Thermogenesis and muscle growth -
Different letters indicate significant differences. Comparison of shivering intensity between A day 1 and B day 5 for the same individual ID 2 of mother I.
the expected frequency range in which shivering should occur in piglets. SERCA activity, as well as SERCA1a and SLN mRNA expressions increased significantly from day 1 to day 5 of the juveniles´ life Fig.
There was generally more variability in the gene expression data at day 5 than at day 1 Fig. Depicted is the range of individual data dots as well as the mean values line. SERCA1a and SLN gene expression were significantly increased on day 5. Statistical test results are reported in the text.
Correlation of SERCA1a and SLN gene expression. Partial regression plots of the effect of A SERCA1a and B SLN gene expression on SERCA activity. This result is clear evidence for an increasing contribution of NST to thermogenesis during cold exposure in piglets over the first days of life.
The finding that SERCA activity and the expression of SERCA1a and SLN were recruited simultaneously points to muscle NST and increased SERCA activity as the principal source of heat production, as we can rule out UCP1- mediated NST in this species.
We observed a high variability in the gene expression data at day 5, suggesting that there is some heterogeneity to the individual response rate to cold exposure. Alternatively, this could reflect different micro-environmental conditions e. Further strong evidence would be provided from measurements of increased SLN protein content.
Unfortunately, in this study, the limitation of sample material available for analyses and the lack of sufficiently high quality antibodies for wild boar precluded us from adding this extra confirmation of the mechanism. Our statistical analysis suggests that the increase in SERCA activity between day 1 and day 5 was mainly due to an SLN controlled upregulation of ATP hydrolysis by SERCA1a instead of an increase in SERCA1a molecules.
However, we cannot exclude that there are also other processes or tissues involved. For example, a study on cold-acclimatization of UCP1-knockout mice has found changes in gene expression levels in inguinal white adipose tissue between cold-adapted wild type and knockout mice, while no change was detected in skeletal muscle However, when kept under cold conditions juvenile mice keep SLN upregulated for improved thermoregulatory capacity 31 , suggesting that both mechanisms of NST are necessary for an effective maintenance of a high T b during cold exposure in newborn rodents.
In adult mice, however, both mechanisms of NST, UCP1-mediated as well as muscle NST, can compensate for the loss of one system Interestingly, in other precocial species, such as sheep and goats that possess functional UCP1, BAT is recruited already before birth 1.
Furthermore, reconstituted function of UCP1 can further improve thermoregulatory function of cold exposed 6-months old Bama pigs, a cold-sensitive pig breed. Our finding that muscle NST is involved in thermoregulation of juvenile wild boars and allows a near stable T b even during short-term cold exposure supports the hypothesis that muscle NST may be the primary mechanism of heat production during cold-exposure in large mammals lacking BAT 2 , While the evolution of BAT has often been related to the ability of small placental mammals to colonize colder habitats 4 , 51 , 52 , a recent study has shown that UCP1-inactivating mutations have occurred in at least eight of the 18 placental mammalian orders, mainly in larger-bodied species 9 , such as pigs.
It therefore appears that the combination of shivering and muscle NST is sufficient for heat production in large mammals. Pigs, for example, likely lost UCP1 function and the ability to use BAT for thermoregulation because of absent or only weak selection for this mechanism in a warm climate 33 ; all species except the wild boar live only in tropical or subtropical habitats.
In addition to heat production via muscle NST, wild boar apparently evolved compensatory mechanisms to cope with adverse thermal conditions in northern habitats, such as larger adult body size 53 , building insulating nests for offspring, and synchronizing reproduction within social groups or enabling piglets to huddle in large groups of combined litters 33 , Behavioural thermoregulation is less energetically costly than NST 55 and a study on winter mortality of juvenile wild boar has shown that the negative effects of cold winters can be compensated by high availability of food resources In addition to our finding that SLN-mediated NST in skeletal muscle is involved in piglet thermoregulation, recent studies on domestic pig breeds suggest that SERCA2b another isoform of SERCA and UCP3 might also influence pig thermoregulation 56 , However, so far the importance of both mechanisms is unclear 2 and the evolution of a compensatory mechanism after the pigs colonized cold habitats is likely 56 , while muscle NST is discussed as a potentially evolutionary old heat production mechanism 2 , Whether and to what extend domestic pig breeds also possess muscle NST remains speculative.
Therefore, it cannot be ruled out that the extreme susceptibility of pigs to cold is partly due to inadvertent selection against high thermogenic capacity during domestication. An earlier study on thermoregulation of young domestic pigs ~13 days old showed that piglets dealt with a five day cold exposure with a decrease in core T b and an increase in insulation by postural changes Previous studies on thermoregulation of juvenile domestic pigs have also found that shivering intensity decreased during the first days after birth while heat production and blood flow to muscles simultaneously increased 59 , 60 , While this was originally attributed to an increase in shivering efficiency 59 , 60 , it seems questionable whether an increased thermogenesis by increased efficiency of shivering is physically possible.
Our data now suggest that the improved thermogenesis found in domestic pigs, similarly to wild boar, was not due to an increase in shivering efficiency, but explained by an increase in muscle NST. Taken together, our data show for the first time that muscle-based NST via SERCA1a plays a role in the thermoregulation of wild type mammals lacking BAT and that muscle NST can replace UCP1-mediated NST.
The function of UCP1 as a thermogenic protein has occurred after the divergence between placental and marsupial mammals 22 , suggesting that the evolution of endothermy in ancestral mammals was independent of heat production in BAT.
Although the earth was likely warmer, ancestral mammals still would have experienced daily and yearly fluctuations in T a , likely similar to temperatures found in tropical areas today, which can get rather cold during the night. Therefore, while heat produced as a by-product of metabolic processes as well as basking 62 would have allowed the establishment of a stable T b during a big part of the day, muscle NST was likely important during the colder night hours.
The study was approved by the institutional ethics and animal welfare committee and the national authority according to §§ 26ff. of Animal Experiments Act, Tierversuchsgesetz — TVG BMWFW Cannon, B.
Brown adipose tissue: function and physiological significance. Article CAS PubMed Google Scholar. Nowack, J. Muscle non-shivering thermogenesis and its role in the evolution of endothermy. Hansen, E. Parallel measurements of heat production and thermogenin content in brown fat cells during cold acclimation of rats.
Heaton, G. Brown-adipose-tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation. Eur J Biochem 82 , — Hayward, J.
Evolution of brown fat: its absence in marsupials and monotremes. Article Google Scholar. Vatnick, I. Regression of brown adipose tissue mitochondrial function and structure in neonatal goats. E Article CAS Google Scholar. Alexander, G. sp Article CAS PubMed PubMed Central Google Scholar. Gilsanz, V.
Relevance of brown adipose tissue in infancy and adolescence. Article PubMed Google Scholar. Gaudry, M. et al. Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades. Article ADS CAS PubMed PubMed Central Google Scholar. Barnett, S. The growth of infant mice at two temperatures.
Bal, N. Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Nature Medicine 18 , — de Meis, L.
Regulation by ADP. M Babu, G. Differential expression of sarcolipin protein during muscle development and cardiac pathophysiology. Increased reliance on muscle-based thermogenesis upon acute minimization of brown adipose tissue function. Pant, M. Sarcolipin: a key thermogenic and metabolic regulator in skeletal muscle.
Anderson, K. Multi-omic analysis of hibernator skeletal muscle and regulation of calcium handling Master of Science thesis, University of Minnesota Proteogenomic analysis of a hibernating mammal indicates contribution of skeletal muscle physiology to the hibernation phenotype.
Rowland, L. The role of skeletal-muscle-based thermogenic mechanisms in vertebrate endothermy. Biol Rev Saito, S. Adaptive evolution of the uncoupling protein 1 gene contributed to the acquisition of novel nonshivering thermogenesis in ancestral eutherian mammals. Periasamy, M.
SERCA pump isoforms: their role in calcium transport and disease. Hasselbach, W. Die Calciumpumpe der Erschlaffungsgrana des Muskels und ihre Abhängigkeit von der ATPSpaltung. Biochemische Zeitschrift , — CAS PubMed Google Scholar.
Mall, S. Asahi, M. Maurya, S. Sarcolipin is a key determinant of the basal metabolic rate, and its overexpression enhances energy expenditure and resistance against diet-induced obesity. MacLennan, D. The regulation of SERCA-type pumps by phospholamban and sarcolipin. x Article ADS CAS PubMed Google Scholar.
Mild cold induced thermogenesis: are BAT and skeletal muscle synergistic partners? Cold adaptation overrides developmental regulation of sarcolipin expression in mice skeletal muscle: SOS for muscle-based thermogenesis?
Article PubMed PubMed Central Google Scholar. Jastroch, M. When pigs fly, UCP1 makes heat. Berg, F. The uncoupling protein 1 gene UCP1 is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets.
Hou, L. Pig has no uncoupling protein 1. R Development Core Team. R: a language and environment for statistical computing R Foundation for Statistical Computing, Vienna, Austria, Lighton, J. Measuring metabolic rates. A manual for scientists. Oxford University Press, Schmidt-Nielsen, K. Animal physiology: adaptation and environment.
Cambridge University Press, Kienzle, P. Signal: signal processing. Spaan, G. Die Frequenz des Kältezitterns bei Tierarten verschiedener Größe. Two processes of metabolism Our metabolism is complex — put simply it has 2 parts, which are carefully regulated by the body to make sure they remain in balance.
They are: Catabolism — the breakdown of food components such as carbohydrates , proteins and dietary fats into their simpler forms, which can then be used to provide energy and the basic building blocks needed for growth and repair.
Anabolism — the part of metabolism in which our body is built or repaired. Anabolism requires energy that ultimately comes from our food.
When we eat more than we need for daily anabolism, the excess nutrients are typically stored in our body as fat. Thermic effect of food also known as thermogenesis — your body uses energy to digest the foods and drinks you consume and also absorbs, transports and stores their nutrients.
Energy used during physical activity — this is the energy used by physical movement and it varies the most depending on how much energy you use each day. Physical activity includes planned exercise like going for a run or playing sport but also includes all incidental activity such as hanging out the washing, playing with the dog or even fidgeting!
Basal metabolic rate BMR The BMR refers to the amount of energy your body needs to maintain homeostasis. Factors that affect our BMR Your BMR is influenced by multiple factors working in combination, including: Body size — larger adult bodies have more metabolising tissue and a larger BMR.
Amount of lean muscle tissue — muscle burns kilojoules rapidly. Crash dieting, starving or fasting — eating too few kilojoules encourages the body to slow the metabolism to conserve energy. Age — metabolism slows with age due to loss of muscle tissue, but also due to hormonal and neurological changes.
Growth — infants and children have higher energy demands per unit of body weight due to the energy demands of growth and the extra energy needed to maintain their body temperature. Gender — generally, men have faster metabolisms because they tend to be larger. Genetic predisposition — your metabolic rate may be partly decided by your genes.
Hormonal and nervous controls — BMR is controlled by the nervous and hormonal systems. Hormonal imbalances can influence how quickly or slowly the body burns kilojoules. Environmental temperature — if temperature is very low or very high, the body has to work harder to maintain its normal body temperature, which increases the BMR.
Infection or illness — BMR increases because the body has to work harder to build new tissues and to create an immune response. Amount of physical activity — hard-working muscles need plenty of energy to burn. Regular exercise increases muscle mass and teaches the body to burn kilojoules at a faster rate, even when at rest.
Drugs — like caffeine or nicotine , can increase the BMR. Dietary deficiencies — for example, a diet low in iodine reduces thyroid function and slows the metabolism. Thermic effect of food Your BMR rises after you eat because you use energy to eat, digest and metabolise the food you have just eaten.
Hot spicy foods for example, foods containing chilli, horseradish and mustard can have a significant thermic effect. Energy used during physical activity During strenuous or vigorous physical activity, our muscles may burn through as much as 3, kJ per hour.
Metabolism and age-related weight gain Muscle tissue has a large appetite for kilojoules. Hormonal disorders of metabolism Hormones help regulate our metabolism. Thyroid disorders include: Hypothyroidism underactive thyroid — the metabolism slows because the thyroid gland does not release enough hormones.
Some of the symptoms of hypothyroidism include unusual weight gain, lethargy, depression and constipation. Hyperthyroidism overactive thyroid — the gland releases larger quantities of hormones than necessary and speeds the metabolism.
Some of the symptoms of hyperthyroidism include increased appetite, weight loss, nervousness and diarrhoea. Genetic disorders of metabolism Our genes are the blueprints for the proteins in our body, and our proteins are responsible for the digestion and metabolism of our food.
Some genetic disorders of metabolism include: Fructose intolerance — the inability to break down fructose, which is a type of sugar found in fruit, fruit juices, sugar for example, cane sugar , honey and certain vegetables. Galactosaemia — the inability to convert the carbohydrate galactose into glucose.
Galactose is not found by itself in nature. It is produced when lactose is broken down by the digestive system into glucose and galactose. The roles of Musclin secreted from these tissues in the regulation of tissue homeostasis and energy metabolism remain to be explored in future studies.
Interestingly, Musclin can be repurposed in primates through the evolutionary acquisition of DNA regulatory elements to regulate neuronal structure and function features that are unique to primates 39 , suggesting a species-specific function of Musclin.
More recently, Musclin was shown to function as an exercise-responsive myokine 40 , to attenuate the pathogenesis and progression of several cardiovascular diseases including heart failure during pathological overload 41 , hypertension 42 , cardiac remodeling and congestive heart failure after myocardial infarction 43 , and chronic doxorubicin-induced cardiotoxicity 44 in animal models.
In addition, low circulating Musclin levels are associated with adverse prognosis of patients undergoing transcatheter aortic valve implantation TAVI 45 , and potential atrial fibrillation in non-diabetic patients Taken together, these findings demonstrate the tissue-specific and context-dependent roles of Musclin under physiological and pathological conditions.
Using SEAP binding assay, we demonstrated that Musclin mainly binds to adipose tissues, especially subcutaneous iWAT, rather than other metabolic tissues, such as skeletal muscle and liver. Further studies revealed that elevated circulating Musclin selectively acts on iWAT to suppress thermogenic gene expression programs, resulting in impairment of beige fat thermogenesis, lower energy expenditure, and augmentation of HFD-induced obesity and metabolic dysfunction.
Consistently, both MCK-Musclin and Musclin-MKO mice used in this study exhibited no detectable differences in muscle morphology, exercise performance, or energy metabolism-related gene expression profiles compared to their respective controls. However, a previous study demonstrated that Musclin whole-body knockout mice displayed impaired oxidative metabolism in skeletal muscle and reduced exercise tolerance compared to controls following five consecutive days of exercise training Three reasons might contribute to this discrepancy.
Firstly, to exclude the potential confounding effects of Musclin on muscle development and bone growth 23 , 36 , we generated mice carrying muscle-specific transgenic expression and knockout of Musclin using MCK promoter 47 and MLC-Cre 48 , respectively. This lies in contrast to the Musclin whole-body knockout mice utilized in their study.
Secondly, different running protocols and testing conditions were used in the two studies. Whilst our evaluation incorporated all the muscle-related parameters in sedentary mice, in their research mice were subjected to 5 consecutive days of exercise training prior to assessment They actually found that differences in endurance running capacity and markers of mitochondrial biogenesis were much less evident in sedentary WT and Musclin-KO mice than those in exercise-trained WT and Musclin-KO mice.
Thirdly, different receptors and associated downstream signal pathways might be another reason for the tissue-specific effects of Musclin on mitochondrial biogenesis in skeletal muscle and adipose tissue. However, in this study, we identified Tfr1 as a receptor for Musclin in adipocytes, and revealed that Tfr1-mediated cAMP signaling plays a pivotal role in the regulation of thermogenic metabolism in subcutaneous white adipose tissue.
However, recent studies have demonstrated distinguishing features and mechanisms underlying the developmental origins and functional regulation of these two cell types 14 , This study demonstrated that Musclin selectively repressed gene expression networks related to lipid and glucose metabolism in the subcutaneous beige fat depot, resulting in the impairment of thermogenesis and augmentation of HFD-induced obesity and metabolic dysfunction.
This highlighted a distinct regulatory mechanism related to beige fat thermogenic metabolism beiging with a unique role in the pathogenesis of obesity and its associated metabolic dysfunction. Receptors are generally indispensable for mediating the vital role of secreted proteins in the crosstalk among tissues.
Musclin has been shown to function as a ligand to Npr3, competing with natriuretic peptides NPs , to amplify the NP signaling 36 , As such, it is possible that Musclin may elicit its effects on adipocyte thermogenesis and metabolism through regulating the circulation levels of NPs and the downstream cGMP levels, a key player in mediating NP-associated responses Consistently, a recent study by Szaroszyk M et al.
Conversely, plasma CNP levels were lower in muscle-specific Musclin deficient mice versus control mice after TAC surgery However, plasma ANP levels were either trending lower or higher in Musclin overexpressing mice after myocardial infarction 43 or TAC surgery 41 , respectively.
Moreover, it has been reported that plasma ANP levels were trending lower, but did not reach statistical significance, in Musclin whole-body KO mice compared to WT controls following exercise training These results revealed an overall modest effect of Musclin overexpression or deficiency on plasma ANP levels in mice.
Consistent with these findings, we observed that plasma ANP levels remained largely unchanged in MCK-Musclin mice compared to control mice in response to cold exposure or HFD feeding Supplementary Fig. Notably, plasma CNP levels were significantly increased in MCK-Musclin mice compared to controls under either cold exposure or HFD feeding conditions Supplementary Fig.
The cGMP, generated by the guanylyl cyclase domain of NPRA receptor for ANP and BNP and NPRB receptor for CNP , is a key mediator of the NP-associated biological responses It has been reported that Musclin could enhance cardiomyocyte cAMP generation through cGMP-mediated inhibition of cAMP-degrading phosphodiesterase 3 PDE3 Previous studies showed that both ANP and CNP could increase the intracellular cGMP levels in adipocytes 51 , Consistently, we also demonstrated that both ANP Supplementary Fig.
However, co-treatment with Musclin exhibited no obvious effect on intracellular cGMP levels in response to increasing amounts of ANP or CNP Supplementary Fig. Interestingly, using the proximity-dependent biotin identification BioID assay and mass spectrometry analysis, we identified Tfr1 as the membrane receptor for Musclin in mediating its inhibitory effect on the thermogenic metabolism in beige adipocytes.
Tfr1, as a member of the transferrin receptor family, is known to play an important role in cellular iron homeostasis through the endocytosis of transferrin-bound iron 53 , One recent study has demonstrated that Tfr1-mediated control of cellular iron levels is important for regulating white adipose tissue homeostasis Intriguingly, accumulating evidence indicates that other iron-independent mechanisms might also be involved in the regulation of biological function by Tfr1 Accordingly, Tfr1 was uncovered to play an essential role in regulating brown and beige fat development and thermogenesis via both iron-dependent and -independent mechanisms 29 , Consistent with the selectivity of Musclin on the thermogenesis of beige adipocytes, we observed that the protein levels of Tfr1 in adipocytes from iWAT were significantly higher than those from eWAT and BAT in WT mice following chronic cold exposure Supplementary Fig.
However, whether iron-dependent and -independent mechanisms are involved in this process warrants further investigation. In summary, this work identifies the myokine Musclin as a critical negative regulator of beige fat thermogenesis that acts in concert with other thermogenesis activators to fine-tune systemic energy balance under both physiological and pathophysiological conditions.
Muscle expression of Musclin is regulated by ambient temperature and is essential for maintaining body temperature upon acute and chronic cold stresses, thus indicating its physiological role in gauging systemic energy expenditure.
Musclin gain-of-function inhibits beige fat thermogenesis and augments HFD-induced obesity and metabolic disorders. More importantly, blocking Musclin actions, either by genetic ablation or neutralizing antibody treatment, promotes energy expenditure and alleviates HFD induced-obesity and metabolic dysfunction.
These findings highlight the therapeutic potential of Musclin inactivation for treating obesity and its associated metabolic disorders.
All animal studies were performed in compliance with the Guide for the Use and Care of Laboratory Animals by the Medical Experimental Animal Care Committee of Zhejiang University.
All animal studies were performed following the protocols approved by the Animal Ethics Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University Approval number: The study on human plasma was approved by the Second Affiliated Hospital of Soochow University Approval number: JD-LK Human biological samples including skeletal muscle from a total of 54 donors; 41 male and 13 female; data shown in Fig.
Sex was comparable between groups, as shown in Supplementary Fig. Sex information for human sample-related results shown in Fig. Only the participants who had provided written informed consent were included in the study.
All participants representing donor tissue samples were subjected to medical history inquiries before hospitalization. Blood biochemical tests were taken after overnight starvation. Subjects with other severe diseases, including malignant tumors and severe obesity-independent cardiovascular disorders, were also excluded.
Moreover, to minimize the confounding effect of previous injury in the current findings, the human muscle samples were only collected from patients who have fully recovered from acute injury with confirmation of the absence of acute inflammation prior to ligament repair or reconstruction treatments.
BMI is the only criterion to divide the human muscle samples into different groups. As such, we assumed that the physical activity should be similar between groups. All the patients for scWAT collection were checked for full recovery from acute injury and confirmation of the absence of inflammation before surgery.
Fat samples from subjects with malignant tumors were excluded. All the tissue samples were freshly collected and immediately frozen in liquid nitrogen. Human blood samples were collected from subjects with differing BMIs, while subjects with malignant tumors, organic lesions, or having previously undergone surgery within several months to half a year were excluded.
Male mice were used for all the experiments unless otherwise indicated. On rare occasions, mice in bad health conditions, such as severe fighting wounds or exhibiting sickness, according to the predefined criteria listed in the Institutional Animal Care and Use Committee IACUC protocol were excluded.
All in vivo experiments were independently repeated at least twice. To generate skeletal muscle-specific Musclin transgenic MCK-Musclin mice by pronuclear microinjection, a full-length Musclin coding sequence was placed downstream of the 4. Liwei Xie Guangdong Institute of Microbiology.
Adipoq-Cre JAX stock mice were generously provided by Dr. Fudi Wang Zhejiang University , and have been previously described 29 , Cre expression in Adipoq-CreERT2 mice was activated through intraperitoneal administration of tamoxifen at the dose of 0.
All mouse strains used in this study were born at the expected Mendelian ratios with normal fertility. Musclin neutralizing Ab preparation was performed by ABclonal Technology Co.
Through epitope prediction, the peptide sequence C-HSKKRFGIP-Nle-DRIGRNR corresponding to aa of Musclin was synthesized and used for immunizing rabbits to generate antibodies. Polyclonal antibodies against Musclin were obtained from inoculated rabbits. Antibodies were purified using affinity chromatography on columns containing the corresponding peptides.
Female mice were only used for the cold exposure study shown in Fig. The mouse age and sex information for each experiment has also been included in the corresponding figure legend.
ExpiF cell line was obtained from Thermo Fisher. AAV cell line was purchased from Agilent. HEKT, ExpiF, and AAV cell lines were authenticated and routinely used in our previous studies 2 , 59 , 60 , HUVEC cell line was kindly provided by Dr. Nan Xu Henan University , and has been authenticated and successfully used in their previous study AAV production and purification were performed by ChuangRui Bio Lianyungang, China.
Culture media were replaced with DMEM plus 0. Cell lysates were gently transferred onto the top layer, and the remaining volume of the ultracentrifuge tube was filled with cell lysis buffer. The viral titer was determined by qPCR assay with a standard curve generated by serial dilutions of the AAV shuttle vector.
Mice were granted free access to pre-chilled food and water during the whole assay. Core body temperature was monitored at indicated time points using a portable intelligent digital thermometer TH During this period, metabolic parameters including mouse body temperature, body weight, blood glucose, as well as plasma TG and NEFA levels were monitored.
Body surface temperature was measured using a thermal imaging camera FLIR Systems, Tsc InfraRed Camera. Mice were anesthetized with isoflurane and quietly laid on a whiteboard with their back up, followed by image capturing with the camera anchored at the same height for all mice in the same batch.
FLIR Tools 5. Food and water were freely accessible to mice. O 2 consumption, CO 2 production, energy expenditure, total locomotor activity, and food intake were monitored under both normal conditions and under adrenergic stimulation.
The metabolic parameters for each mouse were measured two days before CL injection and were continued to be monitored for one more day after CL injection.
Mouse running performance was assessed using the treadmill running system from Columbus Instruments. The inclination angle was level. Total running distance and running time were recorded at the point when the mice reached exhaustion. After full differentiation, myotubes were subject to RNA isolation and gene expression analysis.
ExpiF cells were transfected with pcDNA3. The XF96 microplate was then loaded into a Seahorse XFe96 analyzer for equilibration and determination of the basal respiration rate. OCRs were recorded and the ATP production, maximal respiration, and the spare respiratory capacity-dependent OCRs were calculated.
Cell viability was also quantified using the CCK8 kit Beyotime for normalization. The XF Cell Mito Stress test kit Agilent was used in this assay. Seahorse Wave Desktop and Controller 2. An NMR analyzer NIUMAG, QMNH was applied to measure the body fat and lean mass.
An enzyme-linked immunosorbent assay ELISA kit from Crystal Chem was used to measure plasma insulin concentration. For the detection of Musclin levels in human plasma, a Human Musclin ELISA kit CUSABIO, CSB-Eh was applied.
For the detection of ANP levels in mouse plasma, a mouse ANP ELISA kit Elabscience, E-EL-Mc was used. For the detection of CNP levels in mouse plasma, a Mouse CNP ELISA kit Beijing Sino-UK Institute of Biological Technology, HY-NE was applied.
Mouse tissues including liver, skeletal muscle, BAT, eWAT, and iWAT were dissected, fixed in formalin, followed by embedding in paraffin and cutting for tissue sections at μm for liver, skeletal muscle, and BAT, or μm for eWAT and iWAT.
Tissue sections were then stained with hematoxylin and eosin, and subjected to image acquisition using NIS Elements F 4. The cell size of iWAT was quantified using Image J 1.
The SEAP-binding assay was performed as described Firstly, the vectors expressing SEAP or SEAP-Musclin fusion protein were transiently transfected into HEKT cells. The sections were washed with PBS containing 0. CellSens Standard Olympus was used for image acquisition and data collection.
Total RNA from WAT was isolated using a commercially available kit TIANGEN Biotech, DP Total RNA from other tissues including skeletal muscle, heart, brain, small intestine, liver, BAT, bone, and cultured cells was isolated following the standard method using TRIzol. RNA was then reverse transcribed using HiScript II Q RT SuperMix Vazyme, R , followed by qPCR analysis using SYBR Green Roche.
The qPCR primers used are listed in Supplementary Data. Sequencing libraries were constructed from total RNA using SMART-RNAseq Library Prep Kit Hangzhou KaiTai, AT In brief, mRNA was isolated from total RNA with Sera-Mag Magnetic Olido dT particles, and then chemically fragmented.
And the cDNA libraries were subsequently amplified using the KAPA high-fidelity DNA polymer. Quality of the libraries was validated by the Bioanalyzer Agilent Technologies. Subsequently, high-throughput sequencing was performed using a NovaSeq Illumina. Raw reads were filtered with fastp V0.
Filtered data were then aligned with HISAT2 V2. p13 for human data. The FPKM fragments per kilobase of exon per million fragments mapped was calculated using StringTie V2. For iWAT from cold-acclimated and room temperature-housed control WT mice, RNA sequencing was performed in Majorbio Bio-pharm Biotechnology Co.
Shanghai, China. The library was prepared using TruSeqTM RNA Sample Prep Kit Illumina. Shortly, mRNA was isolated by oligo dT beads and then fragmented.
After quantified by TBS, paired-end RNA sequencing was performed using the NovaSeq Illumina. The raw reads were processed similarly and the TPM transcripts per million reads values were used to determine gene expression levels.
For human scWAT, RNA sequencing was performed in BGI Shenzhen, China. Similarly, mRNA was purified using oligo dT -attached magnetic beads and was then fragmented.
cDNA was generated by random hexamer-primed reverse transcription, and end-repaired through incubation with a-Tailing Mix and RNA Index Adapters. The obtained cDNA fragments were amplified using PCR followed by purification with Ampure XP Beads, which was validated on the Bioanalyzer Agilent Technologies.
Distinctively, further heat-denaturation and circularization by the splint oligo sequence of the PCR products from previous step were needed to get the final library. Pair end base reads were generated for analysis. The sequencing data was filtered with SOAPnuke V1.
p12 with HISAT2 V2. Expression level of gene FPKM value was calculated by RSEM V1. For all these sequencing data, differential expression analysis was performed using the Deseq2 v. Gene Ontology GO and pathway grouping and enrichment studies were performed by clusterProfiler V3.
Html Results were visualized by ggplot2 V3. html 70 and pheatmap V1. Protein lysates from skeletal muscles and adipose tissues, or whole-cell lysates from cultured cells were quantified using BCA protein assay Beyotime.
The final results were visualized with chemiluminescence ECL western blotting substrates. A proximity-dependent biotin identification BioID assay was used to identify the receptors for Musclin on the plasma membrane of adipocytes.
The agarose beads were pelleted by centrifugation and then subjected to five times washing with washing buffer, followed by protein denaturation, SDS-PAGE gel electrophoresis, and Coomassie blue staining or silver staining Pierce Silver Stain Kit , Thermo Fisher.
The gel fragments in pulldown samples containing differential protein bands as compared to control were collected for electrospray ionization tandem M. analysis on a Thermo Finnigan LTQ Orbitrap Instrument Proteome Discoverer version 1. The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE 74 partner repository with the dataset identifier PXD HEKT cells were transiently transfected with plasmids expressing control or Musclin.
Finally, cells and culture media were harvested separately. Cells were subjected to total protein lysate preparation. The obtained protein pellet was washed in cold acetone, air-dried, and finally resuspended in SDS-containing lysis buffer.
The input and IP samples were subjected to immunoblotting using antibodies against Musclin Abcam , Flag Sigma, A, M2 , or HSL Cell signaling, s. Cell proteins were quantified using a BCA protein assay kit Beyotime for normalization.
Tissue weight was used for normalization. A standard chromogen method was used to measure the tissue non-heme iron described as previously described All statistical analyses were performed using GraphPad Prism 9 software.
n values represent biological replicates for cell experiments, or mouse number for in vivo animal studies, or human subject number unless otherwise indicated. Specific details for the n value are noted in each figure legend. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
RNA-Seq data of iWAT from cold-acclimated mice and their controls are available in SRA database under accession code: PRJNA The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 74 partner repository with the dataset identifier PXD , which can be freely accessed.
The images representing human participants, mouse models, skeletal muscle, myotube, adipose tissue, RNA-Sequencing, qPCR analysis, culture dish, and adipocyte shown in Figs. Source data are provided with this paper.
Meng, Z. et al. Glucose sensing by skeletal myocytes couples nutrient signaling to systemic homeostasis. Cell 66 , — e Article CAS PubMed PubMed Central Google Scholar.
Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation. Diabetes 63 , — Uncoupling exercise bioenergetics from systemic metabolic homeostasis by conditional inactivation of Baf60 in skeletal muscle.
Diabetes 67 , 85—97 Article CAS PubMed Google Scholar. Pedersen, B. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Febbraio, M. Who would have thought—myokines two decades on.
Article PubMed Google Scholar. Whitham, M. Extracellular vesicles provide a means for tissue crosstalk during exercise. Cell Metab. Giudice, J.
Muscle as a paracrine and endocrine organ. Article CAS Google Scholar. Priest, C. Inter-organ cross-talk in metabolic syndrome. Ishibashi, J. Beige can be slimming. Science , — Petrovic, N. Chronic peroxisome proliferator-activated receptor gamma PPARgamma activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.
Rosen, E. What we talk about when we talk about fat. Cell , 20—44 Ikeda, K. The common and distinct features of brown and beige adipocytes. Trends Endocrinol.
Wu, J. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell , — Matthew, H. Brown and beige fat: development, function and therapeutic potential. Article Google Scholar. Townsend, K. Brown fat fuel utilization and thermogenesis.
UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis. Med 23 , — Kaisanlahti, A. Browning of white fat: agents and implications for beige adipose tissue to type 2 diabetes.
Biochem 75 , 1—10 Carobbio, S. Brown and beige fat: From molecules to physiology and pathophysiology. Acta Mol. Cell Biol. lipids , 37—50 Blüher, M. Obesity: global epidemiology and pathogenesis.
Wang, Q. The hepatokine Tsukushi gates energy expenditure via brown fat sympathetic innervation. Article PubMed PubMed Central Google Scholar. Xiong, X. Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis.
Cell 75 , — Thomas, G. Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype. Nishizawa, H. Musclin, a novel skeletal muscle-derived secretory factor. Kazak, L. A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat.
Genetic depletion of adipocyte creatine metabolism inhibits diet-induced thermogenesis and drives obesity.
Ablation of adipocyte creatine transport impairs thermogenesis and causes diet-induced obesity. Roux, K. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.
Li, J. CAS PubMed Google Scholar. Tsai, T. Sim, J. P2X1 and P2X4 receptor currents in mouse macrophages. Shen, W. Interaction of rat hormone-sensitive lipase with adipocytelipid-binding protein. Natl Acad. USA 96 , — Article ADS CAS PubMed PubMed Central Google Scholar. Bostrom, P. A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.
Nature , —
Thermogenwsis refers to Tbermogenesis the chemical processes going on continuously Inflammation relief techniques your Ginseng for hair growth that allow life Thermkgenesis normal functioning maintaining normal functioning Thermogenesis and muscle growth the body is called homeostasis. Thermogeensis processes include those that break down nutrients from our food, and those that build and repair our body. Building and repairing the body requires energy that ultimately comes from your food. The amount of energy, measured in kilojoules kJthat your body burns at any given time is affected by your metabolism. Achieving or maintaining a healthy weight is a balancing act. The development of sustained, long-term endothermy was one of Ginseng for hair growth major Ginseng for hair growth in Thermognesis evolution of Thermogenesis and muscle growth. Anx in endotherms does not only occur via shivering Managing type diabetes activity, but also Groth non-shivering thermogenesis Grwoth. Mammalian NST is mediated by TThermogenesis uncoupling protein 1 in the brown muscpe tissue BAT and possibly involves an additional mechanism kuscle NST in skeletal muscle. The existence of muscle based NST has been discussed for a long time and is likely present in all mammals. However, its importance for thermoregulation was demonstrated only recently in mice. Interestingly, birds, which have evolved from a different reptilian lineage than mammals and lack UCP1-mediated NST, also exhibit muscle based NST under the involvement of SERCA, though likely without the participation of sarcolipin. In this review we summarize the current knowledge on muscle NST and discuss the efficiency of muscle NST and BAT in the context of the hypothesis that muscle NST could have been the earliest mechanism of heat generation during cold exposure in vertebrates that ultimately enabled the evolution of endothermy.
Ich kann die Verbannung auf die Webseite suchen, auf der viele Artikel in dieser Frage gibt.
Die Idee gut, ist mit Ihnen einverstanden.
Mir scheint es die ausgezeichnete Phrase
Sie haben Recht.