. Deletion of SIRT6 in adipose tissue impairs the thermogenic function of brown adipocytes, causing a morphological "whitening" of brown fat, reduced oxygen (O2) consumption, obesity, decreased core body temperature, and cold sensitivity. Fat SIRT6-deleted mice exhibit increased blood glucose levels, severe insulin resistance, and hepatic steatosis. Moreover, SIRT6 deficiency inhibits the browning of white adipose tissue (WAT) following cold exposure or β3-agonist treatment. Depletion of SIRT6 expression in brown adipocytes reduces expression of thermogenic genes, causing a reduction in cellular respiration. Conversely, SIRT6 overexpression in primary fat cells stimulates the thermogenic program. Mechanistically, SIRT6 interacts with and promotes phospho-ATF2 binding to the PGC-1α gene promoter to activate its expression. The present study reveals a critical role for SIRT6 in regulating thermogenesis of fat.

Myricetin, a dietary flavonoid, is effective in the treatment of obesity and insulin resistance by increasing glucose transport and lipogenesis in adipocyte and diminishing systemic inflammation in obesity. However, it has not been revealed yet whether myricetin is associated with brown adipose tissue (BAT) activation that tightly mediates systemic energy metabolism. Therefore, this study assessed whether myricetin activated brown adipose tissue in db/db mouse. Myricetin (400 mg/kg) in distilled water was fed daily by oral gavage to leptin receptor-deficient db/db male mice at 4 weeks of age for 14 weeks. Body weight change, glucose intolerance test, blood lipid profile and BAT activation using PET-CT were assessed. After myricetin treatment for 14 weeks, systemic insulin resistance and hepatic steatosis were significantly improved in db/db mice with body weight reduction and myricetin led to decreased adipocity, improved plasma lipid profiles and increased energy expenditure. Myricetin activated BAT by upregulating thermogenic protein expression and activating mitochondrial biogenesis, eventually increasing heat dissipation in skin after cold exposure. In iWAT, myricetin induced beige formation, increased thermogenic protein expression and activated mitochondrial biogenesis. Consistently, thermogenic gene expression was upregulated when myricetin was introduced in C3H10T1/2 cells during brown adipocytes differentiation. Moreover, the expression level of adiponectin was significantly increased in C3H10T1/2 cells, adipose tissues and plasma after myricetin treatment. These results highlight that myricetin prevents obesity and systemic insulin resistance by activating BAT and increasing adiponectin expression in BAT.

Increasing energy expenditure through activation of brown adipose tissue (BAT) is a critical approach to treating obesity and diabetes. In this study, rutin, a natural compound extracted from mulberry and a drug used as a capillary stabilizer clinically for many years without any side effects, regulated whole-body energy metabolism by enhancing BAT activity. Rutin treatment significantly reduced adiposity, increased energy expenditure, and improved glucose homeostasis in both genetically obese (Db/Db) and diet-induced obesity (DIO) mice. Rutin also induced brown-like adipocyte (beige) formation in subcutaneous adipose tissue in both obesity mouse models. Mechanistically, we found that rutin directly bound to and stabilized SIRT1, leading to hypoacetylation of peroxisome proliferator-activated receptor γ coactivator-1α protein, which stimulated Tfam transactivation and eventually augmented the number of mitochondria and UCP1 activity in BAT. These findings reveal that rutin is a novel small molecule that activates BAT and may provide a novel therapeutic approach to the treatment of metabolic disorders.-Yuan, X., Wei, G., You, Y., Huang, Y., Lee, H. J., Dong, M., Lin, J., Hu, T., Zhang, H., Zhang, C., Zhou, H., Ye, R., Qi, X., Zhai, B., Huang, W., Liu, S., Xie, W., Liu, Q., Liu, X., Cui, C., Li, D., Zhan, J., Cheng, J., Yuan, Z., Jin, W. Rutin ameliorates obesity through brown fat activation.

In addition to white adipose tissue (WAT) that stores energy, human and small mammals also have brown adipose tissue (BAT) that dissipates chemical energy for thermogenesis. BAT contains multilocular lipid droplets and much higher numbers of mitochondria than WAT. The mitochondria in BAT uncouple large amounts of fuel oxidation from ATP for heat generation. Accumulating evidences have demonstrated that increased activity and/or amount of BAT can reverse obesity and improve insulin resistance, which highlights that BAT plays an important role in energy metabolism. In this review, we summarized recent findings that shows advantageous effects of BAT activation in metabolic diseases. In addition, we presented the function and role of brown and beige fat cells and regulatory factors for them. Finally, we discussed the potential application of brown adipocytes-based therapy and pharmacological intervention to increase BAT activity for the treatment of obesity and related diseases including insulin resistance, cardiovascular diseases and type 2 diabetes.

ObjectiveDietary fibre has beneficial effects on energy metabolism, and the majority of studies have focused on short-chain fatty acids produced by gut microbiota. Ginseng has been reported to aid in body weight management, however, its mechanism of action is not yet clear. In this study, we focused on the potential modulating effect of ginseng on gut microbiota, aiming to identify specific strains and their metabolites, especially long-chain fatty acids (LCFA), which mediate the anti-obesity effects of ginseng.DesignDb/db mice were gavaged with ginseng extract (GE) and the effects of GE on gut microbiota were evaluated using 16S rDNA-based high throughput sequencing. To confirm the candidate fatty acids, untargeted metabolomics analyses of the serum and medium samples were performed.ResultsWe demonstrated that GE can induce Enterococcus faecalis, which can produce an unsaturated LCFA, myristoleic acid (MA). Our results indicate that E. faecalis and its metabolite MA can reduce adiposity by brown adipose tissue (BAT) activation and beige fat formation. In addition, the gene of E. faecalis encoding Acyl-CoA thioesterases (ACOTs) exhibited the biosynthetic potential to synthesise MA, as knockdown (KD) of the ACOT gene by CRISPR-dCas9 significantly reduced MA production. Furthermore, exogenous treatment with KD E. faecalis could not reproduce the beneficial effects of wild type E. faecalis, which work by augmenting the circulating MA levels.ConclusionsOur results demonstrated that the gut microbiota-LCFA-BAT axis plays an important role in host metabolism, which may provide a strategic advantage for the next generation of anti-obesity drug development.

The development of gut microbiota during infancy is an important event that affects the health status of the host; however, the mechanism governing it is not fully understood. l-Amino acid oxidase 1 (LAO1) is a flavoprotein that catalyzes the oxidative deamination of particular l-amino acids and converts them into keto acids, ammonia, and H

White adipose tissue expansion is associated with both hypertrophy and hyperplasia of adipocytes. Exercise training results in adipocyte hypotrophy by activating lipolysis, but it is poorly understood whether exercise regulates adipogenesis by altering adipogenic gene expression. The purpose of this study was to evaluate the effect of a single bout of swimming exercise on adipogenic gene expression in white adipose tissue (WAT). Male C57BL/6J mice were divided into two groups: a sedentary control group and a 120-minute swimming exercise group. Immediately after acute exercise, adipogenic gene expression in WAT was analysed by RT-PCR, and tdTomato positive cells in WAT from UCP1-cre-tdTomato mice were observed under a confocal microscope. In epididymal white adipose tissue (eWAT), PPARγ2 and C/EBPα expression at the mRNA level was significantly decreased with high induction of Wnt10b and KLFs (KLF2, KLF3, KLF7, KLF6, KLF9 and KLF15), whereas PPARγ2, not C/EBPα, was decreased with high induction of Wnt6 and KLFs (KLF2, KLF3, KLF7, KLF6 and KLF9) in inguinal white adipose tissue (iWAT) after acute exercise. The expression of C/EBPβ and C/EBPδ was upregulated in both WATs with a high level of PGC-1α expression. Expression level of UCP1 was increased only in adipocytes of eWAT, while beige cell specific gene expression was comparable between groups and tdTomato positive cells were not found in WAT of UCP1-cre-tdTomato reporter mouse immediately after acute exercise. These results suggest that acute exercise suppresses adipogenic gene expression and may regulate thermogenesis by activating C/EBPβ, PGC-1α and UCP1 in WAT.

Amino acids participate directly and indirectly in many important biochemical functions in the brain. We focused on one amino acid metabolic enzyme, L-amino acid oxidase (LAO), and investigated the importance of LAO in brain function using LAO1 knockout (KO) mice. Compared to wild-type mice, LAO1 KO mice exhibited impaired fear learning and memory function in a passive avoidance test. This impairment in LAO1 KO mice coincided with significantly reduced hippocampal acetylcholine levels compared to wild-type mice, while treatment with donepezil, a reversible acetylcholine esterase inhibitor, inhibited this reduction. Metabolomic analysis revealed that knocking out LAO1 affected amino acid metabolism (mainly of phenylalanine [Phe]) in the hippocampus. Specifically, Phe levels were elevated in LAO1 KO mice, while phenylpyruvic acid (metabolite of Phe produced largely by LAO) levels were reduced. Moreover, knocking out LAO1 decreased hippocampal mRNA levels of pyruvate kinase, the enzymatic activity of which is known to be inhibited by Phe. Based on our findings, we propose that LAO1 KO mice exhibited impaired fear learning and memory owing to low hippocampal acetylcholine levels. Furthermore, we speculate that hippocampal Phe metabolism is an important physiological mechanism related to glycolysis and may underlie cognitive impairments, including those observed in Alzheimer’s disease.

Saltified rutin enhances microglial Aβ clearance, providing a potential therapeutic avenue for Alzheimer’s disease treatment., The accumulation of aggregated amyloid-β (Aβ) in the brain is the first critical step in the pathogenesis of Alzheimer’s disease (AD), which also includes synaptic impairment, neuroinflammation, neuronal loss, and eventual cognitive defects. Emerging evidence suggests that impairment of Aβ phagocytosis and clearance is a common phenotype in late-onset AD. Rutin (quercetin-3-rutinoside) has long been investigated as a natural flavonoid with different biological functions in some pathological circumstances. Sodium rutin (NaR), could promote Aβ clearance by increasing microglial by increasing the expression levels of phagocytosis-related receptors in microglia. Moreover, NaR promotes a metabolic switch from anaerobic glycolysis to mitochondrial OXPHOS (oxidative phosphorylation), which could provide microglia with sufficient energy (ATP) for Aβ clearance. Thus, NaR administration could attenuate neuroinflammation and enhance mitochondrial OXPHOS and microglia-mediated Aβ clearance, ameliorating synaptic plasticity impairment and eventually reversing spatial learning and memory deficits. Our findings suggest that NaR is a potential therapeutic agent for AD.

Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family Suidae) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous UCP1 locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% (P < 0.01), consequently increasing carcass lean percentage (CLP; P < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production.

Cellulose derivatives are important fine chemicals with a wide range of applications in modern industries, so that cellulose derivatization has been one of hot topics for a long time. However, the existence of numerous hydroxyls in cellulose also generates a well-developed intra- and inter-molecular hydrogen bonding network, thus natural cellulose is neither meltable nor soluble in conventional solvents. Until today, the preparation method for cellulose esters begins with a heterogeneous reaction between cellulose and a large excess of the acylation reagent in the presence of pyridine or sulfuric acid as the catalyst. These heterogeneous reactions cause some problems, such as uneven distribution of substituents, side reactions, time consumption and considerable degradation of cellulose. Homogeneous esterification of cellulose in appropriate media, the alternative procedure for heterogeneous process, has been one focus in cellulose chemistry. It not only provides opportunities to obtain the effective control of degree of substitution (DS), distribution and uniformity of the functional groups along cellulose chains, but also creates more options to induce novel and functional groups. Recently, the advent of ionic liquids that can dissolve cellulose provides a new and versatile platform for the efficient and homogeneous derivatization of cellulose. A variety of well-known, novel and functional cellulose esters have been successfully synthesized in ionic liquids. Meanwhile, development of esterification techniques, utilization of agricultural residues, and pilot scheme of up-scaling esterification in ionic liquids have been in progress continuously. This review summarizes the development in homogeneous synthesis of cellulose esters in ionic liquids published in the recent decade.

Mulberry extract (ME) has been shown to possess beneficial effects towards obesity, but its mechanism is still unclear. In small mammals, mitochondria enriched brown adipose tissue (BAT) is known to convert protein's electrochemical energy to heat and maintain a constant body temperature. Improving the mitochondrial function or increasing the number of mitochondria could promote the metabolism of carbohydrate and fat. Thus, this study was designed to investigate the mitochondrial function regulated by ME and mulberry wine extract (MWE) during the brown adipogenesis. The C3H10T1/2 mesenchymal stem cell was treated with ME and MWE, both of which significantly (p < 0.05) increased the expression levels of fatty acid oxidation related genes such as peroxisome proliferator-activated receptor-γ coactivator-1α, PR domain-containing 16 and carnitine palmitoyltransferase 1α during brown adipogenesis. These changes were accompanied with increases in mitochondrial oxidative complex proteins upon ME and/or MWE exposure. Notably, ME and/or MWE also significantly (p < 0.05) increased the expression of the transcription factor A and the nuclear respiratory factor-1, which are the key transcription factors of mitochondrial biogenesis. In parallel, the mitochondrial copy number and brown adipose tissue specific gene-uncoupling protein-1 expression were dramatically (p < 0.05) elevated after ME or MWE treatment. Cyanidin-3-glucoside (Cy-3-glu) was found to be one of the most abundant anthocyanins in ME and MWE. Therefore, the BAT regulatory activity of ME and MWE might be, at least in part, due to the effect of Cy-3-glu. These results suggested that ME and MWE could ameliorate metabolic disease through an improvement in mitochondrial functions.

cope Obesity develops when energy intake exceeds energy expenditure. Promoting brown adipose tissue (BAT) formation and function increases energy expenditure and may protect against obesity. Cyanidin-3-glucoside (C3G) is an anthocyanin compound which occurs naturally in many fruits and vegetables. In this study, we investigated the effect and mechanism of C3G on the prevention of obesity. Methods and results Db/db mice received C3G dissolved in drinking water for 16 weeks; Drinking water served as the vehicle treatment. The total body weight, energy intake, metabolic rate and physical activity were measured. The lipid droplets, gene expression and protein expression were evaluated by histochemical staining, real-time PCR and western blots. We found that C3G increased energy expenditure, limited weight gain, maintained glucose homeostasis, reversed hepatic steatosis, improved cold tolerance, and enhanced BAT activity in obese db/db mice. C3G also induces brown-like adipocytes (beige) formation in subcutaneous white adipose tissue (sWAT) of db/db mice model. We also found that C3G potently regulates the transcription of UCP1 both in BAT and sWAT through increasing mitochondrial number and function. Conclusion Our results suggest that C3G plays a role in regulating systemic energy balance, which may have potential therapeutic implications for the prevention and control of obesity. This article is protected by copyright. All rights reserved

DJ-1 protein is involved in multiple physiological processes, including Parkinson’s disease. However, the role of DJ-1 in the metabolism is largely unknown. Here we found that DJ-1 maintained energy balance and glucose homeostasis via regulating brown adipose tissue (BAT) activity. DJ-1-deficient mice reduced body mass, increased energy expenditure and improved insulin sensitivity. DJ-1 deletion also resisted high-fat-diet (HFD) induced obesity and insulin resistance. Accordingly, DJ-1 transgene triggered autonomous obesity and glucose intolerance. Further BAT transplantation experiments clarified DJ-1 regulates energy and glucose homeostasis by modulating BAT function. Mechanistically, we found that DJ-1 promoted PTEN proteasomal degradation via an E3 ligase, mind bomb-2 (Mib2), which led to Akt activation and inhibited FoxO1-dependent Ucp1 (Uncoupling protein-1) expression in BAT. Consistently, ablation of Akt1 mitigated the obesity and BAT dysfunction induced by DJ-1 transgene. These findings define a new biological role of DJ-1 protein in regulating BAT function, with an implication of the therapeutic target in the treatment of metabolic disorders.

Pigs lack functional uncoupling protein 1 (UCP1) making them susceptible to cold. Nevertheless, several pig breeds are known to be cold resistant. The molecular mechanism(s) enabling such adaptation are currently unknown. Here, we show that this resistance is not dependent on shivering, but rather depends on UCP3 and white adipose tissue (WAT) browning. In two cold-resistant breeds (Tibetan and Min), but not a cold-sensitive breed (Bama), WAT browning was induced after cold exposure. Beige adipocytes from Tibetan pigs exhibited greater oxidative capacity than those from Bama pigs. Notably, UCP3 expression was significantly increased only in cold-resistant breeds, and knockdown of UCP3 expression in Tibetan adipocytes phenocopied Bama adipocytes in culture. Moreover, the eight dominant pig breeds found across China can be classified into cold-sensitive and cold-resistant breeds based on the UCP3 cDNA sequence. This study indicates that UCP3 has contributed to the evolution of cold resistance in the pig and overturns the orthodoxy that UCP1 is the only thermogenic uncoupling protein.

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy that is characterized by anovulation, hyperandrogenism and polycystic ovary. However, there is a lack of effective treatment for PCOS at present because the pathologic cause of PCOS has not been elucidated. Although it has been known that brown adipose tissue transplantation ameliorates PCOS by activating endogenous BAT, BAT transplantation is not applicable in clinic. Therefore, BAT activation with natural compound could be an effective treatment strategy for PCOS patients. Here, we found that 3 weeks of rutin (a novel compound for BAT activation) treatment increased BAT activation, thereby it improved thermogenesis and systemic insulin sensitivity in dehydroepiandrosterone (DHEA)-induced PCOS rat. In addition, the expression levels of ovarian steroidogenic enzymes such as P450C17, aromatase, 3β-HSD, 17β-HSD and STAR were up-regulated in rutin-treated PCOS rat. Furthermore, acyclicity and the serum level of luteinizing hormone were normalized, and a large number of mature ovulated follicle with a reduction of cystic formation were observed in PCOS rat after rutin treatment. Finally, rutin treatment surprisingly improved fertility and birth defect in PCOS rat. Collectively, our results indicate that rutin treatment significantly improves systemic insulin resistance and ovarian malfunction in PCOS, and our findings in this study provide a novel therapeutic option for the treatment of PCOS by activating BAT with rutin.