Since conventional thermal processing can have detrimental consequences on aroma compounds, non-thermal technologies such as high hydrostatic pressure (HHP) have been explored. HHP may alter the weak chemical bonds of enzymes. These changes can modify the secondary, tertiary, and quaternary structures of key enzymes in the production of aroma compounds. This can result in either an increase or decrease in their content, along with reactions or physical processes associated with a reduction of molecular volume. This article provides a comprehensive review of HHP treatment’s effects on the content of lipid-derived aroma compounds, aldehydes, alcohols, ketones, esters, lactones, terpenes, and phenols, on various food matrices of vegetable and animal origin. The content of aldehydes and ketones in food samples increased when subjected to HHP, while the content of alcohols and phenols decreased, probably due to oxidative processes. Both ester and lactone concentrations appeared to decline due to hydrolysis reactions. There is no clear tendency regarding terpenes concentration when subjected to HHP treatments. Because of the various effects of HHP on aroma compounds, an area of opportunity arises to carry out future studies that allow optimizing and controlling the effect. These vmpounds” . One of mpounds” . Alongside taste, food aroma is the main attribute controlling a consumer’s choice of a product . In a maHHP technology involves using isostatic pressures between 100 and 1000 MPa . HHP proThe present contribution intends to overview the most relevant findings, specifically on how HHP processing affects the concentration of the main types of volatile aroma compounds in food matrices. The performance of HHP treatments is compared to traditional thermal processing methods when applicable to gauge if HHP can be an alternative in the commercial food processing industry in terms of retaining the aroma profile of foods. Aroma can have several functions in food besides stimulating the appetite; it can also convey essential characteristics of the item by alerting when a food item is rancid or unsafe to consume . Chemica3–C5 aldehydes, propanal, butanal, and pentanal, tend to possess a rather chemical/malty/green note to them that is hard to define [6, the aldehydes take on a dual character and have both fruity/floral and fatty descriptors to their aroma, depending on the concentration present, the matrix, and the individual who is perceiving them [Aldehydes are organic compounds containing a terminal carbonyl functional group (“-CHO”), they are extremely common components of any food or flavoring agent, and many possess a low odor threshold . Acetaldo define ,21. As ting them . Unsaturated aldehydes, which can exist in both (E) and (Z) isomers conformations, tend to have higher odor thresholds and are often character impact compounds, with the shorter chain analogs providing green aromas ,24. It iAldehydes containing an aromatic ring such as benzaldehyde, phenylacetaldehyde, and cinnamaldehyde are all important components of foods and flavorings  which doAlcohols are organic compounds that possess at least one hydroxyl (“-OH”) group and are also abundant in foods and flavorings, but their contribution to aroma tends to be significantly less than their corresponding aldehydes due to the considerably higher odor threshold alcohols possess . Straight-chain alcohols are abundant in fruits; they often increase with maturity due to the decrease in the carbonyl content produced by the enzymatic breakdown of unsaturated fatty acids . The terIntroducing a double bond into the chain reduces odor threshold when compared to other alcohols and makes them character impact compounds with earthy and musty tones, such as in the case of mushrooms .Ketones are organic compounds containing a carbonyl center functional group “RC(=O)R”. Most ketones possess a relatively high odor threshold when compared to other lipid-derived aroma compounds such as aldehydes or esters . HoweverStraight-chain methyl ketones, containing one carbonyl group in the 2-position such as 2-heptanone, 2-nonanone, and 2-undecanone impart both blue cheese and a fruity pear aroma depending on the matrix it is encountered ,30. The α-dicarbonyl compounds, such as 2,3-butanedione, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione, have far lower thresholds than other species of ketones and are responsible for buttery and/or creamy notes in many cooked foods or flavorings ,32. Keto9 esters are important components of melon aroma [Esters are acid-derived compounds in which at least one of the structure’s hydroxyl (“OH”) groups is substituted by an “O-R” group, where R symbolizes a carbon-containing substituent. Esters are fundamental to the aroma of most fruits, comprising the major proportion of the volatile compounds in, for example, melons, apples, pineapple, and strawberries ,34,35,36on aroma . Lactones are cyclic or intramolecular esters (“R−C(=O)−O−R”) formed by the intermolecular esterification of the corresponding hydroxy acid. Due to most lactones possessing low odor thresholds, they are considered potent aroma compounds . It is a5H10) and can be linear, cyclic, or polycyclic; however, those that are responsible for odor tend to contain two or three isoprene units  [Terpenes are a type of hydrocarbon, major components of essential oils, and responsible for the characteristic aroma profile of many fruits, particularly citrus fruits such as orange and lemon . They arctively) . Many tectively) .Phenols are a class of organic aroma compounds in which at least one hydroxyl group (“OH”) is directly bonded with an aromatic hydrocarbon ring. Many phenols are particularly odor-active compounds. The methylphenols are particularly phenolic and smoky . It is aEnzymatic and microbial processes liberate low molecular weight volatiles. These types of processes are particularly important in the aroma of fruits and vegetables. An example of this is vanillin, the main aroma component of vanilla extract .Chemical precursors are produced during a fermentation step. Character impact compounds in both cocoa   and breaNon-enzymatic processes result from thermal treatments, such as pasteurization, sterilization, cooking, and roasting ,53. ThesVolatile aroma compounds in food are primarily synthesized by one of three types of processes :EnzymatiOut of the three processes described above, enzymatic and non-enzymatic ones are of particular interest in the food industry due to their roles in two of the major routes to flavor and aroma profile formation and alteration, lipid oxidation and the Maillard reaction, respectively . Lipid oxidation refers to the process that involves the degradation of fatty acids, where hydrogen is abstracted from a lipid to leave a lipid radical, which in turn reacts with molecular oxygen to form a lipid peroxide. In this process, unsaturated fatty acids form stabler radicals when compared to saturated fatty acids, mainly due to conjugation of the radical with the double bonds, hence being more prone to oxidation even at lower temperatures. Saturated fatty acid degradation becomes more important at higher temperatures, such as during thermal processing ,54. LipiThe Maillard reaction is a chemical reaction between amino acids and reducing sugars often also referred to as non-enzymatic browning. From a structural chemistry perspective, the Maillard reaction can be divided into early stages and late stages, where further reactions require more control due to their capacity of forming potentially harmful compounds such as acrylamide, heterocyclic aromatic amines, and advanced glycation end-products ,58. AromWhile other reactions such as caramelization (enols and lactones) ,62, and Food processing is a necessary step to ensure microbiological safety and shelf-life requirements to be met, as well as the generation of multiple volatile aroma compounds characteristic of the matrix they are found in . AlthougEmerging as an alternative to thermal treatments, HHP process is a food preservation method in which the product is subjected to pressures in the range of 100–1000 MPa to inactivate microorganisms and enzymes ,68,69,70a) is a parameter derived from the dependence of the pressure with the reaction rate constant (k). This volume is defined as the difference between the molar partial volume of the transition state and that of the reactants and whose value is determined by evaluating the effect of pressure at constant temperature on k [The effect that HHP has in structural and/or functional properties of the microorganisms, chemical reactions, phase changes, and other products to be pressurized is governed mainly by two principles: the isostatic and the Le Chatelier–Braun principles . Due to ure on k .Lnk = la is negative, the reaction will be favored by pressure (P), and, if it is positive, the opposite will occur. The higher is the absolute value of Va, the greater is the sensitivity of the reaction to pressure, while reactions with Va = 0 are independent of pressure [a will be a decrease, increase or no change if Va < 0, Va > 0, or Va = 0, respectively.In Equation (1), if Vpressure . TherefoHHP has minimal effects on the nutritional quality of foods due to the fact that these levels of pressure are not capable of altering covalent bonds up until pressure ranges of 1000–2000 MPa ,76,77; tAn overview of the main changes in the concentration of aldehydes and alcohols after HHP treatments at 100–600 MPa is shown in 6 aldehyde through cleavage by a hydroperoxide lyase, resulting in the formation of compounds such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal. An enhancement on this reaction due to HHP processing can be observed in kiwifruit pulp [9 aldehydes such as (E)-2-nonenal and -2,6-nonadienal are predominant as in Keitt mango juice [Fatty acids are converted into hydroperoxides, which in turn are easily converted into a Cuit pulp , mulberruit pulp , and greuit pulp  where thuit pulp ,83. As pgo juice . These ago juice ,86. AnotHexanol is an important aroma compound in fruit and vegetable matrices, generally providing grassy and floral notes . It is oAn overview of the main changes in the concentration of ketones, esters, and lactones after HHP treatments at 100–800 MPa is shown in Indica cultivars but at 500 MPa for Japonica cultivars, suggesting the presence of significant interactive effects between pressure and rice cultivars. The effect of HHP treatments on all ketones present on the samples of raw goat cheeses was statistically significant, both when applied at the beginning of the maturation process and at the end of the ripening [Ketones can be products of either lipid oxidation such as aldehydes or amino acid degradation induced by the Strecker reaction where an α-dicarbonyl compound reacts with an amino acid resulting in a carbonyl-amine condensation. Protein-deficient matrices such as Hami melons may remain unaffected to HHP treatments due to the lack of free amino acids necessary to form ketones , and in ripening . TreatmeChanges in ketones due to HHP were not as intense when treatment was applied at the end of ripening but were still deemed to be statistically significant. The authors observed an increase in methyl ketones in goat cheese samples treated at the initial day of the maturation process and attributed it to the fact that ketones did not undergo reduction to alcohols, since lower levels of alcohols were found in HHP-treated cheeses than control ones. It is also inferred that HHP could facilitate the production of methyl ketones from free fatty acids. In green asparagus juice, HHP treatments maintained a higher ketone concentration when compared to traditional thermal treatments .Esters hydrolyze to their respective acids and/or alcohols during HHP treatment, the tendency of esters to decrease during processing can be explained based on a possible hydrolytic effect that HHP causes in the matrices. For red plum puree the levels of most esters present in the matrix decreased during the HHP treatments only (Z)-hex-3-enyl acetate, quantitatively the major ester present, and hex-3-enyl benzoate were not modified after HHP processing, preserving the original aroma of puree . Some esLactones, being a type of ester, share many similarities to the behavior explained in the previous paragraph, but, due to their cyclic nature, there are some properties that set them apart. While ester concentration tends to decrease when submitted to HHP treatments, lactones either increased or remained unaffected after the treatments. Only one matrix presented a decrease in the overall concentration. In red plum puree, only one lactone was identified in the samples, γ-octalactone . No signAn overview of the main changes in the concentration of terpenes and phenols after HHP treatments at 100–600 MPa is shown in trans-Geranylacetone, and β-Damascenone), only β-ionone had its concentration significantly altered by HHP treatments, demonstrating a more noticeable increase at 600 MPa processing conditions [For red plum puree, out of the four terpenes identified in the samples ; if this enzyme is enhanced, the concentration of phenolic compounds is decreased, and, if it is inhibited, the opposite occurs. The concentration of phenolic compounds was found to be greater in thermally treated samples than in HHP samples ,94, likeHHP can both enhance and inhibit chemical reactions occurring in the matrices being subjected, and the consequences it can have on enzymes responsible for these reactions can be both desired and undesired, thus making HHP processing stand out as a unique alternative to conventional thermal processes. Given that many studied samples were stored at refrigerated or room temperature before volatile analysis, it is inferred that the chemistry regarding enzymes responsible for the described processes was affected during this time resulting in the modifications of the concentration of some aroma compounds. Aldehydes and ketones predominantly increased their levels after treatment, probably due to the oxidative processes of alcohols and phenols, which explains the decrease of alcohols and phenols when subjected to HHP. Esters and lactones concentration levels seemed to decrease due to hydrolysis reactions occurring after treatments, and terpene concentration showed mixed results due to the effects that HHP had on enzymes responsible for oxidation and reduction processes. The information presented in this article agrees that HHP treatment in certain processing conditions can preserve select volatile aroma compounds more effectively than thermal processing, proving HHP is an interesting alternative technology as a widespread process that warrants further research that allows both the optimization and control the effect of HHP so that it would be possible to make accurate predictions of the effect it will have on the subjected matrix.


Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects at least 10 million people worldwide and is associated with the development of T-cell lymphoma (TCL). The treatment of TCL remains challenging and new treatment options are urgently needed. With the goal of developing a novel therapeutic approach for TCL, we investigated the activity of the clinical formulation of oncolytic reovirus  in TCL models. Our studies revealed that HTLV-1-negative TCL cells were highly sensitive to Reolysin-induced cell death, but HTLV-1-positive TCL cells were resistant. Consistent with these data, reovirus displayed significant viral accumulation in HTLV-1-negative cells, but failed to efficiently replicate in HTLV-1-positive cells. Transcriptome analyses of HTLV-1-positive vs. negative cells revealed a significant increase in genes associated with retroviral infection including interleukin-13 and signal transducer and activator of transcription 5 (STAT5). To investigate the relationship between HTLV-1 status and sensitivity to Reolysin, we infected HTLV-1-negative cells with HTLV-1. The presence of HTLV-1 resulted in significantly decreased sensitivity to Reolysin. Treatment with the JAK inhibitor ruxolitinib suppressed STAT5 phosphorylation and expression of the key anti-viral response protein MX1 and enhanced the anti-TCL activity of Reolysin in both HTLV-1-positive and negative cells. Our data demonstrate that the inhibition of the JAK/STAT pathway can be used as a novel approach to antagonize the resistance of HTLV-1-positive cells to oncolytic virus therapy. T-cell lymphomas (TCLs) are a heterogeneous group of lymphoid malignancies ,2. AdultOncolytic virus therapy has demonstrated significant potential in a variety of tumor types, and this has resulted in several viruses progressing to clinical testing ,11. ReovGiven the prominent role of HTLV-1 in some TCLs, we hypothesized that oncolytic reovirus therapy may have differential sensitivity in HTLV-1-positive vs. negative cells. Our investigation revealed that HTLV-1-negative TCL cells were highly susceptible to reovirus infection and replication, which resulted in decreased cell viability. In contrast, HTLV-1-positive TCL cells were resistant to productive reovirus infection and did not undergo cell death following Reolysin treatment. We also determined that HTLV-1-positive TCL cells displayed significantly upregulated STAT5 phosphorylation compared to HTLV-1-negative cells, suggesting that they exhibit constitutive anti-viral defense mechanisms. Importantly, treatment with the JAK inhibitor ruxolitinib enhanced sensitivity to Reolysin in both HTLV-1-positive and negative TCL cells. Taken together, our findings provide the foundation for future clinical studies testing Reolysin in combination with JAK inhibitors for patients with TCL.2. HTLV-1-positive HuT-78 and KARPAS-299 cells were created by transfecting cells with HTLV-1 virus isolated from HuT-102 cells. Cell lines were authenticated by the source banks using short tandem repeat (STR) DNA profiling techniques. Cell numbers were counted before initiating each experiment via automated trypan blue exclusion with the assistance of a Vi-Cell XR system .KARPAS-299 cells were obtained from the European Collection of Authenticated Cell Cultures . HuT-78, HuT-102, and MJ cells were purchased from American Type Culture Collection . TCL cells were cultured with medium supplemented with 10% FBS at 37 °C with 5% CORuxolitinib was purchased from SelleckChem . Reolysin was kindly provided by Oncolytics Biotech, Inc. . Reovirus was initially quantified by Oncolytics Biotech, Inc. to meet Food and Drug Administration (FDA) standards for clinical administration to humans. Plaque assays confirmed viral titers. All experiments were performed with the clinical formulation of reovirus (Reolysin). Propidium iodide (PI) and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma .6 cells per ml with 200 µL of media per well and were treated with the indicated concentrations of drugs for 72 h. After drug treatment, MTT was added, and viability was quantified using a microplate reader. The pro-apoptotic effects of ruxolitinib and Reolysin were quantified by propidium iodide staining and fluorescence activated cell sorting (PI-FACS) analysis of sub-G0/G1 DNA and quantification of active caspase-3 positive cells by flow cytometry using a commercial kit .Cell viability was assessed by MTT assay. TCL cells were cultured in 96-well plates at a density of 0.1 × 10HuT-78, KARPAS-299, HuT-102 and MJ cells were treated with 90 plaque forming units (PFU)/cell Reolysin for 48 h and processed for transmission electron microscopy as previously described . The perProtein identification by immunoblotting was performed following an optimized protocol as previously described . AntibodTotal RNAs were isolated from HuT-78, KARPAS-299, HuT-102 and MJ cells using the RNeasy Plus Mini Kit  and treated with the TURBO DNA-free Kit . A total of 300 ng of total RNA per sample was amplified and hybridized to GeneChip Human Gene 1.0 ST arrays  according to the manufacturer’s instructions. Affymetrix CEL files were imported into Partek Genomics Suite 6.4  using the default Partek normalization parameters and the robust multiarray average analysis adjusted for probe sequence and guanine and cytosine robust multiarray average content. Data normalization was performed across all arrays using quantile normalization. Finally, significantly expressed genes between HTLV-1-negative and HTLV-1-positive cells were searched in the DAVID database system to identify the most affected pathways as previously described . Data frIL-13, STAT5, HTLV-1  transcripts were amplified using commercially available TaqManTM Gene expression assays . Relative gene expression was calculated with the 2ΔΔCt method [ß-actin was used as a housekeeping control gene. For quantification of reovirus by qRT-PCR, total RNA was isolated using RNeasy® Plus Mini Kit . First-strand cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit . Reovirus transcripts were amplified by qRT-PCR. Primers were designed to span the Reovirus type 3 (Dearing strain) S3 segment. Primers and TaqMan probe sequences are as follows: Forward primer: 5′-tgtatgacgctggctctacc-3′; Reverse primer: 5′-cgtccacctcacatccatag-3′; Probe: 5′Fam-cgctccaacactgtcagcgga-3′Tamra. Fold change was calculated using the comparative Ct (2−ΔΔCt) method. ß-actin was used as housekeeping gene.Complementary DNA (cDNA) from parental KARPAS-299, HuT-78, MJ, HuT-102 and HTLV-1 transfected KARPAS-299 and HuT-78 cells were used for relative quantification of gene expression by real-time polymerase chain reaction (RT-PCR) analyses. First-strand cDNA synthesis was performed with 1 mg RNA in a 20 µL reaction mixture using the high-capacity cDNA Reverse Transcription Kit . t method . ß-actint-test or the 2-way ANOVA multiple comparison test as appropriate. Differences were considered significant in all experiments at p < 0.05 with two-sided comparisons. All experiments were performed with a minimum of n = 3 from independent experiments.Statistical significance between samples was determined by using the Student’s TCLs are a heterogenous group of lymphoid malignancies that are very aggressive and exhibit a poor prognosis. With the goal of developing a new therapeutic approach, we investigated the replication ability and efficacy of oncolytic reovirus in models of TCL. We initially tested the anti-TCL activity of Reolysin in a panel of TCL cell lines . Reolysin displayed significant anticancer activity against two TCL cell lines (KARPAS-299 and HuT-78), while the other two models (HuT-102 and MJ) demonstrated significant resistance to Reolysin A. We nexTo further investigate the differences in the sensitivity of our TCL cell lines to oncolytic reovirus therapy, we measured reovirus replication by transmission electron microscopy. In agreement with our cell viability and apoptosis data, reovirus replication was only observed in the HuT-78 and KARPAS-299 TCL cell lines A. The peIL-13 and STAT5 were significantly upregulated selectively in the HuT-102 and MJ cell lines  and Signal Transducers and Activators of Transcription (STAT) signaling pathway were significantly altered in both the HuT-102 and MJ cell lines. This may contribute to resistance to oncolytic reovirus therapy by promoting conditions that hinder viral replication. These genes have also been associated with the HTLV-1 Tax protein in that the presence of Tax drives their expression ,22. Consll lines A. This rll lines B. A compll lines C. The exll lines D. CollecHuT-102 and MJ TCL cells have been previously characterized as exhibiting constitutive infection with HTLV-1 ,26. To cTo investigate the mechanistic relationship between HTLV-1 infection and sensitivity to oncolytic reovirus, we evaluated the anticancer activity of Reolysin in parental and HTLV-1-positive HuT-78 and KARPAS-299 cells. Consistent with our prior transcriptome analyses, exposure to HTLV-1 resulted in increased expression of STAT5 as well as STAT5 phosphorylation A. In agrWe next evaluated whether the pharmacological inhibition of JAK/STAT signaling with the JAK inhibitor ruxolitinib could downregulate STAT5 expression and augment Reolysin-mediated cell death. Parental TCL cells were treated with ruxolitinib for 24 h and STAT5 phosphorylation was determined by immunoblotting. Consistent with the drug’s mechanism of action, ruxolitinib dramatically inhibited STAT5 phosphorylation A. MyxoviSelective activity against malignant cells is one of the leading criteria that is prioritized in the development of novel anticancer agents. Accordingly, the therapeutic use of oncolytic viruses represents a promising anticancer approach due to their ability to selectively replicate in tumor cells without harming normal tissue. This distinctive replication profile has resulted in the development of several oncolytic viruses that have been tested in clinical trials for the treatment of various cancer types ,28,29,30We previously determined that Reolysin possesses significant activity in TCL cell lines and, in particular, cells that have developed acquired resistance to histone deacetylase (HDAC) inhibitors . InteresHuT-102 and MJ express high levels of HTLV-1 expression that could not be achieved through the transfection of HTLV-1-negative cell lines HuT-78 and KARPAS-299 with HTLV-1. While the HTLV-1-transfected cells displayed significant resistance to Reolysin, they did not yield the same level of resistance observed in the chronically infected HuT-102 and MJ models. These data indicate that sensitivity to Reolysin may also depend on the degree of HTLV-1 infection.JAK/STAT signaling pathways, particularly STAT5, are frequently dysregulated and play an important role in the pathogenesis and disease progression of TCL ,36,37. SHere, we demonstrate that HTLV-1-infected TCL cells promote resistance to oncolytic reovirus therapy through the upregulation of JAK/STAT signaling. Treatment with the JAK inhibitor ruxolitinib reduces this mechanism of resistance and augments the activity of Reolysin. Our findings provide the rationale for further testing of the combination of Reolysin and JAK inhibitors as a new approach for treating TCL.


Limosilactobacillus fermentum JL-3 strain, isolated from “Jiangshui” (a fermented Chinese food), was investigated. In vitro results showed that JL-3 strain exhibited high degradation capacity and selectivity toward UA. After oral administration to mice for 15 days, JL-3 colonization was continuously detected in the feces of mice. The UA level in urine of mice fed with JL-3 was similar with the control group mice. And the serum UA level of the former was significantly lower (31.3%) than in the control, further confirmed the UA-lowering effect of JL-3 strain. Limosilactobacillus fermentum JL-3 strain also restored some of the inflammatory markers and oxidative stress indicators  related to hyperuricemia, while the gut microbial diversity results showed that JL-3 could regulate gut microbiota dysbiosis caused by hyperuricemia. Therefore, the probiotic Limosilactobacillus fermentum JL-3 strain is effective in lowering UA levels in mice and could be used as a therapeutic adjunct agent in treating hyperuricemia.Recent studies into the beneficial effects of fermented foods have shown that this class of foods are effective in managing hyperuricemia and gout. In this study, the uric acid (UA) degradation ability of  First is an endogenous buildup caused by enzyme deficiencies in the purine metabolism pathway. This deficiency in major purine metabolism enzymes leads to an increased rate of nucleic acid decomposition and UA production, which is under the control of a mechanism whereby an end product control itself by inhibiting the enzyme producing it.13 Another route of UA accumulation is through the intake and exogenous absorption of purine rich foods. A recent study showed that the intake of meat increases the risk of gout by 21%, while sea food intake increases its risk by just 7%.14 Hyperuricemia can be alleviated effectively by external interventions.15 In general, it can also be managed through the excretion of UA in large amounts by the kidneys, while only 30% of the total UA is degraded by the action of intestinal flora burden per time.17 However, the effectiveness of the intestinal flora can be greatly influenced by purine-rich foods, which may disturb the purine metabolism process, thereby elevating serum UA levels.19 It is thus necessary to control UA from external sources to prevent hyperuricemia.3High levels of UA in plasma have long been associated with hyperuricemia.21 An example is the representative UA-lowering drug, Allopurinol, which can competitively bind with the xanthine oxidase (XOD) enzyme to reduce UA production.22 Febuxostat is another novel non-purine selective inhibitor of XOD.23 Both drugs are widely used to treat hyperuricemia and they are relative safety.24 Dietary interventions work by restricting the intake of high-purine foods and alcohol, but can be less effective than drugs because of the difficulty in adhering to dietary restrictions for a long time. Microbial remediation as a cost-effective treatment approach has been greatly accepted by the public due to its minimal site disruptions in the body.25 Thus, several Lactobacillus strains that can utilize purines have been discovered. Among these are the strains found to be effective in lowering UA in rodents. L. brevis (DM9218) and L. gasseri (PA-3) for instance degraded the intermediate products of purine metabolism to ameliorate hyperuricemia.27 For literature, most of these Lactobacillus strains have also been aimed at intestinal degradation and absorption of purines.Currently, several methods are used to treat hyperuricemia, including diet, drugs and biotherapy, aimed at purine uptake and degradation.29 Research has shown that fermented foods such as yogurt are beneficial for type 2 diabetes patients, because of its richness in lactic acid bacteria.30 Probiotics have also been reported to be effective in degrading UA, so we hypothesized that the activity of human intestinal flora can be enhanced by probiotics.31 “Jiangshui” is a traditional fermented food in Northwestern China, which is made with vegetables, such as celery and cabbage. Lactic acid bacteria, acetic acid bacteria and yeast are the main microbes in the Jiangshui fermentation process, especially the facultative anaerobic lactic acid bacteria.32 Thus, the present study is aimed at screening microbial strains from fermented Jiangshui with UA degradation ability to evaluate the effects of these strains on UA-induced hyperuricemia in a mouse model.Fermented foods are rich in lactic acid bacteria, and can produce organic acids to control decaying microorganisms and pathogens.2.2.1.To explore the correlation between the consumption of Jiangshui and the incidence of gout, a questionnaire survey was conducted on 180 residents in Lanzhou city. The results showed that when the consumption frequency of Jiangshui noodle exceeded six times a week, the prevalence of gout displayed a downtrend  solution containing UA (10 mmol/L) for cultivation. Results showed that the JL-3 strain could degrade 40.9% of UA within 24 h, compared to XS-14 which is a m yogurt . Alternam yogurt . This sum yogurt .Figure 22.3.35 Thus, this method was adopted to construct hyperuricemia mice and gavaged with the strain (−6 (P < .001) (P < .0001). However, UA levels of the JL-3 + UA group in urine gradually decreased from day three, and approached the control level on day nine. In serum samples collected on day 15, UA levels of the UA group (249.9 µmol/L) were 2.2 times higher than the control (112.6 µmol/L). After intervention with JL-3, UA concentrations decreased by about 31.3% compared with the UA group (P < .01)  . FurtherP < .01) . CompareP < .01) .Figure 32.4.P < .01) (P < .01). Specifically, IL-1β was reduced by 50.9%, BUN by 66.7% (accounting), MDA by about 40%, and CRE by 73%. In the kidney and liver, IL-1β level in the UA group was significantly increased P < .01). However, it partially dropped in the JL-3 + UA group , creatinine (CRE) and blood urea nitrogen (BUN) were tested, which all showed an increasing trend in the UA mice group (P < .01) , B, C, DUA group , G. AlteUA group . Also, tUA group . HistopaUA group .Figure 42.5.P < .0001). SCFAs detection by GC-MS in the feces of mice on day 15th, revealed that the total SCFAs and acetic acid concentrations in feces of the UA group were significantly lower than the control (P < .01). They were recovered in the JL-3+ UA group, but showed difference with the control group. In the JL-3 group mice feces, the butyric acid concentration also significantly increased compared with the control group (P < .01)  .Limosilactobacillus fermentum JL-3 treated mice. At the end of the experiment, the abundance of microbial flora (Shannon index) was higher in UA+JL-3 group than the UA group, which was regulated by JL-3 treatment (Supplementary Table 1). Alternatively, Principle-coordinates analysis (PCoA) results showed that phylogenetic community structures were markedly different between UA-exposed samples and others, and that the UA group was separated from other groups. It suggested that UA exposure changed the gut microbiota composition, while JL-3 supplement diminished the effect of UA on microbiota changes . Comparing the UA mice group to the control, there was a significant increase in the relative abundance of Bacteroidetes and a reduction in Firmicutes and Proteobacteria. JL-3 treatment attenuated UA-induced increase in Bacteroidetes and reduction in Firmicutes and Acidobacteria (Firmicutes to Bacteroidetes (Firm/Bac ratio) was also deceased on UA exposure mitigated by JL-3 treatment , which was coincident with our results.45 Studies have also shown that Jiangshui has a high degrading capacity of 75%.46 Therefore, we proposed that the effect on gout is due to the action of probiotics present in fermented Jiangshui.Hyperuricemia continues to pose health problems around the world.47 UA has been established as the major etiologic factor in hyperuricemia.48 Research has also found that it can be used as a nitrogen source by many aerobic organisms, such as Escherichia coli. The general catabolic pathway of UA by bacteria is allantoin, which proceeds via three steps, involving the interconversion between 5-hydroxyisouric acid, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline and allantoin, which could easily degrade to ammonia.50 In our study, allantoin was detected as a by-product of JL-3, which is coincident with former reports, as its degradation rate was found to be more significant compared with Bacteroides termitidis UAD-50 (50 µmol/2 h).51This study focused on probiotic treatment for hyperuricemia, while earlier studies mainly concentrated on the purine metabolism, and only a few focus on UA.6 CFU/g, which was in accordance with a previous study, thus indicating that the JL-3 strain continued to survive in the mouse intestine throughout the experiment.52 The beneficial effect of JL-3 on hyperuricemia mice was evidenced by the decreased UA levels, as results showed that the UA levels in JL-3 + UA group significantly decreased to more than 30% in feces and urine compared with the UA group. This was effectively reduced compared with DM9218 (< 30%) and the microbial fermented extracts of Lactobacillus (21.6%).53 Consistent with previous studies, we found a dramatic increase in BUN and CRE after UA injection, which is associated with hyperuricemia, chronic nephritis and renal insufficiency.55 In addition, serum MDA was also increased, as was the IL-1β in the liver and kidney, which is similar to the high-fructose diet.56 However, after JL-3 treatment, the levels of all these parameters were modulated to normal. XOD catalyzes the hydroxylation of hypoxanthine and xanthine, which are the last two steps of urate biosynthesis.57 After intervention with JL-3, increased XOD activity in the liver was restored to normal, suggesting that JL-3 inhibited XOD. Similar XOD-inhibiting effects were reported in microbial fermented extracts of Aspergillus fungi (40.8% – 63.5%), but the underlying mechanism is unclear and deserves further investigation. This suggests that UA-induced oxidative stress and inflammatory response in the liver. The over production of XOD has been demonstrated to cause an increased production of UA to further increase inflammation. In addition, consistent with previous research on L. acidophilus KB27 and L. rhamnosus KB7958, H&E photomicrographs further showed the characteristics of injury in hyperuricemia mice kidney that was significantly reduced after the JL-3 intervention.59After 15 d of intervention, the amount of JL-3 in the feces exceeded 1061 This revealed that JL-3 could restore intestinal metabolism and promote UA degradation.Specifically, the UA degradation capacity of fecal bacteria in UA + JL-3 mice was remarkably higher than that in mice treated solely with UA. This suggested that the UA-degradation ability of fecal bacteria in the UA + JL-3 group was enhanced by the addition of JL-3. The restorative effect of JL-3 on the metabolism of gut microbiota was also evidenced by the recovery of intestinal SCFAs. Studies have shown that SCFAs are important energy and signaling molecules produced by intestinal microorganisms associated with gout. Similar to the acetic acid in the intestine, SCFAs can serve as a central regulator for the intensity and duration of gout inflammation.62 Studies have demonstrated that the gut microbiota might be related to gut inflammatory responses, which participate in a series of diseases.63 However, many external factors that exceed the tolerance capacity of the gut microbiota can lead to dysbiosis.25 As an antioxidant and immune enhancer, UA exerts a great impact on the gut microbiota community.64 UA exposure significantly reduced the diversity and abundance of gut microbiota compared to those of the control group mice, which was regulated by JL-3 treatment, suggesting that JL-3 improves the gut microbiota disturbed by UA exposure. At the phylum level, UA significantly increased the relative abundance of Bacteroidetes, but decreased that of Firmicutes and Acidobacteria, which impart key functions to their host, such as metabolic, developmental, and immunologic properties.65–67 This is similar to a previous research based on the Sprague–Dawley rats with hyperuricemia.57 However, JL-3 pretreatment significantly attenuated UA induced increase in Bacteroidetes and decrease in Firmicutes and Acidobacteria. At the genus level, Prevotella and Bacteroidales were negatively associated with testicular function, while Paraprevotella, Mucispirillum, Oscillospira, Coprococcus and Lactobacillus were positively correlated with that function.68 JL-3 pretreatment reduced the increase of Prevotella and Bacteroidales, and elevated the decrease of Paraprevotella, Mucispirillum, Oscillospira and Lactobacillus in UA-exposed mice by regulating intestinal flora and improving intestinal function to alleviate gout.63 In addition, the low abundance inherent strain of Erysipelatoclostridium that appears in a host colitis by competing for nutrients negatively affected the flora.70 In correspondence with our reports, L. acidophilus KB27 and L. rhamnosus KB79 supplements also confirmed the fact that mice gut microbiota were rich in Firmicutes and Bacteroidetes.58 Thus, it can be concluded that the feed addition of JL-3 can reduce the phylum level disturbance associated with hyperuricemia and maintain the steady-state of the mice intestinal microbial community.Gut microbiota is characterized by temporal stability and resilience.Lactobacillus strains.71 Second, JL-3 can improve bowel movement activity, thus decreasing fecal excretion of UA. Thirdly, JL-3 can modulate the structure and function of gut microbiota.Based on these experimental results, it is hypothesized that JL-3 might exert its effects in multiple ways. First, JL-3 can degrade UA in the gut, thus reducing the amount of UA that accumulates in the intestinal tract, thereby lessening the amount of UA reabsorbed by the intestinal epithelium reentering circulation. The protective mechanism of JL-3 against UA accumulation in mice was also found to be similar to several reports on purine-degrading 3.2.Limosilactobacillus fermentum JL-3, a candidate probiotic strain was derived from fermented dairy products with UA reduction capability. This study explored the degradation of UA by JL-3 using mice as an animal model. The bacteria administered to mice, colonized the intestine, while JL-3 decreased UA levels in mice, and regulated the structure and function of the gut microbial community. Compared to current treatment technologies, probiotics appear to be direct and efficient at repairing hyperuricemia, thus playing an important role in hyperuricemia treatment in humans, which might provide a promising strategy for alleviating hyperuricemia.In this study, 4.4.1.P < 0.05. The difference was statistically significant.A cross-sectional comparison of the questionnaire with a standard chart abstraction-based measure (Pearson Index) was conducted on volunteers among the population in Gansu Province (Supplementary File1). The questionnaire survey was conducted anonymously to count the gout conditions, and statistically investigate the incidence of related behaviors. The prevalence of gout was investigated in 180 residents with eight questions regarding gout. The survey mainly focused on: 1) the frequency of Jiangshui noodle consumption every week; 2) the situation of gout; 3) the frequency of dietary habits such as drinking and eating meat. SPSS v.17.0 software was used to analyze the data at 4.2.2HPO4. 12 H2O, 0.3 g KH2PO4, 0.05 g NaCl, 0.05 g MgSO4.7 H2O, 0.001 g CaCl2, 0.2 g UA and 1.2 g agar, per 100mL) (Supplementary Table 2).72 The selected strains were then transferred to a new medium with UA as the sole carbon source. The grown bacterial culture plates were kept in an incubator at 37°C for 48 h. While the obtained bacterial colonies  were further streaked on new MRS agar plates to acquire a single colony. The new monoclonal colony activated in MRS medium was then stored in 25% glycerol at −80°C.Traditional Jiangshui samples were collected from Lanzhou supermarket . From the collected samples, 20 strains were isolated in a UA medium  was then added to the supernatant and mixed thoroughly to prevent further degradation, after which 20 mL of the mixture was injected into the HPLC device after filtration. The remaining urea and allantoin contents were calculated. The degradation rate of JL-3 strains was also calculated according to the following formula: V = (0.9 C − X)/60, a = [(0.9 C − X)/0.9 C] N100%.To evaluate the UA degradation ability, JL-2, JL-3 and JL-8 were inoculated onto MRS and cultured for 48 h at 37°C under anaerobic conditions. 2 mL of the culture solution was centrifuged at 4,000 ×74 Adenylate-guanylate-neutral potassium phosphate culture solution (900 μL) was mixed with 100 μL of terminator perchloric acid solution. After mixing, 20 μL was aspirated. The external standard method was then used to determine the retention time of UA to generate a quantitative standard curve. The conditions for HPLC were as follows: Water XTERRA MS C18 (250 mm × 4.6 μM), the mobile phase was a gradient of 20 mmol/L potassium dihydrogen phosphate solution (pH 5.0), with a flow rate of 1 mL/min, while column temperature was 25°C, with wavelengths of 254 nm (UA), 190 nm (urea), 190 nm , and an elution time of 40 min.Where V: degrading speed (g/L/min), X: the remaining content of urea or allantoin (g/L).4.4.et al.75 The sequence of potential isolates for species identification was made using the BLAST engine (NCBI) and the nucleotide sequence was submitted to GenBank for reference .76 The morphology of JL-3 was analyzed by scanning electron microscopy , while the multiple sequences of JL-3 and other closely related strains were aligned by MEGA .78TIANamp Bacteria DNA Kit  was used to obtain high quality genomic DNA of strains. The 16S rDNA was amplified as described by Sibley 4.5.Fifty Kunming male mice  which were 4-week-old and weighed 16–20 g, were bought. Before the experiment, mice were habituated for a week. For the experimental procedures, 1 mouse per cage was housed at 22°C ± 1°C and 60%–75% relative humidity. The mice were then fed with standard commercial mouse food. The care and use of laboratory animals in our study was approved by the Lanzhou University Animal Ethics Committee, after that the relevant ethical regulations were followed.79 The control group was then fed with a basal diet, and orally gavaged with 0.5 mL of water, while the UA1 group was given the basal diet along with OP (4%) and UA (1%) injection through the oral gavaged route. The UA2 group was also given the basal diet of OP (4 %) and UA (2 %), which were injected through the oral gavaged route, while the UA3 group was fed the basal diet and intraperitoneally injected with OP (4%) and UA (2%) in 0.5 mL water. The blank group was then fed with a basal diet and intraperitoneal injection of 0.5 mL water. During this period, urine and blood samples were collected to determine whether the model was well established by detecting the UA content. According to an earlier research, different concentrations of UA were selected to feed mice by injection to establish hyperuricemia model mice.34For inducting of UA-induced hyperuricemia, mice were given oteracil potassium (OP) and UA in 0.5 mL water for 14 d. Fifty mice were randomly divided into five groups, where OP and UA were used to construct the hyperuricemia animal model according to literature.8 CFU of JL-3 was dissolved into 0.5 mL of skim milk, which was directly injected into the mouth of each mouse twice a day (9:00 in the morning and 18:00 in the afternoon). Skim milk was prepared using sterilizing skimmed milk powder at 15 min, while 1 mL of bacterial solution was centrifuged at 8,000 ×g for 10 min, after which the suspension of the sterilized milk was used.The 2% concentration of UA and 4% of OP confirmed during the model experiment were used for repair experiments. Forty mice were randomly allocated into four groups (10 mice per group). The control group was fed with the basal diet and sterile skim milk (0.5 mL) administrated by oral gavage every day, while the JL-3 group was also fed with a basal diet and gavage with JL-3 in the sterile skim milk. The UA group, on the other hand, was fed with basal diet plus 2% UA and 4% OP, and orally gavaged sterile skim milk (0.5 mL), while the UA + JL-3 group received a basal diet containing 2% UA and 4% OP, while being orally gavaged with the JL-3 strain in sterile skim milk. About 1×10During the 15 d of treatment, all mice in each group were moved into a clean and empty cage every three days for an hour, and fecal and urine samples were collected. Later, the mice were anesthetized using diethyl ether. Blood was collected from the eye vein and was left undisturbed for 30 min. The collected blood samples were centrifuged at 4000 rpm for 20 min to get the serum. Also, liver, kidney and intestine samples were gathered for the respective groups. The collected samples were then washed with saline, collected in pre-weighted tubes with 1 mL PBS diluent, and stored at −20°C for future use. All samples were preserved at −80°C until analysis, except those used for histopathology, which were stored in 4% paraformaldehyde.4.6.Limosilactobacillus fermentum as reported in previous literatures.81 All primer sequences for qPCR are listed: specific primers of JL-3 , were used to obtain PCR products. Then, semi-quantitative fluorescence analysis of JL-3 was performed using Image J-win v.32.During the experiment, mice feces were collected after 4, 6, 8, and 10 d. PCR was then performed using specific primers of the E. coli, after which the obtained PCR products were cloned into T vector  and transfected into DH-5α, which was then incubated at 37°C for 6 h (100 −140 rpm) by Plasmid Mini Kit I  as the standard sample. The recombinant plasmids were used for qPCR, with 103, 104, 105, 106, 107, 108, 109, and 1010 copies of the plasmids per reaction used for calibration. The target copy numbers (T) were then estimated using the equation: T = (D/(PL × 660)) × 6.022 × 1,023,133, Where D (g/l) and PL (base pairs) were the plasmid DNA concentrations and lengths, respectively. Each standard curve was then generated from at least five 10-fold plasmid dilutions in triplicate. The proportion of JL-3 in the whole bacterial community of bacteria in different samples was calculated based on the standard curve.83On the 22nd day, fresh mouse feces were cultured in the MRS medium, and incubated for 48 h at 37°C in a static incubator, then sub-cultured again. Mouse fecal DNA was then extracted according to the manufacturer’s instructions using TIANamp Stool DNA Kit. To quantify the total bacteria in the feces samples, primers tot-F and tot-R were used for amplifying the 16S rDNA, while was run on a Bio-RAD CFX96 (USA) in a total volume of 10 μL, using SYBR Premix Ex Taq II (Takara) on real-time quantification PCR instrument . All measurements were performed in triplicate. Standard curves were then constructed using PCR products of the 16S rRNA gene of JL-3 and 4.7.2O2), and carbon dioxide. In the presence of horseradish peroxidase, H2O2 reacts with 10- acetyl-3, 7-dihydroxyphenoxazine to produce the highly fluorescent compound resorufin. Resorufin fluorescence was then analyzed at an excitation wavelength of 530 nm and an emission wavelength of 595 nm, after which the concentration of UA in the serum and urine samples were calculated using the equation determined from different doses of standards.53The frozen mouse feces in 1 ml PBS were placed in a 60°C water bath for 10 min, and vortexed for 1 min to break up the stool sample. The solution was collected by centrifugation at 1000 rpm for 5 min. Fresh mouse urine was placed in 60°C water bath and diluted 10 times with distilled water to ensure that no urate deposits were present in the urine sample, after which all samples were strictly detected according to the instructions provided in the UA detection kit . This kit provides a fluorescence-based method for detecting UA. In the assay, uricase transforms UA to allantoin, hydrogen peroxide  fecal suspension. The pH of the suspension was then adjusted to 2–3 by adding 5 M HCl, and then kept at room temperature for 10 min under occasional shaking. The suspension was then transferred into a polypropylene tube and centrifuged for 20 min at 5000 rpm, to give a clear supernatant, while the internal standard, 2-ethylbutyric acid solution (2EB), was spiked into the supernatant at a final concentration of 1 mm and injected in the GC for analysis. The response linearity for fecal samples was tested with the serial solutions prepared by diluting the above serial standard solutions with equal volume of fecal supernatant, after which the concentration of SCFAs were measured by GC-MS chromatographic workstation. N2 was used as the carrier. The concentration of SCFAs in the supernatant was detected by chromatographic column (FFAP 30 m × 0.32 mm × 0.5 µM) and FID detector, and the properties of SCFAs were determined afterward. The content of SCFAs in each sample was then calculated by comparing with the internal standard 2EB.4.9.Fresh liver and kidneys were ground using a handheld grinder  on ice, after which the suspension of samples was obtained at low speeds centrifugation . The levels of XOD and IL-1β in the liver, MDA and IL-1β in kidney, and IL-1β, MDA, creatinine (CRE), and urea nitrogen (BUN) in serum samples were all then detected using commercial kits  according to the protocols provided. At least 5 samples from each group were selected for independent assay.The liver and kidney samples were then rinsed with PBS, after which the liver was taken with a volume of 0.8 × 0.8 cm, while whole kidneys were employed. The samples were fixed with 4 % paraformaldehyde solution at room temperature for more than one day. Later, the samples were sent to the medical college of Lanzhou university for physiological sections and staining with hematoxylin and eosin (HE). The morphology of the sections and the accumulation of UA in mice renal tubules in different groups were observed under a microscope  afterward.4.10.85 and then incubated for 48 h at 37°C in an anaerobic incubator. The samples were then transferred to a new anaerobic incubator for two days. The cell content in the medium was detected using OD600(METASH UV-5100). The media contents were centrifuged at 8,000 × g for 10 min with an equivalent community to obtain cell pellets . The obtained cell pellets were then washed twice with pure sterile PBS and resuspended in pure PBS solution containing UA. Supernatant of the solution was taken from the anaerobic incubator every three days. The UA detection kit  was used to detect the UA concentration changes according to the direction of the manufacturer.86Mice stool samples were collected at different time points. At first, 1 g of stool was cultured in the YCFA medium as reported in literature,4.11.2O using a modified TIANamp Stool DNA Kit  protocol. The obtained DNA was then stored at −20°C until use. According to the selection of sequencing regions, diluted genomic DNA served as templates, with specific primers having barcode and high-fidelity enzymes  to ensure PCR amplification efficiency and accuracy. PCR was performed for 16S v4-v5 region of bacteria using a primer pair 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAA-3′).87 The PCR mixture (25 μl) contained 1x PCR buffer, 1.5 mM MgCl2, and deoxynucleoside triphosphate at 0.4 μM, each primer at 1.0 μM and 0.5 U of Ex Taq  and 10 ng soil genomic DNA. The PCR amplification protocol included initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. Two PCR reactions were conducted for each sample, and combined together after PCR amplification.88 DNA concentration and purity were measured by NanoDrop . The PCR product was detected by 1% agarose gel electrophoresis. The product was recovered from the target strip using the DNA Gel Extraction Kit (OMEGA), and the concentration and quality of the product were determined by Nanodrop. The same amounts of samples were mixed according to the concentration of PCR products. The library was constructed with TruSeq® DNA PCR-Free Sample Preparation Kit and quantified using Qubit and qPCR (PRJNA691734). Later, it was sequenced by an Illumina sequencing platform (Miseq PE250),89 and the resulting sequence read files using the QIIME2 pipeline  and its plugins. Paired-end sequence splicing was also performed using flash .91 The reads of each sample were spliced, and the spliced sequences were raw tags. Singleton was removed from OTUs . The OTU representative sequences were annotated by QIIME2 software, using silva-132-99-515-909-nb-classifier.qza as a reference database. Alternatively, the chloroplast and mitochondrial sequences of eukaryotes were removed, and QIIME2 software used to analyze observed species, evenness, Shannon, Faith’s PD and other diversity indexes were calculated, and dilution curve was drawn afterward. According to OTU table and phylogenetic tree, UniFrac distance matrix was generated and weighted, after which the UniFrac PCoA map was drawn.92The feces were collected from individually housed mice the day before gavage treatment initiation, and stored at −80°C until DNA collection. The stool sample pellets were weighed to 0.2 g and DNA, isolated in ddH4.12.P-value < 0.05 indicated statistical significance. Data were analyzed using one-way analysis of variance (ANOVA). The significant differences between the two groups were analyzed using an independent samples T-test. To determine the composition of the bacterial community, PCoA was performed using Rversion v.3.3.2.Statistical analysis was performed using SPSS v.17.0 software. Click here for additional data file.


Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is  TGF-β signaling is an important cell signaling system through which cells respond to external information. In the TGF-β system, signaling is initiated when a ligand dimer pair binds to a receptor tetramer. Interestingly, in numerous developmental contexts, TGF-β signaling has a greater response to heterodimeric ligands (dimers of multiple ligands), as compared to homomeric ligands (dimers made of two molecules of a single ligand). However, neither the cause of heterodimer signaling primacy, nor the systemic effects of heterodimeric vs homomeric signaling are understood. We use a biophysically-informed computational modeling approach to investigate the system dynamics of heterodimer-heterotetramer BMP signaling, to understand the cause and consequence of the requirement for Bmp2/7-mediated signaling in dorsoventral patterning in zebrafish development. Using our model, we demonstrate that BMP heterodimer signaling complex formation is not kinetically favored over homodimer signaling complexes, suggesting subfunctionalization of BMP receptors may be required to explain heterodimer signaling. Additionally, we show that heterodimer signaling provides a performance advantage via increased range of morphogen interpretation. Our findings provide insight into the systems principles involved in developmental signaling. The differentiation of biological systems is directed by the interpretation of biochemical morphogen gradients in a concentration-dependent manner. The remarkable robustness and precision of developmental patterning reveals careful organization of the underlying developmental signaling systems. These systems are highly evolutionarily conserved, and through the use of mathematical modeling, the foundational principles that guide the evolution of these systems are beginning to be understood. The Bone Morphogenetic Protein (BMP) signaling pathway is an essential signaling system that regulates development as well as a wide range of cellular processes in both vertebrates and invertebrates –4. In caDrosophila development, BMP ligand homologues Decapentaplegic (Dpp) and Screw (Scw) signal through two type I receptors (Saxophone and Thickvein) and two type II receptors (Punt) [Drosophila wing disc and wing vein patterning [Interestingly, in several biological systems BMP ligand heterodimers demonstrate stronger biological activity than BMP ligand homodimers. In s (Punt) . Dpp-Scws (Punt) . Heterodtterning ,8.exclusively mediated by heterodimers [bmp2b or bmp7a is sufficient to induce a complete loss of BMP signaling in the embryo [The primacy of BMP heterodimer signaling is conserved evolutionarily and has been identified in several vertebrate contexts including, bone regeneration, axon guidance, and stem cell differentiation ,2,9–12. rodimers . Mutant e embryo . FurtherDespite the experimental evidence for the centrality of BMP heterodimer-heterotetramer signaling during embryogenesis, the system properties that privilege heterodimer-heterotetramer signaling in the BMP system remain unclear ,2,14. PrIntriguingly, however, BMP heterodimer-heterotetramer signaling in the simulations exhibit important performance advantages over other ligand receptor complexes- suggesting a selective advantage for signaling via heterodimer-heterotetramer in pattern formation or cell signaling control. Specifically, the heterodimer-heterotetramer exhibits a larger dynamic range than all other plausible BMP ligand dimer-receptor tetramer complexes in a broad set of parameter conditions. In other words, the BMP heterodimer-heterotetramer acts as a superior sensor compared to other BMP ligand-receptor combinations because its ligand input concentrations correspond linearly to tetrameric receptor signaling complex levels over a larger concentration range. Recent computational modeling work shows ,17 that 2 heterotetramer signaling, is that the kinetics of Bmp2/7 ligand binding to receptors favors the formation of this species over the other eight tetrameric signaling complexes omplexes . To screomplexes  to steadomplexes . Values omplexes .2-(Type II)2 is the most prevalent, followed by Bmp2/7-(BmpR1)2-(Type II)2, and then Bmp7-(BmpR1)2-(Type II)2. The fourth most-abundant complex is Bmp2/7-Acvr1-BmpR1-(Type II)2, the required signaling complex in embryonic dorsoventral patterning. Even when the homodimer ligands are removed from the model, leaving only the Bmp2/7 ligand, ligand and receptors favor formation of the Bmp2/7 complex with two BmpR1 and two type II receptors 2 and Bmp2/7-(BmpR1)2-(Type II)2 were the most abundant tetramer complexes in 61.89% and 20.01% of parameter conditions 2 homomeric complex formation, except at high ligand concentrations (0.4 to 1 nM of each ligand), where Bmp2/7-Acvr1-BmpR1-(Type II)2 becomes the most prevalent receptor complex 2 complex over a wide range of ligand concentrations 2 and Bmp7/7-(BmpR1)2-(Type II)2 have minimal signaling ability. Analysis of our prevalence screen  of simulations. To further investigate heterodimer-heteromeric receptor signaling in this scenario, we tested the potential contribution of individual receptor levels on expected BMP signaling levels. In model simulations, increasing concentration of Acvr1 leads to increased Bmp2/7 heteromeric receptor signaling at low BmpR1 levels, but has minimal effect at higher BmpR1 concentrations (2) formation; initially greater BmpR1 levels lead to small to moderate increases in heterodimer-homomeric tetramer complexes, which are reversed as BmpR1 concentrations continue to rise  receptor .in silico analysis revealed that the heteromeric complex, Bmp2/7- Acvr1-BmpR1-(Type II)2 is the fourth-most prevalent based on the published kinetic rates. Instead, BmpR1 homomeric complexes Bmp2-(BmpR1)2-(Type II)2, Bmp2/7-(BmpR1)2-(Type II)2, and Bmp7-(BmpR1)2-(Type II)2 are more prevalent. This contrasts with the known signal exclusivity or dominance by the heterodimer, suggesting that the increased signaling must be caused by enhanced kinase activity of the heterotetrameric complex. The simulations also provide insight into potential performance advantages afforded by reliance on heterodimer signaling through the heterotetramer receptor complexes, suggesting potential explanations for their presence in morphogen signaling systems.To summarize, our https://github.com/akmadamanchi/BMPOligomerizationModel. Our full mathematical model is implemented in python using the PySB library which employs the variable coefficient ode solver (VODE), and facilitates implementation on high-performance computing systems and enables large scale screen of parameters [To construct our mathematical model, we devised a biochemical network for BMP ligand-receptor oligomerization that contained all theoretically possible bidirectional interactions between the three ligand-dimers  and the three BMP receptor species . An Ordinary Differential Equation (ODE) was constructed for each of the 51 possible BMP ligand-receptor oligomers; This ODE network simulates each of the 90 possible bidirectional interactions involved in BMP ligand-receptor oligomerization. A schematic of the oligomerization process for each ligand dimer is shown in rameters ,26. Mathnd order enhancement is specifically due to the spatial restriction and greater concentrating effect of the localization [The initial interaction between ligand and receptor occurs between a secreted molecule and a surface localized receptor. Subsequent events take place in a smaller, more restricted two-dimensional environment as the remaining receptors needed for tetramer formation are also surface localized. Ligand-receptor interaction rates which are experimentally observed in 3D must be converted to an increased rate of reaction through a general mechanism known as a reduction of dimensionality that has been used to investigate other aspects of BMP-mediated signaling ,27. We ilization .Dpp oligomerization in Drosophila [To adapt experimental biophysical data for the rectangular geometry of the cell membrane and immediate extracellular region we make a reduction of dimensionality adjustment for the secondary and tertiary receptor recruitment processes that take after the initial ligand binding reaction as previously done by us and others ,29. Addiosophila .The mathematical model we developed is remarkably well supported by biophysical measurements for ligand-receptor binding ,30,31, lNumerous crystal structures of BMP ligands, receptors, and their co-crystals show that BMP homodimers have four receptor binding sites, including two equivalent Type I and two equivalent Type II binding sites ,31–33. BD) of ~0.8 and 6.4nM, respectively [D’s of 47 and 56 nM, respectively [D of~500 nM [D affinity for Bmp7, and no or extremely weak affinity (KD>1000 nM) for Bmp2; similarly, we assume that Bmp2/7 binds to one BmpR1 receptor at a high affinity Bmp2 domain, and binds another BmpR1 receptor at a low affinity Bmp7 domain. The high affinity site is consistent with the measured affinity of Bmp2/7 to BmpR1 [BMP homodimer:receptor binding affinity  results ectively ,31,40. Bectively ,39. The f~500 nM ,35. In ainity KD>00 nM forto BmpR1 ,34S1 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic β2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β 2-adrenergic receptor-dependent signaling may prove beneficial as novel antidiabetic drugs.In this study, the authors demonstrated that agents targeting skeletal muscle metabolism by modulating β Current non-insulin therapies of T2D focus mostly on targeting β-cell dysfunction , hepatic and adipose tissue insulin resistance , and elimination of excess glucose through the kidneys or gut 2. Moreover, SKM insulin resistance is a key metabolic defect in the pathogenesis of T2D2. For these reasons, SKM is a very attractive target for the development of novel classes of antidiabetic drugs. G protein-coupled receptors (GPCRs) represent a highly druggable class of cell surface proteins. In fact, ~35% of all approved pharmaceuticals target specific GPCRs3. SKM expresses dozens of different GPCRs endowed with distinct G protein-coupling properties4. In vitro studies have shown that activation of Gq-coupled GPCRs can regulate glucose uptake into SKM cells7. By using DREADD (Designer Receptor Exclusively Activated by a Designer Drug) technology, we recently demonstrated that receptor-mediated activation of Gq signaling in SKM tissue in vivo leads to greatly improved glucose tolerance8.At present, antidiabetic drugs that directly target skeletal muscle (SKM) are not available, even though SKM is responsible for 80–90% of glucose disposal during a euglycemic-hyperinsulinemic clamp in healthy individuals2-adrenergic receptor (β2-AR) agonist that is approved for the treatment of asthma and chronic obstructive pulmonary disease in several European and Asian countries . Similar to the β1- and β3-AR subtypes, the β2-AR primarily activates the stimulatory G protein Gs, leading to increased intracellular cAMP levels and the activation of protein kinase A(PKA) and other cAMP-dependent effector proteins9. However, several studies have shown that β2-AR can also modulate cellular signaling via stimulation of cAMP inhibitory Gi-type G proteins12. Moreover, activated β2-ARs, such as other GPCRs, can recruit β-arrestins (β-arrestin-1 and -2), which can also act as signaling proteins15.Clenbuterol is a synthetic, highly selective β2-ARs are expressed at high levels in SKM, and clenbuterol and other β2-AR agonists have been reported to prevent or reverse muscle wasting or weakness by promoting SKM growth17. Interestingly, treatment of rodents with clenbuterol has been shown to improve glucose homeostasis20. However, the β2-AR is not only expressed in SKM, but also in many metabolically important tissues, including pancreatic islets and adipose tissues22. For these reasons, it remains unclear which tissues or cell types, and which cellular signaling pathways are involved in mediating the metabolically beneficial actions of clenbuterol in vivo. Specifically, the role of β2-ARs in regulating SKM glucose fluxes remains controversial. Although some studies reported β2-AR-mediated inhibition of glucose uptake into SKM26, other studies reported the opposite effect (stimulation of glucose uptake)27.β2-ARs, and that the coupling of these receptors to SKM Gs is essential for the manifestation of these clenbuterol effects. In agreement with these findings, selective activation of SKM Gs signaling by a Gs-coupled designer GPCR caused similar improvements in glucose homeostasis. Moreover, unbiased metabolomic and RNA-sequencing (RNA-Seq) studies shed light on the molecular mechanisms underlying the clenbuterol-induced improvements in SKM glucose homeostasis following β2-AR stimulation. These findings are of considerable relevance for the development of novel antidiabetic drugs targeting SKM glucose metabolism.In this study, by analyzing several newly developed mouse models, we demonstrate that the beneficial metabolic effects of chronic clenbuterol are mediated by activation of SKM β2-ARs in vivo, we administered clenbuterol in drinking water (30 mg/l) to wild-type (WT) mice for 5 days, followed by a series of metabolic tests. Clenbuterol treatment of lean male and female WT mice led to improved glucose tolerance ) for 5 consecutive days. Seven days after the last STZ injection, the mice developed characteristic signs of β-cell dysfunction including elevated fasting and fed blood glucose levels  treatment protocol known to destroy about 70–80% of mouse β-cells2-ARs are not expressed in adult mouse hepatocytes29, we speculated that the beneficial effects of clenbuterol on glucose homeostasis . The SKM-selective deletion of the β2-AR and Gαs was induced in adult mice via tamoxifen-dependent activation of Cre recombinase in SKM only. Efficient deletion of β2-AR and Gαs in SKM was confirmed via quantitative real-time PCR (qRT-PCR) or western blotting, respectively  mice may be due to the fact that SKM tissue does not only consist of myocytes but also harbors several other cell types30. The lack of SKM β2-ARs in lean mice did not lead to any obvious metabolic phenotypes . Remarkably, in the absence of SKM β2-ARs or SKM Gαs, the ability of clenbuterol treatment to improve glucose tolerance was abolished . During the past decade, DREADDs have been successfully used to study cell type-specific metabolic consequences of activating different functional classes of G proteins31.As βs-linked GPCR in SKM, we generated mice that expressed a Gs-coupled DREADD (GsD)36 under the transcriptional control of the SKM-specific human α-skeletal actin (HSA) promoter. We refer to these mice from hereon simply as SKM-GsD mice. In the absence of CNO, male and female SKM-GsD mice did not show any significant changes in glucose homeostasis, as compared to their control littermates  via the drinking water for 1 week and then subjected to a series of metabolic tests. At the end of the CNO treatment period, male  as a tracer. We found that CNO-treated SKM-GsD mice showed a significant increase of glucose uptake into multiple SKM tissues (quadriceps and gastrocnemius muscles)  Fig. . No chanes) Fig. .To explore the changes in SKM cell metabolism following long-term clenbuterol treatment, we used unbiased high-throughput metabolomics and transcriptomics approaches. Metabolomic analysis of SKM (quadriceps muscle) from lean WT mice treated with clenbuterol or regular water identified a total of 648 metabolites. Principal component analysis showed a clear separation of the two experimental groups based on their biochemical profile -2-bromopalmitate ([3H]-BROMO), a radioactive labeled analog of palmitate that is not a substrate for β-oxidation, but becomes trapped in slower metabolic processes of ω- and α-oxidation37. We did not observe differences in [3H]-BROMO levels in SKM, liver, or adipose tissues of mice treated with clenbuterol water, as compared with mice consuming regular drinking water . We found that 13C enrichment in C16-, C14-, and C12-acylcarnitines was stable at 25 min after [U-13C]-palmitate injection, and that similar enrichment levels were achieved in SKM of control mice, indicative of a pseudo isotopic steady state  drinking water for 5 days . We found that the improvements in glucose homeostasis caused by clenbuterol treatment were significantly reduced in SKM-Rictor-KO mice  was set for the initial analysis. SKM (gastrocnemius muscle) samples derived from control mice treated with clenbuterol showed 2639 differentially expressed genes , as compared to vehicle-treated mice , as compared to SKM samples from vehicle-treated SKM-Rictor-KO mice , respectively) that were differentially expressed only in SKM tissue of clenbuterol-treated control mice or clenbuterol-treated SKM-Rictor-KO mice, respectively  caught our particular attention, because Plin3 represents a regulator of lipid metabolism downstream of the mTORC2 complex38. Previous work has shown that SKM-Rictor-KO mice shift to lipids as energy source by increasing Plin3 expression38. Clenbuterol treatment significantly decreased Plin3 expression in SKM of control mice, but not in SKM-Rictor-KO mice, where SKM Plin3 expression was increased in the absence or presence of clenbuterol  that allowed us to monitor the metabolic outcome of selective activation of SKM Gs signaling. In addition, comprehensive unbiased metabolomic and RNA-Seq studies provided important clues regarding the molecular mechanisms underlying the clenbuterol-induced improvements in SKM glucose homeostasis.The present study has led to several important findings regarding the cellular and molecular mechanisms by which chronic clenbuterol treatment improves glucose homeostasis and other metabolic functions. We provide convincing evidence that the beneficial metabolic effects of chronic clenbuterol depend on the stimulation of SKM β2-AR-KO and SKM-Gs-KO mice , were significantly downregulated in SKM following treatment with clenbuterol . Clenbuterol treatment decreased SKM gene expression levels of glycogenin (Gyg) and Gsk3b  led to an increase in non-oxidative disposal of glucose, e.g., in form of glycogen; however, the cellular mechanisms underlying this effect were not explored54.Long-term treatment of human subjects with a β2-ARs in regulating SKM glycogen metabolism in a glucose/fuel-dependent manner. More detailed studies involving the infusion of stable glucose isotopes will be required to clearly define the fractions of glucose cycled via the glycogen and glycolysis pathways in SKM following clenbuterol treatment.In the present study, clenbuterol treatment significantly affected the inhibitory phosphorylation of GS (pGS) 57. Although our SKM RNA-Seq data did not reveal changes in classic muscle fiber markers like Myh7, Myh1, and Myh2, our unbiased metabolomic and transcriptomic approaches provided evidence for β2-AR-mediated SKM metabolic reprogramming towards enhanced glucose utilization at both the transcriptional and metabolite level, consistent with the outcome of the study be Zeman et al.57.A previous histological study reported that long-term β2-AR is also abundantly expressed in human SKM (https://www.genecards.org/cgi-bin/carddisp.pl?gene=ADRB2), suggesting that clenbuterol may also cause beneficial effects on glucose homeostasis in humans. Clenbuterol and other β2-AR agonists are well known for their therapeutic potential in treating muscle wasting and related SKM disorders16. Moreover, clenbuterol is used for the treatment of asthma and related pulmonary diseases in several European and Asian countries , due to its ability to act on bronchial smooth muscle β2-ARs. Kim et al.58 recently reported that clenbuterol-induced SKM hypertrophy in mice requires SKM β-arrestin-1 signaling. As the beneficial effects of clenbuterol on glucose homeostasis are mediated by SKM Gs signaling, it should be possible to develop novel classes of β2-AR agonists that stimulate SKM Gs but do not recruit β-arrestins. The data by Kim et al.58 suggest that the potential use of such G protein-biased β2-AR agonists for the treatment of SKM insulin resistance will not lead to the development of SKM hypertrophy.The β2-AR agonist (clenbuterol) significantly improves glucose homeostasis under physiological and pathophysiological conditions. We demonstrated that the metabolic improvements observed after a 5-day clenbuterol administration require the activation of the SKM β2-AR/Gs complex and downstream AMPK and mTORC2 signaling. These signaling events eventually lead to a metabolic reprogramming of SKM towards enhanced glucose utilization. No other tissues or G protein signaling pathways appear to be involved in the clenbuterol-dependent metabolic improvements.In conclusion, by analyzing multiple new mouse models and using unbiased multiomic approaches, we provide clear evidence that systemic administration of a selective βs signaling in SKM  or of components of downstream signaling pathways represents an attractive approach to develop novel classes of antidiabetic drugs targeted at modifying SKM metabolism.Our data strongly suggest that activation of G2-ARs. Although we clearly demonstrated that the beneficial metabolic effects seen after a 5-day clenbuterol treatment are dependent on SKM-β2-ARs, we cannot completely exclude the possibility that the clenbuterol-induced changes in SKM  cause secondary changes in other tissues. The static metabolomic analyses that we performed provide a good overview of changes in SKM metabolite levels; however, infusion studies with isotope-labeled metabolite tracers are needed to draw clear conclusions about metabolite fluxes. Moreover, the precise molecular mechanisms through which chronic activation of SKM β2-AR affects AMPK and mTORC2 signaling to alter SKM fuel utilization remain to be explored in future studies.Our study primarily focused on SKM phenotypes and did not explore the effect of systemic clenbuterol on other tissues expressing βThe sources of antibodies, chemicals, assay kits, and mouse lines used in this study are listed in Supplementary Table All mice were fed ad libitum and kept on a 12 h light, 12 h dark cycle at room temperature. Mice were maintained either on a standard mouse chow  or a HFD  for at least 8 weeks.All animal studies were carried out according to the US National Institutes of Health Guidelines for Animal Research and were approved by the NIDDK Institutional Animal Care and Use Committee.T2) mice59. To avoid potential developmental changes, Cre recombinase activity was induced in 7- to 8-week-old HSA-Cre(ERT2)-positive floxed/floxed mice by i.p. injection with tamoxifen (2 mg per day dissolved in corn oil) for 5 consecutive days. Tamoxifen-injected Cre-negative floxed/floxed littermates served as control mice.To generate SKM-specific KO and knock-in mice, floxed mice  for plasma collection and plasma insulin levels were determined using an ultra-sensitive mouse insulin ELISA kit .Unless stated otherwise, all metabolic tests were performed on male littermates (age range: 10–30 weeks) using standard protocols. For i.p. GTTs (ipGTT), mice were fasted overnight for 15 h and blood glucose levels were measured before (0 min) and at defined time points after i.p. injection of normal saline containing glucose . For ITTs, mice were fasted for 4 h and blood glucose levels were measured before (0 min) and at indicated time points after i.p. injection of human insulin . Blood glucose levels were determined using a manual blood glucose meter . To study GSIS, mice were fasted overnight for 15 h and blood samples were collected in heparinized capillary tubes (Fisher Scientific) before (0 min) and 5, 15, and 30 min following the i.p. injection of glucose . Samples were centrifuged  or regular drinking water for 5 days prior to metabolic experiments or tissue collection. During this 5-day period, mice consumed 3.5 ± 0.06 ml of clenbuterol water per day, resulting in an average clenbuterol dose of 104.7 ± 1.2 μg per mouse per day. For chronic clenbuterol studies performed with transgenic mice, baseline ipGTTs were performed with control and the indicated KO mice as described above. Two days later, both control and mutant mice started to consume clenbuterol drinking water. After 5 days on clenbuterol water, mice were subjected to another ipGTT.s signaling in SKM, control and SKM-GsD mice received CNO continuously for 7 days by mixing CNO into the drinking water at a concentration of 250 mg/l, as described previously8.For long-term stimulation of G28, which has been previously shown to destroy about 70–80% of β-cells. Specifically, lean WT mice were fasted for 5 h and injected with a relatively low dose (50 mg/kg i.p.) of STZ for 5 consecutive days. Metabolic studies were initiated seven days after the last STZ injection.To induce β-cell dysfunction, STZ treatment protocol has been used61  or vehicle was provided for the initial 24 h of a 36 h treatment with clenbuterol drinking water. These experimental conditions were chosen to selectively inhibit the mTORC1 complexMouse body mass composition (lean and fat mass) was determined using a 3-in-1 EchoMRI Analyzer .2 consumed/CO2 produced), food intake, and locomotor activity (assayed by beam breaks) were measured simultaneously in mice housed at 22 °C using an Oxymax/CLAMS (Columbus Instruments). Sampling was performed every 13 min, measuring from 12 chambers. Mice were adapted in the chambers for 2 days and then baseline data were collected for 3 days. Water and food were provided ad libitum during the sampling period. For chronic clenbuterol studies, mice consumed clenbuterol (30 mg/l) or regular drinking water for 5 days. Treatment data were recorded for the last 3 days of clenbuterol treatment  (Perkin Elmer). Mice were killed 2 h later, and SKM and other tissues were collected. The tissue content of [14C]2-DG-6-phosphate was determined as a measure of SKM glucose uptake, as described previously62.To measure glucose uptake into individual tissues in vivo, mice were fasted overnight for 14–16 h and then injected i.p. with saline containing 2 g/kg glucose and 10 μCi of [3H]-glucose. Blood samples were collected from the tail vein before and 5, 10, 15, 30, 45, and 60 min post injection. Plasma samples were processed as described previously and glycolysis rate was estimated as the rate of 3H2O appearance63.Overnight (15 h) fasted mice were injected with glucose (2 g/kg i.p.) containing 2.5 mCi/kg  ([3H]-BROMO), a partially metabolizable long-chain fatty acid tracer . Tissues (~150 mg) were collected 1 h later and solubilized in 1 ml of Biosol  for 3 h in a 50 °C water bath, followed by another 1 h incubation in the presence of 0.3 ml 30% hydrogen peroxide. Samples were mixed with 10 ml of scintillation solution  and counted using a Tri-Carb 2910 TR liquid scintillation analyzer . Data were normalized to mg of tissue.In vivo fatty acid uptake was examined in WT male mice treated with clenbuterol (30 mg/l) or regular drinking water for 5 days. Mice were fasted overnight (15 h) and injected i.p. with 200 μl of olive oil containing 2 μCi of (R)-2-bromopalmitic acid -palmitate (50 μg/kg i.p.). The injection solution was prepared as follows: [U-13C]-palmitate powder was dissolved in 50% ethanol and further diluted in sterile 0.9% saline containing bovine serum albumin (BSA) to the final concentration . BSA coupling of palmitate was achieved by shaking the incubation solution overnight at 37 °C. Mice were killed 25 min after the injection and isolated tissues were snap-frozen in liquid nitrogen for metabolite extraction.Lean WT mice  consumed clenbuterol water (30 mg/l) or regular drinking water for 5 days. On the day of the experiment, overnight (15 h) fasted mice were injected with [U-69. UPLC-QTOFMS analysis was performed in a Waters SYNAPT G2-Si in positive and negative electrospray ionization modes. Data were analyzed using Targetlynx XS and the percentage of neutral isotope was calculated as a peak area ratio of complete 13C-labeled metabolite/12C-labeled metabolite × 100%.Quadriceps muscles were homogenized in 14 µl/mg tissue of 3 : 4 water : methanol using a Precellys tissue homogenizer (setting: shaking at 6500 r.p.m. twice for 30 s). Chloroform containing 0.5 µM C17 ceramide  was added to each sample at a ratio of 16 µl/mg tissue. The organic layer was transferred to a glass tube and dried under a gentle stream of nitrogen. Samples were reconstituted in isopropanol/methanol/chloroform (4/2/1) and 5 μl was injected on to a Waters ACQUITY ultra-performance liquid chromatography (UPLC) CSH C18 1.7 μm column (2.1 × 100 mm) maintained at 55 °C. The mobile phase, flow rate, and gradient conditions were as previously describedTissues were isolated quickly, frozen in liquid nitrogen, and stored at −80 °C until use. For western blotting studies, frozen tissues were homogenized in ice-cold RIPA buffer (Sigma Aldrich) and protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific). Protein extracts were separated on NuPAGE 4–12% Bis-Tris or, for high molecular mass protein targets, on 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and blotted onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h at room temperature in TBS-T (0.1%) containing 5% BSA. Membranes were then incubated overnight with primary antibodies at 4 °C. Following three washing steps in TBS-T (0.1%), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After thorough washing, proteins were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) on the c600 Imaging System (Azure Biosystems). Immunoreactive bands were quantified using Image J Software (NIH). Uncropped images are provided in the Source Data file.t-test, as appropriate. A p-value of <0.05 was considered statistically significant. The specific statistical tests that were used are indicated in the figure legends. Precise p-values are provided in the Source Data File.Data are expressed as mean ± SEM for the indicated number of observations. Data were assessed for statistical significance by two-way ANOVA tests, followed by the indicated post hoc tests, or by using a two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary